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Ischemic cardiomyopathy (ICM) is the clinical endpoint of coronary heart disease and a leading cause of heart failure. Despite growing demands to develop personalized approaches to treat ICM, progress is limited by inadequate knowledge of its pathogenesis. Since epigenetics has been implicated in the development of other chronic diseases, the current study was designed to determine whether transcriptional and/or epigenetic changes are sufficient to distinguish ICM from other etiologies of heart failure. Specifically, we hypothesize that genome-wide DNA methylation encodes transcriptional reprogramming in ICM. RNA-sequencing analysis was performed on human ischemic left ventricular tissue obtained from patients with end-stage heart failure, which enriched known targets of the polycomb methyltransferase EZH2 compared to non-ischemic hearts. Combined RNA sequencing and genome-wide DNA methylation analysis revealed a robust gene expression pattern consistent with suppression of oxidative metabolism, induced anaerobic glycolysis, and altered cellular remodeling. Lastly, KLF15 was identified as a putative upstream regulator of metabolic gene expression that was itself regulated by EZH2 in a SET domain-dependent manner. Our observations therefore define a novel role of DNA methylation in the metabolic reprogramming of ICM. Furthermore, we identify EZH2 as an epigenetic regulator of KLF15 along with DNA hypermethylation, and we propose a novel mechanism through which coronary heart disease reprograms the expression of both intermediate enzymes and upstream regulators of cardiac metabolism such as KLF15. Human ischemic cardiomyopathy is defined by DNA hypermethylation, methyltransferase EZH2 induction, and transcription factor KLF15 suppression. Together these changes may mediate a gene expression pattern reflecting decreased oxidative phosphorylation and increased cellular remodeling. This study therefore identifies a novel mechanism through which coronary heart disease may be regulated.

Ischemic cardiomyopathy (ICM) is the clinical endpoint of coronary heart disease and a leading cause of heart failure. Despite growing demands to develop personalized approaches to treat ICM, progress is limited by inadequate knowledge of its pathogenesis. Since epigenetics has been implicated in the development of other chronic diseases, the current study was designed to determine whether transcriptional and/or epigenetic changes are sufficient to distinguish ICM from other etiologies of heart failure. Specifically, we hypothesize that genome-wide DNA methylation encodes transcriptional reprogramming in ICM. RNA-sequencing analysis was performed on human ischemic left ventricular tissue obtained from patients with end-stage heart failure, which enriched known targets of the polycomb methyltransferase EZH2 compared to non-ischemic hearts. Combined RNA sequencing and genome-wide DNA methylation analysis revealed a robust gene expression pattern consistent with suppression of oxidative metabolism, induced anaerobic glycolysis, and altered cellular remodeling. Lastly, KLF15 was identified as a putative upstream regulator of metabolic gene expression that was itself regulated by EZH2 in a SET domain-dependent manner. Our observations therefore define a novel role of DNA methylation in the metabolic reprogramming of ICM. Furthermore, we identify EZH2 as an epigenetic regulator of KLF15 along with DNA hypermethylation, and we propose a novel mechanism through which coronary heart disease reprograms the expression of both intermediate enzymes and upstream regulators of cardiac metabolism such as KLF15.

Previous studies showed that endocardial activation during long-duration ventricular fibrillation (VF) exhibits organized activity. We identified and quantified the different types of organized activity.

Background:N6-methyladenosine (m6A) methylation is one of the important posttranscriptional modification of RNA, which affects RNA splicing, translation and stability. These modifications are dynamically regulated by writers, readers and erasers, and their alteration affects m6A modification and have shown to play key regulatory role in numerous biological processes. Fat mass- and obesity-associated (FTO) gene, known as a nucleic acid demethylase removes the methyl group from RNA and has been shown to be associated with progression of heart diseases. In this study, we determined m6A methylation in human failing heart, and evaluated if FTO regulates m6A-methylation of transcripts related to inflammation in cultured rat cardiomyocytes under various stress conditions.Methods and Results:m6A RNA-methylation was increased in human failing hearts as compared to heart samples from non-failing subjects. Interestingly, failing hearts were associated with significant decrease in FTO mRNA and protein expression. Furthermore, cultured rat cardiomyoblasts (H9C2) cells exposed to stressors (hypoxia or isoproterenol) for 24hrs showed significant decrease of FTO and an associated increase in m6A-RNA methylation (dot blot analysis). To further test if stress-induced increase in m6A-RNA modification is mediated through FTO, we knocked-down FTO, treated with isoproterenol for 24hrs and evaluated m6A methylation status of RNA. Interestingly, dot blot analysis showed significant increase in m6A methylation levels and proinflammatory cytokines (IL6 and TNFa) in FTO knockdown cells as compared to scramble, both at basal level and after exposure to stress. Furthermore, we evaluated the effect of FTO on methylation of RNA transcripts that encode inflammatory cytokines. MeRIP assay (Methylated RNA immunoprecipitation using m6A antibody and analysis of m6A-transcript by qRT-PCR) demonstrated that the gene expression of proinflammatory cytokines, IL6 and TNF-alpha was significantly increased in FTO knockdown cells as compared to scramble control cells .Conclusion:This data suggests stress-induced loss of FTO in the heart might mediate m6A-RNA methylation and therefore potential leading to upregulation of inflammatory response in the myocardium.

Congestive heart failure (HF) is characterized by contractile dysfunction and a high incidence of sudden death from nonreentrant ventricular arrhythmias, both of which involve altered intracellular calcium handling. The focus of this article is on the critical role of the NaCa exchanger. We demonstrate that upregulation of NaCa exchanger unloads the sarcoplasmic reticulum (SR), leading to contractile dysfunction. At the same time, NaCa exchanger underlies the arrhythmogenic transient inward current (Iti) in HF. Preserved ?-adrenergic responsiveness in HF plays a crucial role in increasing SR Ca load, leading to SR Ca release and activation of Iti. In addition, decreased IK1(inward rectifier) current in HF destabilizes resting membrane potential (Em) and further enhances arrhythmogenesis mediated by the upregulated NaCa exchanger. We thus propose a new paradigm in which upregulated NaCa exchanger in HF plays a dual role in underlying both the contractile dysfunction and arrhythmogenesis in the failing heart. Therapeutic approaches to the treatment of HF will need to balance increasing SR Ca load with the arrhythmogenic effects of SR Ca overload that involve activation of Iticarried by NaCa exchanger.

Intracellular [Na+] ([Na+]i) is centrally involved in regulation of cardiac Ca2+and contractility via Na+‐Ca2+exchange (NCX) and Na+‐H+exchange (NHX). Previous work has indicated that [Na+]iis higher in rat than rabbit ventricular myocytes. This has major functional consequences, but the reason for the higher [Na+]iin rat is unknown. Here, resting [Na+]iwas measured using the fluorescent indicator SBFI, with both traditional calibration and a novel null‐point method (which circumvents many limitations of prior methods). In rabbit, resting [Na+]iwas 4.5 ± 0.4 mm(traditional calibration) and 4.4 mm(null‐point). Resting [Na+]iin rat was significantly higher using both the traditional calibration (11.1 ± 0.7 mm) and the null‐point approach (11.2 mm). The rate of Na+transport by the Na+pump was measured as a function of [Na+]iin intact cells. Rat cells exhibited a higher Vmaxthan rabbit (7.7 ± 1.1 vs.4.0 ± 0.5 mmmin−1) and a higher Km(10.2 ± 1.2 vs.7.5 ± 1.1 mm). This results in little difference in pump activity for a given [Na+]ibelow 10 mm, but at measured resting [Na+]ilevels the pump‐mediated Na+efflux is much higher in rat. Thus, Na+pump rate cannot explain the higher [Na+]iin rat. Resting Na+influx rate was two to four times higher in rat, and this accounts for the higher resting [Na+]i. Using tetrodotoxin, HOE‐642 and Ni2+to block Na+channels, NHX and NCX, respectively, we found that all three pathways may contribute to the higher resting Na+influx in rat (albeit differentially). We conclude that resting [Na+]iis higher in rat than in rabbit, that this is caused by higher resting Na+influx in rat and that a higher Na+,K+‐ATPase pumping rate in rat is a consequence of the higher [Na+]i.

The inhibitory and bactericidal effects of gentamicin, amikacin, netilmicin (Sch 20569), and carbenicillin were tested against 55 clinical isolates of Serratia marcescensthat had been subtyped into 26 strains by biotyping and serotyping. Three major patterns of resistance to gentamicin, netilmicin, and carbenicillin were recognized among these isolates. (i) Most of the 27 isolates that were susceptible to gentamicin (minimal bactericidal concentration [MBC] ≤6.25 μg/ml) were susceptible to carbenicillin (MBC ≤125 μg/ml) and resistant to netilmicin (MBC ≥12.5 μg/ml). (ii) Most of the 11 isolates with moderate resistance to gentamicin (MBC of 12.5 to 25 μg/ml) were also susceptible to carbenicillin and resistant to netilmicin. (iii) The 17 isolates with high-level resistance to gentamicin (MBC ≥ 50 μg/ml) were all highly resistant to carbenicillin (MBC ≥8,000 μg/ml) but susceptible to netilmicin (MBC ≤6.25 μg/ml). The susceptibility to amikacin was unpredictable among these groups of isolates but, overall, 80% of the isolates were killed by 25 μg of amikacin/ml, which is within the range of peak serum concentrations used therapeutically. Clinically attainable subinhibitory concentrations of carbenicillin enhanced the activity of the three aminoglycosides against all isolates with MBCs of carbenicillin ≤2,000 μg/ml. The 17 isolates with high-level resistance to carbenicillin and gentamicin, as well as the four isolates with high-level resistance to carbenicillin but not to gentamicin, were not susceptible to such enhancement of aminoglycoside activity by carbenicillin.

To define the mechanism of ventricular tachycardia (VT) induced by programmed electrical stimulation in the setting of congestive heart failure, 3-dimensional cardiac mapping was performed in six dogs with ischemic cardiomyopathy induced by multiple intracoronary embolizations with microspheres. Ejection fraction (EF) progressively decreased from 54±2 to 30±5% (p<0.005) after an average of 6 weekly embolizations. Four months later (EF=28±5%), each dog underwent a thoracotomy with insertion of plungeneedle electrodes into the heart under pentobarbital anesthesia. Continuous recording from 232 intramural sites throughout the left and right ventricles and the interventricular septum was performed during spontaneous rhythm and during programmed stimulation in both the absence and presence of isoproterenol (Iso) (0.1mg/kg/min). Three dogs (1 at baseline, 2 during Iso) developed sustained VT (SuVT) during programmed stimulation with up to 3 extrastimuli. After the last extrastimulus, the first beat of SuVT (T1) initiated in the subendocardium by a focal mechanism, based on the absence of electrical activity from the termination of the last extrastimulus to the initiation of T1despite the presence of multiple intermediate electrode sites. Maintenance of SuVT was also due to a focal mechanism arising in the subendocardium, with a total activation time (TAT) of subsequent SuVT beats (99±14msec, n=61 that was comparable to that of initiating beats (85±8msec. n=6, p=0.08). There was no evidence of macroreentry. Episodes of SuVT (n=3) exhibited TATs of the last extrastimulus before SuVT (106±11 msec) and coupling intervals of T1 (196±70 msec) that were comparable to those observed in nonsustained VT (n=3) (138±26 msec, p=0.19; 166±26msec, p=0.59). Conduction delay during sinus rhythm in dogs with SuVT (TAT 47±5 msec, n=10) was comparable to that in dogs without SuVT (TAT 47±4 msec, n=10, p=0.96) and was unchanged by Iso (TAT 50±4 msec, n=7, p=0.40). In the 2 dogs with inducible SuVT on Iso only, TATs of the last extrastimulus (124±22 msec) and of T1(110±36 msec) at baseline were unchanged by infusion of Iso (108±13 msec, p=0.59; 79±1 msec, p=0.49). Thus, inducible sustained VT in a model of ischemic cardiomyopathy is due to a focal mechanism, as opposed to macroreentry, and this focal mechanism is enhanced by β-adrenergic stimulation.

We have previously demonstrated a significant antifibrillatory effect of -adrenergic blockade during myocardial ischaemia and reperfusion, enhanced -adrenergic responsivity and increased ₁-adrenergic receptors in ischaemic myocardium. β-adrenergic blockade is also antiarrhythmic in ischaemic hearts and may decrease the ultimate area of necrotic tissue after infarction. The present study was performed to assess the relative β- and -adrenergic blocking potency of labetalol in the chloralose anaesthetised cat and correlate this to its antiarrhythmic efficacy during LAD coronary occlusion for 35 min followed by reperfusion. Based on the dose ratio₁₀ (DR₁₀) for isoprenaline-induced tachycardia (β) and phenylephrine-induced increase in mean systemic arterial pressure (), labetalol was 3.1 times less potent than d,l-propranolol (β) and 6.5 times less potent than phentolamine (). Labetalol was found to be 11.5 times more potent as a β-adrenergic blocking agent than as an -adrenergic blocking agent. Labetalol (1 mg·kg–1) failed significantly to reduce either the number of premature ventricular complexes (PVCs) or incidence of ventricular fibrillation (VF) during occlusion (827± 150 to 852±246 PVCs and 31% to 20% VF) or reperfusion. In contrast, labetalol at 2 mg·kg–1 and 5 mg·kg–1 significantly (P<0.05) reduced the number of PVCs during occlusion (320±94 and 466± 137, respectively) as well as the incidence of VF (9% and 7%, respectively). During, reperfusion, labetalol (5 mg·kg–1) failed to reduce the number of PVCs but abolished mortality due to VF. The antifibrillatory effect of labetalol was not due to alterations in systemic arterial pressure, heart rate, LVEDP, cardiac output, stroke work or total peripheral resistance. Thus, labetalol, an agent well-tolerated clinically, induces profound antiarrhythmic effectiveness during both experimental myocardial ischaemia and reperfusion and may prove to be an effective agent in the prevention and treatment of malignant ventricular dysrhythmia associated with acute myocardial infarction in man.