Heart failure with preserved ejection fraction (HFpEF) accounts for nearly half of all heart failure cases. In addition to diastolic dysfunction, patients with HFpEF have a substantial risk of sudden cardiac death, suggesting a potential contribution of malignant ventricular arrhythmias. Recently, small conductance Ca2+-activated K+ (SK) channels have emerged as potential modulators of mitochondria-dependent production of harmful reactive oxygen species (ROS). This study aimed to evaluate SK channel enhancement as a novel strategy to reverse proarrhythmic changes in intracellular Ca2+ cycling and in mitochondrial redox and Ca2+ homeostasis in ventricular myocytes using obese ZSF1 rat model of HFpEF. Confocal imaging of Ca2+ and ROS was performed in ventricular myocytes isolated from lean and obese ZSF1 rats. Mitochondrial matrix Ca2+ and ROS levels were measured using matrix-targeted biosensors mtRCamp1h and MLS-HyPer7, respectively. SK channel activity was enhanced by adenoviral overexpression of rat SK channel type 2 and by pharmacological activators NS309 and Riluzole. Ventricular myocytes from obese ZSF1 rats, when periodically paced and exposed to the β-adrenergic agonist isoproterenol, showed increased pro-arrhythmic diastolic sarcoplasmic reticulum (SR) Ca2+ release, elevated mitochondrial ROS production, and marked mitochondrial Ca2+ overload. SK channel enhancement prevented mitochondrial Ca2+ overload, reduced ROS emission, and improved cytosolic Ca2+ cycling by suppressing diastolic SR Ca2+ release These findings identify SK channel activation as a potential antiarrhythmic therapeutic strategy in HFpEF by limiting mitochondrial Ca2+ uptake, reducing oxidative stress and stabilizing intracellular Ca2+ dynamics.

BACKGROUND:Mitochondrial dysfunction caused by abnormally high RyR2 (ryanodine receptor) activity is a common finding in cardiovascular diseases. Mechanisms linking RyR2 gain of function with mitochondrial remodeling remain elusive. We hypothesized that RyR2 hyperactivity in cardiac disease increases [Ca2+] in the mitochondrial intermembrane space (IMS) and activates the Ca2+-sensitive protease calpain, driving remodeling of mitochondrial cristae architecture through cleavage of structural protein OPA1 (optic atrophy protein 1). METHODS:We generated a highly arrhythmogenic rat model of catecholaminergic polymorphic ventricular tachycardia, induced by RyR2 gain-of-function mutation S2236L(Ser2336Leu)(+/-). We created a new biosensor to measure IMS-[Ca2+] in adult cardiomyocytes with intact Ca2+ cycling. We used ex vivo whole heart optical mapping, confocal and electron microscopy, as well as in vivo/in vitro gene editing techniques to test the effects of calpain in the IMS. RESULTS:We found altered mitochondrial cristae structure, increased IMS-[Ca2+], reduced OPA1 expression, and augmented mito-reactive oxygen species emission in catecholaminergic polymorphic ventricular tachycardia myocytes. We show that calpain-mediated OPA1 cleavage led to disrupted cristae organization and, thereby, decreased electron transport chain supercomplex assembly, resulting in accelerated reactive oxygen species production. Genetic inhibition of calpain activity in IMS reversed mitochondria structural defects in catecholaminergic polymorphic ventricular tachycardia myocytes and reduced arrhythmic burden in ex vivo optically mapped hearts. CONCLUSIONS:Our data suggest that RyR2 hyperactivity contributes to mitochondrial structural damage by promoting an increase in IMS-[Ca2+], sufficient to activate IMS-residing calpain. Calpain activation leads to proteolysis of OPA1 and cristae widening, thereby decreasing assembly of electron transport chain components into supercomplexes. Consequently, excessive mito-reactive oxygen species release critically contributes to RyR2 hyperactivation and ventricular tachyarrhythmia. Our new findings suggest that targeting IMS calpain may be beneficial in patients at risk for sudden cardiac death.

Small-conductance Ca2⁺-activated K⁺ (SK) channels have emerged over the past decade as compelling antiarrhythmic targets. All three isoforms, SK1, SK2 and SK3, are expressed in both atrial and ventricular cardiomyocytes, where they are exclusively gated by intracellular Ca2⁺ via constitutively bound calmodulin. Sarcolemmal SK channels uniquely translate elevations in intracellular Ca2+ concentration into action potential repolarization. In doing so they mitigate pro-arrhythmic disturbances in membrane potential caused by pathological spontaneous Ca2⁺ release from sarcoplasmic reticulum, thereby reducing Ca2+-mediated arrhythmia triggers such as early and delayed afterdepolarizations. However the role of SK channels in arrhythmogenesis is complex. Although their activation can be protective against triggered activity, additional repolarizing force under certain conditions may shorten the action potential excessively and create a substrate for re-entrant arrhythmias. Furthermore SK channels have recently been found in cardiac mitochondria, where they appear to regulate mitochondrial Ca2⁺ handling and reactive oxygen species (ROS) production, suggesting a prominent role in cardioprotection. The contribution of mitochondrial SK (mito-SK) channels to cardiac arrhythmogenesis, however, remains incompletely understood. In this review we summarize current advances in understanding the therapeutic potential of SK channels as an antiarrhythmic target, with a particular focus on the contribution of mito-SK channels to cardioprotection and mitochondrial ROS production.

Mitochondrial dysfunction caused by abnormally high RyR2 (ryanodine receptor) activity is a common finding in cardiovascular diseases. Mechanisms linking RyR2 gain of function with mitochondrial remodeling remain elusive. We hypothesized that RyR2 hyperactivity in cardiac disease increases [Ca 2+ ] in the mitochondrial intermembrane space (IMS) and activates the Ca 2+ -sensitive protease calpain, driving remodeling of mitochondrial cristae architecture through cleavage of structural protein OPA1 (optic atrophy protein 1). We generated a highly arrhythmogenic rat model of catecholaminergic polymorphic ventricular tachycardia, induced by RyR2 gain-of-function mutation S2236L(Ser2336Leu) ( +/-) . We created a new biosensor to measure IMS-[Ca 2+ ] in adult cardiomyocytes with intact Ca 2+ cycling. We used ex vivo whole heart optical mapping, confocal and electron microscopy, as well as in vivo/in vitro gene editing techniques to test the effects of calpain in the IMS. We found altered mitochondrial cristae structure, increased IMS-[Ca 2+ ], reduced OPA1 expression, and augmented mito-reactive oxygen species emission in catecholaminergic polymorphic ventricular tachycardia myocytes. We show that calpain-mediated OPA1 cleavage led to disrupted cristae organization and, thereby, decreased electron transport chain supercomplex assembly, resulting in accelerated reactive oxygen species production. Genetic inhibition of calpain activity in IMS reversed mitochondria structural defects in catecholaminergic polymorphic ventricular tachycardia myocytes and reduced arrhythmic burden in ex vivo optically mapped hearts. Our data suggest that RyR2 hyperactivity contributes to mitochondrial structural damage by promoting an increase in IMS-[Ca 2+ ], sufficient to activate IMS-residing calpain. Calpain activation leads to proteolysis of OPA1 and cristae widening, thereby decreasing assembly of electron transport chain components into supercomplexes. Consequently, excessive mito-reactive oxygen species release critically contributes to RyR2 hyperactivation and ventricular tachyarrhythmia. Our new findings suggest that targeting IMS calpain may be beneficial in patients at risk for sudden cardiac death.

Sarcolemmal small conductance Ca2+-activated K+ channels have the unique capacity to translate intracellular Ca2+ signal into repolarization, while mitochondrial SK channels can link Ca2+ cycling to mitochondrial function. We hypothesize that pharmacological enhancement of SK channels can be protective against malignant cardiac arrhythmias associated with disturbances in Ca2+ handling machinery. A mouse CASQ2 KO (calsequestrin type 2 knockout) model of catecholaminergic polymorphic ventricular tachycardia (CPVT) was used for in vivo ECG recordings and for cell electrophysiology, Ca2+, and reactive oxygen species imaging in isolated ventricular myocytes (VMs). Bidirectional and polymorphic ventricular tachycardias in CASQ2 KO mice induced by stress challenge (epinephrine+caffeine cocktail) were attenuated by injection of NS309, a specific SK channel enhancer. Voltage-clamp experiments in isolated VMs treated with β-adrenergic agonist isoproterenol showed a reduction of sarcolemmal SK channel current (ISK) density in CPVT VMs. Application of NS309 to CPVT VMs increased ISK. Current-clamp experiments demonstrated a significant reduction of arrhythmogenic delayed afterdepolarizations and spontaneous Ca2+ waves in isoproterenol-challenged CPVT VMs pretreated with NS309. Importantly, subsequent application of membrane-impermeable SK channel inhibitor apamin did not reverse the protective effects of NS309, suggesting important roles of mitochondrial SK channels in intracellular Ca2+ handling rescue. SK channel enhancement reversed the increased rate of reactive oxygen species production by mitochondria in CPVT VMs. It also reversed increased cardiac RyR2 (ryanodine receptor 2) oxidation measured in samples from CPVT hearts of the animals after the stress challenge. Electron microscopy studies showed a significant widening of mitochondria cristae in the ventricular tissue from CPVT mice, which led to a decrease in quaternary supercomplexes of electron transport chain, resulting in impairment of ATP production in VMs under β-adrenergic stimulation. Application of NS309 facilitated cristae flattening in CPVT ventricular tissue and restored supercomplexes and ATP production in VMs from diseased animals. Sarcolemmal SK channel enhancement reduces arrhythmic potential by restoring repolarization force in CPVT VMs. Activation of mitochondrial SK channels attenuates mitochondria structural changes in CPVT, restoring more efficient electron transport chain assembly into supercomplexes and reducing mito-reactive oxygen species production. This decreases RyR2 oxidation and thus channel activity, reducing spontaneous Ca2+ waves underlying arrhythmogenic delayed afterdepolarizations.

Gain-of-function mutations of the sarcoplasmic reticulum (SR) Ca 2+ release channel, the ryanodine receptor (RyR2), are linked to the inherited arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Increasing evidence suggests that RyR2 gain-of-function not only disturbs intracellular Ca 2+ homeostasis but drives remodeling of cell signaling and ultrastructure that markedly contributes to the arrhythmogenic phenotype. It is well established that disturbed SR Ca 2+ homeostasis can activate the endoplasmic reticulum (ER) stress response, yet whether RyR2 gain-of-function in CPVT drives ER stress is yet to be explored. The goal of our study was to determine the contribution of ER stress evoked by RyR2 gain-of-function to cardiac arrhythmogenesis. To test this, we created a new rat model of CPVT induced by RyR2-S2222L (+/-) mutation. Simultaneous whole cell patch clamp and Ca 2+ imaging demonstrated that under β-adrenergic stimulation, CPVT ventricular myocytes (VMs) exhibit a high propensity to spontaneous Ca 2+ waves (SCWs) and delayed afterdepolarizations. Importantly, CPVT VMs showed increased XBP1 splicing as a marker of ER stress, as well as increased intra-SR redox stress measured using biosensor ER_roGFPiE. Assessment of ER stress proteins that contribute to SR redox status revealed upregulation of H 2 0 2 -producer enzyme ERO1α, with no compensatory change in expression of H 2 O 2 -degrader Peroxiredoxin-4 (PRDX4). Adenoviral overexpression of PRDX4 in CPVT VMs not only normalized SR redox status but attenuated Ca 2+ mishandling, reducing RyR2 activity and the incidence of proarrhythmic spontaneous Ca 2+ waves. Of note, immunofluorescence and biochemical studies suggest a direct interaction between RyR2 and PRDX4. To test whether targeting ER stress was protective at the whole heart level, we delivered AAV9-αMHC-PRDX4 to CPVT rats and performed ex vivo optical mapping. While CPVT hearts exhibited triggered activity and increased incidence of VT, sustained VT was prevented in PRDX4-injected hearts. Collectively, these data strongly suggest that ER stress contributes to the arrhythmogenic phenotype of CPVT. Targeting ER stress protein PRDX4 has promising therapeutic potential to normalize SR homeostasis, stabilize RyR2 activity and attenuate Ca 2+ -dependent arrhythmogenesis.

Atrial fibrillation (AF) is the most prevalent form of cardiac arrhythmia associated with a significant reduction in quality of life and increased morbidity and mortality. Frontline therapies such as beta blockers and Ca2+ channels blockers help control cardiac rhythm to reduce the symptoms, but do not prevent AF. Therefore, the discovery of novel approaches to treat AF is imperative. Enhancement of small conductance Ca2+ activated K+ (SK) channel activity has recently emerged as a potential strategy to reduce ventricular arrhythmogenicity in acquired and hereditary cardiac diseases. However, the role of SK channels in pathogenesis of AF remains controversial given both loss- and gain-of-function of SK channels were linked to increased atrial arrhythmogenesis in various genetic mouse models and in GWAS association studies. We previously showed that synchronized diastolic Ca2+ release is involved in Ca2+-dependent atrial arrhythmias in catecholaminergic ventricular polymorphic tachycardia (CPVT). The objective of the present study was to examine the effects of inhibition and stimulation of SK activity on diastolic Ca2+ release in atrial myocytes using well-established calsequestrin knockout (CSQ-KO) mouse model of CPVT. Pacing protocol along with exposure to beta-adrenergic agonist isoproterenol (100 nM) were used to induce self-sustained repetitive diastolic Ca2+ release triggering action potentials (APs) in atrial myocytes from CSQ-KO hearts. Pharmacological enhancement of SK channels with NS309 (6 μM) completely blocked extra-systolic Ca2+ transients and APs, whereas inhibition of SK channels with apamine (100 nM) did not produce any effect on arrhythmogenic Ca2+ release. Therefore, as we showed previously using ventricular myocytes from CSQ-KO mice, pharmacological enhancement of SK channels CPVT appears to be an effective strategy to reduce both atrial and ventricular Ca2+-dependent arrhythmia in mouse model of CPVT.

Calmodulin, a protein that affords Ca2+-sensitivity to multiple enzymes and ion channels, is encoded by three distinct genes. Recent studies revealed that mutations in only one out of six alleles can underlie cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). Calm CPVT mutations are thought to disrupt RyR2 function, while LQTS mutations were linked to a reduction in L-type Ca2+-channel Ca2+-dependent inactivation. Surprisingly, mutations in different Calm genes resulting in identical protein products can lead to different arrhythmia phenotypes.

Inherited gain-of-function (GOF) mutations of the sarcoplasmic reticulum Ca2+ release channel, RyR2, are linked to the malignant arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Growing evidence suggests that RyR2 GOF not only results in perturbed SR Ca2+ release, but can lead to secondary changes in cell physiology and signaling that markedly contribute to the arrhythmic phenotype. However, whether RyR2 GOF drives ER stress remodeling is yet to be explored. The goal of our study was to define the contribution of ER stress evoked by Ca2+ mishandling to cardiac arrhythmogenesis.

Heart failure is the leading cause of death in the postindustrial world. While many effective therapies are available to treat heart failure with reduced ejection fraction (HFrEF), these approaches fail to improve outcomes in heart failure with preserved ejection fraction (HFpEF). Small conductance Ca2+-activated K+ (SK) channels have recently emerged as an attractive target to improve defective mitochondrial function and reduce emission of damaging reactive oxygen species (ROS) by the organelle. The goal of the present study was to test enhancement of SK as a new strategy to restore abnormal intracellular Ca2+ cycling and mitochondrial redox and Ca2+ homeostasis in ventricular myocytes from HFpEF rat hearts. To achieve this goal as a model of HFpEF we employed ZSF1 obese rats that demonstrate diastolic dysfunction, unchanged fractional shortening and ejection fraction. The confocal Ca2+ and ROS imaging experiments were carried out in ventricular myocytes isolated from lean and obese ZSF1 rats. Mitochondrial matrix [Ca2+] and ROS were assessed in ventricular myocytes expressing matrix-targeted biosensors mtRCamp1h and MLS-HyPer-7, respectively. To increase SK channel activity, we employed adenovirus-mediated overexpression of rat SK channel type 2 and pharmacological SK channel enhancer NS309 . Periodically paced ventricular myocytes isolated from obese ZSF1 rat hearts exhibited enhanced propensity to spontaneous sarcoplasmic reticulum (SR) Ca2+ release, increased mito-ROS production and a dramatic increase in mitochondrial [Ca2+] when exposed to beta-adrenergic agonist isoproterenol. Enhancement of SK channels in ZSF1 obese myocytes prevented mitochondrial Ca2+ overload, reduced mito-ROS emission to the control levels, and improved cytosolic Ca2+ cycling, reducing diastolic SR Ca2+ release. In summary, enhancement of SK channels shows high therapeutic potential in HFpEF by limiting mitochondrial Ca2+ uptake, thereby reducing oxidative stress restraining excessive SR Ca2+ release during diastole.

Hereditary RyR2 gain-of function mutations are linked to the highly malignant arrhythmia syndrome, catecholaminergic polymorphic tachycardia (CPVT). A mutation-mediated increase in RyR2 activity not only results in unstable sarcoplasmic reticulum Ca2+ release, but also alters mitochondrial structure and function, leading to excessive emission of damaging reactive oxygen species (ROS) by the organelle. However, the exact mechanisms underlying RyR2 hyperactivity-dependent mitochondrial damage remain elusive. We hypothesized that RyR2 hyperactivity activates the intermembrane space (IMS)-residing mitochondrial Ca2+-dependent protease calpain, driving proteolysis of OPA1, a protein responsible for tight cristae arrangement. Concomitant changes in cristae architecture promotes mito-ROS production, thereby aggravating the CPVT phenotype. To test this, we generated a unique CPVT RyR2-S2222L(+/-) rat model. Electron microscopy demonstrated significantly increased mitochondrial cristae diameter in tissue slices from CPVT rat hearts. Biochemical analysis showed reduced OPA1 expression in isolated CPVT myocytes, suggesting that mitochondrial ultrastructural changes in CPVT could be driven by Ca2+-dependent proteolysis of OPA1 by calpain. Importantly, specific inhibition of calpain via adeno-associated virus mediated expression of IMS-targeted calpastatin reduced the incidence of ventricular tachycardia in CPVT rats. At the cellular level, adenoviral expression of IMS-CAST restored OPA1 expression levels and significantly improved intracellular Ca2+ homeostasis in CPVT, indicative of normalized RyR2 function. Furthermore, IMS-calpain inhibition attenuated the reduction in mitochondrial matrix Ca2+ and increase in mito-ROS emission observed in CPVT myocytes using genetic probes mtRCamp1h and MLS-HyPer7. All protective effects were lost in myocytes with additional shRNA-mediated knockdown of OPA1. We conclude that RyR2 hyperactivity promotes mitochondrial structural damage via IMS-calpain-mediated degradation of OPA1 and contributes to proarrhythmic remodeling in CPVT.

Cardiac stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry (SOCE), is a known determinant of cardiomyocyte pathological growth in hypertrophic cardiomyopathy. We examined the role of STIM1 and SOCE in response to exercise-dependent physiological hypertrophy. Wild-type (WT) mice subjected to exercise training (WT-Ex) showed a significant increase in exercise capacity and heart weight compared with sedentary (WT-Sed) mice. Moreover, myocytes from WT-Ex hearts displayed an increase in length, but not width, compared with WT-Sed myocytes. Conversely, exercised cardiac-specific STIM1 knock-out mice (cSTIM1KO-Ex), although displaying significant increase in heart weight and cardiac dilation, evidenced no changes in myocyte size and displayed a decreased exercise capacity, impaired cardiac function, and premature death compared with sedentary cardiac-specific STIM1 knock-out mice (cSTIM1KO-Sed). Confocal Ca2+ imaging demonstrated enhanced SOCE in WT-Ex myocytes compared with WT-Sed myocytes with no measurable SOCE detected in cSTIM1KO myocytes. Exercise training induced a significant increase in cardiac phospho-Akt Ser473 in WT mice but not in cSTIM1KO mice. No differences were observed in phosphorylation of mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) in exercised versus sedentary cSTIM1KO mice hearts. cSTIM1KO-Sed mice showed increased basal MAPK phosphorylation compared with WT-Sed that was not altered by exercise training. Finally, histological analysis revealed exercise resulted in increased autophagy in cSTIM1KO but not in WT myocytes. Taken together, our results suggest that adaptive cardiac hypertrophy in response to exercise training involves STIM1-mediated SOCE. Our results demonstrate that STIM1 is involved in and essential for the myocyte longitudinal growth and mTOR activation in response to endurance exercise training.NEW & NOTEWORTHY Store-operated Ca2+ entry (SOCE) has been implicated in pathological cardiac hypertrophy; however, its role in physiological hypertrophy is unknown. Here we report that SOCE is also essential for physiological cardiac hypertrophy and functional adaptations in response to endurance exercise. These adaptations were associated with activation of AKT/mTOR pathway and curtailed cardiac autophagy and degeneration. Thus, SOCE is a common mechanism and an important bifurcation point for signaling paths involved in physiological and pathological hypertrophy.

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with β-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.