Aims: Deregulation of epigenetic processes and aberrant gene expression are important mechanisms in heart failure. Here we studied the potential relevance of m6A RNA methylation in heart failure development. Methods and results: We analysed m6A RNA methylation via next-generation sequencing. We found that approximately one quarter of the transcripts in the healthy mouse and human heart exhibit m6A RNA methylation. During progression to heart failure we observed that changes in m6A RNA methylation exceed changes in gene expression both in mouse and human. RNAs with altered m6A RNA methylation were mainly linked to metabolic and regulatory pathways, while changes in RNA expression level mainly represented changes in structural plasticity. Mechanistically, we could link m6A RNA methylation to altered RNA translation and protein production. Interestingly, differentially methylated but not differentially expressed RNAs showed differential polysomal occupancy, indicating transcription-independent modulation of translation. Furthermore, mice with a cardiomyocyte restricted knockout of the RNA demethylase Fto exhibited an impaired cardiac function compared to control mice. Conclusions: We could show that m6A landscape is altered in heart hypertrophy and heart failure. m6A RNA methylation changes lead to changes in protein abundance, unconnected to mRNA levels. This uncovers a new transcription-independent mechanisms of translation regulation. Therefore, our data suggest that modulation of epitranscriptomic processes such as m6A methylation might be an interesting target for therapeutic interventions.

Introduction: Pronounced extracellular acidosis reduces both cardiac contractility and the β-adrenergic response. In the past, this was shown in some studies using animal models. However, few data exist regarding how the human end-stage failing myocardium, in which compensatory mechanisms are exhausted, reacts to acute mild metabolic acidosis. The aim of this study was to investigate the effect of mild metabolic acidosis on contractility and the β-adrenergic response of isolated trabeculae from human end-stage failing hearts. Methods: Intact isometrically twitching trabeculae isolated from patients with end-stage heart failure were exposed to mild metabolic acidosis (pH 7.20). Trabeculae were stimulated at increasing frequencies and finally exposed to increasing concentrations of isoproterenol (0 to 1 × 10^-6M). Results: A mild metabolic acidosis caused a depression in twitch-force amplitude of 26% (12.1 ± 1.9 to 9.0 ± 1.5 mN/mm²; n = 12; P < 0.01) as compared with pH 7.40. Force-frequency relation measurements yielded no further significant differences of twitch force. At the maximal isoproterenol concentration, the force amplitude was comparable in each of the two groups (pH 7.40 versus pH 7.20). However, the half-maximal effective concentration (EC₅₀) was significantly increased in the acidosis group, with an EC₅₀ of 5.834 × 10^-8M (confidence interval (CI), 3.48 × 10^-8 to 9.779 × 10^-8; n = 9), compared with the control group, which had an EC₅₀ of 1.056 × 10^-8M (CI, 2.626 × 10^-9 to 4.243 × 10^-8; n = 10; P < 0.05), indicating an impaired β-adrenergic force response. Conclusions: Our data show that mild metabolic acidosis reduces cardiac contractility and significantly impairs the β-adrenergic force response in human failing myocardium. Thus, our results could contribute to the still-controversial discussion about the therapy regimen of acidosis in patients with critical heart failure.

Aim We have previously reported that early phase (1 week) of experimental volume overload (VO) has an adaptive phenotype while wall stress-matched pressure overload (PO) is maladaptive. Here we investigate the transition from adaptation to heart failure (HF) in long-term VO. Methods and results FVB/N wild-type mice were subjected to VO induced by aortocaval shunt, and were followed by serial echocardiography until in vivo left ventricular ejection fraction was below <50% (135 ± 35 days). Heart failure was evident from increased lung and liver weight and increased mortality compared with sham. Maladaptive remodelling resulted in significantly reduced sarcomeric titin phosphorylation (causing increased sarcomeric stiffness), whereas interstitial fibrosis was not increased. This was paralleled by re-expression of the fetal gene program, activation of calcium/calmodulin-dependent protein kinase II (CaMKII), decreased protein kinase B (Akt) phosphorylation, high oxidative stress, and increased apoptosis. Consistently, development of HF and mortality were significantly aggravated in Akt-deficient mice. Conclusion Transition to HF in VO is associated with decreased Akt and increased CaMKII signalling pathways together with increased oxidative stress and apoptosis. Lack of interstitial fibrosis together with sarcomeric titin hypophosphorylation indicates an increased stiffness at the sarcomeric but not matrix level in VO-induced HF (in contrast to PO). Transition to HF may result from myocyte loss and myocyte dysfunction owing to increased stiffness.

In heart failure, intracellular Ca²⁺ leak from cardiac ryanodine receptors (RyR2s) leads to a loss of Ca²⁺ from the sarcoplasmic reticulum (SR) potentially contributing to decreased function. Experimental data suggest that the 1,4-benzothiazepine K201 (JTV-519) may stabilise RyR2s and thereby reduce detrimental intracellular Ca²⁺ leak. Whether K201 exerts beneficial effects in human failing myocardium is unknown. Therefore, we have studied the effects of K201 on muscle preparations from failing human hearts. K201 (0.3 μM; extracellular [Ca²⁺]_e 1.25 mM) showed no effects on contractile function and micromolar concentrations resulted in negative inotropic effects (K201 1 μM; developed tension −9.8 ± 2.5% compared to control group; P < 0.05). Interestingly, K201 (0.3 μM) increased the post-rest potentiation (PRP) of failing myocardium after 120 s, indicating an increased SR Ca²⁺ load. At high [Ca²⁺]_e concentrations (5 mmol/L), K201 increased PRP already at shorter rest intervals (30 s). Strikingly, treatment with K201 (0.3 μM) prevented diastolic dysfunction (diastolic tension at 5 mmol/L [Ca²⁺]_e normalised to 1 mmol/L [Ca²⁺]_e: control 1.26 ± 0.06, K201 1.01 ± 0.03, P < 0.01). In addition at high [Ca²⁺]_e, K201 (0.3 μM) treatment significantly improved systolic function [developed tension +27 ± 8% (K201 vs. control); P < 0.05]. The beneficial effects on diastolic and systolic functions occurred throughout the physiological frequency range of the human heart rate from 1 to 3 Hz. Upon elevated intracellular Ca²⁺ concentration, systolic and diastolic contractile functions of terminally failing human myocardium are improved by K201.

Increased sarcoplasmic reticulum (SR) Ca²⁺ leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca²⁺ leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36– versus placebo-treated mice. Reduction of SR Ca²⁺ leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca²⁺ leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca²⁺ leak–independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca²⁺ waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca²⁺ transient, SR Ca²⁺ load, SR Ca²⁺ adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca²⁺ leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.

Induction of the cell cycle is emerging as an intervention to treat heart failure. Here, we tested the hypothesis that enhanced cardiomyocyte renewal in transgenic mice expressing cyclin D2 would be beneficial during hemodynamic overload. We induced pressure overload by transthoracic aortic constriction (TAC) or volume overload by aortocaval shunt in cyclin D2–expressing and WT mice. Although cyclin D2 expression dramatically improved survival following TAC, it did not confer a survival advantage to mice following aortocaval shunt. Cardiac function decreased following TAC in WT mice, but was preserved in cyclin D2–expressing mice. On the other hand, cardiac structure and function were compromised in response to aortocaval shunt in both WT and cyclin D2–expressing mice. The preserved function and improved survival in cyclin D2–expressing mice after TAC was associated with an approximately 50% increase in cardiomyocyte number and exaggerated cardiac hypertrophy, as indicated by increased septum thickness. Aortocaval shunt did not further impact cardiomyocyte number in mice expressing cyclin D2. Following TAC, cyclin D2 expression attenuated cardiomyocyte hypertrophy, reduced cardiomyocyte apoptosis, fibrosis, calcium/calmodulin–dependent protein kinase IIδ phosphorylation, brain natriuretic peptide expression, and sustained capillarization. Thus, we show that cyclin D2–induced cardiomyocyte renewal reduced myocardial remodeling and dysfunction after pressure overload but not after volume overload.