Shanna Hamilton

Small‐conductance Ca ² ⁺‐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 Ca ² ⁺ via constitutively bound calmodulin. Sarcolemmal SK channels uniquely translate elevations in intracellular Ca ²⁺ concentration into action potential repolarization. In doing so they mitigate pro‐arrhythmic disturbances in membrane potential caused by pathological spontaneous Ca ² ⁺ release from sarcoplasmic reticulum, thereby reducing Ca ²⁺ ‐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 Ca ² ⁺ 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. image

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 [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). 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-[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. RESULTS: 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. CONCLUSIONS: 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.

BACKGROUND: Sarcolemmal small conductance Ca ²⁺ -activated K ⁺ channels have the unique capacity to translate intracellular Ca ²⁺ signal into repolarization, while mitochondrial SK channels can link Ca ²⁺ cycling to mitochondrial function. We hypothesize that pharmacological enhancement of SK channels can be protective against malignant cardiac arrhythmias associated with disturbances in Ca ²⁺ handling machinery. METHODS: 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, Ca ²⁺ , and reactive oxygen species imaging in isolated ventricular myocytes (VMs). RESULTS: 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 (I _SK ) density in CPVT VMs. Application of NS309 to CPVT VMs increased I _SK . Current-clamp experiments demonstrated a significant reduction of arrhythmogenic delayed afterdepolarizations and spontaneous Ca ²⁺ 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 Ca ²⁺ 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. CONCLUSIONS: 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 Ca ²⁺ waves underlying arrhythmogenic delayed afterdepolarizations.

The sarcoplasmic reticulum (SR) plays the key role in cardiac function as the major source of Ca²⁺ that activates cardiomyocyte contractile machinery. Disturbances in finely-tuned SR Ca²⁺ release by SR Ca²⁺ channel ryanodine receptor (RyR2) and SR Ca²⁺ reuptake by SR Ca²⁺-ATPase (SERCa2a) not only impair contraction, but also contribute to cardiac arrhythmia trigger and reentry. Besides being the main Ca²⁺ storage organelle, SR in cardiomyocytes performs all the functions of endoplasmic reticulum (ER) in other cell types including protein synthesis, folding and degradation. In recent years ER stress has become recognized as an important contributing factor in many cardiac pathologies, including deadly ventricular arrhythmias. This brief review will therefore focus on ER stress mechanisms in the heart and how these changes can lead to pro-arrhythmic defects in SR Ca²⁺ handling machinery.

Background: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca ²⁺ homeostasis, impairing luminal Ca ²⁺ regulation of the sarcoplasmic reticulum (SR) Ca ²⁺ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process. Methods: A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca ²⁺ and reactive oxygen species imaging in isolated ventricular myocytes (VMs). Results: The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca ²⁺ transient amplitude and SR Ca ²⁺ content, and reduced proarrhythmic spontaneous Ca ²⁺ waves in TAB VMs under β-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca ²⁺ -dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca ²⁺ -channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. Conclusions: A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca ²⁺ release and Ca ²⁺ -dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

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.

Key points: Small‐conductance Ca²⁺‐activated K⁺ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca²⁺] in a biphasic manner. Submicromolar [Ca²⁺] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca²⁺] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function. PKA phosphorylation attenuates I_SK rectification by reducing the Ca²⁺/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca²⁺]_i. This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Small‐conductance Ca²⁺‐activated K⁺ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L^–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L^–1), which was eliminated by inhibition of PKA (1 μmol L^–1). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L^–1) evoked I_SK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca²⁺ imaging experiments established that PKA phosphorylation lessens rectification of I_SK via reduction Ca²⁺/voltage‐dependent inhibition of the channels at high [Ca²⁺] without affecting their sensitivity to activation by Ca²⁺ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465.

In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca²⁺ uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca²⁺ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca²⁺-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca²⁺ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca²⁺ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na⁺/Ca²⁺ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca²⁺ accumulation monitored by mitochondrial-targeted Ca²⁺ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca²⁺ imaging showed that accelerated Ca²⁺ accumulation reduced Ca²⁺ transient amplitude and promoted generation of spontaneous Ca²⁺ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca²⁺ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca²⁺ imaging demonstrated that enhancement of mitochondrial Ca²⁺ accumulation further perturbed global Ca²⁺ handling, increasing the number of cells exhibiting spontaneous Ca²⁺ waves, shortening RyR refractoriness and decreasing SR Ca²⁺ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca²⁺ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca²⁺ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca²⁺ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca²⁺ accumulation can be protective.

A rapid growth in the incidence of diabetes and obesity has transpired to a major heath issue and economic burden in the postindustrial world, with more than 29 million patients affected in the United States alone. Cardiovascular defects have been established as the leading cause of mortality and morbidity of diabetic patients. Over the last decade, significant progress has been made in delineating mechanisms responsible for the diminished cardiac contractile function and enhanced propensity for malignant cardiac arrhythmias characteristic of diabetic disease. Rhythmic cardiac contractility relies upon the precise interplay between several cellular Ca²⁺ transport protein complexes including plasmalemmal L-type Ca²⁺ channels (LTCC), Na⁺-Ca²⁺ exchanger (NCX1), Sarco/endoplasmic Reticulum (SR) Ca²⁺-ATPase (SERCa2a) and ryanodine receptors (RyR2s), the SR Ca²⁺ release channels. Here we provide an overview of changes in Ca²⁺ homeostasis in diabetic ventricular myocytes and discuss the therapeutic potential of targeting Ca²⁺ handling proteins in the prevention of diabetes-associated cardiomyopathy and arrhythmogenesis.