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.

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.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca ²⁺ and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca ²⁺ homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca ²⁺ leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca ²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca ²⁺ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α–induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca ²⁺ leak trough normalization of RyR2 function is cardioprotective.

Rationale: Epidemiologic studies associate atmospheric carbon monoxide (CO) pollution with adverse cardiovascular outcomes and increased cardiac mortality risk. However, there is a lack of data regarding cellular mechanisms in healthy individuals. Objectives: To investigate the chronic effects of environmentally relevant CO levels on cardiac function in a well-standardized healthy animal model. Methods: Wistar rats were exposed for 4 weeks to filtered air (CO < 1 ppm) or air enriched with CO (30 ppm with five peaks of 100 ppm per 24-h period), consistent with urban pollution. Myocardial function was assessed by echocardiography and analysis of surface ECG and in vitro by measuring the excitation-contraction coupling of single left ventricular cardiomyocytes. Measurements and Main Results: Chronic CO pollution promoted left ventricular interstitial and perivascular fibrosis, with no change in cardiomyocyte size, and had weak, yet significant, effects on in vivo cardiac function. However, both contraction and relaxation of single cardiomyocytes were markedly altered. Several changes occurred, including decreased Ca2+ transient amplitude and Ca2+ sensitivity of myofilaments and increased diastolic intracellular Ca2+ subsequent to decreased SERCA-2a expression and impaired Ca2+ reuptake. CO pollution increased the number of arrhythmic events. Hyperphosphorylation of Ca2+-handling and sarcomeric proteins, and reduced responses to β-adrenergic challenge were obtained, suggestive of moderate CO-induced hyperadrenergic state. Conclusions: Chronic CO exposure promotes a pathological phenotype of cardiomyocytes in the absence of underlying cardiomyopathy. The less severe phenotype in vivo suggests a role for compensatory mechanisms. Arrhythmia propensity may derive from intracellular Ca2+ overload.

TRPM4 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. We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4^-/-) model. Morpho-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. TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Rationale:: Revascularization of injured, ischemic, and regenerating organs is essential to restore organ function. In the postinfarct heart, however, the mechanisms underlying the formation of new coronary arteries are poorly understood. Objective:: To study vascular remodeling of coronary arteries after infarction. Methods and Results:: We performed permanent left coronary ligation on Connexin40-GFP mice expressing green fluorescent protein ( GFP ) in endothelial cells of coronary arteries but not veins, capillaries, or endocardium. GFP ⁺ endothelial foci were identified within the endocardium in the infarct zone. These previously undescribed structures, termed endocardial flowers, have a distinct endothelial phenotype (Cx40 ⁺ , VEGFR2 ⁺ , and endoglin ⁻ ) to the surrounding endocardium (Cx40 ⁻ , VEGFR2 ⁻ , and endoglin ⁺ ). Endocardial flowers are contiguous with coronary vessels and associated with subendocardial smooth muscle cell accumulation. Genetic lineage tracing reveals extensive endothelial plasticity in the postinfarct heart, showing that endocardial flowers develop by arteriogenesis of Cx40 ⁻ cells and by outgrowth of pre-existing coronary arteries. Finally, endocardial flowers exhibit angiogenic features, including early VEGFR2 expression and active proliferation of adjacent endocardial and smooth muscle cells. Conclusions:: Arterial endothelial foci within the endocardium reveal extensive endothelial cell plasticity in the infarct zone and identify the endocardium as a site of endogenous arteriogenesis and source of endothelial cells to promote vascularization in regenerative strategies.