WO2020022891A2 - Biomarkers for atrial fibrillation - Google Patents

Biomarkers for atrial fibrillation Download PDF

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Publication number
WO2020022891A2
WO2020022891A2 PCT/NL2019/050480 NL2019050480W WO2020022891A2 WO 2020022891 A2 WO2020022891 A2 WO 2020022891A2 NL 2019050480 W NL2019050480 W NL 2019050480W WO 2020022891 A2 WO2020022891 A2 WO 2020022891A2
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Prior art keywords
suffering
mitochondrial
individual
nucleic acid
atrial fibrillation
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WO2020022891A3 (en
Inventor
Bianca Johanna Josephina Maria Brundel
Marit WIERSMA
Natasja Mireille Silvia DE GROOT
Deli ZHANG
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Erasmus University Medical Center
Vrije Universiteit Medisch Centrum VUMC
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Erasmus University Medical Center
Vrije Universiteit Medisch Centrum VUMC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to methods of typing an individual as suffering from, or being at risk of suffering from, atrial fibrillation. Furthermore, the invention relates to treatment of an individual that was typed as suffering from, or being at risk of suffering from, atrial fibrillation according to the methods of the invention.
  • Atrial fibrillation is the most common sustained clinical
  • proteostasis i.e. the homeostasis of protein synthesis, folding, assembly, trafficking, function and degradation.
  • proteases Activation of proteases (Brundel et al., 2002.
  • Circulation 129: 346-358 contribute to degradation of contractile and structural proteins, resulting in proteostasis derailment and structural remodeling.
  • in vitro AF initiation leads to increased RhoA activation, changes in structural proteins and failure to mount the heat shock response (Ke et al., 2011. PLoS One 6: e20395; Meijering et al., 2015. PLoS One 10: e0133553).
  • mitochondrial dysfunction is detrimental for the heart. This is exemplified by the vast amount of cardiac diseases caused or worsened by mitochondrial dysfunction. Therefore, we examined the mitochondrial function in experimental models of AF remodeling and in AF patients. Furthermore, we determined whether cell-free circulating mitochondrial DNA represents a biomarker in a cohort of
  • the invention provides a method of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of providing a bodily fluid, preferably blood, from said individual, quantifying a level of long non coding RNA (lncRNA), mitochondrial nucleic acid and/or a level of DNA damage in said bodily fluid, preferably of lncRNA and mitochondrial nucleic acid, comparing said quantified lncRNA, mitochondrial nucleic acid and/or level of DNA damage to a reference and typing said individual as suffering from, or at risk of suffering from, atrial fibrillation if said quantified lncRNA is altered, compared to the reference, if the quantified mitochondrial nucleic acid is altered, compared to the reference, and/or if said quantified level of DNA damage is altered compared to the reference.
  • lncRNA long non coding RNA
  • an increase of a level of lncRNA, a mitochondrial nucleic acid and/or a level of DNA damage when compared to a healthy individual, can be used to identify an individual suffering from self-terminating stages of atrial fibrillation (AF) who is at risk of progressing to more persistent AF, while a decrease of a mitochondrial nucleic acid and a level of DNA damage, when compared to a healthy individual, can be used to identify an individual suffering from persistent stages of AF who is at risk of progressing to long-standing persistent AF.
  • AF atrial fibrillation
  • the level of DNA damage is preferably quantified by determining a level of 8- oxoguanine.
  • Said lncRNA preferably is selected from Sarrah, UCA1, CDR1AS and
  • the mitochondrial nucleic acid in methods of the invention preferably is or comprises mitochondrial DNA.
  • Said mitochondrial nucleic acid preferably comprises nucleic acid from a cytochrome C oxidase 3 gene and/or a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene.
  • a preferred method of the invention comprises quantifying mitochondrial DNA from a cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L,
  • a further preferred method of the invention comprises quantifying a level of lncRNA, a level of nucleic acid from a mitochondrial cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene, and quantifying a level of DNA damage in said bodily fluid.
  • a method of the invention may further comprise determining a level of heat shock protein the bodily fluid of the individual, preferably of HSPB7.
  • the invention further provides a method for assigning standard-of-care therapy to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention; and assigning a standard-of-care therapy to the individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation.
  • the standard-of-care therapy preferably is selected from anti-arrhythmic drug therapy aimed at rate and/or rhythm control, electrical or chemical cardioversion, and/or ablative therapy.
  • Said standard-of-care therapy preferably is combined with an antiplatelet and/or anticoagulant.
  • Said antiplatelet and/or anticoagulant preferably is selected from aspirin, warfarin, and a direct- acting oral anticoagulant such as dabigi trail, rivaroxaban, edoxaban and apixaban.
  • Said rhythm controller preferably is an ion channel blocker, such as a sodium channel blocker such as flecainide, propafenone and/or quinidine, and/or a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
  • a sodium channel blocker such as flecainide, propafenone and/or quinidine
  • a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
  • the invention further provides a standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to the method the invention.
  • Tachypacing induces mitochondrial dysfunction.
  • OCR consumption rate showing the mitochondrial respiration.
  • E Representative CaTmito of HL-1 eardiomyocytes after normal pacing (NP) or tachypacing (TP).
  • F Quantified data showing mitochondrial membrane potential during tachypacing. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 versus NP, #P ⁇ 0.05, ##P ⁇ 0.01 versus NP.
  • Tachypacing induces mitochondrial network fragmentation and stress.
  • Figure 3 Inhibition of the MCU protects against mitochondrial stress and dysfunction.
  • FIG. 1 Mitochondrial changes are due to the MCU.
  • A) Top panel represent Western blot of MCU and GAPDH and lower panel reveals quantified date of MCU normalized for basal GAPDH levels.
  • NP neuropeptide
  • C Representative CaTmito of NP or TP HL-1 cardiomyocytes either non-transfected or transiently transfected with MCU, generating MCU overexpression (OE).
  • D Quantified CaTmito amplitude of NP and 6 hour TP HL-1 cardiomyocytes either non-transfected (C) or transfected with MCU.
  • E Quantified CaTmito amplitude of NP and 6 hour TP HL-1 cardiomyocytes either non-transfected (C) or transfected with MCU siRNA with 60% or 20% reduced MCU expression (high and low, respectively).
  • F Representative CaTmito of NP or TP HL-1 cardiomyocytes either non-transfected or transiently transfected with MCU siRNA.
  • G Quantified data showing heart wall contraction rates from Drosophila melanogaster from each group as indicated. White bars represent normal paced (NP in HL-1
  • cardiomyocytes or spontaneous heart rate (SR in Drosophila ) and black bars represent tachypaced HL-1 cardiomyocytes or Drosophila.
  • FIG. 5 AF patients show mitochondrial dysfunction.
  • C Cellular ATP levels in patients in SR and AF.
  • RAA right atrial appendages
  • FIG. 6 Cell-free circulating mitochondri l DNA is a potential biomarker for AF. Quantitative real-time PCR of the mitochondrial-transcribed cytochrome c oxidase subunit 3 (COX3) and NADH dehydrogenase subunit 1 (ND1) genes in serum of control and AF patients in different stages of AF. A+B) per stage of AF, C+D) per stage of AF and divided by gender, E+F) per stage of AF and divided whether patients had a recurrence within 1 year or not, G) correlation between COX3 and ND1. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 versus Control (male), #P ⁇ 0.05 versus control female. Figure 7.
  • COX3 mitochondrial-transcribed cytochrome c oxidase subunit 3
  • ND1 NADH dehydrogenase subunit 1
  • Tachypacing induces PARP activation, DNA damage and NAD+ depletion in HL-1 cardiomyocytes.
  • B) Relative NAD+ levels in HL-1 cardiomyocytes during time-course of TP (2 h-8 h) compared to control (0 h). *P ⁇ 0.05 vs 0 h. N 2 independent experiments and scalebar is 15 pm. Data are all expressed as mean ⁇ s.e.m.. Individual group mean differences were evaluated with the two-tailed Student’s t-test.
  • PARP1 is the key enzyme mediating tachypaeing- induced contractile dysfunction in HL-1 cardiomyocytes and Drosophila.
  • CTL non-paced
  • TP tachypaced
  • PARPli PARP1 siRNA
  • PARP2i PARP2 siRNA
  • B Arrhythmicity index in milliseconds (ms).
  • FIG. 9 PARP1 inhibitor prevents and reverses tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes.
  • Atrial fibrillation refers to an irregular and often rapid heart rate during which the atria beat irregularly and out of coordination with the two ventricles. Atrial fibrillation symptoms often include heart palpitations, shortness of breath and weakness. Atrial fibrillation is a risk facture for stroke, heart failure and other heart-related diseases.
  • paroxysmal atrial fibrillation specifically refers to self- terminating stages of atrial fibrillation, lasting less than 7 days. Later stages of atrial fibrillation are termed persistent atrial fibrillation, when atrial fibrillation occurs for more than a week, and long-standing persistent atrial fibrillation, when atrial fibrillation occurs for more than a year and rhythm control therapy is chosen.
  • mitochondrial DNA molecule refers to a double- stranded circular mitochondrial DNA molecule as well as RNA products, including transfer RNA (tRNA), rihosomal RNA (rRNA) and messenger RNA (mRNA) products of the mitochondrial DNA molecule.
  • Said mitochondrial DNA molecule is present in mitochondria of eukaryotic cells and comprises a total of 37 genes.
  • DNA damage refers in general to ROS-induced oxidative DNA damage involving single- or double-strand DNA breaks, purine and pyrimidine or deoxyribose modifications as well as DNA cross links.
  • a prominent marker that preferably is used to determine a level of DNA damage is provided by 7,8-dihydro-8-oxoguanine (8-oxoguanine).
  • heat shock protein refers to a protein that is produced by cells in response to exposure to stress such as heat, cold, UV light, wound healing or tissue remodeling. Heat shock proteins perform a chaperone function by stabilizing proteins to ensure correct folding. Heat-shock proteins are named according to their molecular weight.
  • bodily fluid refers to blood, urine, milk, cerebrospinal fluid, interstitial fluid, lymph, amniotic fluid, bile, cerumen, feces, female ejaculate, gastric juice, mucus pericardial fluid, pleural fluid, pus, saliva, semen, smegma, sputum, synovial fluid, sweat, tears, vaginal secretion, and vomit.
  • a preferred bodily fluid is blood.
  • blood includes reference to serum and plasma.
  • serum and plasma both refer to blood components without cells, whereby serum also excludes clotting factors such as fibrinogen.
  • blood may, for example, be centrifuged to remove cellular components.
  • the thus obtained plasma may be coagulated followed by, for example, centrifugation to remove the clotting factors.
  • the resulting serum is a most preferred bodily fluid.
  • the term“quantifying”, as is used herein, refers to determining a quantity of the level of mitochondrial nucleic acid and/or heat shock protein in a bodily fluid, preferably in blood.
  • Said quantity preferably refers to the presence and level of mitochondrial DNA in a bodily fluid, preferably in blood, most preferably in serum.
  • Methods to determine presence and quantify of DNA in a bodily fluid are known to a skilled person and include, but are not limited to, quantitative PCR, microarray analysis and DNA sequencing, especially next generation sequencing.
  • the determined DNA levels preferably are normalized for differences in the total amounts of nucleic acid molecules between two separate samples by comparing the level of mitochondrial DNA, for example to the level of nuclear DNA molecules of which the level is known not to differ between different bodily fluid samples.
  • the term“reference”, as is used herein, refers to a level of mitochondrial nucleic acid and/or a level of DNA damage in a bodily fluid of an individual that is known to suffer from, or, preferably, known not to suffer from, atrial fibrillation.
  • Said reference preferably is a value, preferably an average value, that is obtained from pooled, multiple individuals known to suffer from, or known not to suffer from atrial fibrillation. It is preferred that said reference is pooled from more than 10 individuals, more preferred more than 20 individuals, more preferred more than 30 individuals, more preferred more than 40 individuals, most preferred more than 50 individuals.
  • said reference may be a value, preferably average value, that is obtained from pooled, mixed individuals known to suffer from and/or known not to suffer from atrial fibrillation.
  • standard-of-care therapy refers to medical treatment to control heart rate and/or rhythm.
  • ablative therapy and/or cardioconversion either chemical or electrical, have emerged as an effective therapy for AF treatment.
  • Ablative therapy involves the generation of lesions in the atrial tissue to disrupt cells that provide superfluous electrical pulses.
  • Said standard-of-care therapy may be combined with an anticoagulant to reduce a risk of a stroke.
  • lncRNA long non-coding RNA or lncRNA
  • lncRNAs are now known to exhibit diverse functions through a wide array of mechanisms, and have been associated with diseases including cancer.
  • An overview of human lncRNA species has recently been published (Uszczynska-Ratajczak et ah, 2018. Nature Reviews Genetics 19: 535-548).
  • Preferred LncRNA include Sarrah (OXCT1-AS1; HGNC: 40423), urothelial carcinoma- associated- 1 (UCA1; HGNC:37126), CDR1 anti sense (CDR1AS; HGNC:48926) and long intergenic non-coding RNA predicting cardiac remodeling (LIPCAR; HGNC:50279).
  • the invention provides methods of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of providing a bodily fluid from said individual and quantifying mitochondrial nucleic acid and/or a level of DNA damage in said bodily fluid, comparing said quantified
  • mitochondrial nucleic acid and/or a level of DNA damage to a reference typing said individual as suffering from, or at risk of suffering from, atrial fibrillation if the quantified mitochondrial nucleic acid is increased or decreased, compared to the reference, and/or if said quantified level of DNA damage is increased or decreased compared to the reference.
  • the present invention provides general methods of diagnosing and/or prognosticating a subject as suffering from, or at risk of suffering from, atrial fibrillation using said general methods.
  • any and all of these embodiments are referred to, except if explicitly indicated otherwise.
  • a method of the invention can be performed on any suitable bodily fluid, such as, for example, blood, urine, mucus, especially mouth or cheek mucus, tear fluid and vitreous fluid.
  • a blood sample of a subject can be obtained by any standard method, for instance by venous extraction.
  • a bodily fluid, preferably blood, is preferably collected in a tube that is coated with anticoagulants such as EDTA, sodium citrate, and/or heparin.
  • anticoagulants such as EDTA, sodium citrate, and/or heparin.
  • the amount of a bodily fluid, preferably blood, that is required is not limited. Depending on the methods employed, the skilled person will be capable of establishing the amount of sample required to perform the various steps of the methods of the present invention and obtain sufficient nucleic acid for analysis. Generally, such amounts will comprise a volume ranging from 0.01 m ⁇ to 100 ml, preferably between 1 m ⁇ to 10 ml, more preferably about 1 ml.
  • a bodily fluid preferably blood
  • the bodily fluid may be treated to remove nucleic acid degrading enzymes like RNases and DNases, in order to prevent destruction of the nucleic acids.
  • specific cells for example anucleated cells such as thrombocytes, may be isolated from the bodily fluid, preferably blood, followed by quantifying mitochondrial nucleic acid and/or a heat shock protein in said isolated anucleated cells. It has been reported that anucleated cells such as thrombocytes may absorb genetic material secreted by diseased cells such as cardiac myocytes, serving as an attractive platform for diagnostics of diseases such as atrial fibrillation.
  • the bodily fluid preferably blood
  • the bodily fluid may be analyzed immediately following withdrawal of the sample.
  • analysis according to the method of the invention may be performed on a stored bodily fluid.
  • the body fluid for testing may be preserved using methods and apparatuses known in the art.
  • the bodily fluid preferably blood
  • the bodily fluid can be prepared and stored at -70°C until processed for sample preparation.
  • storage is performed under conditions that preserve the quality of the nucleic acid content of the bodily fluid.
  • preservative conditions are fixation using e.g.
  • aqueous solutions such as Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect (HOPE; DE10021390), and RCL2 (Alphelys; WO04083369), and the addition of non-aquous solutions such as
  • Methods to quantify mitochondrial nucleic acid and/or a level of DNA damage in said bodily fluid include, but are not limited to, quantitative PCR, microarray analysis, nucleic acid sequencing, and enzyme- linked immunosorbent assay (ELISA). It is preferred that said quantification is determined simultaneously. Simultaneous analyses can be performed, for example, by quantitative amplification, nucleic acid sequencing procedures, microarray analysis, and/or ELISA.
  • RNA ribonucleic acid
  • RNA may be isolated from said bodily fluid by any technique known in the art, including but not limited to Trizol (Invitrogen; Carlsbad, California), RNAqueous® (Applied
  • RNA-Bee® Tel-Test.
  • RNA isolation procedure involves the use of Qiazol® (Qiagen, Hilden, Germany), especially a miRNeasy Kit.
  • a reverse transcriptase preferably is used to convert RNA into complementary DNA (cDNA) which may subsequently be amplified.
  • Suitable reverse transcriptase enzymes include human immunodeficiency virus (HIV), Moloney murine leukemia virus (M- MuLV), and avian myeloblastosis virus (AMV) reverse transcriptase, as is known to a person skilled in the art.
  • RNA expression product of a mitochondrial nucleic acid or of a lncRNA can be determined by any method known in the art. Methods to determine RNA levels are known to a skilled person and include, but are not limited to, Northern blotting, quantitative polymerase chain reaction (qPCR), also termed real time PCR (rtPCR), microarray analysis and next generation RNA sequencing.
  • qPCR quantitative polymerase chain reaction
  • rtPCR real time PCR
  • microarray analysis next generation RNA sequencing.
  • DNA desoxyribonucleic acid
  • DNA may be isolated, for example from blood, or from pretreated blood as is indicated herein above.
  • DNA may be purified from a bodily fluid, preferably blood, or from pretreated blood samples as is indicated herein above, using, for instance, a combination of physical and chemical methods.
  • NucleoSpin® Tissue kit Machery-Nagel
  • QIAamp blood mini kit columns Qiagen, Venlo, The Netherlands
  • Said mitochondrial nucleic acid preferably includes nucleic acid comprising at least part of a MT-C03 gene which encodes a cytochrome c oxidase subunit 3 (COX3); at least part of a MT-ND1 gene which encodes a NADH-ubiquinone oxidoreductase chain 1, at least part of a MT-ATP8 gene which encodes a ATP synthase protein 8; at least part of a MT-CYB gene, which encodes a cytochrome b; at least part of a MT-ND2 gene which encodes a NADH dehydrogenase 2; at least part of MT-ND3 gene which encodes a NADH dehydrogenase 3; at least part of a MT-ND4L gene which encodes a NADH-ubiquinone oxidoreductase chain 4L; at least part of a MT-ND4 gene which encodes a NADH-ubiquinone oxidor
  • nucleic acid comprising at least part of MT-ATP6 gene, MT-COl gene, MT-C02 gene, MT-ND1 gene, MT- humanin encoding gene, transfer RNA encoding genes including MT-TA gene, MT- TR gene, MT-TN gene, MT-TD gene, MT-TC gene, MT-TE gene, MT-TQ gene, MT- TG gene, MT-TH gene, MT-TI gene, MT-TL1 gene, MT-TL2 gene, MT-TK gene, MT-TM gene, MT-TF gene, MT-TP gene, MT-TSlgene, MT-TS2 gene, MT-TT gene, MT-TW gene, MT-TY gene, MT-TV gene, and ribosomal RNA encoding genes including MT-RNR1 gene and/or MT-RNR2 gene.
  • PCR Polymerase Chain Reaction
  • rolling circle amplification nucleic acid sequence-based amplification
  • transcription mediated amplification A preferred amplification method is PCR, especially real-time PCR.
  • PCR is a technology that relies on thermal cycling, consisting of cycles of repeated heating and cooling of a reaction for DNA melting into single stranded molecules and enzymatic replication of the DNA.
  • Primers containing sequences that specifically hybridizes to the target region, and a DNA polymerase are key components to enable selective and repeated amplification.
  • the amplified DNA product that is generated is itself used as a template for replication, resulting in a chain reaction in which the DNA template is exponentially amplified.
  • a preferred DNA polymerase is a thermostable polymerase, preferably a thermostable recombinant polymerase.
  • Preferred commercially available DNA polymerases include AptaTaq Fast DNA Polymerase and LightCycler® FastStart Enzyme (Roche Diagnostics, Almere, The Netherlands).
  • Real-time PCR also called quantitative PCR (qPCR) is a technique which is used to amplify and simultaneously quantify a template DNA molecule.
  • the detection of the amplification products can in principle be accomplished by any suitable method known in the art.
  • the amplified products may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents.
  • Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.
  • the amplified product may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments.
  • Detection labels which may be associated with nucleotide bases include, for example, fluorescein, cyanine dye and BrdUrd.
  • a primer or the probe is preferably labelled with a detectable label, preferably a fluorescent label.
  • Preferred labels for use in this invention comprise fluorescent labels, preferably selected from Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto 647N (ATTO-TEC GmbH, Siegen, Germany), YakimaYellow (Epoch
  • a probe is preferably labeled at
  • a primer such as a Scorpion primer, or a probe preferably has a fluorescent label at one end and a quencher of fluorescence at the opposite end of the probe.
  • the close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser.
  • An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.
  • Quenchers for example tetramethylrhodamine
  • TAMRA dihydrocyclopyrroloindole tripeptide minor groove binder
  • Preferred quenchers are Black Hole Quenchers- 1 (BHQ1) and BHQ2, which are available from Biosearch Technologies, Petaluma, CA, USA).
  • BHQ1 dark quencher has strong absorption from 480 nm to 580 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as FAM, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, and Quasar® 570 dyes.
  • the BHQ2 dark quencher has strong absorption from 599 nm to 670 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as Quasar® 570, TAMRA, CAL Fluor® Red 590, CAL Fluor Red 610, ROX, CAL Fluor Red 635, Pulsar® 650, Quasar 670 and Quasar 705 dyes.
  • BHQ1 and BHQ2 may quench fluorescence by both FRET and static quenching mechanisms.
  • hybridizing refers to a nucleic acid molecule that is capable of hybridizing specifically under stringent hybridization conditions to a target nucleic acid template that is obtained or derived from mitochondrial nucleic acid and/or a heat shock protein nucleic acid.
  • stringency and stringent hybridization refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, and the like. These conditions are empirically optimized to maximize specific binding and minimize non- specific binding of primer or probe to its target nucleic acid sequence.
  • the terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background).
  • Stringent conditions may be sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tin) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer.
  • Tin thermal melting point
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer.
  • Hybridization procedures are well known in the art and are described by e.g. Ausubel et ak, 1998. Current Protocols in Molecular Biology, John Wiley, New York; and Sambrook et a , 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.
  • An oligonucleotide primer or probe, or an oligonucleotide mimic primer or probe, is able to hybridize to a target nucleic acid template when the length of the molecule is or resembles at least 15 bases.
  • the length of the primer or probe is preferably less than 100 bases.
  • a preferred length of a primer or probe is between 15 and 50 bases, preferably between 16 and 30 bases.
  • a primer or probe is able to hybridize to a target nucleic acid template when the percentage of sequence identity of the molecule is at least 90% over substantially the whole length, more preferred at least 91%, more preferred at least 92%, more preferred at least 93%, more preferred at least 94%, more preferred at least 95%, more preferred at least 96%, more preferred at least 97%, more preferred at least 98%, more preferred at least 99%, more preferred 100% identical to a nucleic acid that is obtained or derived from said target nucleic acid template over substantially the whole length of the primer or probe.
  • the term “substantially the whole length” is used to indicate that the probe may comprise additional nucleotide sequences, for example at the 5’ and/or 3’ ends that are not present in the gene or region described herein above.
  • Efficient real-time PGR reactions are dependent upon high quality primer and probe design.
  • Rules of thumbs for the design of primers include the selection of primers having a Tm between 58°C and 65°C while keeping the annealing temperatures of the primers as close as possible, having no more than two G's or C's in the last 5 bases at the 3’ end, and the selection of primer pairs with minim l number of potential primer dimers and primer hairpins.
  • Rides of thumbs for the design of probes include the selection of probes that have a Tm between 68°C and 72°C, have no Gs on the 5’ end, resemble a strand that has more C than G bases, and are as short as possible, without being shorter than 13 nucleotides.
  • the primers and probes are preferably tested in single nucleic acid
  • Microarray-based analysis involves the use of selected biomolecules that are immobilized on a solid surface, an array.
  • a microarray usually comprises nucleic acid molecules, termed probes, which are able to hybridize to gene expression products. The probes are exposed to labeled sample nucleic acid, hybridized, where after the abundance of gene expression products in the sample that are
  • the probes on a microarray may comprise DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA.
  • the probes may also comprise DNA and/or RNA analogues such as, for example, nucleotide analogues or peptide nucleic acid molecules (PNA), or combinations thereof.
  • the sequences of the probes may be full or partial fragments of genomic DNA.
  • the sequences may also be in vitro synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
  • a probe preferably is specific for a particular gene.
  • a probe is specific when it comprises a continuous stretch of nucleotides that are completely complementary to a nucleotide sequence of the gene.
  • a probe can also be specific when it comprises a continuous stretch of nucleotides that are partially complementary to a nucleotide sequence of a gene. Partially means that a maximum of 5% from the nucleotides in a continuous stretch of at least 20 nucleotides differs from the corresponding nucleotide sequence of said gene.
  • the term complementary is known in the art and refers to a sequence that is related by base-pairing rules to the sequence that is to be detected. It is preferred that the sequence of the probe is carefully designed to minimize nonspecific hybridization to said probe.
  • the probe is, or mimics, a single stranded nucleic acid molecule.
  • the length of said complementary continuous stretch of nucleotides can vary between 15 bases and several kilo bases, and is preferably between 20 bases and about 60 nucleotides.
  • a most preferred probe comprises at least 20, preferably at least 25, nucleotides that are identical to a nucleotide sequence of a gene.
  • mitochondrial gene nucleic acid products in the sample are preferably labeled, either directly or indirectly, and contacted with probes on the array under conditions that favor duplex formation between a probe and a complementary molecule in the labeled gene nucleic acid products in the sample.
  • the amount of label that remains associated with a probe after washing of the microarray can be determined and is used as a measure for the level of a mitochondrial gene nucleic acid product that is complementary to said probe.
  • a further preferred method for quantifying gene nucleic acid levels is by sequencing techniques, preferably high throughput sequencing techniques, also termed next generation sequencing (NGS) techniques. These techniques include Illumina® sequencing; Roche 454 pyrosequencing®; ion torrent and ion proton sequencing; and ABI SOLiD®) sequencing. NGS techniques allow sequencing of fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads. Each base is sequenced multiple times, providing high depth to deliver accurate data and an insight into unexpected DNA variation. NGS can be used to sequence a complete exome including all or small numbers of individual genes. Such high throughput sequencing techniques include sequencing-by synthesis, such as provided by Illumina® sequencing and by ion torrent and ion proton sequencing. Sequencing-by-synthesis or cycle sequencing can be
  • nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in U.S. Patent No. 7,427,673; U.S. Patent No. 7,414, 116; WO 04/018497; WO 91/06678; WO
  • pyrosequencing techniques may be employed. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi et ah, Analytical Biochemistry 242(l):84-9 (1996); Ronaghi, M. Genome Res. 11(1):3- 11 (2001); Ronaghi, M. et ah, Science 281:5375, 363 (1998); U.S. Patent No. 6,210,891 ; U.S. Patent No.
  • PPi inorganic pyrophosphate
  • released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons.
  • ATP adenosine triphosphate
  • Sequencing techniques also include sequencing by ligation techniques, such as provided by ABI SO Li I ) n sequencing. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides and are inter alia described in U.S. Patent No 6,969,488 ; U.S. Patent No.
  • Further sequencing techniques include, for example, fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS).
  • FISSEQ fluorescent in situ sequencing
  • MPSS Massively Parallel Signature Sequencing
  • a level of DNA damage may be determined, for example, by determining an amount of oxidative stress and/or DNA lesions. Guanine is a base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is a most common lesion. Hence, a level of DNA damage preferably is determined by determining a level of 8-oxoG in a bodily fluid, preferably blood.
  • a level of 8-oxoG may be determined by, for example, high performance liquid chromatography coupled to electrochemical detection and/or gas chromatography separation followed by mass spectrometry, and/or by an ELISA kit, for example an Oxiselect oxidative DNA damage ELISA kit (Cell Biolabs, San Diego, CA), or an 8-hydroxy 2 deoxyguanosine ELISA Kit (Abeam, Cambridge, MA).
  • an ELISA kit for example an Oxiselect oxidative DNA damage ELISA kit (Cell Biolabs, San Diego, CA), or an 8-hydroxy 2 deoxyguanosine ELISA Kit (Abeam, Cambridge, MA).
  • a method of the invention may be further comprise determining a level of heat shock protein in the bodily fluid of the individual.
  • Said heat shock protein preferably includes at least part of a HSP60 gene product, which is a protein that is implicated in mitochondrial protein import and macromolecular assembly; at least part of a HSP Family B (Small) Member 7 (HSPB7) gene product, which may function as a cardioprotective chaperone that is involved in overcoming stress; at least part of a HSPB1 gene product, which is a HSP27 protein that may function in thermotolerance; and/or at least part of a HSP70 gene product, which is a HSP70 protein that may help to protect cells from stress.
  • HSP60 gene product which is a protein that is implicated in mitochondrial protein import and macromolecular assembly
  • HSPB7 HSP Family B
  • HSPB7 HSP Family B
  • HSP70 gene product which is a HSP70 protein that may help to protect cells from stress.
  • Methods to isolate proteins from a bodily fluid are known in the art, including, for example, the Total Protein Extraction Kit (BioCat GmbH, Heidelberg, Germany), or the NucleoSpin® Tissue kit (Maehery-Nagel GmbH & Co. KG, Bethlehem, PA).
  • heat shock protein levels in a bodily fluid may be directly quantified using ELISA.
  • Suitable ELISA kits are known in the art and include, for example a DuoSet IC ELISA kit, for example the Human Total HSP60 DuoSet IC ELISA DYC 1800-2 (R&D Systems, Minneapolis, MN), a HSP60 ELISA kit (ENZO Life Sciences BVBA, Brussels, Belgium), a Human Heat shock protein beta-7(HSPB7) ELISA kit (Cusabio Technology LLC, Houston, TX), a HSP27 Human ELISA Kit (ThermoFisher Scientific, Waltham, MA), and/or a Human HSP70 ELISA Kit (Abeam; Cambridge MA).
  • a DuoSet IC ELISA kit for example the Human Total HSP60 DuoSet IC ELISA DYC 1800-2 (R&D Systems, Minneapolis, MN), a HSP60 ELISA kit (ENZO Life Sciences BVBA, Brussels, Belgium), a Human Heat shock protein
  • the quantified mitochondrial nucleic acid and said level of DNA damage and, when determined, said level of heat shock protein are preferably normalized.
  • Normalization refers to a method for adjusting or correcting a systematic error in the measurements for determining expression levels.
  • Systemic bias may result from variation by differences in overall performance, differences in isolation efficiency of nucleic acid and/or proteins resulting in differences in purity of the isolated products, and to variation between nucleic acid or protein samples, which can be due for example to variations in purity.
  • Systemic bias can be introduced during the handling of the sample during the quantification of the nucleic acid and/or protein products. Typing of a sample can be performed in various ways.
  • the quantified lncRNA, mitochondrial nucleic acid and/or level of DNA damage is compared to a reference and the individual is typed as suffering from, or at risk of suffering from, atrial fibrillation if the level of quantified lncRNA is altered, compared to the reference, if the level of quantified mitochondrial nucleic acid is altered, compared to the reference, and/or if said quantified level of DNA damage is altered, when compared to the reference.
  • the reference provides a level for a lncRNA molecule, a mitochondrial nucleic acid and/or of DNA damage that functions as a threshold to determine whether the individual may suffer from, or may be at risk of suffering from, atrial fibrillation.
  • a coefficient is determined that is a measure of a similarity or dissimilarity of a quantified mitochondrial nucleic acid and/or level of DNA damage, with a reference.
  • a reference may include one or more lncRNAs, multiple mitochondrial nucleic acids and/or a levels of DNA damage, for example employing different markers for determining a level of DNA damage, and can be referred to as a profile template.
  • Typing of an individual can be based on its (dis)similarity to a single profile template or based on multiple profile templates.
  • the profile templates may be representative of individuals that (i) are known to suffer from atrial fibrillation, or (ii) are known not to suffer from atrial fibrillation.
  • a number of different coefficients can be used for determining a correlation between the determined lncRNAs, mitochondrial nucleic acids and/or level of DNA damage, and a profile template.
  • Preferred methods are parametric methods which assume a normal distribution of the data.
  • One of these methods is the Pearson product-moment correlation coefficient, which is obtained by dividing the covariance of the two variables by the product of their standard deviations.
  • Preferred methods comprise cosine-angle, un-centered correlation and, more preferred, cosine correlation (Fan et a , Conf Proc IEEE Eng Med Biol Soc. 5:4810- 3 (2005)).
  • a similarity score is a measure of the average correlation of the quantified mitochondrial nucleic acids and/or level of DNA damage in a bodily fluid from an individual and a profile template.
  • Said similarity score can be, but does not need to be, a numerical value between +1, indicative of a high correlation between the quantified lncRNA, mitochondrial nucleic acid and/or level of DNA damage in a bodily fluid from an individual and a profile template, and -1, which is indicative of an inverse correlation.
  • a threshold can be used to differentiate between quantified lncRNA, mitochondrial nucleic acids and/or level of DNA damage from an individual who is known to suffer from atrial fibrillation and an individual that is known not to suffer from atrial fibrillation.
  • Said threshold is an arbitrary value that allows for discrimination between an individual who is known to suffer from atrial fibrillation and an individual that is known not to suffer from atrial fibrillation.
  • a similarity threshold value is employed, it is preferably set at a value at which an acceptable number of individuals who are known to suffer from atrial fibrillation would score as false negatives, and an acceptable number of individuals who are known not to suffer from atrial fibrillation would score as false positives.
  • a similarity score is preferably displayed or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.
  • the methods of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to the invention are specifically suited for identifying an individual that is suffering from, or expected to suffer from, paroxysmal atrial fibrillation and who will likely develop persistent atrial fibrillation, and for identifying an individual who is at risk of recurrence of AF after treatment, including anti arrhythmic drug therapy, ablative therapy, and/or cardioversion.
  • an increase in a level of lncRNA, mitochondrial nucleic acid and a level of DNA damage when compared to a reference from individuals not suffering from AF, can be used to identify an individual suffering from paroxysmal AF who is at risk of progressing to more persistent AF
  • a decrease of a lncRNA, mitochondrial nucleic acid and a decrease of a level of DNA damage when compared to a reference from individuals not suffering from AF, can be used to identify an individual suffering from persistent AF who is at risk of progressing to long-standing persistent AF, and/or at risk of recurrence of AF after treatment.
  • a decrease in a level of heat shock protein when compared to a reference from individuals not suffering from AF, can be used to identify an individual suffering from paroxysmal AF who is at risk of progressing to more persistent AF.
  • lncRNAs, mitochondrial nucleic acid and DNA damage markers in a bodily fluid, preferably blood are increased in an individual that is suffering from, or expected to suffer from, paroxysmal atrial fibrillation, and is likely to progress to persistent AF, while the same lncRNAs and mitochondrial nucleic acid are decreased in a bodily fluid, preferably blood, from an individual that suffers from persistent atrial fibrillation and is likely to progress to long standing persistent atrial fibrillation and/or at risk of recurrence of AF after treatment.
  • the methods of the invention may in particular be suited for typing a female as suffering from, or at risk of suffering from, atrial fibrillation. It was found that the relative increase of IncRNA, mitochondrial nucleic acid and DNA damage markers, relative to a healthy reference, is higher in females when compared to males. For this reason, said reference preferably is gender-matched.
  • a IncRNA that is specifically suited for typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, especially paroxysmal atrial fibrillation is represented by Sarrah, UCA1, CDR1AS and/or LIPCAR. These markers are especially enhanced in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, while these markers are decreased in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation. However, all other lncRNAs showed a similar trend: increase in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, and decrease in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation.
  • a mitochondrial nucleic acid that is specifically suited for typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, especially paroxysmal atrial fibrillation, is represented by COX3, ND1 and/or ND2. These markers are especially enhanced in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, while these markers are decreased in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation.
  • all other mitochondrial nucleic acid showed a similar trend: increase in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, and decrease in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation.
  • heat shock protein in a bodily fluid, preferably blood is decreased in an individual that is suffering from, or expected to suffer from, atrial fibrillation, especially paroxysmal atrial fibrillation.
  • a heat shock protein that is specifically suited for typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, especially paroxysmal atrial fibrillation is represented by HSPB7, which marker was also found to be decreased in an individual suffering from persistent atrial fibrillation and long-standing persistent atrial fibrillation.
  • LncRNAs and mitochondrial DNA markers seem the best option to predict AF stage and recurrence after AF treatment.
  • the methods of the invention are particularly suited to identify an individual that has a low risk of recurrence after initial treatment such as pulmonary vein isolation (PVI) and electrical
  • ECV cardioversion
  • Preferred markers for identifying an individual that has an altered, i.e high or low, risk of recurrence after initial treatment include lncRNA, especially UCA1, CD IAS, and LIPCAR; and/or mitochondrial markers, especially ND1 and COX3.
  • Preferred markers are UCA1 and ND1, UCA1 and COX3, UCA1, CD IAS and ND1, UCA1, CD1AS and COX3, CD IAS, LIPCAR and ND1, CD IAS, LIPCAR and COX3, UCA1, CD IAS, LIPCAR and ND1, UCA1, CD1AS, LIPCAR and COX3, and UCA1, CD IAS, LIPCAR, ND1 and COX3.
  • the invention further provides a method for assigning a standard-of-care therapeutic agent to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of assigning a standard-of-care therapy to an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention.
  • Said standard-of-care therapy preferably comprises an inhibitor of poly(ADP- ribose) polymerase, ablative therapy, electrical or chemical cardioversion, and/or the administration of anti-arrhythmic drugs aimed at controlling rhythm or rate.
  • Said inhibitor of poly(ADP-ribose) polymerase also termed PARP-inhihitor, preferably is a specific PARP1 inhibitor.
  • PARP1 is the key PARP enzyme instigating tachypacing-induced contractile dysfunction in ardiomyocytes. As is shown herein below, PARP1 inhibition prevents NAD+ depletion and functional loss and accelerates recovery after cessation of tachypacing.
  • a PARP-inhihitor such as nicotinamide and/or ABT-888 may halt or delay onset of AF and/or progression to paroxysmal AF and to persistent AF.
  • Preferred PARP inhibitors include nicotinamide, 2-[(2R)-2-Methylpyrrolidin- 2-yl] - lH-benzimidazole-4-carboxamide dihydrochloride benzimidazole carboxamide (ABT-888), 3-aminobenzamide, 4-(3-(l-(cyclopropanecarbonyl)piperazine-4- carbonyl)-4-fluorobenzyl)phthalazin- l(2H)-one (AZD-2281), 8-fluoro-2- ⁇ 4- [(methylamino)methyl]phenyl ⁇ - 1,3,4, 5-tetr ahydro-6H-pyrrolo[4, 3,2- ef][2]benzazepin-6-one phosphate (1:1) (AG014699), (8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9-d
  • Ablative therapy is a procedure aimed at isolating electrical activity causing atrial fibrillation by scarring or destroying specific areas or cells.
  • Ablative therapy is preferably performed by catheters that generate heat or extreme cold in order to destroy cells in the atrium.
  • AV node ablation in which cells of the atrioventricular node are ablated to prevent the atria from propagating electrical activity at a too high rate to the ventricles; or by performing a modified maze procedure, in which ablative therapy is performed during open-heart surgery.
  • Said electrical or chemical cardioversion is aimed at restoring a normal rhythm.
  • Chemical cardioversion comprises administration, preferably intravenous administration, of an antiarrhythmic drug such as procainamide (4-amino-N-[2- (diethylamino)ethyl]benzamide), a class Ic agent such as flecainide (N-(piperidin-2- ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide) or propafenone (l-[2-[2-hydroxy- 3-(propylamino)propoxy]phenyl]-3-phenylpropan-l-one), amiodarone ((2-butyl-l- benzofuran-3-yl)-[4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl]methanone) and/or ibutilide (N - [4- [4- [ethyl (hep tyl) amino] - l-hydroxyhutyl]phenyl]me thane
  • a dose preferably is between 20 and 250 mg. preferably about 100 mg, every 5 minutes until a maximum dose of about 1 g is reached for
  • procainamide between 10 and 100 mg every 12 hours, preferably about 50 mg orally every 12 hours, for flecainide, between 50 and 250 mg every 8 hours, preferably about 150 mg orally every 8 hours, for propafenone, a bolus of between 100 and 400 mg/30 minutes, preferably about 300 mg/30 mins, for amiodarone, and/or one or two doses of 1 mg for ibutilide.
  • Electrical cardioversion preferably is performed by applying a current across the chest of a patient.
  • Said current preferably is applied with increasing shock energies of between 100 and 360 J in an anterior-posterior electrode position.
  • Said current may have a monophasic or biphasic waveform, preferably has a biphasic waveform.
  • both methods may be applied successively, meaning that chemical conversion may be followed by electrical cardioversion if chemical conversion was not successful; and that electrical cardioversion may be followed by chemical cardioversion if electrical conversion was not successful.
  • Said rate control therapy preferably comprises a beta blocker, a calcium channel blocker and/or a cardiac glycoside.
  • Preferred beta blockers also termed adrenergic beta-antagonists, include atenolol (2- [4- [2-hydroxy-3-(propan-2-ylamino)propoxy]phenyl] acetamide), bisoprolol (l-(propan-2-ylamino)-3-[4-(2-propan-2- yloxyethoxymethyl)phenoxy]propan-2-ol), earvedilol (l-(9H-carbazol-4-yloxy)-3- [2- (2-methoxyphenoxy)ethylamino]propan-2-ol), metoprolol (l-[4-(2- methoxyethyl)phenoxy]-3-(propan-2-ylamino)propan-2-ol), nadolol ((2R,3S)-5-[3- (tert-butylamino)-2-hydroxypropoxy]-l,2,3,4-tetrahydronaphthalene-2,3-
  • Recommended dosages for a beta blocker are between 50 and 200 mg daily, preferably about 100 mg daily, for atenolol, between 2.5 and 20 mg daily, preferably about 5 mg daily, for bisoprolol, between 1 and 25 mg twice a day, preferably about 12.5 mg twice daily, for carvedilol, between 50 and 450 mg daily, preferably about 100 mg daily, for metoprolol, between 40 and 320 mg daily, preferably about 80 mg daily, for nadolol, between 40 and 640 mg daily, preferably about 160 mg daily, for propranolol, between 5 and 30 mg twice daily, preferably about 10 mg twice daily, for timolol, and between 80 and 640 mg twice daily, preferably about 240 mg twice daily, for sotalol.
  • a preferred cardiac glycoside is a cardenolide, preferably digoxin (3-
  • a recommended dosage for digoxin is between 0.125 mg and 0.375 mg orally or intravenously, every two hours up to a total of 1.5 mg, or between 0.125 mg and 0.375 mg orally once daily.
  • Preferred calcium channel blockers include diltiazem ([(2S,3S)-5-[2- (dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3-dihydro- l,5-benzothiazepin- 3-yl] acetate;hydrochloride) and verapamil (2-(3,4-dimethoxyphenyl)-5-[2-(3,4- dimethoxyphenyl)ethyl-methylamino] -2-propan-2-ylpentanenitrile) .
  • recommended dosage for diltiazem is an initial intravenous bolus of 0.25 mg/kg, followed by infusion with 10 mg/hr to 15 mg/hr for a maximum duration of 24 hours for diltiazem, and between 80 mg and 480 mg orally once daily, preferably about 160 mg once daily, for verapamil.
  • Said standard-of-care therapeutic agent rhythm control therapy preferably comprises an ion channel blocker, more preferably a sodium channel blocker such as flecainide (N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide), propafenone (1- [2- [2-hydroxy-3-(propylamino)propoxy]phenyl] -3-phenylpropan- 1- one) and/or quinidine (S)-[(2R,4S,5R)-5-ethenyl-l-azabicyelo[2.2.2]octan-2-yl]-(6- methoxyquinolin-4-yl)methanol), a potassium channel blocker such as amiodarone ((2-butyl-l-benzofuran-3-yl)-[4-[2-(diethylamino)ethoxy]-3,5- diiodophenyljmethanone), sotalol (N-[4
  • Recommended dosages for a rhythm controller are between 50 and 200 mg twice daily, preferably about 100 mg twice daily, for flecainide, between 150 and 300 mg three times daily, preferably about 150 mg three time daily, for
  • propafenone between 80 and 600 mg every 4 to 6 hours, preferably about 200 mg every 4 to 6 hours, for quinidine, between 400 and 1600 mg daily, preferably about 800 mg orally daily, for amiodarone, between 80 and 320 mg twice daily, preferably about 160 mg twice daily, for sotalol, between 125 and 500 mg twice daily, preferably about 250 mg twice daily, for dofetdide, and between 2.5 and 20 mg daily, preferably about 5 mg daily, for dronedarone.
  • Said standard-of-care therapy may be combined with an antiplatelet and/or anticoagulant.
  • Said antiplatelet and/or anticoagulant preferably is selected from aspirin, warfarin, and a direct-acting oral anticoagulant.
  • said standard-of-care therapeutic agent comprises only an antiplatelet and/or an anticoagulant as active
  • ingredient(s) This may be the case where a decrease of a mitochondrial nucleic acid and a decrease of a level of DNA damage, when compared to a reference from individuals not suffering from AF, is observed and further treatment of the individual suffering from AF is not recommended.
  • Said antiplatelet therapy includes the administration of acetylsalicylic acid, dipyridamole and/or clopidogrel.
  • Acetylsalicylic acid is known to inhibit the COX-1 enzyme.
  • a recommended dosage is between 75 and 325 mg daily, preferably about 200 mg daily.
  • Dipyridamole (2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-l- yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol) acts as a platelet aggregation inhibitor.
  • a recommended dosage is between 10 and 100 mg daily, preferably about 50 mg daily.
  • Clopidogrel (methyl (2S)-2-(2-chlorophenyl)-2-(6,7- dihydro-4H-thieno[3,2-c]pyridin-5-yl)acetate) also acts as a platelet aggregation inhibitor.
  • a recommended dosage is between 25 and 300 mg daily, preferably about 75 mg daily.
  • Said anticoagulant preferably is a 4-hydroxycoumarin derivative that reduces vitamin K1 levels.
  • Such coumarin derivatives include warfarin (4-hydroxy-3-(3-oxo- l-phenylbutyl)chromen-2-one), acenocoumarol (4-hydroxy-3-[l-(4-nitrophenyl)-3- oxobutyl]chromen-2-one) and bishydroxycoumarin (4-hydroxy-3-[(4-hydroxy-2- oxochromen-3-yl)methyl]chromen-2-one).
  • a recommended dosage of warfarin is between 1 and 10 mg daily, preferably about 5 mg daily.
  • a recommended dosage of acenocoumarol is between 1 and 20 mg daily, preferably about 12 mg daily.
  • a recommended dosage of bishydroxycoumarin is between 100 and 200 mg daily, preferably about 150 mg daily
  • Said direct-acting oral anticoagulant preferably is a direct thrombin inhibitor such as dabigatran (ethyl N-[(2- > [(4- ⁇ N'-j(hexyloxy)earbonyI]oarbamimidoyl ⁇ phenyI)amino]methyj)-l-niefhyl-lH-benzimidazoI-5-yI)carbony!]-N-pyfidin-2-yl- beta-alaninate), and/or a direct Factor Xa inhibitor such as rivaroxaban (5-chloro- N-[[(5S)-2-oxo-3-[4-(3-oxomorphobn-4-yl)phenyl]-l,3-oxazobdin-5- yl]methyl]thiophene-2-carboxamide), edoxaban (N’-(5-chloropyridin-2-yl)-N- [(lS,2R,
  • dabigatran may be provided as a precursor named dabigatran etexilate, which is converted after absorption to the active substance.
  • Recommended dosages for the active inhibitor, or its precursor are between 200 and 400 mg daily, preferably about 150 mg orally twice a day, for dabigatran, between 5 and 30 mg daily, preferably about 20 mg daily, for rivaroxaban, between 10 and 60 mg daily, preferably about 30 mg daily, for edoxaban, between 1 and 10 mg daily, preferably about 2.5 mg daily, for apixaban.
  • the invention further provides a standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention.
  • Said standard-of- care therapy is as defined herein above.
  • the invention further provides a method for determining the efficacy of a standard-of-care therapy in an individual, comprising the steps of analyzing a bodily fluid, preferably blood, of an individual for the presence of a biomarker using a method of typing according to the invention at a first time point to thereby provide a first value for the level of said biomarker in said subject, analyzing a bodily fluid, preferably blood, of said individual for the presence of said biomarker using a method of typing according to the invention at a second time point that is earlier or later, preferably later, than said first time point, to thereby provide a second value for the level of said biomarker in said individual, wherein said individual has been subjected to a standard-of-care therapy between said first and second time point, and comparing said first and second value to determine the efficacy of said standard-of-care therapy in said individual.
  • a decrease of the quantified mitochondrial nucleic acid at the second time point, compared to the first time point, is indicative that the standard-of-care therapy has been effective
  • an increase in the quantified heat shock protein nucleic at the second time point, compared to the first time point is indicative that the standard-of-care therapy has been effective.
  • Said biomarker is one or more of a mitochondrial nucleic acid and/or a heat shock protein as defined herein above.
  • aspects of the invention include a method of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of:
  • a bodily fluid from said individual quantifying a level of DNA damage and/or mitochondrial nucleic acid in said bodily fluid; comparing said quantified level of DNA damage and/or mitochondrial nucleic acid to a reference; and typing said individual as suffering from, or at risk of suffering from, atrial fibrillation it said quantified level of DNA damage is altered, compared to the reference and/or if the quantified mitochondrial nucleic acid is altered, compared to the reference.
  • Said bodily fluid preferably is blood.
  • the level of DNA damage in a preferred method of the invention is quantified by determining a level of 8-oxoguanine.
  • the mitochondrial nucleic acid in a preferred method of the invention is mitochondrial DNA.
  • Said mitochondrial nucleic acid preferably comprises nucleic acid from a cytochrome C oxidase 3 gene and/or a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene.
  • a preferred method of the invention comprises quantifying a level of DNA damage and quantifying mitochondrial DNA from a cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene.
  • Said method preferably further comprises quantifying a heat shock protein level, preferably heat shock protein B7, in said bodily fluid.
  • the invention further provides a method for assigning standard-of-care therapy to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of any one of claims 1-7; and assigning a standard-of-care therapy to the individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation.
  • Said standard-of-care therapy preferably is ablative therapy, electrical or chemical cardioversion, and/or the administration of anti -arrhythmic drugs aimed at controlling rhythm or rate.
  • Said standard-of-care therapy preferably is combined with an antiplatelet and/or anticoagulant.
  • Said antiplatelet and/or anticoagulant preferably is selected from aspirin, warfarin, and a direct-acting oral anticoagulant such as dabigitran, rivaroxaban, edoxaban and apixaban.
  • Said standard-of-care therapy preferably is an ion channel blocker including a sodium channel blocker such as flecainide, propafenone and/or quinidine, and/or a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
  • a sodium channel blocker such as flecainide, propafenone and/or quinidine
  • a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
  • the invention further provides a standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention.
  • the invention further provides a method for determining the efficacy of a standard-of-care therapy of an individual, comprising the steps of analysing a bodily fluid, preferably blood, of an individual for the presence of a biomarker using a method of typing according to the method of any one of claims 1-7, at a first time point to thereby provide a first value for the level of said biomarker in said subject, analysing a bodily fluid, preferably blood, of said individual for the presence of said biomarker using a method of typing according to the invention at a second time point that is earlier or later, preferably later, than said first time point, to thereby provide a second value for the level of said biomarker in said individual, wherein said individual has been subjected to a standard-of-care therapy between said first and second time point
  • HL-1 atrial cardiomyocytes derived from adult mouse atria, were obtained from Dr. William Claycomb (Louisiana State University, New Jersey, USA; Claycomb, et a , 1998. PNAS 95: 2979-2984). To induce AF, HL-1 atrial cardiomyocytes were subjected to 6Hz (tachypacing), 40V and 20ms pulses, for maximal 8 hours via the C-PacelOO TM-culture pacer (IonOptix Corporation, The Netherlands). HL-1 cardiomyocytes were transiently transfected with pDEST40-MCU-V5-HIS
  • MCU knockdown was accomplished by transiently transfecting the cardiomyocytes with Mission MCU esiRNA (EMU213891, Sigma, The Netherlands) by the use of Lipofectamin RNAiMAX (Life Technologies, The Netherlands). Ru360 was purchased from Millipore (USA) and dissolved in according to manufacturer’s instructions. Treatment (5 pM) was started 30 minutes prior to and was continued during tachypacing.
  • Mission MCU esiRNA EMU213891, Sigma, The Netherlands
  • Lipofectamin RNAiMAX Life Technologies, The Netherlands.
  • Ru360 was purchased from Millipore (USA) and dissolved in according to manufacturer’s instructions. Treatment (5 pM) was started 30 minutes prior to and was continued during tachypacing.
  • TMRM tetramethyl rhodamine methylester
  • OCR mitochondrial oxygen consumption rate
  • Mitochondrial morphology was measured by incubating HL-1 atrial cardiomyocytes with 100 nM Mitotracker Deep Red (Life Technologies, The Netherlands) and thereafter excited by a 647 nm laser with emission at 665 nm and were visually recorded with a 63x-objective, using a Solamere-Nipkow-Confocal-Live-Cell- Imaging system (based on a Leica DM IRE2 inverted microscope). Ten random fields containing at least 15 cardiomyocytes were recorded and the mitochondrial morphology per cardiomyocyte was scored as tubular, intermediate or fragmented (Lanters et ah, 2015. J Transl Med 13: 347) by an investigator blinded for the treatment conditions.
  • Mitochondrial calcium transients were measured by incubating HL-1 atrial cardiomyocytes with 5 pM of the mitochondrial Ca2+-sensitive dye Rhod-2 AM33 (Abeam, Cambridge, MA) and thereafter excited by a 600 nm laser with emission at 605 nm and CaTmito were recorded with the Myocyte Calcium and Contractility System (IonOptix Corporation, The Netherlands).
  • the live recording of the CaTmito was performed at 1Hz stimulation (normal pacing) at 37 °C.
  • HL-1 atrial cardiomyocytes or human tissue samples were lysed in radioimmunoprecipitation assay buffer and Western blot analysis was performed as described before (Zhang D et ah, 2014. Circulation 129: 346-358).
  • the following primary antibodies were used: anti-HSP60 (ADI-SPA-805, Enzo Life Sciences, USA), anti-TOM20 (MCA4300Z, Bio-Rad, The Netherlands), anti-MCU (14997S, Cell Signaling Technology, The Netherlands), OXPHOS Antibody Cocktail (MS604, Abeam, Cambridge MA) and anti-GAPDH (10R-G109a, Fitzgerald, USA).
  • RNA was isolated from HL-1 atrial cardiomyocytes using the Nucleospin RNA isolation kit (Maehery-Nagel, The Netherlands). First strand cDNA was generated by M-MLV reverse transcriptase (Promega, The Netherlands) and random hexamers primers (Promega, The Netherlands). Subsequently, the cDNA was used as a template for quantitative real-time PCR.
  • mRNA levels were expressed in relative units on the basis of a standard curve (serial dilutions of a calibrator cDNA mixture).
  • Total DNA was isolated from patient and control serum utilizing the Nucleospin Tissue kit (Machery-Nagel, The Netherlands), according to manufacturer’s instructions. Isolated DNA was used to determine mitochondrial DNA levels utilizing the CFX384 Real-time system C1000 Thermocycler (Bio-Rad, The
  • Mitochondrial DNA levels were adjusted for nuclear DNA levels and analyzed using the ACT method.
  • HSP levels HSP60, HSPB7, HSP27 and HSP70 were measured in patient and control serum utilizing the DuoSet IC ELISA kit (R&D systems), according to manufacturer’s instructions.
  • DNA damage 80X0G was measured in patient and control serum utilizing the Oxiselect oxidative DNA damage ELISA kit (Cell Biolabs, USA), according to manufacturers instructions.
  • HSP and DNA damage levels were determined by using a standard curve, as provided by manufacturer. Drosophila heart wall contraction measurement
  • Drosophila melanogaster heart wall contraction measurements were performed with the wlll8 strains (Genetic Services Inc, USA), which were maintained at 25 °C on standard medium.
  • Adult Drosophila’s were removed after fertilization and the medium, containing the fly embryos, was supplied with Ru360 (20, 50 and 100 pM), freshly dissolved in demineralized water. Controls were subjected to demineralized water only.
  • Transparent prepupae were selected, placed on a 1% agarose gel in PBS and subjected to taehypacing (5Hz for 20 minutes, 20V and 5ms pulses) with a C-PacelOOTM-culture pacer (IonOptix Corporation, The
  • Results are expressed as mean ⁇ SEM of at least two independent experiments. Multiple -group comparisons were obtained by a one-way ANOVA with a Bonferroni correction or a Kruskal- Wallis test followed by a Mann-Whitney U test. Individual group-mean differences were evaluated with a Student’s t-test. Correlations were estimated using Pearson correlation. All P-values were two-sided. A value of P.0.05 was considered statistically significant. SPSS version 22 was used for all statistical evaluations. Results
  • Tachypacing induces mitochondrial stress and dysfunction
  • Tachypacing for 2h initially increased basal oxygen consumption rate and spare respiratory capacity.
  • tachypacing beyond 2h progressively decreased the basal oxygen consumption rate and very markedly inhibited spare respiratory capacity (Figure IB), which accounts for a less adequate response to cellular stress or increased cardiac workload (Desler et a , 2012. J Aging Res 2012: 192503).
  • Figure 1C protein expression of complex I, II, III and V of the respiratory chain did not change during tachypacing ( Figure 1C), indicating that the decreased respiration during tachypacing is not due to diminished respiratory chain protein expression.
  • Cytosolic Ca2+ overload constitutes the most obvious mechanism underlying the tachypaced-induced mitochondrial dysfunction.
  • Ca2+ overload in both cytosol and ER will result in Ca2+ buffering by mitochondria (Babcock et a , 1997. J Cell Biol 136: 833-844; Maack et ah, 2006. Circ Res 99: 172-182).
  • the subsequent excessive mitochondrial Ca2+ buffering leads to mitochondrial Ca2+ overload, and consequently mitochondrial swelling, dysfunction (Rizzuto et ah, 2012. Nat Rev Mol Cell Biol 13: 566-578) and reduced mitochondrial Ca2+ uptake.
  • Reduced mitochondrial Ca2+ uptake may be a trigger for AF, as it encounters for enhanced cytosolic Ca2+ levels.
  • CaTmito mitochondrial calcium transients
  • Tachypacing beyond 2h significantly reduced CaTmito, mainly characterized by the reduction in amplitude ( Figure ID and E).
  • LYhhIo mitochondrial membrane potential
  • TMRM fluorescent probe
  • Table 1 Demographic and clinical characteristics of patients with AF and control patients in Sinus Rhythm, used for Western blot analysis of atrial appendages.
  • CAD coronary artery disease
  • VHD valvular heart disease
  • ASD atrial septal defect
  • Aorta aneurysm aorta ascendens
  • CHD congenital heart defect
  • AT congenital heart defect
  • Atrial tachycardia LS: longstanding
  • LA left atrium
  • LVF left ventricular function
  • NYHA New York Health Association for exercise tolerance
  • tachypacing also induces mitochondrial stress by measuring levels of mitochondrial chaperones upregulated upon mitochondrial stress. Tachypacing resulted in a significant and progressive upregulation of mRNA of both HSP60 and HSP10 ( Figure 2C and D).
  • tachypacing affected the number of mitochondria by measuring the amount of mitochondrial DNA (mt.DNA) and TOM20 levels. Both cellular mtDNA and TOM20 levels showed no changes upon tachypacing ( Figure 2E and F), suggesting that the number of mitochondria did not change upon tachypacing. Therefore, the mitochondrial dysfunction upon tachypacing is not due to a decreased number of mitochondria.
  • a mitochondrial Ca2+ uniporter inhibitor protects from tachypacing-induced mitochondrial stress and dysfunction.
  • the findings reveal that tachypacing induces impaired mitochondrial function.
  • CaTmito the effect of a compound targeting the MCU (by inhibition; Ying et ah, 1991.
  • Ru360 was explored.
  • the effect of RU360 to counteract tachypacing-induced loss of CaTmito was examined after 6 hours of pacing, when the CaTmito are significantly reduced ( Figure ID and E).
  • Ru360 treatment also ameliorated mitochondrial stress and dysfunction.
  • Ru360 treatment normalized cellular ATP levels, transcription levels of HSP60 and HSP10 and CaTmito to non- treated control levels ( Figure 3B-E).
  • Ru360 treatment even enhanced cellular ATP levels and CaTmito significantly in normal-paced cardiomyocytes (Figure 3B and 3E).
  • the MCU mediates tachypacing-induced mitochondrial changes. To determine whether tachypacing-induced mitochondrial changes are specifically mediated by the MCU, we first examined its protein and mRNA levels.
  • tachypaced cardiomyocytes (MCU siRNA high, Figure 4E and F). These results suggest that a small reduction in MCU, not affecting normal mitochondrial Ca2+ handling, is beneficial to counteract tachypaeing effects. However, a larger reduction of MCU levels seems detrimental, likely due to an imp ir ent of physiological mitochondrial Ca2+ influx, which is already observed under baseline conditions. To confirm the importance of the MCU, the effect of Ru360 was explored in tachypaced Drosophila melanogaster (Zhang D et ah, 2014. Circulation 129: 346-358; Zhang et ah, 2011. J Mol Cell Cardiol 51: 381-389).
  • Mitochondrial dysfunction is further evidenced in AF patients by increased protein expression of HSP60 and MCU in LAA, while there is no change in expression of TOM20 (Figure 5D-G). Changes in expression were only present in LAA, as observed before (Zhang D et ah, 2014. Circulation 129: 346-358; Li et al awkward 2001. Circulation 104: 2608-2614; Voigt et ah, 2010. Circ Arrhythm Electrophysiol 3: 472-480) as the expression of HSP60 and MCU in RAA is similar to SR. These results suggest that mitochondrial dysfunction is not only found in an in vitro model of AF, but is also present in AF patients.
  • Mitochondrial dysfunction can lead to the release of mitochondrial DNA into the circulation of patients, where it acts as a damage associated molecular pattern (Krysko et ah, 2011. Trends Immunol 32: 157-164; Nakahira et ah, 2015. Antioxid Redox Signal 23: 1329-1350), increased levels might lead to inflammatory responses, organ injury (Nakahira et ah, 2015. Antioxid Redox Signal 23: 1329- 1350; Zhang et ah, 2010. Nature 464: 104-107) and increased mortality (45.
  • mitochondrial DNA is primarily used to restore homeostasis, but prolonged exposure leads to detrimental changes (Palmai-Pallag and Bachrati, 2014.
  • COX3 cytochrome c oxidase subunit 3
  • ND1 DNA ND1 DNA in serum of control patients (without any atrial disease) or patients with different stages of AF (paroxysmal (PAF), persistent (PeAF) and longstanding persistent (FS-PeAF)) (See Table 3).
  • AF paroxysmal
  • PeAF persistent persistent
  • FS-PeAF longstanding persistent
  • tachypacing to induce substantial mitochondrial dysfunction, including failure of respiration, most likely resulting from enhanced Ca2+ influx through the MCU, consequently impairing mitochondrial calcium transients.
  • Our data demonstrate that tachypacing induces mitochondrial stress, as exemplified by increased transcription of the mitochondrial stress chaperones HSP60 and HSP10 and fragmentation of the mitochondrial network.
  • mitochondrial changes including decreased cellular ATP levels and increased HSP60 expression, are present in AF patients, which also show myolysis and fragmented and dispersed mitochondrial localization.
  • Treatment with Ru360, an inhibitor of the MCU, or modest MCU downregulation restored these detrimental mitochondrial changes upon tachypacing.
  • Ru360 treatment protected against contractile dysfunction in a Drosophila model for AF.
  • our data indicates that cell-free circulating mitochondrial DNA in serum may be a potential biomarker of AF.
  • HL-1 cardiomyocytes derived from adult mouse atria were obtained from Dr. William Clayeomb (Louisiana State University, New Jersey) and cultured in complete Clayeomb medium (Sigma) supplemented with 10% FBS (PAA).
  • HL-1 cardiomyocytes were cultured on cell culture plastics or on glass coverslips coated with 0.02% gelatin (Sigma) in a humidified atmosphere o f 5% C02 at 37oC.
  • the cardiomyocytes which have a basal spontaneous contraction rate of ⁇ 0.5-1 Hz4, were subjected by tachypacing (TP) to a 5-10 fold rate increase as observed in clinical AF (5 Hz, 40 V, pulse duration of 20 ms) with a C-PacelOO culture pacer (IonOptix) for 12 h unless stated otherwise.
  • HL-1 cardiomyocytes followed the pacing rate.
  • Ca2+ transients (CaT) were imaged by Solamere-Nipkow-Confocal-Live-Cell-Imaging system (based on a Leica DM IRE2 Inverted microscope).
  • Ca2+-sensitive Fluo-4-AM dye (Invitrogen) was loaded into HL-1 cardiomyocytes by 45 min incubation, followed by 3 times washing with PBS. Ca2+ loaded cardiomyocytes were excited by 488 nm and emitted at 500-550 nm and visually recorded with a 40* -objective. Calcium transient (CalT) measurements were performed in a blinded manner by selection of normal shaped cardiomyocytes with the use of bright field settings, followed by a switch to the fluorescent filter to determine the CaT.
  • Ca2+-sensitive Fluo-4-AM dye Invitrogen
  • HL-1 cardiomyocytes Prior to 12 h TP, HL-1 cardiomyocytes were treated for 12 h with the PAftP inhibitors 3-aminobenzamide (3-AB, Sigma-Aldrich), ABT-888 (Selleckchem), olaparib (Selleckchem), beta-nicotinamide adenine dinucleotide hydrate (NAD+, Sigma-Aldrich) or transfected with scrambled siRNA (control, Ambion) PARP1 siRNA (Ambion), or PARP2 siRNA (Santa Cruz) to study the specific role of PARP1 and PARP2, respectively.
  • the atria were cut off the heart and rinsed in isolation solution containing (in mM): 100 NaCl, 5 Hepes, 20 D-glucose, 10 KC1, 5 MgS04, 1.2 KH2P04, 50 Taurin, 0.5% bovine serum albumin (BSA) (pH 7.4), transferred to a 15-ml tube containing 10 ml of isolation solution plus 0.02 mM CaCL and 0.02 U per ml DNase, gently triturated for 7 min, and subsequently filtered through a 200pm mesh filter into another 15-ml tube, followed by centrifugation for 1 min at 700x g. The supernatant was removed and the pellet containing atrial
  • cardiomyocytes was resuspended carefully in 10 ml of isolation solution plus 0.02 mM CaC42. Next, the Ca2+ concentration was increased in 5-min steps from 0.1,
  • Atrial cardiomyocytes were left to sink for 20 min and transferred into laminin-coated plates in plating medium (M199 medium plus 5% fetal calf serum) for 2 h followed by replacement with M199 medium plus Insulin- Transferrin-Sodium Selenite Supplement (Sigma).
  • the isolated adult rat atrial cardiomyocytes have a basal spontaneous contraction rate of - 0.5-1 Hz in vitro.
  • Atrial cardiomyocytes Prior to tachypacing, atrial cardiomyocytes were treated for 2 h with the PARP inhibitors ABT-888 (Selleckchem) or olaparib (Selleckchem), followed by 2 h tachypacing at 5 Hz, 30 V with a pulse duration of 2 ms.
  • ABT-888 Selleckchem
  • olaparib Selleckchem
  • cardiomyocytes were either non-paced (NP) or paced for 2 h at 1Hz, 30 V and pulse duration of 2 ms. Atrial cardiomyocytes followed the pacing rate.
  • Atrial cardiomyocytes were washed twice with M199 medium, incubated with the Ca2+ dye Fluo-4 (1 u per ml) in M199 medium for 15 min, and rinsed twice again with M199 medium.
  • the Fluo-4-loaded cardiomyocytes were excited at 488 nm and the light emitted at 500-550 nm and recorded with a high speed confocal microscope (Nikon AIR). Bright field settings were used to randomly select normal-shaped cardiomyocytes, followed by a switch to the fluorescent filter to determine the CaT. As such, CaT measurements were conducted in a blinded manner.
  • the wild-type Drosophila melanogaster strain wlll8 strain was used for all drug screening (PARP inhibitors or NAD+ ) experiments. Hereto, female and male adult flies were crossed. After 3 days, flies were removed from the embryos- eontaining tubes and drugs or the same amount of vehicle (DMSO) were added to the food. Drosophila were incubated at 25 °C for 48 h, with larvae consuming the drug/vehicle prior to entering the prepupae stage. The Drosophila prepupae were collected and subjected to tachypacing for 20 min (4 Hz, 20 V, pulse duration of 5 ms) and heart wall functions were measured as described in detail below.
  • PARP inhibitors or NAD+ drug screening
  • RNAi lines were crossed with a Hand-GAF4 driver strain (kind gift of Prof. Dr. Achim Paululat; Sellin et a , 2006. Gene Expr. Patterns 6: 360-375).
  • wild-type flies wlll8 were crossed with Hand-GAE4 driver flies.
  • Prepupae of FI offspring were tachypaced as previously described (Zhang et al., 2014. Circulation 129: 346- 358).
  • Heart wall contractions were measured utilizing high-speed digital video imaging (100 frames per s) before and after tachypacing in at least duplicated 10 s- movies. Movies were used to prepare heart wall traces and M-mode cardiography. Hereto, 1-pixel width lines were drawn across the heart wall, followed by determination of Plot-Z axis profile (based on contrast changes) to generate heart wall traces or kymographs (via the kymograph plugin of Image J) for M-mode cardiography. To determine the heart rate and arrhythmicity index (defined as the standard deviation of the heart period normalized to the median heart period of each fly followed by averaging across flies; Fink et al., 200.
  • RAA Right atrial appendages
  • LAA left atrial appendages
  • HL-1 cardiomyocytes or human tissue samples were lysed in
  • SDS-polyacrylamide gel electrophoresis SDS- PAGE
  • PARP1 1:500, Santa Cruz, sc-25780
  • Y H2AX 1:1000, Millipore, 05-636
  • Cavl.2 (1:200, Alomone Labs, ACC-003)
  • Kvll.l (1:400, Alomone Labs, APC-062)
  • Kir3.1 (1:200, Alomone Labs, APC-005)
  • B-actin (1:1000, Santa Cruz, se-47778)
  • GAPDH 1:5000, Fitzgerald, 10R-G109A
  • Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Dako). Signals were detected by the ECL detection method (Amersham) and quantified by densitometry (Syngene, Genetools).
  • NAD and NADH levels were measured according the manufacturer’s instructions of the assay kit (Abeam, ab65348).
  • HL-1 cardiomyocyt.es were lysed in NAD extraction buffer and the protein concentration was determined (BioRad Laboratories).
  • 50 m ⁇ of each sample was mixed with 100 m ⁇ NAD cycling buffer and incubated at room temperature (RT) for 5 min to convert NAD+ to NADH, followed by the addition of 10 m ⁇ NADH developer buffer and 2 h incubation at RT.
  • RT room temperature
  • NAD/NADH levels were measured at 450 nm (BioTek Synergy 4 plate reader).
  • NAD+ in each sample was decomposed by incubation at 600C for 30 min before measurement.
  • the NADH amount in cultured cardiomyocytes and tissue was below the detection limit59. Therefore, the NAD+ amount per ug of total protein was used as endpoint.
  • HL-1 cardiomyocytes were t.rypsinized, harvested by centrifugation, suspended at 2x105 cells per ml in phosphate-buffered saline (PBS), combined with 45 pl melted LAM agarose at ratio of 1:10 (v:v) and immediately pipetted onto CometSlides. Slides were dried for 30 min at 4°C, incubated firstly in lysis solution for 1 h and then in freshly prepared alkaline unwinding solution (pH>13) for 1 h.
  • PBS phosphate-buffered saline
  • HL-1 atrial cardiomyocytes received 10 Gy and rat atrial cardiomyocytes 40 Gy of irradiation with a dose rate of 0.0562 Gy per second by utilizing a cobalt-60 gamma-source (Gammacell 220 Research Irradiator, MDS Nordion, Canada).
  • HL-1 and rat atrial cardiomyocytes were treated with 40 mM ABT-888 (12 h) or 5 mM ABT-888 (2 h), respectively, prior to the irradiation.
  • cardiomyocytes were either prepared for Western blot analyses, NAD+ level measurements or CaT recordings.
  • OxyBlot protein oxidation detection kit (Millipore, S7510) was used, following the company’s instructions.
  • cardiomyocytes were lysed in RIPA buffer containing 1% beta-mercapto- ethanol (Sigma). 10 pg of protein was denatured in 6% SDS, derivatized by incubation for 15 min in 2,4-dinitrophenylhydrazine (DNPH) solution, followed by the addition of neutralization solution. After neutralization, protein samples were subjected to SDS-PAGE, transferred onto nitrocellulose membranes and probed with anti-dinitrophenyl (DNP) antibody (1: 150) for 1 h at RT.
  • DNP anti-dinitrophenyl
  • HRP-conjugated goat anti-rabbit IgG (1:300) was used as secondary antibody. All reagents were included in the kit. Signals were detected by the ECL detection method (Amersham) and quantified by densitometry (Synge ne, Gene tools).
  • GAPDH F CATCAAGAAGGTGGTGAAGC and R: AC C AC C CT GTT GCT GTAG .
  • ACTB F GGCTGTATTCCCCTCCATCG and R: C C AGTT GGT AAC AAT G C CAT GT .
  • Primer pairs used in Drosophila included PARP1 F:
  • HL-1 cardiomyocytes were grown on coverslips until 80% confluence and subjected to TP for various time periods, with or without drug treatment.
  • cardiomyocytes were rinsed in PBS and fixed with 4% formaldehyde for 15 min, rinsed twice with PBS, permeabilized by incubation with 0.1% Triton X-100 in PBS for 10 min, rinsed twice in PBS and blocked with blocking solution (0.5% BSA and 0.15% glycine in PBS) for 10 min. After blocking, cardiomyocytes were incubated with primary antibodies for 2 h at RT. After rinsing the cardiomyocytes three times with blocking solution, cardiomyocytes were incubated with secondary antibodies for 45 min at RT shielded from light, followed by rinsing with blocking solution three times and PBS twice. Lastly,
  • cardiomyocytes were incubated with mounting media containing DAPI
  • Antibodies used were: anti-YH2AX (1: 100, Millipore, 05-636), anti- PAR (1:200, BD Bioscience, 551813), anti-PARPl (1:200, Santa Cruz, sc-25780), anti-oxoguanine 8 (1:100, Abeam, ab64548), goat anti-rabbit FITC (1:200,
  • the frozen RAA samples of SR and AF patients were used for staining of gH2AC and 53BP1.
  • Frozen sections were cut into 5 mih slices. Sections were air dried for 30 min, fixed in 4% formaldehyde for 10 min at RT, washed 3 times with PBS for 10 min, then permeabilized with 0.3% Triton X-100 (in PBS) for 10 min at RT and washed 3 times for 5 min with PBS. After blocking of the sections with 1% BSA blocking solution for 30 min at RT, sections were incubated with primary antibodies directed against gH2AC (1:100; Millipore, 05-636) or 53BP1 (1:100;
  • the nuclear shape of cardiomyocytes in RAAs of SR and AF patients was determined by measuring its circularity (form factor) with Image J 1.48 software (US National Institute of Health).
  • 8-bit images of DAPI-stained nuclei were converted to binary photos by the method of“make binary” in Image J, traced by hand and the circularity was calculated by the formula 4n*A per P2, in which A denotes the surface area and P the perimeter.
  • the circularity of a perfect round circle and a line segment are 1 and 0 respectivelyGO.
  • Results are expressed as mean ⁇ standard error of the mean (SEM).
  • PAKP Upon activation, PAKP consumes NAD+ to synthesize PAR. Therefore, progressive and excessive activation of PARP results in reductions in NAD+ levels, which finally results in the energy loss and functional impairment of
  • NAD+ is an important constituent for proper cell function and health
  • Tachypacing resulted in a significant calcium transient (CaT) loss, which was dose-dependently abrogated by preserving cellular NAD+ levels through exogenous supplementation (Fig. 7C).
  • CaT calcium transient
  • PARPl is the key PARP enzyme driving tachypacing-induced contractile dysfunction
  • PARPl expression was suppressed specifically in the heart of Drosophila in two RNAi lines, as confirmed by Western blotting (data not shown).
  • suppression of PARPl resulted in protection against tachypacing-induced heart wall dysfunction (Fig.
  • PARP1 inhibitors including the general inhibitors, nicotinamide and 3-AB, and the specific PARP1/2 inhibitors ABT-888 and olaparib. Both general and specific inhibition of PARP1/2 precluded
  • tachypacing-induced PARylation of proteins and decrease in NAD+ levels Fig. 8C.
  • the PARP1 inhibitors ABT-888 and olaparib also significantly attenuated tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes and Drosophila without influencing the baseline contractile function in
  • tachypacing of HL-1 cardiomyocytes resulted in significant electrophysiological deteriorations, including alterations in action potential duration (APD), increased APD dispersions, decreased area of excitability and ion channel remodeling. All tachypacing-induced electrophysiological alterations were prevented by PARP1 inhibitors olaparib and/or ABT-888 (data not shown). Since AF is a progressive disease, it is of interest to study whether PARP1 inhibition accelerates recovery from tachypacing- induced NAD+ depletion and contractile dysfunction.
  • HL-1 cardiomyocytes were tachypaced, followed by 24 h recovery under no pacing conditions.
  • vehicle treated cardiomyocytes no recovery from tachypacing induced CaT loss, NAD+ depletion or increased PAR levels was observed.
  • tachypaced HL-1 cardiomyocytes post-treated with ABT-888 revealed accelerated recovery at all endpoints (Fig. 9A).
  • gH2AC levels were significantly increased in patients with AF compared to SR (Fig. 9C).
  • a significant positive correlation was found between the amount of PAR and gH2AC (Fig. 9C), indicating that AF patients with high levels of PAR also reveal more DNA damage.
  • the amount of another DNA damage marker, 53BP1 was significantly increased in AF patients compared to control SR patients (data not shown).
  • nuclear circularity a marker for oxidative stress-induced DNA damage (Barascu et ak, 2012. EMBO J 31: 1080-1094), showing that nuclear circularity was significantly decreased in patients with AF compared to controls in SR (data not shown).
  • RNA from human serum and human tissue was extracted by use of miRNeasy Serum/Plasma Advanced Kit (Qiagen, German) and TRIzol reagent (Invitrogen, Carlsbad, CA), respectively. The purity and concentration of the RNA were determined by a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).
  • cDNA was synthesized by utilizing iScript cDNA synthesis kit (BioRad, CA) following the manufacturer’s instructions. RT reactions were performed with a total volume of 10 ul with random primers. RT procedures were: 25°C for 5 min, 46°C for 20 min, 95°C for 1 min, followed by storage at 4°C.
  • Plasma levels of lncRNAs were determined by quantitative real time polymerase chain reaction (qRT-PCR) on a Bio-Rad CFX384 real time system using SYBR green dye (Bio-Rad, CA). qRT-PCR procedure: 95°C for 3 min, 45 cycles of 95°C for 10 s, and 60°C for 30 s, followed by 95°C for 10 s, melt curve 65°C to 95°C increment of 0.5°C for 10 s + plate-read. Gene expression was corrected for levels of the reference gene values (18S/GAPDH). Relative expression was calculated by relative quantification (2-AACt) method. Values of Ct over 35 were considered to be negative. Primer sequences are listed below:
  • hSarrah forward CCTGGACTGCGTTCACGTTT and hSarrah reverse:
  • hUCAl forward ACGCTAACTGGCACCTTGTT and hUCAl reversed:
  • hLIPCAR forward TAAAGGATGCGTAGGGATGG
  • hLIPCAR reverse TAAAGGATGCGTAGGGATGG
  • DNA damage 8-hydroxydeoxyguanosine (8-OHdG), which is induced by DNA damage and is a marker of DNA oxidation.
  • Long non-coding RNAs have a length >200 nucleotides, are not translated into proteins and are implicated in the regulation of gene expression. These long non-coding RNAs include Sarrah, Urothelial carcinoma-associated- 1 (UCA1), CDR1AS and long intergenie non-coding RNA predicting cardiac remodeling (LIPCAR).
  • UCA1 Urothelial carcinoma-associated- 1
  • CDR1AS CDR1AS
  • LIPCAR long intergenie non-coding RNA predicting cardiac remodeling
  • Mitochondrial DNA cell-free circulating mitochondrial DNA can act as a damage-associated pattern, hut is also implicated as an biomarker for several applications, including cancer progression, ICU mortality, dengue severity, cardiac arrest survival and diabetes mellitus. Mitochondrial DNA levels were determined for cytochrome C oxidase subunit III (COX3) and NADH dehydrogenase subunit I (ND1).
  • COX3 cytochrome C oxidase subunit III
  • ND1 NADH dehydrogenase subunit I
  • DNA damage for example 8-OHdG
  • DNA damage showed increased levels in all the AF stages, it did not correlate with any of the other measured stress markers.
  • several of the long non-coding RNAs did correlate with each other and the mitochondrial DNA markers (see Tables 4 and 5).
  • the long-non coding RNAs and mitochondrial DNA markers also correlate with each other in patients with recurrence of AF after treatment.

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Abstract

The invention relates to methods of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of providing a bodily fluid from said individual and quantifying a biomarker in said bodily fluid. The invention further relates to methods for assigning standard-of-care therapy to an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to the methods of typing of the invention, and to a standard- of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to the methods of the invention.

Description

Title: Biomarkers for atrial fibrillation
FIELD
The invention relates to methods of typing an individual as suffering from, or being at risk of suffering from, atrial fibrillation. Furthermore, the invention relates to treatment of an individual that was typed as suffering from, or being at risk of suffering from, atrial fibrillation according to the methods of the invention.
1 INTRODUCTION
Atrial fibrillation (AF) is the most common sustained clinical
tachyarrhythmia and is associated with increased mortality and morbidity (Dobrev et a , 2012. Nat Rev Drug Discov 11: 275-291; Heijman and Dobrev. 2015. Basic Res Cardiol 110: 41). Its incidence is age-related and expected to rise due to the aging population, which will increase hospitalization and medical costs, contributing significantly to the socioeconomic burden (Mozaffarian et ah, 2015. Circulation 131:e29-3223). Due to its progressive nature, patients undergo transition from paroxysmal AF to persistent and longstanding persistent AF (Heijman and Dobrev. 2015. Basic Res Cardiol 110: 41). Each stage of AF is caused by a certain degree of structural and electrical remodeling. Progressive electrical remodeling is associated with an increase in conduction abnormalities, which favor AF progression (De Groot et a , 2010. Circulation 122:1674-1682; Dobrev et ah, 2012. Nat Rev Drug Discov 11: 275-291; Heijman and Dobrev. 2015. Basic Res Cardiol 110: 41).
Electrical remodeling is defined by shortening of the action potential duration and the effective refractory period, due to alterations in ion channel expression, increased cytosolic calcium levels and atrial hypocontractility, but is often reversible ((Dobrev et a , 2012. Nat Rev Drug Discov 11: 275-291; Heijman and Dobrev. 2015. Basic Res Cardiol 110: 41). Structural remodeling, however, is irreversible and includes atrial hypertrophy, fibrosis and myolysis (Dobrev et ah, 2012. Nat Rev Drug Discov 11: 275-291; Thijssen et a , 2001. Cardiovasc Res 52: 14-24). Current therapies have limited efficacy, especially in longstanding persistent AF, likely due to the lack of knowledge about the underlying molecular mechanisms of AF-related cardiac structural remodeling (Heijman and Dobrev. 2015. Basic Res Cardiol 110: 41). Therefore, recent research is directed at revealing the pathways leading to AF-induced cardiac structural remodeling in order to develop more mechanism-related AF therapies.
One of the mechanisms underlying AF-induced structural remodeling is derailment of proteostasis, i.e. the homeostasis of protein synthesis, folding, assembly, trafficking, function and degradation (Labbadia and Morimoto, 2015. Annu Rev Biochem 84: 435-464). Activation of proteases (Brundel et al., 2002.
Cardiovasc Res 54: 380-389; Brundel et al., 2004. Cardiovasc Res 62: 521-528; Ke et al., 2008. J Mol Cell Cardiol 45: 685-693) and histone deacetylases (Zhang D et al., 2014. Circulation 129: 346-358) contribute to degradation of contractile and structural proteins, resulting in proteostasis derailment and structural remodeling. In addition, in vitro AF initiation leads to increased RhoA activation, changes in structural proteins and failure to mount the heat shock response (Ke et al., 2011. PLoS One 6: e20395; Meijering et al., 2015. PLoS One 10: e0133553). In accord, induction of the heat shock response, a primary defense mechanism against derailment of proteostasis, attenuates cardiomyoeyte remodeling and preserves the cardiomyocyte contractile function (Ke et al., 2011. PLoS One 6: e20395; Brundel et al, 2006. Circ Res 99: 1394-1402; Brundel et ah, 2006. J Mol Cell Cardiol 41: 555- 562; Zhang et al., 2011. J Mol Cell Cardiol 51: 381-389). In addition, activation of macroautophagy, in response to endoplasmic reticulum (ER) stress, was recently found to constitute an important route involved in degradation of structural proteins in AF (Wiersma et al., 2017. J Amer Heart Assoc 6: e006458). As the ER is in close contact with mitochondria through so called mitochondria-associated membranes, which promote the exchange of metabolites, including lipids and Ca2+, mitochondria respond with stress to ER stress (Rainbolt et al., 2014. Trends Endocrinol Metab 25: 528-537).
Interestingly, there are indications that mitochondrial stress contributes to AF pathogenesis. One of the most direct indications comprises a study
demonstrating the association between decreased mitochondrial respiration and expression of respiratory chain proteins and the incidence of post-operative AF in obese patients (Montaigne et al., 2013. J Am Coll Cardiol 62: 1466-1473). Likewise, protein expression profiling in a small cohort of valvular disease patients demonstrated differential expression of important energy metabolism-related proteins between sinus rhythm and AF patients (Tu et ah, 2014. Circ J 78: 993- 1001). Moreover, the presence of oxidative stress in in vivo models of AF (Lenaerts et ah, 2013. Europace 15: 754-760) and in AF patients (Mihm et ah, 2001.
Circulation 104: 174-180; Kim et al., 2003. Exp Mol Med 35: 336-349; Rodrigo et al., 2013. J Am Coll Cardiol 62: 1457-1465) indicates disruption of normal respiration. Despite these indications of aberrant mitochondrial function in AF, characterization of these changes, putative mechanism and contribution to AF pathogenesis has not been studied. This is striking, as mitochondrial ATP production is vital for cardiac contraction. Moreover, on a theoretical level, the relationship between mitochondrial ATP production and Ca2+ influx from the ER (I’rou nd et al., 2013. J Biol Chem 288: 18975-18986) immediately links cellular Ca2+ overload in AF to mitochondrial dysfunction. Following cellular Ca2+ overload, which is toxic for the cardiomyocytes, excessive Ca2+ is stored in the ER and mitochondria, thereby causing an ER stress response and swelling and dysfunction of mitochondria, respectively (Bround et al., 2013. J Biol Chem 288: 18975-18986; Rizzuto et al., 2012. Nat Rev Mol Cell Biol 13: 566-578; Neef et al., 2010. Circ Res 106: 1134-1144; Ribeiro et al, 2000. Cell Calcium 27: 175-185;
Hoyer-Hansen and Jaattela, 2007. Cell Death Differ 14: 1576-1582). As dysfunction of mitochondria induces release of its components from the cardiomyocytes (Krysko et al., 2011. Trends Immunol 32: 157-164; Nakahira et al., 2015. Antioxid Redox Signal 23: 1329-1350; Kwong and Molkentm, 2015. Cell Metab 21: 206-214), cell- free circulating mitochondrial DNA might represent a biomarker for AF in patients. As mitochondria comprise approximately 30% (Harris and Das, 1991. Biochem J 280: 561-573) of the cardiomyocyte volume and account for 90% (Harris and Das, 1991. Biochem J 280: 561-573) of the provided cardiac contraction energy, mitochondrial dysfunction is detrimental for the heart. This is exemplified by the vast amount of cardiac diseases caused or worsened by mitochondrial dysfunction. Therefore, we examined the mitochondrial function in experimental models of AF remodeling and in AF patients. Furthermore, we determined whether cell-free circulating mitochondrial DNA represents a biomarker in a cohort of
approximately 400 AF patients in different stages (paroxysmal, persistent and longstanding persistent) of AF. 2 SUMMARY OF THE INVENTION
The invention provides a method of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of providing a bodily fluid, preferably blood, from said individual, quantifying a level of long non coding RNA (lncRNA), mitochondrial nucleic acid and/or a level of DNA damage in said bodily fluid, preferably of lncRNA and mitochondrial nucleic acid, comparing said quantified lncRNA, mitochondrial nucleic acid and/or level of DNA damage to a reference and typing said individual as suffering from, or at risk of suffering from, atrial fibrillation if said quantified lncRNA is altered, compared to the reference, if the quantified mitochondrial nucleic acid is altered, compared to the reference, and/or if said quantified level of DNA damage is altered compared to the reference.
As at present, there is no technique to detect onset of atrial fibrillation and/or progression. The identification of a biomarker to identify patients at risk for AF onset, progression and/or recurrence after treatment is of paramount importance to prevent AF-related complications such as stroke and heart failure.
It was found that an increase of a level of lncRNA, a mitochondrial nucleic acid and/or a level of DNA damage, when compared to a healthy individual, can be used to identify an individual suffering from self-terminating stages of atrial fibrillation (AF) who is at risk of progressing to more persistent AF, while a decrease of a mitochondrial nucleic acid and a level of DNA damage, when compared to a healthy individual, can be used to identify an individual suffering from persistent stages of AF who is at risk of progressing to long-standing persistent AF.
The level of DNA damage is preferably quantified by determining a level of 8- oxoguanine.
Said lncRNA preferably is selected from Sarrah, UCA1, CDR1AS and
LIPCAR.
The mitochondrial nucleic acid in methods of the invention preferably is or comprises mitochondrial DNA. Said mitochondrial nucleic acid preferably comprises nucleic acid from a cytochrome C oxidase 3 gene and/or a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene. A preferred method of the invention comprises quantifying mitochondrial DNA from a cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L,
4, 5, 6) gene in a bodily fluid, preferably blood, from an individual.
A further preferred method of the invention comprises quantifying a level of lncRNA, a level of nucleic acid from a mitochondrial cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene, and quantifying a level of DNA damage in said bodily fluid.
A method of the invention may further comprise determining a level of heat shock protein the bodily fluid of the individual, preferably of HSPB7.
The invention further provides a method for assigning standard-of-care therapy to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention; and assigning a standard-of-care therapy to the individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation.
The standard-of-care therapy preferably is selected from anti-arrhythmic drug therapy aimed at rate and/or rhythm control, electrical or chemical cardioversion, and/or ablative therapy.
Said standard-of-care therapy preferably is combined with an antiplatelet and/or anticoagulant. Said antiplatelet and/or anticoagulant preferably is selected from aspirin, warfarin, and a direct- acting oral anticoagulant such as dabigi trail, rivaroxaban, edoxaban and apixaban.
Said rhythm controller preferably is an ion channel blocker, such as a sodium channel blocker such as flecainide, propafenone and/or quinidine, and/or a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
The invention further provides a standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to the method the invention.
3 FIGURE LEGENDS
Figure 1. Tachypacing induces mitochondrial dysfunction. A) Quantified data showing reduced cellular ATP levels during tachypacing. B) The oxygen
consumption rate (OCR) showing the mitochondrial respiration. 1-33 minutes: basal respiration, 33-56 minutes: addition of oligomycin to inhibit ATP synthesis, 56-96 minutes: addition of FCCP for the maximal respiratory capacity, 96-119 minutes: addition of rote none and antimycin A for the non-mitochondrial respiration. C) Top panel represent Western blot of respiratory chain complexes I, II, III and V. Lower panels reveal quantified data of the respiratory chain complexes normalized for basal GAPDH protein level. D) Quantified data showing mitochondrial calcium transients (CaTmito) during tachypacing. E) Representative CaTmito of HL-1 eardiomyocytes after normal pacing (NP) or tachypacing (TP). F) Quantified data showing mitochondrial membrane potential during tachypacing. *P<0.05, **P<0.01, ***P<0.001 versus NP, #P<0.05, ##P<0.01 versus NP.
Figure 2. Tachypacing induces mitochondrial network fragmentation and stress. A) Quantified data showing the transition of the mitochondrial network from tubular to fragmented during tachypacing. B) Representative confocal images of tachypaeed HL-1 eardiomyocytes of the mitochondrial network morphology, for the period as indicated. Quantitative real-time PCR of mitochondrial stress markers C) HSP60 and D) HSP10 in response to tachypacing for the indicated duration. E) Quantified data showing no changes in mitochondrial DNA during tachypacing. F) Top panel represent Western blot and lower panel reveal quantified data of TOM20 normalized for basal GAPDH protein level. *P<0.05, ***p<0.001 versus NP.
Figure 3. Inhibition of the MCU protects against mitochondrial stress and dysfunction. A) Quantified CaTmito amplitude of normal paced (NP) and 6h tachypaeed (TP) HL-1 atrial eardiomyocytes treated with different concentrations of RU360. Quantified data showing protection of Ru360 treatment on B) cellular ATP levels, C) HSP60 mRNA levels, D) HSP10 mRNA levels and E) CaTmito after NP or TP. Black bars represent non-treated HL-1 eardiomyocytes; white bars represent Ru360-treated eardiomyocytes. *P<0.05, **P<0.01, ***P<0.()01 versus NP C no treatment (black bar), #P<0.05, ##P<0.01, ###P<0.001, &P<0.05, &&P<0.01, &&&P<0.001 versus NP C Ru360 (white bar).
Figure 4. Mitochondrial changes are due to the MCU. A) Top panel represent Western blot of MCU and GAPDH and lower panel reveals quantified date of MCU normalized for basal GAPDH levels. B) Quantitative real-time PCR of MCU in response to tachypacing (TP) for the indicated duration relative to normal pacing ί
(NP). C) Representative CaTmito of NP or TP HL-1 cardiomyocytes either non- transfected or transiently transfected with MCU, generating MCU overexpression (OE). D) Quantified CaTmito amplitude of NP and 6 hour TP HL-1 cardiomyocytes either non-transfected (C) or transfected with MCU. E) Quantified CaTmito amplitude of NP and 6 hour TP HL-1 cardiomyocytes either non-transfected (C) or transfected with MCU siRNA with 60% or 20% reduced MCU expression (high and low, respectively). F) Representative CaTmito of NP or TP HL-1 cardiomyocytes either non-transfected or transiently transfected with MCU siRNA. G) Quantified data showing heart wall contraction rates from Drosophila melanogaster from each group as indicated. White bars represent normal paced (NP in HL-1
cardiomyocytes) or spontaneous heart rate (SR in Drosophila ) and black bars represent tachypaced HL-1 cardiomyocytes or Drosophila. *P<0.05, **P<0.01, ***P<0.001 versus control NP or SR, #P<0.05, ###P<0.001 versus control TP.
Figure 5. AF patients show mitochondrial dysfunction. A) Image of left atrial appendage of a patient in sinus rhythm (SR), showing normal sarcomere structures and mitochondrial localization along the sarcomeres, as indicated by the black arrows. B) Electron microscopic image of left atrial appendage of a patient with persistent atrial fibrillation (PeAF), showing myolysis and dispersed mitochondria as indicated by the black arrows. C) Cellular ATP levels in patients in SR and AF. D) Representative Western blot of the mitochondrial markers as indicated in right atrial appendages (RAA) of control patients in sinus rhythm and left (LAA) and RAA of AF patients. Quantified data of mitochondrial markers E) HSP60, F) TOM20 and G) MCU in RAA of SR patients and LAA and RAA of AF patients. *P<0.05 versus SR RAA, #P<0.05, ##P<0.01 versus AF LAA.
Figure 6. Cell-free circulating mitochondri l DNA is a potential biomarker for AF. Quantitative real-time PCR of the mitochondrial-transcribed cytochrome c oxidase subunit 3 (COX3) and NADH dehydrogenase subunit 1 (ND1) genes in serum of control and AF patients in different stages of AF. A+B) per stage of AF, C+D) per stage of AF and divided by gender, E+F) per stage of AF and divided whether patients had a recurrence within 1 year or not, G) correlation between COX3 and ND1. *P<0.05, **P<0.01, ***P<0.001 versus Control (male), #P<0.05 versus control female. Figure 7. Tachypacing induces PARP activation, DNA damage and NAD+ depletion in HL-1 cardiomyocytes. A) Representative Western blot of PAR and PARP1 levels in control non-paced (0 h) and tachypaced (TP) HL-1 cardiomyocytes for durations as indicated. B) Relative NAD+ levels in HL-1 cardiomyocytes during time-course of TP (2 h-8 h) compared to control (0 h). *P<0.05 vs 0 h. N=2 independent experiments and scalebar is 15 pm. Data are all expressed as mean ± s.e.m.. Individual group mean differences were evaluated with the two-tailed Student’s t-test. C) Representative CaT traces of control non-paced (NP) and tachypaced (TP) HL-1 cardiomyocytes pretreated with or without different doses of NAD+ (0.25 mM, 0.5 mM, 1 mM).
Figure 8. PARP1, not PARP2, is the key enzyme mediating tachypaeing- induced contractile dysfunction in HL-1 cardiomyocytes and Drosophila. A) Representative CaT traces in control non-paced (NP) or tachypaced (TP) HL-1 cardiomyocytes transfected with scrambled siRNA (CTL), PARP1 siRNA (PARPli), PARP2 siRNA (PARP2i). B) Arrhythmicity index in milliseconds (ms).
Arrhythmicity index was defined as the standard deviation of the heart periodicity. *P<0.05, **P<0.01, ***P<0.001 vs WT NP, #P<0.05 vs WT TP, N=26 Drosophila prepupae for WT, N=20 Drosophila prepupae for PARPli. Data are expressed as mean ± s.e.m.. Individual group mean differences were evaluated with the two- tailed Student’s t-test. C) Quantified CaT amplitude data in control non-paced (NP) or tachypaced (TP) HL-1 cardiomyocytes transfected with scrambled siRNA (CTL), PARP1 siRNA (PARPli), PARP2 siRNA (PARP2i). **P<0.01 vs CTL NP, ##P<0.01 vs CTL TP.
Figure 9. PARP1 inhibitor prevents and reverses tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes. A) Quantified data showing significant reduction of CaT in TP and TP+Ree groups, which were prevented or reversed by ABT-888 treatment, respectively. N=21 cardiomyocytes for NP, N=20 for TP, N=13 for TP+ABT-888, N=20 for TP+Rec, N=42 for TP+Rec+ABT-888. *P<0.05, **P<0.01, ***P<0.001, vs NP, #P<0.05, ##P<0.01, ###P<0.001 TP/TP+Rec without ABT treatment vs TP/TP+Rec with ABT-888 treatment, respectively. Data are presented as mean ± s.e.m. and two-tailed t-test was used to evaluate differences between groups. B) Representative immunofluorescence staining of oxidative DNA damage marker 8-oxoguine (8-OxoG). N=ll images from over 1000 cardiomyocytes. **P<0.01 vs NP CTL, ##P<0.01 vs CTL TP. C) Quantified data of positive nuclear gH2AC staining of RAA from SR and AF patients. N=4 for SR, N=5 for AF. D) PARP1 activity (PAR) correlates significantly with DNA damage (gH2AC positive nuclei). N=4 for each group. SR: open circle and AF: filled circle.
4 DETAILED DESCRIPTION OF THE INVENTION
4.1 Definitions
The term“atrial fibrillation”, as is used herein, refers to an irregular and often rapid heart rate during which the atria beat irregularly and out of coordination with the two ventricles. Atrial fibrillation symptoms often include heart palpitations, shortness of breath and weakness. Atrial fibrillation is a risk facture for stroke, heart failure and other heart-related diseases. The term paroxysmal atrial fibrillation specifically refers to self- terminating stages of atrial fibrillation, lasting less than 7 days. Later stages of atrial fibrillation are termed persistent atrial fibrillation, when atrial fibrillation occurs for more than a week, and long-standing persistent atrial fibrillation, when atrial fibrillation occurs for more than a year and rhythm control therapy is chosen.
The term“mitochondrial nucleic acid”, as is used herein, refers to a double- stranded circular mitochondrial DNA molecule as well as RNA products, including transfer RNA (tRNA), rihosomal RNA (rRNA) and messenger RNA (mRNA) products of the mitochondrial DNA molecule. Said mitochondrial DNA molecule is present in mitochondria of eukaryotic cells and comprises a total of 37 genes.
The term“DNA damage”, as is used herein, refers in general to ROS-induced oxidative DNA damage involving single- or double-strand DNA breaks, purine and pyrimidine or deoxyribose modifications as well as DNA cross links. A prominent marker that preferably is used to determine a level of DNA damage is provided by 7,8-dihydro-8-oxoguanine (8-oxoguanine).
The term“heat shock protein”, as is used herein, refers to a protein that is produced by cells in response to exposure to stress such as heat, cold, UV light, wound healing or tissue remodeling. Heat shock proteins perform a chaperone function by stabilizing proteins to ensure correct folding. Heat-shock proteins are named according to their molecular weight. The term“bodily fluid”, as is used herein, refers to blood, urine, milk, cerebrospinal fluid, interstitial fluid, lymph, amniotic fluid, bile, cerumen, feces, female ejaculate, gastric juice, mucus pericardial fluid, pleural fluid, pus, saliva, semen, smegma, sputum, synovial fluid, sweat, tears, vaginal secretion, and vomit. A preferred bodily fluid is blood.
The term“blood”, as is used herein, includes reference to serum and plasma. The terms serum and plasma both refer to blood components without cells, whereby serum also excludes clotting factors such as fibrinogen. As is known to a person skilled in the art blood may, for example, be centrifuged to remove cellular components. The thus obtained plasma may be coagulated followed by, for example, centrifugation to remove the clotting factors. The resulting serum is a most preferred bodily fluid.
The term“quantifying”, as is used herein, refers to determining a quantity of the level of mitochondrial nucleic acid and/or heat shock protein in a bodily fluid, preferably in blood. Said quantity preferably refers to the presence and level of mitochondrial DNA in a bodily fluid, preferably in blood, most preferably in serum. Methods to determine presence and quantify of DNA in a bodily fluid are known to a skilled person and include, but are not limited to, quantitative PCR, microarray analysis and DNA sequencing, especially next generation sequencing. The determined DNA levels preferably are normalized for differences in the total amounts of nucleic acid molecules between two separate samples by comparing the level of mitochondrial DNA, for example to the level of nuclear DNA molecules of which the level is known not to differ between different bodily fluid samples.
The term“reference”, as is used herein, refers to a level of mitochondrial nucleic acid and/or a level of DNA damage in a bodily fluid of an individual that is known to suffer from, or, preferably, known not to suffer from, atrial fibrillation. Said reference preferably is a value, preferably an average value, that is obtained from pooled, multiple individuals known to suffer from, or known not to suffer from atrial fibrillation. It is preferred that said reference is pooled from more than 10 individuals, more preferred more than 20 individuals, more preferred more than 30 individuals, more preferred more than 40 individuals, most preferred more than 50 individuals. As an alternative, said reference may be a value, preferably average value, that is obtained from pooled, mixed individuals known to suffer from and/or known not to suffer from atrial fibrillation.
The term“standard-of-care therapy”, as is used herein, refers to medical treatment to control heart rate and/or rhythm. In addition, or as an alternative, ablative therapy and/or cardioconversion, either chemical or electrical, have emerged as an effective therapy for AF treatment. Ablative therapy involves the generation of lesions in the atrial tissue to disrupt cells that provide superfluous electrical pulses. Said standard-of-care therapy may be combined with an anticoagulant to reduce a risk of a stroke.
The term“long non-coding RNA or lncRNA”, as is used herein, refers to transcripts with lengths exceeding 200 nucleotides that are not translated into protein. Although first regarded as transcriptional noise, lncRNAs are now known to exhibit diverse functions through a wide array of mechanisms, and have been associated with diseases including cancer. An overview of human lncRNA species has recently been published (Uszczynska-Ratajczak et ah, 2018. Nature Reviews Genetics 19: 535-548). Preferred LncRNA include Sarrah (OXCT1-AS1; HGNC: 40423), urothelial carcinoma- associated- 1 (UCA1; HGNC:37126), CDR1 anti sense (CDR1AS; HGNC:48926) and long intergenic non-coding RNA predicting cardiac remodeling (LIPCAR; HGNC:50279).
4.2 Method of typing
The invention provides methods of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of providing a bodily fluid from said individual and quantifying mitochondrial nucleic acid and/or a level of DNA damage in said bodily fluid, comparing said quantified
mitochondrial nucleic acid and/or a level of DNA damage to a reference; typing said individual as suffering from, or at risk of suffering from, atrial fibrillation if the quantified mitochondrial nucleic acid is increased or decreased, compared to the reference, and/or if said quantified level of DNA damage is increased or decreased compared to the reference.
The present invention provides general methods of diagnosing and/or prognosticating a subject as suffering from, or at risk of suffering from, atrial fibrillation using said general methods. When reference is herein made to a method of the invention, any and all of these embodiments are referred to, except if explicitly indicated otherwise.
A method of the invention can be performed on any suitable bodily fluid, such as, for example, blood, urine, mucus, especially mouth or cheek mucus, tear fluid and vitreous fluid. A blood sample of a subject can be obtained by any standard method, for instance by venous extraction.
A bodily fluid, preferably blood, is preferably collected in a tube that is coated with anticoagulants such as EDTA, sodium citrate, and/or heparin. The amount of a bodily fluid, preferably blood, that is required is not limited. Depending on the methods employed, the skilled person will be capable of establishing the amount of sample required to perform the various steps of the methods of the present invention and obtain sufficient nucleic acid for analysis. Generally, such amounts will comprise a volume ranging from 0.01 mΐ to 100 ml, preferably between 1 mΐ to 10 ml, more preferably about 1 ml.
A bodily fluid, preferably blood, may suitably be processed, for instance, it may be purified, or digested, or specific compounds may be extracted therefrom.
For example, the bodily fluid may be treated to remove nucleic acid degrading enzymes like RNases and DNases, in order to prevent destruction of the nucleic acids. In addition, specific cells, for example anucleated cells such as thrombocytes, may be isolated from the bodily fluid, preferably blood, followed by quantifying mitochondrial nucleic acid and/or a heat shock protein in said isolated anucleated cells. It has been reported that anucleated cells such as thrombocytes may absorb genetic material secreted by diseased cells such as cardiac myocytes, serving as an attractive platform for diagnostics of diseases such as atrial fibrillation.
The bodily fluid, preferably blood, may be analyzed immediately following withdrawal of the sample. Alternatively, analysis according to the method of the invention may be performed on a stored bodily fluid. The body fluid for testing may be preserved using methods and apparatuses known in the art.
For preservation, the bodily fluid, preferably blood, can be prepared and stored at -70°C until processed for sample preparation. Preferably, storage is performed under conditions that preserve the quality of the nucleic acid content of the bodily fluid. Examples of preservative conditions are fixation using e.g.
formalin, the addition of aqueous solutions such as Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect (HOPE; DE10021390), and RCL2 (Alphelys; WO04083369), and the addition of non-aquous solutions such as
Universal Molecular Fixative (Sakura Finetek USA Inc.; US7138226).
Methods to quantify mitochondrial nucleic acid and/or a level of DNA damage in said bodily fluid are known to a skilled person and include, but are not limited to, quantitative PCR, microarray analysis, nucleic acid sequencing, and enzyme- linked immunosorbent assay (ELISA). It is preferred that said quantification is determined simultaneously. Simultaneous analyses can be performed, for example, by quantitative amplification, nucleic acid sequencing procedures, microarray analysis, and/or ELISA.
For quantification of mitochondrial nucleic acid and/or long non-coding RNAs, ribonucleic acid (RNA) may be isolated from said bodily fluid. RNA may be isolated from said bodily fluid by any technique known in the art, including but not limited to Trizol (Invitrogen; Carlsbad, California), RNAqueous® (Applied
Biosystems/Ambion, Austin, Tx), Qiazol® (Qiagen, Hilden, Germany), Agilent Total RNA Isolation Lits (Agilent; Santa Clara, California), RNA-Bee® (Tel-Test.
Friendswood, Texas), and Maxwell™ 16 Total RNA Purification Kit (Promega; Madison, Wisconsin).
A preferred RNA isolation procedure involves the use of Qiazol® (Qiagen, Hilden, Germany), especially a miRNeasy Kit. Following RNA isolation, a reverse transcriptase preferably is used to convert RNA into complementary DNA (cDNA) which may subsequently be amplified. Suitable reverse transcriptase enzymes include human immunodeficiency virus (HIV), Moloney murine leukemia virus (M- MuLV), and avian myeloblastosis virus (AMV) reverse transcriptase, as is known to a person skilled in the art.
The level of a RNA expression product of a mitochondrial nucleic acid or of a lncRNA can be determined by any method known in the art. Methods to determine RNA levels are known to a skilled person and include, but are not limited to, Northern blotting, quantitative polymerase chain reaction (qPCR), also termed real time PCR (rtPCR), microarray analysis and next generation RNA sequencing.
For quantification of mitochondrial nucleic acid in said bodily fluid, desoxyribonucleic acid (DNA) may be isolated, for example from blood, or from pretreated blood as is indicated herein above. DNA may be purified from a bodily fluid, preferably blood, or from pretreated blood samples as is indicated herein above, using, for instance, a combination of physical and chemical methods. Very suitably commercially available systems for DNA isolation are the NucleoSpin® Tissue kit (Machery-Nagel), QIAamp blood mini kit columns (Qiagen, Venlo, The Netherlands), the NucliSENS® easyMAG® nucleic acid extraction system
(bioMerieux, Marcy l'Etoile, France) or the MagNA Pure 96 System (Roche
Diagnostics, Almere, The Netherlands).
Said mitochondrial nucleic acid preferably includes nucleic acid comprising at least part of a MT-C03 gene which encodes a cytochrome c oxidase subunit 3 (COX3); at least part of a MT-ND1 gene which encodes a NADH-ubiquinone oxidoreductase chain 1, at least part of a MT-ATP8 gene which encodes a ATP synthase protein 8; at least part of a MT-CYB gene, which encodes a cytochrome b; at least part of a MT-ND2 gene which encodes a NADH dehydrogenase 2; at least part of MT-ND3 gene which encodes a NADH dehydrogenase 3; at least part of a MT-ND4L gene which encodes a NADH-ubiquinone oxidoreductase chain 4L; at least part of a MT-ND4 gene which encodes a NADH-ubiquinone oxidoreductase chain 4; at least part of a MT-ND5 gene which encodes a NADH-ubiquinone oxidoreductase chain; and/or at least part of a MT-ND6 gene which encodes a NADH-ubiquinone oxidoreductase chain 6.
Further preferred mitochondrial nucleic acid includes nucleic acid comprising at least part of MT-ATP6 gene, MT-COl gene, MT-C02 gene, MT-ND1 gene, MT- humanin encoding gene, transfer RNA encoding genes including MT-TA gene, MT- TR gene, MT-TN gene, MT-TD gene, MT-TC gene, MT-TE gene, MT-TQ gene, MT- TG gene, MT-TH gene, MT-TI gene, MT-TL1 gene, MT-TL2 gene, MT-TK gene, MT-TM gene, MT-TF gene, MT-TP gene, MT-TSlgene, MT-TS2 gene, MT-TT gene, MT-TW gene, MT-TY gene, MT-TV gene, and ribosomal RNA encoding genes including MT-RNR1 gene and/or MT-RNR2 gene.
Different amplification methods, known to a skilled artisan, can be employed for amplification, including but not limited to Polymerase Chain Reaction (PCR), rolling circle amplification, nucleic acid sequence-based amplification, and transcription mediated amplification. A preferred amplification method is PCR, especially real-time PCR. PCR is a technology that relies on thermal cycling, consisting of cycles of repeated heating and cooling of a reaction for DNA melting into single stranded molecules and enzymatic replication of the DNA. Primers containing sequences that specifically hybridizes to the target region, and a DNA polymerase are key components to enable selective and repeated amplification. As PCR progresses, the amplified DNA product that is generated is itself used as a template for replication, resulting in a chain reaction in which the DNA template is exponentially amplified.
A preferred DNA polymerase is a thermostable polymerase, preferably a thermostable recombinant polymerase. Preferred commercially available DNA polymerases include AptaTaq Fast DNA Polymerase and LightCycler® FastStart Enzyme (Roche Diagnostics, Almere, The Netherlands).
Real-time PCR, also called quantitative PCR (qPCR), is a technique which is used to amplify and simultaneously quantify a template DNA molecule. The detection of the amplification products can in principle be accomplished by any suitable method known in the art. The amplified products may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium monoazide or Hoechst dyes.
Alternatively, the amplified product may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments. Detection labels which may be associated with nucleotide bases include, for example, fluorescein, cyanine dye and BrdUrd.
When using for example Scorpion primers or a probe-based detection system, a primer or the probe is preferably labelled with a detectable label, preferably a fluorescent label. Preferred labels for use in this invention comprise fluorescent labels, preferably selected from Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto 647N (ATTO-TEC GmbH, Siegen, Germany), YakimaYellow (Epoch
Biosciences Inc, Bothell, WA, USA), CalGlO (BioSearch Technologies, Petaluma,
CA, USA), Cal635 (BioSearch Technologies, Petaluma, CA, USA), FAM (Thermo Fisher Scientific Inc., Waltham, MA USA), TET (Thermo Fisher Scientific Inc., Waltham, MA USA), HEX ((Thermo Fisher Scientific Inc., Waltham, MA USA), cyanine dyes such as Cy5, Cy5.5, Cy3, Cy3.5, Cy7 (Thermo Fisher Scientific Inc., Waltham, MA USA), Alexa dyes (Thermo Fisher Scientific Inc., Waltham, MA USA), Tamra (Thermo Fisher Scientific Inc., Waltham, MA USA), ROX (Thermo Fisher Scientific Inc., Waltham, MA USA), JOE (Thermo Fisher Scientific Inc., Waltham, MA USA), fluorescein isothiocyanate (FITC, Thermo Fisher Scientific Inc., Waltham, MA USA), and tetramethylrhodamine (TRITC, Thermo Fisher Scientific Inc., Waltham, MA USA). A probe is preferably labeled at the 5’ end with a detectable label, preferably a fluorescent label.
A primer such as a Scorpion primer, or a probe preferably has a fluorescent label at one end and a quencher of fluorescence at the opposite end of the probe.
The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Quenchers, for example tetramethylrhodamine
TAMRA, dihydrocyclopyrroloindole tripeptide minor groove binder, are known in the art. Preferred quenchers are Black Hole Quenchers- 1 (BHQ1) and BHQ2, which are available from Biosearch Technologies, Petaluma, CA, USA). The BHQ1 dark quencher has strong absorption from 480 nm to 580 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as FAM, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, and Quasar® 570 dyes. The BHQ2 dark quencher has strong absorption from 599 nm to 670 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as Quasar® 570, TAMRA, CAL Fluor® Red 590, CAL Fluor Red 610, ROX, CAL Fluor Red 635, Pulsar® 650, Quasar 670 and Quasar 705 dyes. BHQ1 and BHQ2 may quench fluorescence by both FRET and static quenching mechanisms.
The term“specifically hybridizing” refers to a nucleic acid molecule that is capable of hybridizing specifically under stringent hybridization conditions to a target nucleic acid template that is obtained or derived from mitochondrial nucleic acid and/or a heat shock protein nucleic acid. The terms "stringency" and "stringent hybridization" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, and the like. These conditions are empirically optimized to maximize specific binding and minimize non- specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions may be sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tin) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer. Hybridization procedures are well known in the art and are described by e.g. Ausubel et ak, 1998. Current Protocols in Molecular Biology, John Wiley, New York; and Sambrook et a , 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.
An oligonucleotide primer or probe, or an oligonucleotide mimic primer or probe, is able to hybridize to a target nucleic acid template when the length of the molecule is or resembles at least 15 bases. The length of the primer or probe is preferably less than 100 bases. A preferred length of a primer or probe is between 15 and 50 bases, preferably between 16 and 30 bases.
In general, a primer or probe is able to hybridize to a target nucleic acid template when the percentage of sequence identity of the molecule is at least 90% over substantially the whole length, more preferred at least 91%, more preferred at least 92%, more preferred at least 93%, more preferred at least 94%, more preferred at least 95%, more preferred at least 96%, more preferred at least 97%, more preferred at least 98%, more preferred at least 99%, more preferred 100% identical to a nucleic acid that is obtained or derived from said target nucleic acid template over substantially the whole length of the primer or probe. The term “substantially the whole length” is used to indicate that the probe may comprise additional nucleotide sequences, for example at the 5’ and/or 3’ ends that are not present in the gene or region described herein above.
Efficient real-time PGR reactions are dependent upon high quality primer and probe design. Rules of thumbs for the design of primers include the selection of primers having a Tm between 58°C and 65°C while keeping the annealing temperatures of the primers as close as possible, having no more than two G's or C's in the last 5 bases at the 3’ end, and the selection of primer pairs with minim l number of potential primer dimers and primer hairpins.
Rides of thumbs for the design of probes include the selection of probes that have a Tm between 68°C and 72°C, have no Gs on the 5’ end, resemble a strand that has more C than G bases, and are as short as possible, without being shorter than 13 nucleotides.
Methods for the design of primers and probes are known in the art. For example, Premier Biosoft (Palo Alto, CA, USA) offers Allolel I) i< and Beacon Designer™ to design probes for real-time PCR assays that are free of dimers, repeats and runs and ensure signal fidelity. In addition, Primer3
(http://primer3.sourceforge.net) and Integrated DNA Technologies, Inc.
(www.idtdna.com/Scitools/Applications/Primerquest) provide online tools for the design of primers and probes for real-time PCR assays. Hence, the skilled person is able to design primers and probes for real-time PCR analyses of one or more target nucleic acid templates.
The primers and probes are preferably tested in single nucleic acid
amplification reactions (monoplex) and combined nucleic acid amplification reactions (multiplex) to determine optimal combinations of specific nucleic acid amplification reactions.
Microarray-based analysis involves the use of selected biomolecules that are immobilized on a solid surface, an array. A microarray usually comprises nucleic acid molecules, termed probes, which are able to hybridize to gene expression products. The probes are exposed to labeled sample nucleic acid, hybridized, where after the abundance of gene expression products in the sample that are
complementary to a probe is determined. The probes on a microarray may comprise DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA. The probes may also comprise DNA and/or RNA analogues such as, for example, nucleotide analogues or peptide nucleic acid molecules (PNA), or combinations thereof. The sequences of the probes may be full or partial fragments of genomic DNA. The sequences may also be in vitro synthesized nucleotide sequences, such as synthetic oligonucleotide sequences.
A probe preferably is specific for a particular gene. A probe is specific when it comprises a continuous stretch of nucleotides that are completely complementary to a nucleotide sequence of the gene. A probe can also be specific when it comprises a continuous stretch of nucleotides that are partially complementary to a nucleotide sequence of a gene. Partially means that a maximum of 5% from the nucleotides in a continuous stretch of at least 20 nucleotides differs from the corresponding nucleotide sequence of said gene. The term complementary is known in the art and refers to a sequence that is related by base-pairing rules to the sequence that is to be detected. It is preferred that the sequence of the probe is carefully designed to minimize nonspecific hybridization to said probe. It is preferred that the probe is, or mimics, a single stranded nucleic acid molecule. The length of said complementary continuous stretch of nucleotides can vary between 15 bases and several kilo bases, and is preferably between 20 bases and about 60 nucleotides. A most preferred probe comprises at least 20, preferably at least 25, nucleotides that are identical to a nucleotide sequence of a gene.
To determine a quantity of a mitochondrial gene nucleic acid in a sample comprising a bodily fluid by micro-arraying, mitochondrial gene nucleic acid products in the sample are preferably labeled, either directly or indirectly, and contacted with probes on the array under conditions that favor duplex formation between a probe and a complementary molecule in the labeled gene nucleic acid products in the sample. The amount of label that remains associated with a probe after washing of the microarray can be determined and is used as a measure for the level of a mitochondrial gene nucleic acid product that is complementary to said probe.
A further preferred method for quantifying gene nucleic acid levels is by sequencing techniques, preferably high throughput sequencing techniques, also termed next generation sequencing (NGS) techniques. These techniques include Illumina® sequencing; Roche 454 pyrosequencing®; ion torrent and ion proton sequencing; and ABI SOLiD®) sequencing. NGS techniques allow sequencing of fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads. Each base is sequenced multiple times, providing high depth to deliver accurate data and an insight into unexpected DNA variation. NGS can be used to sequence a complete exome including all or small numbers of individual genes. Such high throughput sequencing techniques include sequencing-by synthesis, such as provided by Illumina® sequencing and by ion torrent and ion proton sequencing. Sequencing-by-synthesis or cycle sequencing can be
accomplished by stepwise addition of nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in U.S. Patent No. 7,427,673; U.S. Patent No. 7,414, 116; WO 04/018497; WO 91/06678; WO
07/123744; and U.S. Patent No. 7,057,026, all of which are incorporated herein by reference.
Alternatively, pyrosequencing techniques may be employed. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi et ah, Analytical Biochemistry 242(l):84-9 (1996); Ronaghi, M. Genome Res. 11(1):3- 11 (2001); Ronaghi, M. et ah, Science 281:5375, 363 (1998); U.S. Patent No. 6,210,891 ; U.S. Patent No.
6,258,568 ; and U.S. Patent No. 6,274,320, which are all incorporated herein by reference. In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons.
Sequencing techniques also include sequencing by ligation techniques, such as provided by ABI SO Li I ) n sequencing. Such techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides and are inter alia described in U.S. Patent No 6,969,488 ; U.S. Patent No.
6, 172,218 ; and U.S. Patent No. 6,306,597.
Further sequencing techniques include, for example, fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS).
A level of DNA damage may be determined, for example, by determining an amount of oxidative stress and/or DNA lesions. Guanine is a base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is a most common lesion. Hence, a level of DNA damage preferably is determined by determining a level of 8-oxoG in a bodily fluid, preferably blood. As is known to a person skilled in the art, a level of 8-oxoG may be determined by, for example, high performance liquid chromatography coupled to electrochemical detection and/or gas chromatography separation followed by mass spectrometry, and/or by an ELISA kit, for example an Oxiselect oxidative DNA damage ELISA kit (Cell Biolabs, San Diego, CA), or an 8-hydroxy 2 deoxyguanosine ELISA Kit (Abeam, Cambridge, MA).
A method of the invention may be further comprise determining a level of heat shock protein in the bodily fluid of the individual. Said heat shock protein preferably includes at least part of a HSP60 gene product, which is a protein that is implicated in mitochondrial protein import and macromolecular assembly; at least part of a HSP Family B (Small) Member 7 (HSPB7) gene product, which may function as a cardioprotective chaperone that is involved in overcoming stress; at least part of a HSPB1 gene product, which is a HSP27 protein that may function in thermotolerance; and/or at least part of a HSP70 gene product, which is a HSP70 protein that may help to protect cells from stress. Methods to isolate proteins from a bodily fluid are known in the art, including, for example, the Total Protein Extraction Kit (BioCat GmbH, Heidelberg, Germany), or the NucleoSpin® Tissue kit (Maehery-Nagel GmbH & Co. KG, Bethlehem, PA).
As an alternative, or in addition, heat shock protein levels in a bodily fluid, preferably in blood, most preferably in serum, may be directly quantified using ELISA. Suitable ELISA kits are known in the art and include, for example a DuoSet IC ELISA kit, for example the Human Total HSP60 DuoSet IC ELISA DYC 1800-2 (R&D Systems, Minneapolis, MN), a HSP60 ELISA kit (ENZO Life Sciences BVBA, Bruxelles, Belgium), a Human Heat shock protein beta-7(HSPB7) ELISA kit (Cusabio Technology LLC, Houston, TX), a HSP27 Human ELISA Kit (ThermoFisher Scientific, Waltham, MA), and/or a Human HSP70 ELISA Kit (Abeam; Cambridge MA).
The quantified mitochondrial nucleic acid and said level of DNA damage and, when determined, said level of heat shock protein are preferably normalized.
Normalization refers to a method for adjusting or correcting a systematic error in the measurements for determining expression levels. Systemic bias may result from variation by differences in overall performance, differences in isolation efficiency of nucleic acid and/or proteins resulting in differences in purity of the isolated products, and to variation between nucleic acid or protein samples, which can be due for example to variations in purity. Systemic bias can be introduced during the handling of the sample during the quantification of the nucleic acid and/or protein products. Typing of a sample can be performed in various ways. In one method, the quantified lncRNA, mitochondrial nucleic acid and/or level of DNA damage is compared to a reference and the individual is typed as suffering from, or at risk of suffering from, atrial fibrillation if the level of quantified lncRNA is altered, compared to the reference, if the level of quantified mitochondrial nucleic acid is altered, compared to the reference, and/or if said quantified level of DNA damage is altered, when compared to the reference. In this method, the reference provides a level for a lncRNA molecule, a mitochondrial nucleic acid and/or of DNA damage that functions as a threshold to determine whether the individual may suffer from, or may be at risk of suffering from, atrial fibrillation.
In a further preferred method, a coefficient is determined that is a measure of a similarity or dissimilarity of a quantified mitochondrial nucleic acid and/or level of DNA damage, with a reference. Such reference may include one or more lncRNAs, multiple mitochondrial nucleic acids and/or a levels of DNA damage, for example employing different markers for determining a level of DNA damage, and can be referred to as a profile template. Typing of an individual can be based on its (dis)similarity to a single profile template or based on multiple profile templates. For example, the profile templates may be representative of individuals that (i) are known to suffer from atrial fibrillation, or (ii) are known not to suffer from atrial fibrillation.
A number of different coefficients can be used for determining a correlation between the determined lncRNAs, mitochondrial nucleic acids and/or level of DNA damage, and a profile template. Preferred methods are parametric methods which assume a normal distribution of the data. One of these methods is the Pearson product-moment correlation coefficient, which is obtained by dividing the covariance of the two variables by the product of their standard deviations.
Preferred methods comprise cosine-angle, un-centered correlation and, more preferred, cosine correlation (Fan et a , Conf Proc IEEE Eng Med Biol Soc. 5:4810- 3 (2005)).
Said correlation with a profile template is used to produce an overall similarity score for the mitochondrial nucleic acids and/or level of DNA damage that are used. A similarity score is a measure of the average correlation of the quantified mitochondrial nucleic acids and/or level of DNA damage in a bodily fluid from an individual and a profile template. Said similarity score can be, but does not need to be, a numerical value between +1, indicative of a high correlation between the quantified lncRNA, mitochondrial nucleic acid and/or level of DNA damage in a bodily fluid from an individual and a profile template, and -1, which is indicative of an inverse correlation. A threshold can be used to differentiate between quantified lncRNA, mitochondrial nucleic acids and/or level of DNA damage from an individual who is known to suffer from atrial fibrillation and an individual that is known not to suffer from atrial fibrillation. Said threshold is an arbitrary value that allows for discrimination between an individual who is known to suffer from atrial fibrillation and an individual that is known not to suffer from atrial fibrillation. If a similarity threshold value is employed, it is preferably set at a value at which an acceptable number of individuals who are known to suffer from atrial fibrillation would score as false negatives, and an acceptable number of individuals who are known not to suffer from atrial fibrillation would score as false positives. A similarity score is preferably displayed or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.
The methods of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to the invention are specifically suited for identifying an individual that is suffering from, or expected to suffer from, paroxysmal atrial fibrillation and who will likely develop persistent atrial fibrillation, and for identifying an individual who is at risk of recurrence of AF after treatment, including anti arrhythmic drug therapy, ablative therapy, and/or cardioversion.
As is indicated herein above, an increase in a level of lncRNA, mitochondrial nucleic acid and a level of DNA damage, when compared to a reference from individuals not suffering from AF, can be used to identify an individual suffering from paroxysmal AF who is at risk of progressing to more persistent AF, while a decrease of a lncRNA, mitochondrial nucleic acid and a decrease of a level of DNA damage, when compared to a reference from individuals not suffering from AF, can be used to identify an individual suffering from persistent AF who is at risk of progressing to long-standing persistent AF, and/or at risk of recurrence of AF after treatment. Similarly, a decrease in a level of heat shock protein, when compared to a reference from individuals not suffering from AF, can be used to identify an individual suffering from paroxysmal AF who is at risk of progressing to more persistent AF.
It was found that all tested lncRNAs, mitochondrial nucleic acid and DNA damage markers in a bodily fluid, preferably blood, are increased in an individual that is suffering from, or expected to suffer from, paroxysmal atrial fibrillation, and is likely to progress to persistent AF, while the same lncRNAs and mitochondrial nucleic acid are decreased in a bodily fluid, preferably blood, from an individual that suffers from persistent atrial fibrillation and is likely to progress to long standing persistent atrial fibrillation and/or at risk of recurrence of AF after treatment.
The methods of the invention may in particular be suited for typing a female as suffering from, or at risk of suffering from, atrial fibrillation. It was found that the relative increase of IncRNA, mitochondrial nucleic acid and DNA damage markers, relative to a healthy reference, is higher in females when compared to males. For this reason, said reference preferably is gender-matched.
A IncRNA that is specifically suited for typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, especially paroxysmal atrial fibrillation, is represented by Sarrah, UCA1, CDR1AS and/or LIPCAR. These markers are especially enhanced in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, while these markers are decreased in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation. However, all other lncRNAs showed a similar trend: increase in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, and decrease in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation.
A mitochondrial nucleic acid that is specifically suited for typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, especially paroxysmal atrial fibrillation, is represented by COX3, ND1 and/or ND2. These markers are especially enhanced in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, while these markers are decreased in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation. However, all other mitochondrial nucleic acid showed a similar trend: increase in an individual suffering from, or at risk of suffering from, paroxysmal atrial fibrillation, and decrease in an individual suffering from persistent atrial fibrillation or long-standing persistent atrial fibrillation.
It was further found that heat shock protein in a bodily fluid, preferably blood, is decreased in an individual that is suffering from, or expected to suffer from, atrial fibrillation, especially paroxysmal atrial fibrillation. A heat shock protein that is specifically suited for typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, especially paroxysmal atrial fibrillation, is represented by HSPB7, which marker was also found to be decreased in an individual suffering from persistent atrial fibrillation and long-standing persistent atrial fibrillation.
LncRNAs and mitochondrial DNA markers seem the best option to predict AF stage and recurrence after AF treatment. The methods of the invention are particularly suited to identify an individual that has a low risk of recurrence after initial treatment such as pulmonary vein isolation (PVI) and electrical
cardioversion (ECV). An individual with a low risk of recurrence may be selected for ablative therapy, over an individual with a high risk of recurrence.
Preferred markers for identifying an individual that has an altered, i.e high or low, risk of recurrence after initial treatment include lncRNA, especially UCA1, CD IAS, and LIPCAR; and/or mitochondrial markers, especially ND1 and COX3.
Preferred markers are UCA1 and ND1, UCA1 and COX3, UCA1, CD IAS and ND1, UCA1, CD1AS and COX3, CD IAS, LIPCAR and ND1, CD IAS, LIPCAR and COX3, UCA1, CD IAS, LIPCAR and ND1, UCA1, CD1AS, LIPCAR and COX3, and UCA1, CD IAS, LIPCAR, ND1 and COX3.
4.3 Methods of treatment
The invention further provides a method for assigning a standard-of-care therapeutic agent to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of assigning a standard-of-care therapy to an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention. Said standard-of-care therapy preferably comprises an inhibitor of poly(ADP- ribose) polymerase, ablative therapy, electrical or chemical cardioversion, and/or the administration of anti-arrhythmic drugs aimed at controlling rhythm or rate.
Said inhibitor of poly(ADP-ribose) polymerase, also termed PARP-inhihitor, preferably is a specific PARP1 inhibitor. PARP1 is the key PARP enzyme instigating tachypacing-induced contractile dysfunction in ardiomyocytes. As is shown herein below, PARP1 inhibition prevents NAD+ depletion and functional loss and accelerates recovery after cessation of tachypacing. These results indicate that a PARP-inhihitor such as nicotinamide and/or ABT-888 may halt or delay onset of AF and/or progression to paroxysmal AF and to persistent AF.
Preferred PARP inhibitors include nicotinamide, 2-[(2R)-2-Methylpyrrolidin- 2-yl] - lH-benzimidazole-4-carboxamide dihydrochloride benzimidazole carboxamide (ABT-888), 3-aminobenzamide, 4-(3-(l-(cyclopropanecarbonyl)piperazine-4- carbonyl)-4-fluorobenzyl)phthalazin- l(2H)-one (AZD-2281), 8-fluoro-2-{4- [(methylamino)methyl]phenyl}- 1,3,4, 5-tetr ahydro-6H-pyrrolo[4, 3,2- ef][2]benzazepin-6-one phosphate (1:1) (AG014699), (8S,9R)-5-fluoro-8-(4- fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2- de]phthalazin-3(7H)-one (BMN-673), 8-fluoro-2-{4-[(methylamino)methyl]phenyl}- l,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one (AG 014699 ), (S)-2-(4- (piperidin-3-yl)phenyl)-2H-indazole-7-carhoxamide hydrochloride (MK-4827), (R)-2- fluoro-10a-methyl-5,8,9, 10, 10a, ll-hexahydro-5,6,7a, ll-tetraazacyclohepta[ det]cyclopenta[a]fluoren-4(7H)-one maleate (BGB-290), ll-methoxy-2-((4- methylpiperazin-l-yl)methyl)-4,5,6,7-tetrahydro-lH-eyclopenta[a]pyrrolo[3,4- c]carbazole-l,3(2H)-dione (CEP 9722) and 10- ((4-Fly droxypiperidin-1 - yl)methyl)chromeno[4,3,2-de]phthalazin-3(2H)-one (E7016). A most preferred PARP inhibitor is ABT-888.
Ablative therapy is a procedure aimed at isolating electrical activity causing atrial fibrillation by scarring or destroying specific areas or cells. Ablative therapy is preferably performed by catheters that generate heat or extreme cold in order to destroy cells in the atrium. In case of AV node ablation, in which cells of the atrioventricular node are ablated to prevent the atria from propagating electrical activity at a too high rate to the ventricles; or by performing a modified maze procedure, in which ablative therapy is performed during open-heart surgery. Said electrical or chemical cardioversion is aimed at restoring a normal rhythm. Chemical cardioversion comprises administration, preferably intravenous administration, of an antiarrhythmic drug such as procainamide (4-amino-N-[2- (diethylamino)ethyl]benzamide), a class Ic agent such as flecainide (N-(piperidin-2- ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide) or propafenone (l-[2-[2-hydroxy- 3-(propylamino)propoxy]phenyl]-3-phenylpropan-l-one), amiodarone ((2-butyl-l- benzofuran-3-yl)-[4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl]methanone) and/or ibutilide (N - [4- [4- [ethyl (hep tyl) amino] - l-hydroxyhutyl]phenyl]me thane
sulfonamide). A dose preferably is between 20 and 250 mg. preferably about 100 mg, every 5 minutes until a maximum dose of about 1 g is reached for
procainamide, between 10 and 100 mg every 12 hours, preferably about 50 mg orally every 12 hours, for flecainide, between 50 and 250 mg every 8 hours, preferably about 150 mg orally every 8 hours, for propafenone, a bolus of between 100 and 400 mg/30 minutes, preferably about 300 mg/30 mins, for amiodarone, and/or one or two doses of 1 mg for ibutilide.
Electrical cardioversion preferably is performed by applying a current across the chest of a patient. Said current preferably is applied with increasing shock energies of between 100 and 360 J in an anterior-posterior electrode position. Said current may have a monophasic or biphasic waveform, preferably has a biphasic waveform. To restore a normal rhythm, both methods may be applied successively, meaning that chemical conversion may be followed by electrical cardioversion if chemical conversion was not successful; and that electrical cardioversion may be followed by chemical cardioversion if electrical conversion was not successful.
Said rate control therapy preferably comprises a beta blocker, a calcium channel blocker and/or a cardiac glycoside.
Preferred beta blockers, also termed adrenergic beta-antagonists, include atenolol (2- [4- [2-hydroxy-3-(propan-2-ylamino)propoxy]phenyl] acetamide), bisoprolol (l-(propan-2-ylamino)-3-[4-(2-propan-2- yloxyethoxymethyl)phenoxy]propan-2-ol), earvedilol (l-(9H-carbazol-4-yloxy)-3- [2- (2-methoxyphenoxy)ethylamino]propan-2-ol), metoprolol (l-[4-(2- methoxyethyl)phenoxy]-3-(propan-2-ylamino)propan-2-ol), nadolol ((2R,3S)-5-[3- (tert-butylamino)-2-hydroxypropoxy]-l,2,3,4-tetrahydronaphthalene-2,3-diol), propranolol (1-naphthalen- l-yloxy-3-(propan-2-ylamino)propan-2-ol), timolol ((2S)- l-(tert-butylamino)-3-[(4-morpholin-4-yl- l,2,5-thiadiazol-3-yl)oxy]propan-2-ol) and sotalol (N-[4-[l-hydroxy-2-(propan-2-ylamino)ethyl]phenyl]methanesulfonamide).
Recommended dosages for a beta blocker are between 50 and 200 mg daily, preferably about 100 mg daily, for atenolol, between 2.5 and 20 mg daily, preferably about 5 mg daily, for bisoprolol, between 1 and 25 mg twice a day, preferably about 12.5 mg twice daily, for carvedilol, between 50 and 450 mg daily, preferably about 100 mg daily, for metoprolol, between 40 and 320 mg daily, preferably about 80 mg daily, for nadolol, between 40 and 640 mg daily, preferably about 160 mg daily, for propranolol, between 5 and 30 mg twice daily, preferably about 10 mg twice daily, for timolol, and between 80 and 640 mg twice daily, preferably about 240 mg twice daily, for sotalol.
A preferred cardiac glycoside is a cardenolide, preferably digoxin (3-
[(3S,5R,8R,9S, 10S, 12R, 13S, 14S, 17R)-3-[(2R,4S,5S,6R)-5-[(2S,4S,5S,6R)-5-
[(2S,4S,5S,6R)-4,5-dihydroxy-6-methyloxan-2-yl]oxy-4-hydiOxy-6-methyloxan-2- yl]oxy-4-hydroxy-6-methyloxan-2-yl]oxy- 12, 14-dihydroxy- 10, 13-dimethyl- 1,2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 15, 16, 17-tetradecahydrocyclopenta[a]phenanthren-17-yl]- 2H-furan-5-one). A recommended dosage for digoxin is between 0.125 mg and 0.375 mg orally or intravenously, every two hours up to a total of 1.5 mg, or between 0.125 mg and 0.375 mg orally once daily.
Preferred calcium channel blockers include diltiazem ([(2S,3S)-5-[2- (dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3-dihydro- l,5-benzothiazepin- 3-yl] acetate;hydrochloride) and verapamil (2-(3,4-dimethoxyphenyl)-5-[2-(3,4- dimethoxyphenyl)ethyl-methylamino] -2-propan-2-ylpentanenitrile) . A
recommended dosage for diltiazem is an initial intravenous bolus of 0.25 mg/kg, followed by infusion with 10 mg/hr to 15 mg/hr for a maximum duration of 24 hours for diltiazem, and between 80 mg and 480 mg orally once daily, preferably about 160 mg once daily, for verapamil.
Said standard-of-care therapeutic agent rhythm control therapy preferably comprises an ion channel blocker, more preferably a sodium channel blocker such as flecainide (N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide), propafenone (1- [2- [2-hydroxy-3-(propylamino)propoxy]phenyl] -3-phenylpropan- 1- one) and/or quinidine (S)-[(2R,4S,5R)-5-ethenyl-l-azabicyelo[2.2.2]octan-2-yl]-(6- methoxyquinolin-4-yl)methanol), a potassium channel blocker such as amiodarone ((2-butyl-l-benzofuran-3-yl)-[4-[2-(diethylamino)ethoxy]-3,5- diiodophenyljmethanone), sotalol (N-[4-[l-hydroxy-2-(propan-2- ylamino)ethyl]phenyl]methanesulfonamide) and/or dofetdide (N-[4-[2-[2-[4- (methanesulfonamido)phenoxy]ethyl- methylamino]ethyl]phenyl]methanesulfonamide), and/or a multichannel blocker such as dronedarone (N-[2-butyl-3-[4-[3-(dibutylamino)propoxy]benzoyl]-l- benzofuran-5-yl]methanesulfonamide).
Recommended dosages for a rhythm controller are between 50 and 200 mg twice daily, preferably about 100 mg twice daily, for flecainide, between 150 and 300 mg three times daily, preferably about 150 mg three time daily, for
propafenone, between 80 and 600 mg every 4 to 6 hours, preferably about 200 mg every 4 to 6 hours, for quinidine, between 400 and 1600 mg daily, preferably about 800 mg orally daily, for amiodarone, between 80 and 320 mg twice daily, preferably about 160 mg twice daily, for sotalol, between 125 and 500 mg twice daily, preferably about 250 mg twice daily, for dofetdide, and between 2.5 and 20 mg daily, preferably about 5 mg daily, for dronedarone.
Said standard-of-care therapy may be combined with an antiplatelet and/or anticoagulant. Said antiplatelet and/or anticoagulant preferably is selected from aspirin, warfarin, and a direct-acting oral anticoagulant.
In a specific embodiment of the invention, said standard-of-care therapeutic agent comprises only an antiplatelet and/or an anticoagulant as active
ingredient(s). This may be the case where a decrease of a mitochondrial nucleic acid and a decrease of a level of DNA damage, when compared to a reference from individuals not suffering from AF, is observed and further treatment of the individual suffering from AF is not recommended.
Said antiplatelet therapy includes the administration of acetylsalicylic acid, dipyridamole and/or clopidogrel. Acetylsalicylic acid is known to inhibit the COX-1 enzyme. A recommended dosage is between 75 and 325 mg daily, preferably about 200 mg daily. Dipyridamole (2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-l- yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol) acts as a platelet aggregation inhibitor. A recommended dosage is between 10 and 100 mg daily, preferably about 50 mg daily. Clopidogrel (methyl (2S)-2-(2-chlorophenyl)-2-(6,7- dihydro-4H-thieno[3,2-c]pyridin-5-yl)acetate) also acts as a platelet aggregation inhibitor. A recommended dosage is between 25 and 300 mg daily, preferably about 75 mg daily.
Said anticoagulant preferably is a 4-hydroxycoumarin derivative that reduces vitamin K1 levels. Such coumarin derivatives include warfarin (4-hydroxy-3-(3-oxo- l-phenylbutyl)chromen-2-one), acenocoumarol (4-hydroxy-3-[l-(4-nitrophenyl)-3- oxobutyl]chromen-2-one) and bishydroxycoumarin (4-hydroxy-3-[(4-hydroxy-2- oxochromen-3-yl)methyl]chromen-2-one).
A recommended dosage of warfarin is between 1 and 10 mg daily, preferably about 5 mg daily. A recommended dosage of acenocoumarol is between 1 and 20 mg daily, preferably about 12 mg daily. A recommended dosage of bishydroxycoumarin is between 100 and 200 mg daily, preferably about 150 mg daily
Said direct-acting oral anticoagulant preferably is a direct thrombin inhibitor such as dabigatran (ethyl N-[(2->[(4-{N'-j(hexyloxy)earbonyI]oarbamimidoyl} phenyI)amino]methyj)-l-niefhyl-lH-benzimidazoI-5-yI)carbony!]-N-pyfidin-2-yl- beta-alaninate), and/or a direct Factor Xa inhibitor such as rivaroxaban (5-chloro- N-[[(5S)-2-oxo-3-[4-(3-oxomorphobn-4-yl)phenyl]-l,3-oxazobdin-5- yl]methyl]thiophene-2-carboxamide), edoxaban (N’-(5-chloropyridin-2-yl)-N- [(lS,2R,4S)-4-(dimethylcarbamoyl)-2-[(5-methyl-6,7-dihydro-4H-[l,3]thiazolo[5,4- c]pyridine-2-carbonyl)amino]cyclohexyl]oxamide) and apixaban (l-(4- methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-l-yl)phenyl]-4,5-dihydropyrazolo[3,4- c] p yr idine - 3 - c arboxamide) .
A person skilled in the art is aware that some direct inhibitors preferably are provided as a prodrug in an inactive precursor form. For example, dabigatran may be provided as a precursor named dabigatran etexilate, which is converted after absorption to the active substance.
Recommended dosages for the active inhibitor, or its precursor, are between 200 and 400 mg daily, preferably about 150 mg orally twice a day, for dabigatran, between 5 and 30 mg daily, preferably about 20 mg daily, for rivaroxaban, between 10 and 60 mg daily, preferably about 30 mg daily, for edoxaban, between 1 and 10 mg daily, preferably about 2.5 mg daily, for apixaban.
The invention further provides a standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention. Said standard-of- care therapy is as defined herein above.
The invention further provides a method for determining the efficacy of a standard-of-care therapy in an individual, comprising the steps of analyzing a bodily fluid, preferably blood, of an individual for the presence of a biomarker using a method of typing according to the invention at a first time point to thereby provide a first value for the level of said biomarker in said subject, analyzing a bodily fluid, preferably blood, of said individual for the presence of said biomarker using a method of typing according to the invention at a second time point that is earlier or later, preferably later, than said first time point, to thereby provide a second value for the level of said biomarker in said individual, wherein said individual has been subjected to a standard-of-care therapy between said first and second time point, and comparing said first and second value to determine the efficacy of said standard-of-care therapy in said individual. According to this method, a decrease of the quantified mitochondrial nucleic acid at the second time point, compared to the first time point, is indicative that the standard-of-care therapy has been effective, while an increase in the quantified heat shock protein nucleic at the second time point, compared to the first time point, is indicative that the standard-of-care therapy has been effective. Said biomarker is one or more of a mitochondrial nucleic acid and/or a heat shock protein as defined herein above. The skilled artisan will understand that standard-of-care therapy prior to the first time point and a subsequent measurement at a second, later, time point without any standard-of-care therapy having occurred between said time points, is included in aspects of the invention for determining the efficacy of a standard-of-care therapy.
4.4 Miscellaneous
Aspects of the invention include a method of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of:
providing a bodily fluid from said individual; quantifying a level of DNA damage and/or mitochondrial nucleic acid in said bodily fluid; comparing said quantified level of DNA damage and/or mitochondrial nucleic acid to a reference; and typing said individual as suffering from, or at risk of suffering from, atrial fibrillation it said quantified level of DNA damage is altered, compared to the reference and/or if the quantified mitochondrial nucleic acid is altered, compared to the reference.
Said bodily fluid preferably is blood.
The level of DNA damage in a preferred method of the invention is quantified by determining a level of 8-oxoguanine.
The mitochondrial nucleic acid in a preferred method of the invention is mitochondrial DNA. Said mitochondrial nucleic acid preferably comprises nucleic acid from a cytochrome C oxidase 3 gene and/or a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene.
A preferred method of the invention comprises quantifying a level of DNA damage and quantifying mitochondrial DNA from a cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene. Said method preferably further comprises quantifying a heat shock protein level, preferably heat shock protein B7, in said bodily fluid.
The invention further provides a method for assigning standard-of-care therapy to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of any one of claims 1-7; and assigning a standard-of-care therapy to the individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation. Said standard-of-care therapy preferably is ablative therapy, electrical or chemical cardioversion, and/or the administration of anti -arrhythmic drugs aimed at controlling rhythm or rate. Said standard-of-care therapy preferably is combined with an antiplatelet and/or anticoagulant. Said antiplatelet and/or anticoagulant preferably is selected from aspirin, warfarin, and a direct-acting oral anticoagulant such as dabigitran, rivaroxaban, edoxaban and apixaban.
Said standard-of-care therapy preferably is an ion channel blocker including a sodium channel blocker such as flecainide, propafenone and/or quinidine, and/or a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
The invention further provides a standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of the invention. The invention further provides a method for determining the efficacy of a standard-of-care therapy of an individual, comprising the steps of analysing a bodily fluid, preferably blood, of an individual for the presence of a biomarker using a method of typing according to the method of any one of claims 1-7, at a first time point to thereby provide a first value for the level of said biomarker in said subject, analysing a bodily fluid, preferably blood, of said individual for the presence of said biomarker using a method of typing according to the invention at a second time point that is earlier or later, preferably later, than said first time point, to thereby provide a second value for the level of said biomarker in said individual, wherein said individual has been subjected to a standard-of-care therapy between said first and second time point, and comparing said first and second value to determine the efficacy of said standard-of-care therapy in said individual.
The content of publications mentioned herein are incorporated herein by reference.
For the purpose of clarity and a concise description, features may be described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.
Examples
Example 1
Methods
Cell culture
HL-1 atrial cardiomyocytes derived from adult mouse atria, were obtained from Dr. William Claycomb (Louisiana State University, New Orleans, USA; Claycomb, et a , 1998. PNAS 95: 2979-2984). To induce AF, HL-1 atrial cardiomyocytes were subjected to 6Hz (tachypacing), 40V and 20ms pulses, for maximal 8 hours via the C-PacelOO TM-culture pacer (IonOptix Corporation, The Netherlands). HL-1 cardiomyocytes were transiently transfected with pDEST40-MCU-V5-HIS
(Addgene, USA) by the use of Lipofectamin 2000 (Life Technologies, The
Netherlands). MCU knockdown was accomplished by transiently transfecting the cardiomyocytes with Mission MCU esiRNA (EMU213891, Sigma, The Netherlands) by the use of Lipofectamin RNAiMAX (Life Technologies, The Netherlands). Ru360 was purchased from Millipore (USA) and dissolved in according to manufacturer’s instructions. Treatment (5 pM) was started 30 minutes prior to and was continued during tachypacing.
Mitochondrial measurements
Cellular ATP levels were measured according to the manufacturer’s instructions of the ATP Bioluminescence Assay Kit CLS II (Roche, The Netherlands),
mitochondrial membrane potential was determined by incubating the HL-1 atrial cardiomyocytes with 100 nM tetramethyl rhodamine methylester (TMRM, Sigma, The Netherlands), resuspended in 1% BSA in PBS and mean fluorescence intensity of 10.000 cardiomyocytes was analyzed by the LSR-II flow cytometer (BD
Biosciences, USA) and mitochondrial oxygen consumption rate (OCR) was measured utilizing the XF24 extracellular flux analyzer (Seahorse Bioscience,
USA), according to manufacturer’s instructions.
Mitochondrial morphology was measured by incubating HL-1 atrial cardiomyocytes with 100 nM Mitotracker Deep Red (Life Technologies, The Netherlands) and thereafter excited by a 647 nm laser with emission at 665 nm and were visually recorded with a 63x-objective, using a Solamere-Nipkow-Confocal-Live-Cell- Imaging system (based on a Leica DM IRE2 inverted microscope). Ten random fields containing at least 15 cardiomyocytes were recorded and the mitochondrial morphology per cardiomyocyte was scored as tubular, intermediate or fragmented (Lanters et ah, 2015. J Transl Med 13: 347) by an investigator blinded for the treatment conditions.
Mitochondrial calcium transients (CaTmito) were measured by incubating HL-1 atrial cardiomyocytes with 5 pM of the mitochondrial Ca2+-sensitive dye Rhod-2 AM33 (Abeam, Cambridge, MA) and thereafter excited by a 600 nm laser with emission at 605 nm and CaTmito were recorded with the Myocyte Calcium and Contractility System (IonOptix Corporation, The Netherlands). The live recording of the CaTmito was performed at 1Hz stimulation (normal pacing) at 37 °C. The relative value of fluorescent signals was determined utilizing the following calculation: Fcal=Fl/F0, where FI is the fluorescent dye signal at any given time and F0 is the fluorescent signal at rest.
Protein and mRNA measurements HL-1 atrial cardiomyocytes or human tissue samples were lysed in radioimmunoprecipitation assay buffer and Western blot analysis was performed as described before (Zhang D et ah, 2014. Circulation 129: 346-358). The following primary antibodies were used: anti-HSP60 (ADI-SPA-805, Enzo Life Sciences, USA), anti-TOM20 (MCA4300Z, Bio-Rad, The Netherlands), anti-MCU (14997S, Cell Signaling Technology, The Netherlands), OXPHOS Antibody Cocktail (MS604, Abeam, Cambridge MA) and anti-GAPDH (10R-G109a, Fitzgerald, USA).
Horseradish peroxidase -conjugated anti-mouse or anti-rabbit (Dako, Denmark) were used as secondary antibodies, depending on the species origin of the primary antibody. Total RNA was isolated from HL-1 atrial cardiomyocytes using the Nucleospin RNA isolation kit (Maehery-Nagel, The Netherlands). First strand cDNA was generated by M-MLV reverse transcriptase (Promega, The Netherlands) and random hexamers primers (Promega, The Netherlands). Subsequently, the cDNA was used as a template for quantitative real-time PCR. Relative changes in transcription level were determined utilizing the CFX384 Real-time system C1000 Thermocycler (Bio-Rad, The Netherlands) in combination with SYBR green ROX- mix (Westburg, The Netherlands). mRNA levels were expressed in relative units on the basis of a standard curve (serial dilutions of a calibrator cDNA mixture).
DNA isolation, measurement and analysis
Total DNA was isolated from patient and control serum utilizing the Nucleospin Tissue kit (Machery-Nagel, The Netherlands), according to manufacturer’s instructions. Isolated DNA was used to determine mitochondrial DNA levels utilizing the CFX384 Real-time system C1000 Thermocycler (Bio-Rad, The
Netherlands) in combination with SYBR green ROX-mix (Westburg, The
Netherlands). Mitochondrial DNA levels were adjusted for nuclear DNA levels and analyzed using the ACT method.
HSP levels and DNA damage
HSP levels (HSP60, HSPB7, HSP27 and HSP70) were measured in patient and control serum utilizing the DuoSet IC ELISA kit (R&D systems), according to manufacturer’s instructions. DNA damage (80X0G) was measured in patient and control serum utilizing the Oxiselect oxidative DNA damage ELISA kit (Cell Biolabs, USA), according to manufacturers instructions. HSP and DNA damage levels were determined by using a standard curve, as provided by manufacturer. Drosophila heart wall contraction measurement
Drosophila melanogaster heart wall contraction measurements were performed with the wlll8 strains (Genetic Services Inc, USA), which were maintained at 25 °C on standard medium. Adult Drosophila’s were removed after fertilization and the medium, containing the fly embryos, was supplied with Ru360 (20, 50 and 100 pM), freshly dissolved in demineralized water. Controls were subjected to demineralized water only. Transparent prepupae were selected, placed on a 1% agarose gel in PBS and subjected to taehypacing (5Hz for 20 minutes, 20V and 5ms pulses) with a C-PacelOOTM-culture pacer (IonOptix Corporation, The
Netherlands). Before and after taehypacing, the heart wall contractions in whole prepupae were measured for a period of 30 seconds and analyzed with the Myocyte Calcium and Contractility System (IonOptix Corporation, The Netherlands).
Patient material
Before surgery, patient characteristics were assessed (Table 1 and 2) as described before (Ke et ah, 2008. J Mol Cell Cardiol 45: 685-693). Right and left atrial appendages (RAA and LAA, respectively) were obtained from patients with AF, and RAA were obtained from control patients in sinus rhythm (SR) as described in the Halt and Reverse study (EMC-MEC 2014-393) (Lanters et ah, 2015. J Transl Med 13: 347). Both AF and SR patients groups had underlying heart disease. After excision, the atrial appendages were immediately snap-frozen in liquid nitrogen and stored at -80 °C. The study conforms to the principles of the Declaration of Helsinki. The institutional review board of the Erasmus Medical Center approved the study and patient gave written informed consent.
Statistical analysis
Results are expressed as mean ±SEM of at least two independent experiments. Multiple -group comparisons were obtained by a one-way ANOVA with a Bonferroni correction or a Kruskal- Wallis test followed by a Mann-Whitney U test. Individual group-mean differences were evaluated with a Student’s t-test. Correlations were estimated using Pearson correlation. All P-values were two-sided. A value of P.0.05 was considered statistically significant. SPSS version 22 was used for all statistical evaluations. Results
Tachypacing induces mitochondrial stress and dysfunction
To examine the influence of tachypacing on mitochondrial function, we first determined cellular ATP levels in tachypaced HL-1 cardiomyocytes. Tachypacing for 2h induced a large decrease in cellular ATP levels, which gradually reduced further upon extended pacing (Figure 1A). As the decrease in cellular ATP may have been caused by excessive consumption and/or impaired production, we next examined mitochondrial respiration. To that end, oxygen consumption was measured in whole cardiomyocytes under basal conditions and after addition of oligomycin to inhibit ATP synthesis, FCCP to determine the xim l respiratory capacity and rote none/an timycin A to determine non-mitochondrial respiration (Figure IB; 1, 33, 56 and 96 minutes, respectively). Tachypacing for 2h initially increased basal oxygen consumption rate and spare respiratory capacity. However, tachypacing beyond 2h progressively decreased the basal oxygen consumption rate and very markedly inhibited spare respiratory capacity (Figure IB), which accounts for a less adequate response to cellular stress or increased cardiac workload (Desler et a , 2012. J Aging Res 2012: 192503). However, protein expression of complex I, II, III and V of the respiratory chain did not change during tachypacing (Figure 1C), indicating that the decreased respiration during tachypacing is not due to diminished respiratory chain protein expression.
Together, these results demonstrate that tachypacing induces mitochondrial dysfunction, likely leading to a progressive impairment of ATP synthesis.
Cytosolic Ca2+ overload constitutes the most obvious mechanism underlying the tachypaced-induced mitochondrial dysfunction. Ca2+ overload in both cytosol and ER, as encountered in tachypacing and AF, will result in Ca2+ buffering by mitochondria (Babcock et a , 1997. J Cell Biol 136: 833-844; Maack et ah, 2006. Circ Res 99: 172-182). The subsequent excessive mitochondrial Ca2+ buffering leads to mitochondrial Ca2+ overload, and consequently mitochondrial swelling, dysfunction (Rizzuto et ah, 2012. Nat Rev Mol Cell Biol 13: 566-578) and reduced mitochondrial Ca2+ uptake. Reduced mitochondrial Ca2+ uptake may be a trigger for AF, as it encounters for enhanced cytosolic Ca2+ levels. Thus, we examined mitochondrial calcium transients (CaTmito) during normal pacing at 1Hz and during a time course of tachypacing. Tachypacing beyond 2h significantly reduced CaTmito, mainly characterized by the reduction in amplitude (Figure ID and E). Both the mitochondrial respiration and Ca2+ influx are dependent on the mitochondrial membrane potential (LYhhIo) (Babcock et ah, 1997. J Cell Biol 136: 833-844), therefore we measured LYthίIo by the fluorescent probe TMRM, which is readily sequestered in polarized mitochondria. Tachypacing strongly and progressively reduced LY m i to as from 2h onwards (Figure IF).
Next, we examined the morphology of the mitochondrial network, as dysfunction of respiratory chain complexes is accompanied by network fragmentation, either as a cause (Liot et a , 2009. Cell Death Differ 16: 899-909; Benard et ah, 2007. J Cell Sci 120: 838-848) or as a consequence (Chen et ah, 2005. J Biol Chem 280: 26185- 26192. Indeed, tachypacing as early as 2h induced a transition from a tubular to a fragmented mitochondrial network (Figure 2A and B). Fragmentation of the network was progressive over time, as evidenced by the time-dependent decrease and the time- dependent increase in a tubular and fragmented mitochondrial network, respectively.
Table 1 Demographic and clinical characteristics of patients with AF and control patients in Sinus Rhythm, used for Western blot analysis of atrial appendages.
SR (N=8) AF (N=ll)
Figure imgf000040_0001
Underlying heart disease (N, %)
CAD 8 (100)
VHD 6 (55)
ASD 1 (9)
Aorta 1 (9)
CAD+CHD 2 (18)
Surgical MAZE 1 (9)
Figure imgf000040_0002
CAD: coronary artery disease, VHD: valvular heart disease, ASD: atrial septal defect, Aorta: aneurysm aorta ascendens, CHD: congenital heart defect, AT:
atrial tachycardia, LS: longstanding, LA: left atrium, LVF: left ventricular function NYHA: New York Health Association for exercise tolerance
In addition, we examined whether tachypacing also induces mitochondrial stress by measuring levels of mitochondrial chaperones upregulated upon mitochondrial stress. Tachypacing resulted in a significant and progressive upregulation of mRNA of both HSP60 and HSP10 (Figure 2C and D). Finally, we examined whether tachypacing affected the number of mitochondria by measuring the amount of mitochondrial DNA (mt.DNA) and TOM20 levels. Both cellular mtDNA and TOM20 levels showed no changes upon tachypacing (Figure 2E and F), suggesting that the number of mitochondria did not change upon tachypacing. Therefore, the mitochondrial dysfunction upon tachypacing is not due to a decreased number of mitochondria. Together, these data demonstrate tachypacing to progressively impair mitochondrial function of cultured cardiomyocytes, characterized by a very early reduction in cellular ATP, loss of DYhiίΐo and fragmentation of the mitochondrial network, followed by dysfunction of the respiratory chain, impaired mitochondrial Ca2+ handling and induction of mitochondrial stress chaperones.
A mitochondrial Ca2+ uniporter inhibitor protects from tachypacing-induced mitochondrial stress and dysfunction. The findings reveal that tachypacing induces impaired mitochondrial function. As we showed a significantly reduced CaTmito, the effect of a compound targeting the MCU (by inhibition; Ying et ah, 1991.
Biochem 30: 4949-4952), Ru360, was explored. The effect of RU360 to counteract tachypacing-induced loss of CaTmito was examined after 6 hours of pacing, when the CaTmito are significantly reduced (Figure ID and E). Ru360 significantly and concentration-dependently protected against CaTmito reduction, induced by tachypacing (Figure 3A). Next, we explored whether Ru360 treatment also ameliorated mitochondrial stress and dysfunction. Ru360 treatment normalized cellular ATP levels, transcription levels of HSP60 and HSP10 and CaTmito to non- treated control levels (Figure 3B-E). Interestingly, Ru360 treatment even enhanced cellular ATP levels and CaTmito significantly in normal-paced cardiomyocytes (Figure 3B and 3E). Furthermore, Ru360 treatment protected the mitochondrial network from tachypacing-induced fragmentation (data not shown). These results strongly suggest that inhibition of the MCU-mediated Ca2+ influx into the mitochondria protects against mitochondrial stress and dysfunction and preserves cellular function in tachypaced cardiomyocytes.
The MCU mediates tachypacing-induced mitochondrial changes. To determine whether tachypacing-induced mitochondrial changes are specifically mediated by the MCU, we first examined its protein and mRNA levels.
Tachypacing did not affect protein expression of MCU, but significantly reduced MCU mRNA (Figure 4A and B). Next, we manipulated MCU levels by
overexpression and siRNA treatment. Overexpression of MCU did not affect tachypacing-induced loss of CaTmito (Figure 4C and D). In contrast, reducing MCU expression by 20% protected against tachypacing-induced CaTmito loss, without affecting CaTmito in normal-paced cardiomyocyt.es (MCU siRNA low, Figure 4E and F). Interestingly, an approximate 60% reduction of MCU expression lowered CaTmito in normal-paced cardiomyocytes and conferred no protection in
tachypaced cardiomyocytes (MCU siRNA high, Figure 4E and F). These results suggest that a small reduction in MCU, not affecting normal mitochondrial Ca2+ handling, is beneficial to counteract tachypaeing effects. However, a larger reduction of MCU levels seems detrimental, likely due to an imp ir ent of physiological mitochondrial Ca2+ influx, which is already observed under baseline conditions. To confirm the importance of the MCU, the effect of Ru360 was explored in tachypaced Drosophila melanogaster (Zhang D et ah, 2014. Circulation 129: 346-358; Zhang et ah, 2011. J Mol Cell Cardiol 51: 381-389). Comparable to findings in tachypaced HL-1 cardiomyocytes, Ru360 conferred a dose-dependent protection against tachypacing-induced decrease of heart wall contraction in Drosophila (Figure 4G). The optimal concentration of Ru360 needed, 50 pM in Drosophila as opposed to 5 pM in HL-1 cardiomyocytes, corresponds well with previous experiments in which concentrations needed to confer protection in Drosophila are generally lOx higher than in HL-1 cardiomyocytes (Zhang et ah, 2011. J Mol Cell Cardiol 51: 381-389).
Markers of mitochondrial dysfunction are present in AF patients. To extend our findings to human AF, we examined mitochondrial dysfunction in left and right atrial appendages (LAA and RAA, respectively) in patients with AF and control patients in sinus rhythm (SR). Upon electron microscopic examination, patients in SR show mitochondrial localization along the entire length of the sarcomeres. In contrast, in AF patients the mitochondria are fragmented and dispersed and sarcomeres are degraded (myolysis), the latter being absent in SR patients (Figure 5A and B). Besides divergent distribution of mitochondria, cellular ATP levels are also significantly lower in AF patients (Figure 5C). Mitochondrial dysfunction is further evidenced in AF patients by increased protein expression of HSP60 and MCU in LAA, while there is no change in expression of TOM20 (Figure 5D-G). Changes in expression were only present in LAA, as observed before (Zhang D et ah, 2014. Circulation 129: 346-358; Li et al„ 2001. Circulation 104: 2608-2614; Voigt et ah, 2010. Circ Arrhythm Electrophysiol 3: 472-480) as the expression of HSP60 and MCU in RAA is similar to SR. These results suggest that mitochondrial dysfunction is not only found in an in vitro model of AF, but is also present in AF patients.
Cell-free circulating mitochondrial DNA as a potential biomarker for AF. Mitochondrial dysfunction can lead to the release of mitochondrial DNA into the circulation of patients, where it acts as a damage associated molecular pattern (Krysko et ah, 2011. Trends Immunol 32: 157-164; Nakahira et ah, 2015. Antioxid Redox Signal 23: 1329-1350), increased levels might lead to inflammatory responses, organ injury (Nakahira et ah, 2015. Antioxid Redox Signal 23: 1329- 1350; Zhang et ah, 2010. Nature 464: 104-107) and increased mortality (45.
Simmons et ah, 2013. Ann Surg 258: 591-598). Importantly, release of
mitochondrial DNA is primarily used to restore homeostasis, but prolonged exposure leads to detrimental changes (Palmai-Pallag and Bachrati, 2014.
Microbes Infect 16: 822-832). As circulating mitochondrial DNA increases with age (Pinti et ah, 2014. Eur J Immunol 44: 1552-1562) the increased risk of
inflammation might lead to (age-related) diseases, including cardiac diseases such as heart failure and AF (Oka et ah, 2012. Nature 485: 251-255; Akdag et ah, 2015. Ther Clin Risk Manag 11: 1675-1681; Zhang et ah, 2015. Med Sci Monit 21: 3505- 3513). This makes cell-free circulating mitochondrial DNA in serum an interesting biomarker. The potential of cell-free circulating mitochondrial DNA in serum as a biomarker has been evaluated in a variety of diseases, including cancer, viral infections, neurodegenerative and cardiac diseases (Ellinger et ah, 2012. Urol Oncol 30: 509-515; Hou et ah, 2013. Clin Biochem 46: 1474-1477; Wang et ah, 2015. Coron Artery Dis 26: 296-300; Xia et ah, 2014. Asian Pac J Cancer Prev 15: 1339- 1344; Yu, 2012. Mitochondrial DNA 23: 329-332), where it could be associated with disease progression (Hou et ah, 2013. Clin Biochem 46: 1474-1477). Thus, we examined the potential of cell-free circulating mitochondrial DNA in serum as a biomarker for AF. We measured levels of COX3 (cytochrome c oxidase subunit 3) and ND1 DNA in serum of control patients (without any atrial disease) or patients with different stages of AF (paroxysmal (PAF), persistent (PeAF) and longstanding persistent (FS-PeAF)) (See Table 3). Expression of both COX3 and ND1 showed significant increased levels in PAF and decreased levels in FS-PeAF compared to Table 2 Demographic and clinical characteristics of patients with AF and control patients in SR, used for mitochondrial DNA analysis in serum.
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
control patients (Figure 6A and B; Table 3). Interestingly, male and female AF patients show differences in COX3 and ND1 expression (Figure 6C and D). In addition, patients with recurrence of AF within 1 year after treatment show different levels of COX3 and ND1, compared to control and patients that did not get an AF recurrence within 1 year (Figure 6 E and F). Figure 6G shows that the levels of COX3 and ND1 are highly correlated, suggesting that these cell-free circulating mitochondrial DNA markers in serum may be a promising biomarker for AF.
In conclusion, it is shown tachypacing to induce substantial mitochondrial dysfunction, including failure of respiration, most likely resulting from enhanced Ca2+ influx through the MCU, consequently impairing mitochondrial calcium transients. Our data also demonstrate that tachypacing induces mitochondrial stress, as exemplified by increased transcription of the mitochondrial stress chaperones HSP60 and HSP10 and fragmentation of the mitochondrial network. Moreover, mitochondrial changes, including decreased cellular ATP levels and increased HSP60 expression, are present in AF patients, which also show myolysis and fragmented and dispersed mitochondrial localization. Treatment with Ru360, an inhibitor of the MCU, or modest MCU downregulation restored these detrimental mitochondrial changes upon tachypacing. Furthermore, Ru360 treatment protected against contractile dysfunction in a Drosophila model for AF. In addition, our data indicates that cell-free circulating mitochondrial DNA in serum may be a potential biomarker of AF.
Example 2
Methods
HL-1 cardiomyocytes derived from adult mouse atria were obtained from Dr. William Clayeomb (Louisiana State University, New Orleans) and cultured in complete Clayeomb medium (Sigma) supplemented with 10% FBS (PAA
Laboratories GmbH, Austria), 100 U per ml penicillin (GE Healthcare), 100 pg per ml streptomycin (GE Healthcare), 4 mM L-glutmaine (Gibco), 0.3 mM L-aseorbic acid (Sigma) and 100 mM norepinephrine (Sigma). HL-1 cardiomyocytes were cultured on cell culture plastics or on glass coverslips coated with 0.02% gelatin (Sigma) in a humidified atmosphere o f 5% C02 at 37oC. The cardiomyocytes, which have a basal spontaneous contraction rate of ~ 0.5-1 Hz4, were subjected by tachypacing (TP) to a 5-10 fold rate increase as observed in clinical AF (5 Hz, 40 V, pulse duration of 20 ms) with a C-PacelOO culture pacer (IonOptix) for 12 h unless stated otherwise. HL-1 cardiomyocytes followed the pacing rate. Ca2+ transients (CaT) were imaged by Solamere-Nipkow-Confocal-Live-Cell-Imaging system (based on a Leica DM IRE2 Inverted microscope). 2 mM of the Ca2+-sensitive Fluo-4-AM dye (Invitrogen) was loaded into HL-1 cardiomyocytes by 45 min incubation, followed by 3 times washing with PBS. Ca2+ loaded cardiomyocytes were excited by 488 nm and emitted at 500-550 nm and visually recorded with a 40* -objective. Calcium transient (CalT) measurements were performed in a blinded manner by selection of normal shaped cardiomyocytes with the use of bright field settings, followed by a switch to the fluorescent filter to determine the CaT.
Prior to 12 h TP, HL-1 cardiomyocytes were treated for 12 h with the PAftP inhibitors 3-aminobenzamide (3-AB, Sigma-Aldrich), ABT-888 (Selleckchem), olaparib (Selleckchem), beta-nicotinamide adenine dinucleotide hydrate (NAD+, Sigma-Aldrich) or transfected with scrambled siRNA (control, Ambion) PARP1 siRNA (Ambion), or PARP2 siRNA (Santa Cruz) to study the specific role of PARP1 and PARP2, respectively.
Adult Wistar rats (~ 200 g) were injected with heparin 15 min before atrial cardiomyocyte isolation, followed by anesthetisation (2% isoflurane and 98% CL). Hearts were excised and placed in ice-cold, oxygenated buffer solution containing (in mM) 134 NaCl, 10 HEPES, 4 KC1, 1.2 MgS04, 1.2 Na2HP04, and 11 D-glucose (pH 7.4). Freshly excised rat hearts were mounted on a Langendorff setup and perfused retrogradely through the aorta for 30 min with oxygenated buffer solution of 37°C, to which 66.7 mg perL librase (Roche) was added. Following Langendorff perfusion, the atria were cut off the heart and rinsed in isolation solution containing (in mM): 100 NaCl, 5 Hepes, 20 D-glucose, 10 KC1, 5 MgS04, 1.2 KH2P04, 50 Taurin, 0.5% bovine serum albumin (BSA) (pH 7.4), transferred to a 15-ml tube containing 10 ml of isolation solution plus 0.02 mM CaCL and 0.02 U per ml DNase, gently triturated for 7 min, and subsequently filtered through a 200pm mesh filter into another 15-ml tube, followed by centrifugation for 1 min at 700x g. The supernatant was removed and the pellet containing atrial
cardiomyocytes was resuspended carefully in 10 ml of isolation solution plus 0.02 mM CaC42. Next, the Ca2+ concentration was increased in 5-min steps from 0.1,
0.2 mM to 0.4 mM Ca2+. Atrial cardiomyocytes were left to sink for 20 min and transferred into laminin-coated plates in plating medium (M199 medium plus 5% fetal calf serum) for 2 h followed by replacement with M199 medium plus Insulin- Transferrin-Sodium Selenite Supplement (Sigma). The isolated adult rat atrial cardiomyocytes have a basal spontaneous contraction rate of - 0.5-1 Hz in vitro.
The rat experiments complied with all relevant ethical regulations and theVUmc approved the study protocol (DEC FYS 14-06).
Prior to tachypacing, atrial cardiomyocytes were treated for 2 h with the PARP inhibitors ABT-888 (Selleckchem) or olaparib (Selleckchem), followed by 2 h tachypacing at 5 Hz, 30 V with a pulse duration of 2 ms. Control atrial
cardiomyocytes were either non-paced (NP) or paced for 2 h at 1Hz, 30 V and pulse duration of 2 ms. Atrial cardiomyocytes followed the pacing rate. CaT
measurement was performed according to previous studies with minor changes2,
54. In short, atrial cardiomyocytes were washed twice with M199 medium, incubated with the Ca2+ dye Fluo-4 (1 u per ml) in M199 medium for 15 min, and rinsed twice again with M199 medium. The Fluo-4-loaded cardiomyocytes were excited at 488 nm and the light emitted at 500-550 nm and recorded with a high speed confocal microscope (Nikon AIR). Bright field settings were used to randomly select normal-shaped cardiomyocytes, followed by a switch to the fluorescent filter to determine the CaT. As such, CaT measurements were conducted in a blinded manner.
The wild-type Drosophila melanogaster strain wlll8 strain was used for all drug screening (PARP inhibitors or NAD+ ) experiments. Hereto, female and male adult flies were crossed. After 3 days, flies were removed from the embryos- eontaining tubes and drugs or the same amount of vehicle (DMSO) were added to the food. Drosophila were incubated at 25 °C for 48 h, with larvae consuming the drug/vehicle prior to entering the prepupae stage. The Drosophila prepupae were collected and subjected to tachypacing for 20 min (4 Hz, 20 V, pulse duration of 5 ms) and heart wall functions were measured as described in detail below.
To create the knockdown of PARP1 in Drosophila, two PARP1 UAS-RNAi Drosophila lines, from the Vienna Drosophila RNAi Center (VDRC, ID:330230) and Bloomington Drosophila Stock Center (BDSC, ID:34888), were utilized. Both RNAi lines were crossed with a Hand-GAF4 driver strain (kind gift of Prof. Dr. Achim Paululat; Sellin et a , 2006. Gene Expr. Patterns 6: 360-375). As control, wild-type flies wlll8 were crossed with Hand-GAE4 driver flies. Prepupae of FI offspring were tachypaced as previously described (Zhang et al., 2014. Circulation 129: 346- 358).
Heart wall contractions were measured utilizing high-speed digital video imaging (100 frames per s) before and after tachypacing in at least duplicated 10 s- movies. Movies were used to prepare heart wall traces and M-mode cardiography. Hereto, 1-pixel width lines were drawn across the heart wall, followed by determination of Plot-Z axis profile (based on contrast changes) to generate heart wall traces or kymographs (via the kymograph plugin of Image J) for M-mode cardiography. To determine the heart rate and arrhythmicity index (defined as the standard deviation of the heart period normalized to the median heart period of each fly followed by averaging across flies; Fink et al., 200. BioTechniques 46: 101- 113), the heart wall traces were further analysed with the use of Drosan software, which was modified from the software originally developed to determine human heart rate and arrhythmicity (Greaves-Lord et al., 2010. Psychiatry Res 179: 187- 193; Nolte et al., 2017. Nat. Commun 8: 16140).
Right atrial appendages (RAA) and/or left atrial appendages (LAA) tissue samples were obtained from patients with coronary artery and/or valvular heart disease having sinus rhythm (SR) or (longstanding) persistent AF. After excision, atrial appendages were immediately snap-frozen in liquid nitrogen and stored at -80°C. The study conformed to the principles of the Declaration of Helsinki and complied with all relevant ethical regulations. The Erasmus Medical Center Review Board approved the study (MEC-2014-393), and all patients gave written informed consent.
HL-1 cardiomyocytes or human tissue samples were lysed in
radioimmunoprecipitation assay (RIPA) buffer containing PBS, Igecal ca-630, eoxycholic acid and SDS (Ke et al., 2008. J Mol Cell Cardiol 45: 685-693; Zhang et al., 2014. Circulation 129: 346-358). In short, equal amounts of protein
homogenates were separated by SDS-polyacrylamide gel electrophoresis (SDS- PAGE), transferred onto nitrocellulose membranes, and probed with antibodies directed against poly (ADP-Ribose) (PAR, 1:1000, BD bioscience, 551813), PARP1 (1:500, Santa Cruz, sc-25780), YH2AX (1:1000, Millipore, 05-636), Cavl.2 (1:200, Alomone Labs, ACC-003), Kvll.l (1:400, Alomone Labs, APC-062), Kir3.1 (1:200, Alomone Labs, APC-005), B-actin (1:1000, Santa Cruz, se-47778), or GAPDH (1:5000, Fitzgerald, 10R-G109A). Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Dako). Signals were detected by the ECL detection method (Amersham) and quantified by densitometry (Syngene, Genetools).
NAD and NADH levels, in which NAD represents the sum of NAD+ and NADH, were measured according the manufacturer’s instructions of the assay kit (Abeam, ab65348). In short, HL-1 cardiomyocyt.es were lysed in NAD extraction buffer and the protein concentration was determined (BioRad Laboratories). To measure NAD, after equalizing the protein concentration, 50 mΐ of each sample was mixed with 100 mΐ NAD cycling buffer and incubated at room temperature (RT) for 5 min to convert NAD+ to NADH, followed by the addition of 10 mΐ NADH developer buffer and 2 h incubation at RT. NAD/NADH levels were measured at 450 nm (BioTek Synergy 4 plate reader). To measure NADH, NAD+ in each sample was decomposed by incubation at 600C for 30 min before measurement. Notably, in accordance with previousl findings, the NADH amount in cultured cardiomyocytes and tissue was below the detection limit59. Therefore, the NAD+ amount per ug of total protein was used as endpoint.
To evaluate DNA damage in cardiomyocytes, an alkaline comet assay kit (Trevigen) was utilized according to the manufacturer’s instructions with minor changes. HL-1 cardiomyocytes were t.rypsinized, harvested by centrifugation, suspended at 2x105 cells per ml in phosphate-buffered saline (PBS), combined with 45 pl melted LAM agarose at ratio of 1:10 (v:v) and immediately pipetted onto CometSlides. Slides were dried for 30 min at 4°C, incubated firstly in lysis solution for 1 h and then in freshly prepared alkaline unwinding solution (pH>13) for 1 h. After placing the slides in 4°C alkaline electrophoresis solution, electrophoresis at 21 V for 30 min was performed. After incubation for 2 times 5 min in demineralized H2O and once for 5 min in 70% ethanol, slides were dried at 37°C, stained with SYBR Gold for 30 min at RT in the dark, rinsed in water and dried again at 37°C. Finally, comets were visualized after excitation at 496 nm by fluorescence microscopy (Leica Microsystems) at 522 nm. DNA damage was quantified by scoring the percentage of DNA in the tail, using the Image J Marco“Comet_Assay” based on an NIH Image Comet Assay developed by Herbert M. Geller (1997).
To induce DNA damage, HL-1 atrial cardiomyocytes received 10 Gy and rat atrial cardiomyocytes 40 Gy of irradiation with a dose rate of 0.0562 Gy per second by utilizing a cobalt-60 gamma-source (Gammacell 220 Research Irradiator, MDS Nordion, Canada). HL-1 and rat atrial cardiomyocytes were treated with 40 mM ABT-888 (12 h) or 5 mM ABT-888 (2 h), respectively, prior to the irradiation. After irradiation, cardiomyocytes were either prepared for Western blot analyses, NAD+ level measurements or CaT recordings.
To evaluate oxidative stress in cardiomyocytes, OxyBlot protein oxidation detection kit (Millipore, S7510) was used, following the company’s instructions. In short, cardiomyocytes were lysed in RIPA buffer containing 1% beta-mercapto- ethanol (Sigma). 10 pg of protein was denatured in 6% SDS, derivatized by incubation for 15 min in 2,4-dinitrophenylhydrazine (DNPH) solution, followed by the addition of neutralization solution. After neutralization, protein samples were subjected to SDS-PAGE, transferred onto nitrocellulose membranes and probed with anti-dinitrophenyl (DNP) antibody (1: 150) for 1 h at RT. HRP-conjugated goat anti-rabbit IgG (1:300) was used as secondary antibody. All reagents were included in the kit. Signals were detected by the ECL detection method (Amersham) and quantified by densitometry (Synge ne, Gene tools).
Total RNA was isolated from HL- 1 cardiomyocytes utilizing the nucleospin RNA isolation kit (Machery-Nagel). First strand cDNA was generated by M-MLV reverse transcriptase (Invitrogen) and random hexamer primers (Promega).
Relative changes in transcription level were determined using the CFX384 Real time system C1000 thermocycler in combination with SYBR green supermix (both from BioRad Laboratories). Calculations were performed using the comparative CT method according to User Bulletin 2 (Applied Biosystems). Fold inductions were adjusted for GAPDH levels. Primer pairs used included PARP1 F:
CACCTTCCAGAAGCAGGAGA and R: GCAAGAAATGCAGCGAGAGT; PARP2 F: TCCTCTGGGCATCATCTTCT and R: AAGCTGGGAAAGGCTCATGT. CACNA1C F: CAAACAACAGGTTCCGCCTG and R: ATCTTTAGAGCAATTTCAATGGTGA. KCNQ1 F: G C CT C ACT CAT C GAG ACT G C and R: GGAC AGAAGC GTGTG ACT C C . KCNH2 F: GGCGTACAGACAAGGACACA and R: CAGGGCCCTCATCTTCACTG. KCNJ3 F: TT CAT C CT C C AAC AG C AC C C and R: GGCCATAGCTGCTTGCTAGA. GAPDH F: CATCAAGAAGGTGGTGAAGC and R: AC C AC C CT GTT GCT GTAG . ACTB F: GGCTGTATTCCCCTCCATCG and R: C C AGTT GGT AAC AAT G C CAT GT . Primer pairs used in Drosophila included PARP1 F:
TGGTTTGCGTCAGGTGAAGA and R: TCGCGAAACCTGAAGTAGGC; Actin5C: F: GAGCAC GGT AT C GTGAC C AA and R: GC CT C C ATT C C C AAGAAC GA. HL-1 cardiomyocytes were grown on coverslips until 80% confluence and subjected to TP for various time periods, with or without drug treatment.
Immediately after pacing, cardiomyocytes were rinsed in PBS and fixed with 4% formaldehyde for 15 min, rinsed twice with PBS, permeabilized by incubation with 0.1% Triton X-100 in PBS for 10 min, rinsed twice in PBS and blocked with blocking solution (0.5% BSA and 0.15% glycine in PBS) for 10 min. After blocking, cardiomyocytes were incubated with primary antibodies for 2 h at RT. After rinsing the cardiomyocytes three times with blocking solution, cardiomyocytes were incubated with secondary antibodies for 45 min at RT shielded from light, followed by rinsing with blocking solution three times and PBS twice. Lastly,
cardiomyocytes were incubated with mounting media containing DAPI
(Veetashield), sealed with nail polish and used for fluorescent microscopy (Leica Microsystems). Antibodies used were: anti-YH2AX (1: 100, Millipore, 05-636), anti- PAR (1:200, BD Bioscience, 551813), anti-PARPl (1:200, Santa Cruz, sc-25780), anti-oxoguanine 8 (1:100, Abeam, ab64548), goat anti-rabbit FITC (1:200,
Invitrogen, 65-6111), and goat anti-mouse TRITC (1:200, Southern Biotech, 1021- OS). For quantification, Image Pro software was used to calculate the total fluorescent (green for FITC and red for TRITC) signal per image as well as the DAPI signal. The total fluorescent signals, corresponding to the expression of PARPl, PAR or gH2AC, were divided by the respective blue signals (DAPI), representing the cell number.
The frozen RAA samples of SR and AF patients were used for staining of gH2AC and 53BP1. Frozen sections were cut into 5 mih slices. Sections were air dried for 30 min, fixed in 4% formaldehyde for 10 min at RT, washed 3 times with PBS for 10 min, then permeabilized with 0.3% Triton X-100 (in PBS) for 10 min at RT and washed 3 times for 5 min with PBS. After blocking of the sections with 1% BSA blocking solution for 30 min at RT, sections were incubated with primary antibodies directed against gH2AC (1:100; Millipore, 05-636) or 53BP1 (1:100;
Santa Cruz Biotechnology, sc-22760) overnight at 40C. After washing with PBS for 3 times 10 min, slides were incubated with secondary antibodies and 1% human serum, TRITC labeled goat anti-mouse (1:200; Southern Biotech, 1021-03) and FITC labeled goat anti-rabbit (1:200, Invitrogen, 65-6111) for 1 h at RT and protected from light. Following 3 washes of 10 min, DAPI mounting medium (Veetashield) was applied to the sections, after which they were covered with coverslips and sealed. Slides were stored at 40C for a few h and subsequently used for fluorescent microscopy (Leica Microsystems). gH2AC and 53BP1 positive nuclei were expressed as the percentage of the total number of nuclei (typically about 200).
The nuclear shape of cardiomyocytes in RAAs of SR and AF patients was determined by measuring its circularity (form factor) with Image J 1.48 software (US National Institute of Health). Hereto, 8-bit images of DAPI-stained nuclei were converted to binary photos by the method of“make binary” in Image J, traced by hand and the circularity was calculated by the formula 4n*A per P2, in which A denotes the surface area and P the perimeter. The circularity of a perfect round circle and a line segment are 1 and 0 respectivelyGO.
Results are expressed as mean ± standard error of the mean (SEM).
Biochemical analyses were performed at least in duplicate. Individual group mean differences were evaluated with the two-tailed Student’s t-test. Correlation was determined with the Spearman correlation test. To compare continuous variables with a skewed distribution, the Mann-Whitney U test was applied. All P values were 2 sided. Values of P<0.05 were considered statistically significant. SPSS version 20 (IBM Analytics) was used for all statistical evaluations.
Results
As nicotinamide is known to inhibit activation of PARP (Thiemermann et ah, 1997. Proc Natl Acad Sci USA 94: 679-683; Pacher et ah, 2007. Cardiovasc Drug Rev 25: 235-260), we tested the level of PARP activity by measuring the amount of PAR synthesis in normal and tachypaced cardiomyocytes. A gradual increase in PAR levels was observed upon tachypacing, which reached significance after 8 h of tachypacing and remained increased afterwards, while PARP1 protein expression was unchanged during tachypacing (Fig. 7A). This observation indicates that tachypacing induces PAR synthesis, suggesting induction of PARP activation.
Since PARP gets activated by single and double strand breaks in the DNA (Hassa and Hottiger, 2008, Front Biosci 13: 3046-3082), the level of DNA damage was determined by comet assay (single-cell gel electrophoresis; Collins, 2004. Mol Biotechnol 26: 249-252) and by measurement of phosphorylation of the Ser-139 residue of the histone variant H2AX, forming gH2AC. Four hours of tachypacing significantly increased both the amount of DNA in the comet tail and gH2AC levels of cardiomyocytes (data not shown).
Upon activation, PAKP consumes NAD+ to synthesize PAR. Therefore, progressive and excessive activation of PARP results in reductions in NAD+ levels, which finally results in the energy loss and functional impairment of
cardiomyocytes. To study whether tachypaeing-indueed PARP activation depleted NAD+ levels in HL-1 cardiomyocytes, NAD+ levels were measured in HL-1 cardiomyocytes in the course of tachypacing. Eight hours of tachypacing induced a significant reduction in NAD+ levels (Figure 7B). Normal pacing at 1 Hz did not reveal changes at PAR, gH2AC, or NAD+ levels (data not shown). Together, these findings reveal that tachypacing induces substantial DNA damage and
consequently the activation of PARP, resulting in depletion of the cellular content of NAD+ in cardiomyocytes.
Since NAD+ is an important constituent for proper cell function and health, we next investigated whether the decline in NAD+ levels is a driving mechanism for functional loss by testing the effect of replenishment of NAD+ on contractile function in tachypaced HL-1 cardiomyocytes. Tachypacing resulted in a significant calcium transient (CaT) loss, which was dose-dependently abrogated by preserving cellular NAD+ levels through exogenous supplementation (Fig. 7C). This observation was confirmed in tachypaced Drosophila prepupae, where tachypacing resulted in loss of heart wall contractions and an increase of arrhythmia incidence, which was dose-dependently prevented by replenishment of NAD+ (data not shown). Next, we examined whether PARP mediates the NAD+ depletion, since particularly PARP1 and to a lesser extent PARP2 isoforms consume NAD+ (Hassa and Hottiger, 2008, Front Biosci 13: 3046-3082). Hereto, HL-1 cardiomyocytes were transfected with siRNA targeting PARP1 or PARP2, resulting in specific and effective suppression of their expression in the cardiomyocytes. Subsequent tachypacing of siRNA treated cardiomyocytes demonstrated that downregulation of PARPl significantly protected cardiomyocytes against CaT loss, whereas downregulation of PARP2 did not (Fig. 8A).
To confi m that PARPl is the key PARP enzyme driving tachypacing-induced contractile dysfunction, PARPl expression was suppressed specifically in the heart of Drosophila in two RNAi lines, as confirmed by Western blotting (data not shown). In line with the findings in HL-1 cardiomyocytes, suppression of PARPl resulted in protection against tachypacing-induced heart wall dysfunction (Fig.
8B).
These data were confirmed by PARP1 inhibitors including the general inhibitors, nicotinamide and 3-AB, and the specific PARP1/2 inhibitors ABT-888 and olaparib. Both general and specific inhibition of PARP1/2 precluded
tachypacing-induced PARylation of proteins and decrease in NAD+ levels (Fig. 8C). Furthermore, the PARP1 inhibitors ABT-888 and olaparib also significantly attenuated tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes and Drosophila without influencing the baseline contractile function in
cardiomyocytes (data not shown), as was previously observed for nicotinamide (Zhang et al., 2014. Circulation 129: 346-358). In addition, tachypacing of HL-1 cardiomyocytes resulted in significant electrophysiological deteriorations, including alterations in action potential duration (APD), increased APD dispersions, decreased area of excitability and ion channel remodeling. All tachypacing-induced electrophysiological alterations were prevented by PARP1 inhibitors olaparib and/or ABT-888 (data not shown). Since AF is a progressive disease, it is of interest to study whether PARP1 inhibition accelerates recovery from tachypacing- induced NAD+ depletion and contractile dysfunction. Hereto, HL-1 cardiomyocytes were tachypaced, followed by 24 h recovery under no pacing conditions. In vehicle treated cardiomyocytes, no recovery from tachypacing induced CaT loss, NAD+ depletion or increased PAR levels was observed. In contrast, tachypaced HL-1 cardiomyocytes post-treated with ABT-888 revealed accelerated recovery at all endpoints (Fig. 9A). These findings demonstrate that PARP1 inhibitors not only prevent PARP1 activation, NAD+ depletion, CaT loss, and electrophysiological and ion channel deteriorations, but also accelerate recovery after cessation of tachypacing.
Since NAD+ depletion is associated with the induction of oxidative stress, which may in turn leads to (further) DNA damage, we tested whether PARP1 inhibition protects by reducing oxidative stress-induced DNA damage. Tachypacing of HL-1 cardiomyocytes resulted in significant induction of oxidative damage to proteins and DNA (data not shown), as evidenced by formation of 8-oxoguanine, a biomarker for oxidative DNA damage (Bruner et al., 2000. Nature 403: 859-866). Inhibition of PARP1 by ABT-888 prevented tachypacing-induced oxidative protein and DNA damage (Fig. 9B). In addition, the tachypacing-induced gH2AC levels were partly reduced by ABT-888 treatment (data not shown). Together, these data indicate that PARP1 inhibition precludes the initiation of a vicious circle in which advanced PARP1 activation is driven by depletion of NAD+, causing further DNA damage.
To study whether PARP activation is the cause of NAD+ depletion and contractile dysfunction in cardiomyocytes, cardiomyocytes were gamma-irradiated to induce DNA damage and thereby PARP activation. As expected, irradiation resulted in a significant induction of DNA damage and consequently an increase in PAR levels, reduction in NAD+ levels, and finally loss in CaT in both HL- 1 and rat atrial cardiomyocytes (data not shown). The PARP1 inhibitor ABT-888 prevented the increase in PAR levels, NAD+ depletion and CaT loss (data not shown). These findings confirm that DNA damage-mediated PARP activation is the cause of NAD+ depletion and CaT remodeling in atrial cardiomyocytes.
To extend our findings to clinical AF, we measured DNA damage and PARP1 activation in right and/or left atrial samples (RAA and/or LAA) of (longstanding) persistent AF patients and controls in SR. Compared to SR, AF patients demonstrate a significant increase in PAR formation in both RAA and LAA, while both groups show similar PARP1 protein expression (data not shown).
Furthermore, gH2AC levels were significantly increased in patients with AF compared to SR (Fig. 9C). Moreover, a significant positive correlation was found between the amount of PAR and gH2AC (Fig. 9C), indicating that AF patients with high levels of PAR also reveal more DNA damage. In addition, the amount of another DNA damage marker, 53BP1, was significantly increased in AF patients compared to control SR patients (data not shown). Finally, we examined nuclear circularity, a marker for oxidative stress-induced DNA damage (Barascu et ak, 2012. EMBO J 31: 1080-1094), showing that nuclear circularity was significantly decreased in patients with AF compared to controls in SR (data not shown). Thus, patients with (longstanding) persistent AF showed an increase in levels of PAR, indicative for PARP1 activation, markers of DNA damage including gH2AC, 53BP1 and reduced levels of nuclear circularity. The features found in patients thus match the observations in tachypaced cardiomyocytes and Drosophila, indicating the clinical significance of PARP1 activation in (longstanding) persistent AF. Example 3
Materials and method
Total RNA from human serum and human tissue was extracted by use of miRNeasy Serum/Plasma Advanced Kit (Qiagen, German) and TRIzol reagent (Invitrogen, Carlsbad, CA), respectively. The purity and concentration of the RNA were determined by a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). cDNA was synthesized by utilizing iScript cDNA synthesis kit (BioRad, CA) following the manufacturer’s instructions. RT reactions were performed with a total volume of 10 ul with random primers. RT procedures were: 25°C for 5 min, 46°C for 20 min, 95°C for 1 min, followed by storage at 4°C.
Plasma levels of lncRNAs were determined by quantitative real time polymerase chain reaction (qRT-PCR) on a Bio-Rad CFX384 real time system using SYBR green dye (Bio-Rad, CA). qRT-PCR procedure: 95°C for 3 min, 45 cycles of 95°C for 10 s, and 60°C for 30 s, followed by 95°C for 10 s, melt curve 65°C to 95°C increment of 0.5°C for 10 s + plate-read. Gene expression was corrected for levels of the reference gene values (18S/GAPDH). Relative expression was calculated by relative quantification (2-AACt) method. Values of Ct over 35 were considered to be negative. Primer sequences are listed below:
hSarrah forward: CCTGGACTGCGTTCACGTTT and hSarrah reverse:
CTGCAAGCCTTGTTGCTCAC;
hUCAl forward: ACGCTAACTGGCACCTTGTT and hUCAl reversed:
TGGGGATTACTGGGGTAGGG;
hLIPCAR forward: TAAAGGATGCGTAGGGATGG, hLIPCAR reverse:
TTCATGATCACGCCCTCATA;
18S reversed: TAGTAGCGACGGGCGGTGTG;
U6 forward: GCTTCGGCAGCACATATACTAAAAT and U6 reversed:
C G CTT C AC G AATTT G C GT GT CAT ;
P0 forward: TCGACAATGGCAGCATCTAC and P0 reverse:
AT C C GT CT C C AC AG AC AAG G .
Data are shown as means ± SEM. Statistical analysis was performed by using the SPSS version 19.0 program (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7. Statistical differences between two or more groups were analyzed by using a one way analysis of variance (ANOVA) with Turkey post-hoc. Before ANOVA, all data were tested for normal distribution and similarity of variance. The area under the ROC curve (AUC) analysis was used to assess the predictive power. Pearson correlation was obtained to analyze correlations between each quantitative parameter. P values of < 0.05 was considered as significant.
Results
We have measured the levels of different stress markers, including DNA damage, long non-coding RNAs and mitochondrial DNA, in the serum of control persons and patients with different stages of atrial fibrillation, i.e paroxysmal (PAF), persistent (PeAF) and longstanding persistent (LS)) atrial fibrillation (AF). AF patients underwent either pulmonary vein isolation (PVI), electrical cardioversion (ECV) as AF treatment, or surgery for an underlying heart disease.
After PVI or ECV treatment, we recorded whether the patient had an AF recurrence within 1 year after the treatment and determined whether we could predict the recurrence on the level(s) of the determined stress marker.
The stress markers that were determined included:
DNA damage: 8-hydroxydeoxyguanosine (8-OHdG), which is induced by DNA damage and is a marker of DNA oxidation.
Long non-coding RNAs: have a length >200 nucleotides, are not translated into proteins and are implicated in the regulation of gene expression. These long non-coding RNAs include Sarrah, Urothelial carcinoma-associated- 1 (UCA1), CDR1AS and long intergenie non-coding RNA predicting cardiac remodeling (LIPCAR).
Mitochondrial DNA: cell-free circulating mitochondrial DNA can act as a damage-associated pattern, hut is also implicated as an biomarker for several applications, including cancer progression, ICU mortality, dengue severity, cardiac arrest survival and diabetes mellitus. Mitochondrial DNA levels were determined for cytochrome C oxidase subunit III (COX3) and NADH dehydrogenase subunit I (ND1).
The results are shown in Tables 4 and 5. It is apparent that the different stress markers have divergent expression patterns between the different stages of AF. Nevertheless, the markers within 1 group (mitochondrial DNA and long non coding RNAs) show approximately the same expression pattern (data not shown).
Although DNA damage, for example 8-OHdG, showed increased levels in all the AF stages, it did not correlate with any of the other measured stress markers. On the other hand, several of the long non-coding RNAs did correlate with each other and the mitochondrial DNA markers (see Tables 4 and 5). In addition, the long-non coding RNAs and mitochondrial DNA markers also correlate with each other in patients with recurrence of AF after treatment.
Table 3. Overview of biomarker levels in various groups.
Figure imgf000061_0001
*P<0.05, ** P<0.01 vs Contro
Table 4. Overview of biomarkers after AF treatment (PVI or ECV) in patients without recurrence.
Figure imgf000062_0001
Table 5. Overview of biomarkers after AF treatment (PVI or ECV) in patients with recurrence.
Figure imgf000063_0001

Claims

Claims
1. A method of typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of:
- providing a bodily fluid from said individual;
- quantifying a level of long non-coding RNA (lncRNA), DNA damage and/or mitochondrial nucleic acid in said bodily fluid, preferably of lncRNA and mitochondrial nucleic acid;
- comparing said quantified level of lncRNA, DNA damage and/or
mitochondrial nucleic acid to a reference; and
- typing said individual as suffering from, or at risk of suffering from, atrial fibrillation if said quantified level of lncRNA is altered, compared to the reference, if the quantified level of DNA damage is altered, compared to the reference and/or if the quantified mitochondrial nucleic acid is altered, compared to the reference.
2. The method according to claim 1, wherein said bodily fluid is blood.
3. The method according to claim 1 or claim 2, the level of DNA damage is quantified by determining a level of 8-oxoguanine.
4. The method according to any one of the previous claims, wherein said lncRNA is selected from Sarrah, UCA1, CDR1AS and LIPCAR.
5. The method according to any one of the previous claims, wherein said mitochondrial nucleic acid is mitochondrial DNA.
6. The method according to any one of the previous claims, wherein said mitochondrial nucleic acid comprises nucleic acid from a cytochrome C oxidase 3 gene and/or a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene.
7. The method according to any one of the previous claims, comprising quantifying a level of lncRNA and quantifying mitochondrial DNA from a cytochrome C oxidase 3 gene and a NADH dehydrogenase 1 (2, 3, 4L, 4, 5, 6) gene.
8. The method according to claim 7, further comprising quantifying a heat shock protein level, preferably heat shock protein B7, in said bodily fluid.
9. A method for assigning standard-of-care therapy to an individual suffering from, or at risk of suffering from, atrial fibrillation, comprising the steps of:
- typing an individual as suffering from, or at risk of suffering from, atrial fibrillation, according to a method of any one of claims 1-8; and
- assigning a standard-of-care therapy to the individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation.
10. The method of claim 9, wherein the standard-of-care therapy is ablative therapy, electrical or chemical cardioversion, and/or the administration of anti- arrhythmic drugs aimed at controlling rhythm or rate.
11. The method of claim 9 or claim 10, wherein the standard-of-care therapy is combined with an antiplatelet and/or anticoagulant.
12. The method of claim 11, wherein the antiplatelet and/or anticoagulant is selected from aspirin, warfarin, and a direct-acting oral anticoagulant such as dabigitran, rivaroxaban, edoxaban and apixaban.
13. The method of any one of claims 9-12, wherein the standard-of-care therapy is an ion channel blocker.
14. The method of any one of claims 9-13, wherein the standard-of-care therapy is a sodium channel blocker such as flecainide, propafenone and/or quinidine, and/or a potassium channel blocker such as amiodarone, sotalol and/or dofetilide.
15. A standard-of-care therapy for use in a method of treating an individual that is typed as suffering from, or at risk of suffering from, atrial fibrillation, according to the method of any one of claims 1-8.
16. A method for determining the efficacy of a standard-of-care therapy of an individual, comprising the steps of: analysing a bodily fluid, preferably blood, of an individual for the presence of a biomarker using a method of typing according to the method of any one of claims 1-8, at a first time point to thereby provide a first value for the level of said biomarker in said subject,
analysing a bodily fluid, preferably blood, of said individual for the presence of said biomarker using a method of typing according to the invention at a second time point that is earlier or later, preferably later, than said first time point, to thereby provide a second value for the level of said biomarker in said individual,
wherein said individual has been subjected to a standard-of-care therapy between said first and second time point, and
comparing said first and second value to determine the efficacy of said standard-of-care therapy in said individual.
PCT/NL2019/050480 2018-07-26 2019-07-24 Biomarkers for atrial fibrillation Ceased WO2020022891A2 (en)

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