Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca 2+ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in "leaky" RyR2 channels and a diastolic SR Ca 2+ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca 2+ channel stabilizer S107 ("rycal") inhibited the SR Ca 2+ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca 2+ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca 2+ leak prevents fatal sudden cardiac arrhythmias in DMD.calcium | excitation-contraction coupling | heart | sudden cardiac death | myopathy

The mouse is the animal model principally used to study biological processes in mammals. The mutation, overexpression or knockout of one or several genes can provide insight into human disease. In cardiovascular research, evaluation of autonomic nervous function is an essential tool for a better understanding of the pathophysiological conditions in which cardiomyopathy arises and develops. Analysis of heart rate variability is the least invasive method to evaluate the sympathovagal balance on the sino-atrial level. The need to perform this technique on freely moving mice emerged in the 1990s, but despite previous studies it has been difficult to set up and standardize a common protocol. The multitudes of techniques used, plus subtle differences in methodology, impede the comparison and clear interpretation of results. This article aims to make a survey of heart rate variability analysis and to establish a standardized protocol for the assessment of the autonomic neural regulation of heart rate in mice.

RationaleTRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.ObjectivesWe aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 -/-) model.Methods and ResultsMorpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 -/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 -/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 -/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase.ConclusionsTRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Rationale: Clinical reports describe life-threatening cardiac arrhythmias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown. Objectives: To understand the cellular basis of CO-induced arrhythmias and to indentify an effective therapeutic approach. Methods: Patch-clamp electrophysiology and confocal Ca 21 and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo. Measurements and Main Results: CO increased the sustained (late) component of the inward Na 1 current, resulting in prolongation of the action potential and the associated intracellular Ca 21 transient. In more than 50% of myocytes these changes progressed to early after-depolarization-like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na 1 channel, Na v 1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor L-NAME, and reversed by ranolazine, an inhibitor of the late Na 1 current. Ranolazine also corrected QT variability and arrhythmias induced by CO in vivo, as monitored by telemetry. Conclusions: Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitrosylation of Na V 1.5 and to induction of the late Na 1 current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.