MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1 , have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation–contraction coupling and Ca ²⁺ cycling in rat ventricular myocytes using methods of electrophysiology, Ca ²⁺ imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca ²⁺ current, flattening of Ca ²⁺ transients voltage dependence, and enhanced frequency of spontaneous Ca ²⁺ sparks while reducing the sarcoplasmic reticulum Ca ²⁺ content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1 –overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca ²⁺ , events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca ²⁺ /calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56α involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation–contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels.

Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor (RyR2). The transfer of Ca²⁺ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca²⁺ leak, via altered SR-mitochondrial Ca²⁺ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca²⁺ probe mtRCamp1h revealed diminished net mitochondrial [Ca²⁺] with enhanced SR Ca²⁺ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca²⁺ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca²⁺ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca²⁺ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca²⁺ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca²⁺ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.

Aging of the heart is associated with a blunted response to sympathetic stimulation, reduced contractility, and increased propensity for arrhythmias, with the risk of sudden cardiac death significantly increased in the elderly population. The altered cardiac structural and functional phenotype, as well as age-associated prevalent comorbidities including hypertension and atherosclerosis, predispose the heart to atrial fibrillation, heart failure, and ventricular tachyarrhythmias. At the cellular level, perturbations in mitochondrial function, excitation-contraction coupling, and calcium homeostasis contribute to this electrical and contractile dysfunction. Major determinants of cardiac contractility are the intracellular release of Ca²⁺ from the sarcoplasmic reticulum by the ryanodine receptors (RyR2), and the following sequestration of Ca²⁺ by the sarco/endoplasmic Ca²⁺-ATPase (SERCa2a). Activity of RyR2 and SERCa2a in myocytes is not only dependent on expression levels and interacting accessory proteins, but on fine-tuned regulation via post-translational modifications. In this paper, we review how aberrant changes in intracellular Ca²⁺ cycling via these proteins contributes to arrhythmogenesis in the aged heart.

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

Sudden cardiac death due to ventricular tachyarrhythmias remains the major cause of mortality in the world. Heart failure, diabetic cardiomyopathy, old age-related cardiac dysfunction and inherited disorders are associated with enhanced propensity to malignant cardiac arrhythmias. Both defective mitochondrial function and abnormal intracellular Ca2+ homeostasis have been established as the key contributing factors in the pathophysiology and arrhythmogenesis in these conditions. This article reviews current advances in understanding of bidirectional control of ryanodine receptor-mediated sarcoplasmic reticulum Ca2+ release and mitochondrial function, and how defects in crosstalk between these two organelles increase arrhythmic risk in cardiac disease.

Defective intracellular Ca²⁺ homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca²⁺ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca²⁺ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca²⁺ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.