Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia.

BACKGROUND: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+ homeostasis, impairing luminal Ca2+ regulation of the sarcoplasmic reticulum (SR) Ca2+ 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 Ca2+ 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 Ca2+ transient amplitude and SR Ca2+ content, and reduced proarrhythmic spontaneous Ca2+ waves in TAB VMs under ß-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-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 Ca2+-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 Ca2+ release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes.

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 ß-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.

Sarcoplasmic reticulum-mitochondria communication; implications for cardiac arrhythmia.

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.

MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca2+ release in hypertrophic rat cardiomyocytes.

Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca2+ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca2+]). Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca2+ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca2+ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca2+] restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2+] using genetic probe mtRCamp1h revealed that mito-[Ca2+] in TAB VMs paced at 2 Hz under ß-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca2+]. However, it failed to improve cytosolic Ca2+ handling and reduce proarrhythmic spontaneous Ca2+ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca2+], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca2+ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca2+ mishandling. These data establish nonlinear mito-[Ca2+]/mito-ROS relationship, whereby partial restoration of mito-[Ca2+] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca2+ uptake exacerbates damaging mito-ROS emission.NEW & NOTEWORTHY 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.

Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS.

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 ß-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.

The role of spatial organization of Ca2+ release sites in the generation of arrhythmogenic diastolic Ca2+ release in myocytes from failing hearts.

In heart failure (HF), dysregulated cardiac ryanodine receptors (RyR2) contribute to the generation of diastolic Ca2+ waves (DCWs), thereby predisposing adrenergically stressed failing hearts to life-threatening arrhythmias. However, the specific cellular, subcellular, and molecular defects that account for cardiac arrhythmia in HF remain to be elucidated. Patch-clamp techniques and confocal Ca2+ imaging were applied to study spatially defined Ca2+ handling in ventricular myocytes isolated from normal (control) and failing canine hearts. Based on their activation time upon electrical stimulation, Ca2+ release sites were categorized as coupled, located in close proximity to the sarcolemmal Ca2+ channels, and uncoupled, the Ca2+ channel-free non-junctional Ca2+ release units. In control myocytes, stimulation of ß-adrenergic receptors with isoproterenol (Iso) resulted in a preferential increase in Ca2+ spark rate at uncoupled sites. This site-specific effect of Iso was eliminated by the phosphatase inhibitor okadaic acid, which caused similar facilitation of Ca2+ sparks at coupled and uncoupled sites. Iso-challenged HF myocytes exhibited increased predisposition to DCWs compared to control myocytes. In addition, the overall frequency of Ca2+ sparks was increased in HF cells due to preferential stimulation of coupled sites. Furthermore, coupled sites exhibited accelerated recovery from functional refractoriness in HF myocytes compared to control myocytes. Spatially resolved subcellular Ca2+ mapping revealed that DCWs predominantly originated from coupled sites. Inhibition of CaMKII suppressed DCWs and prevented preferential stimulation of coupled sites in Iso-challenged HF myocytes. These results suggest that CaMKII- (and phosphatase)-dependent dysregulation of junctional Ca2+ release sites contributes to Ca2+-dependent arrhythmogenesis in HF.

Decreased RyR2 refractoriness determines myocardial synchronization of aberrant Ca2+ release in a genetic model of arrhythmia.

Dysregulated intracellular Ca(2+) signaling is implicated in a variety of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia. Spontaneous diastolic Ca(2+) release (DCR) can induce arrhythmogenic plasma membrane depolarizations, although the mechanism responsible for DCR synchronization among adjacent myocytes required for ectopic activity remains unclear. We investigated the synchronization mechanism(s) of DCR underlying untimely action potentials and diastolic contractions (DCs) in a catecholaminergic polymorphic ventricular tachycardia mouse model with a mutation in cardiac calsequestrin. We used a combination of different approaches including single ryanodine receptor channel recording, optical imaging (Ca(2+) and membrane potential), and contractile force measurements in ventricular myocytes and intact cardiac muscles. We demonstrate that DCR occurs in a temporally and spatially uniform manner in both myocytes and intact myocardial tissue isolated from cardiac calsequestrin mutation mice. Such synchronized DCR events give rise to triggered electrical activity that results in synchronous DCs in the myocardium. Importantly, we establish that synchronization of DCR is a result of a combination of abbreviated ryanodine receptor channel refractoriness and the preceding synchronous stimulated Ca(2+) release/reuptake dynamics. Our study reveals how aberrant DCR events can become synchronized in the intact myocardium, leading to triggered activity and the resultant DCs in the settings of a cardiac rhythm disorder.

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1.

AIMS: Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. METHODS AND RESULTS: In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex. CONCLUSIONS: Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients.

Alternating membrane potential/calcium interplay underlies repetitive focal activity in a genetic model of calcium-dependent atrial arrhythmias.

KEY POINTS: Atrial fibrillation is often initiated and perpetuated by abnormal electrical pulses repetitively originating from regions outside the heart's natural pacemaker. In this study we examined the causal role of abnormal calcium releases from the sarcoplasmic reticulum in producing repetitive electrical discharges in atrial cells and tissues. Calsequestrin2 is a protein that stabilizes the closed state of calcium release channels, i.e. the ryanodine receptors. In the atria from mice predisposed to abnormal calcium releases secondary to the absence of calsequestrin2, we observed abnormal repetitive electrical discharges that may lead to atrial fibrillation. Here, we report a novel pathological rhythm generator. Specifically, abnormal calcium release leads to electrical activation, which in turn results in another abnormal calcium release. This process repeats itself and thus sustains the repetitive electrical discharges. These results suggest that improving the stability of ryanodine receptors might be useful to treat atrial fibrillation. ABSTRACT: Aberrant diastolic calcium (Ca) release due to leaky ryanodine receptors (RyR2s) has been recently associated with atrial fibrillation (AF) and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, it remains unclear how diastolic Ca release contributes to the rising of rapid repetitive focal activity, which is considered as a common AF triggering mechanism. To address this question, we conducted simultaneous voltage/Ca optical mapping in atrial tissue and one-/two-dimensional confocal imaging in atrial tissue and myocytes from wild-type (WT, n = 15) and CPVT mice lacking calsequestrin 2 (Casq2(-/-), n = 45), which promotes diastolic Ca release. During ß-adrenergic stimulation (100 nM isoproterenol), only Casq2(-/-) atrial myocytes showed pacing-induced self-sustained repetitive activity (31 ± 21 s vs. none in WT). Importantly, in atrial tissue, this repetitive activity could translate to Ca-dependent focal arrhythmia. Ectopic action potential (AP) firing during repetitive activity occurred only when diastolic Ca release achieved a sufficient level of synchronization. The AP, in turn, synchronized subsequent diastolic Ca release by temporally aligning multiple sources of Ca waves both within individual myocytes and throughout the atrial tissue. This alternating interplay between AP and diastolic Ca release perpetuates the self-sustaining repetitive activity. In fact, pharmacological disruption of synchronized diastolic Ca release (by ryanodine) prevented aberrant APs; and vice versa, the inhibition of AP (by TTX or 0 Na, 0 Ca solution) de-synchronized diastolic Ca release. Taken together, these results suggest that a cyclical interaction between synchronized diastolic Ca release and AP forms a pathological rhythm generator that is involved in Ca-dependent atrial arrhythmias in CPVT.

Ibandronate and ventricular arrhythmia risk.

INTRODUCTION: Bisphosphonates, including ibandronate, are used in the prevention and treatment of osteoporosis. METHODS AND RESULTS: We report a case of suspected ibandronate-associated arrhythmia, following a single dose of ibandronate in a 55-year-old female. ECG at presentation revealed frequent ectopy and QT/QTc interval prolongation; at follow-up 9 months later the QT/QTc intervals were normalized. Proarrhythmic potential of ibandronate was assessed with a combination of in vivo and in vitro approaches in canines and canine ventricular myocytes. We observed late onset in vivo repolarization instability after ibandronate treatment. Myocytes superfused with ibandronate exhibited action potential duration (APD) prolongation and variability, increased early afterdepolarizations (EADs) and reduced Ito (P < 0.05), with no change in IKr . Ibandronate-induced APD changes and EADs were prevented by inhibition of intracellular calcium cycling. Ibandronate increased sarcoplasmic reticulum calcium load; during washout there was an increase in calcium spark frequency and spontaneous calcium waves. Computational modeling was used to examine the observed effects of ibandronate. While reductions in Ito alone had modest effects on APD, when combined with altered RyR inactivation kinetics, the model predicted effects on APD and SR Ca(2+) load consistent with observed experimental results. CONCLUSION: Ibandronate may increase the susceptibility to ventricular ectopy and arrhythmias. Collectively these data suggest that reduced Ito combined with abnormal RyR calcium handling may result in a previously unrecognized form of drug-induced proarrhythmia.

Shortened Ca2+ signaling refractoriness underlies cellular arrhythmogenesis in a postinfarction model of sudden cardiac death.

RATIONALE: Diastolic spontaneous Ca(2+) waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when [Ca(2+)] in sarcoplasmic reticulum ([Ca(2+)](SR)) reaches a certain threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca(2+). OBJECTIVE: We investigated the mechanisms of DCW generation in myocytes from normal and diseased hearts, using a canine model of post-myocardial infarction ventricular fibrillation (VF). METHODS AND RESULTS: The frequency of DCWs, recorded during periodic pacing in the presence of a ß-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final [Ca(2+)](SR), DCWs arose with a distinct time delay after attaining steady [Ca(2+)](SR) in both experimental groups. Although the rate of [Ca(2+)](SR) recovery after the SR Ca(2+) release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the [Ca(2+)](SR) at DCW initiation was lower. The restitution of depolarization-induced Ca(2+) transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca(2+) cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca(2+) release in VF myocytes. CONCLUSIONS: The attainment of a certain threshold [Ca(2+)](SR) is not sufficient for the generation of DCWs. Postrelease Ca(2+) signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca(2+) signaling refractoriness due to RyR2 phosphorylation and oxidation is responsible for the increased rate of DCWs observed in VF myocytes and could provide a substrate for synchronization of arrhythmogenic events at the tissue level in hearts prone to VF.

Store-dependent deactivation: cooling the chain-reaction of myocardial calcium signaling.

In heart cells, Ca(2+) released from the internal storage unit, the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR2) channels is the predominant determinant of cardiac contractility. Evidence obtained in recent years suggests that SR Ca(2+) release is tightly regulated not only by cytosolic Ca(2+) but also by intra-store Ca(2+) concentration. Specifically, Ca(2+)-induced Ca(2+) release (CICR) that relies on auto-catalytic action of Ca(2+) at the cytosolic side of RyR2s is precisely balanced and counteracted by RyR2 deactivation dependent on a reciprocal decrease of Ca(2+) at the luminal side of RyR2s. Dysregulation of this inherently unstable Ca(2+) signaling is considered to be an underlying cause of triggered arrhythmias, and is associated with genetic and acquired forms of sudden cardiac death. In this article, we present an overview of recent advances in our understanding of the regulatory role luminal Ca(2+) plays in Ca(2+) handling, with a particular emphasis on the role of Ca(2+)release refractoriness in aberrant Ca(2+) release.

Functional consequences of stably expressing a mutant calsequestrin (CASQ2D307H) in the CASQ2 null background.

The role of calsequestrin (CASQ2) in cardiac sarcoplasmic reticulum (SR) calcium (Ca(2+)) transport has gained significant attention since point mutations in CASQ2 were reported to cause ventricular arrhythmia. In the present study, we have critically evaluated the functional consequences of expressing the CASQ2(D307H) mutant protein in the CASQ2 null mouse. We recently reported that the mutant CASQ2(D307H) protein can be stably expressed in CASQ2 null hearts, and it targets appropriately to the junctional SR (Kalyanasundaram A, Bal NC, Franzini-Armstrong C, Knollmann BC, Periasamy M. J Biol Chem 285: 3076-3083, 2010). In this study, we found that introduction of CASQ2(D307H) protein in the CASQ2 null background partially restored triadin 1 levels, which were decreased in the CASQ2 null mice. Despite twofold expression (relative to wild-type CASQ2), the mutant protein failed to increase SR Ca(2+) load. We also found that the Ca(2+) transient decays slower in the CASQ2 null and CASQ2(D307H) cells. CASQ2(D307H) myocytes, when rhythmically paced and challenged with isoproterenol, exhibit spontaneous Ca(2+) waves similar to CASQ2 null myocytes; however, the stability of Ca(2+) cycling was increased in the CASQ2(D307H) myocytes. In the presence of isoproterenol, Ca(2+)-transient amplitude in CASQ2(D307H) myocytes was significantly decreased, possibly indicating an inherent defect in Ca(2+) buffering capacity and release from the mutant CASQ2 at high Ca(2+) concentrations. We also observed polymorphic ventricular tachycardia in the CASQ2(D307H) mice, although lesser than in the CASQ2 null mice. These data suggest that CASQ2(D307H) point mutation may affect Ca(2+) buffering capacity and Ca(2+) release. We propose that poor interaction between CASQ2(D307H) and triadin 1 could affect ryanodine receptor 2 stability, thereby increasing susceptibility to delayed afterdepolarizations and triggered arrhythmic activity.

miR-1 overexpression enhances Ca(2+) release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and causing CaMKII-dependent hyperphosphorylation of RyR2.

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 B56alpha 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.

Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 µM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a ß-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to -25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation-contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

Effects of dietary omega-3 fatty acids on ventricular function in dogs with healed myocardial infarctions: in vivo and in vitro studies.

Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.

Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure.

Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

Assessment of temporal functional changes and miRNA profiling of human iPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been developed for cardiac cell transplantation studies more than a decade ago. In order to establish the hiPSC-CM-based platform as an autologous source for cardiac repair and drug toxicity, it is vital to understand the functionality of cardiomyocytes. Therefore, the goal of this study was to assess functional physiology, ultrastructural morphology, gene expression, and microRNA (miRNA) profiling at Wk-1, Wk-2 & Wk-4 in hiPSC-CMs in vitro. Functional assessment of hiPSC-CMs was determined by multielectrode array (MEA), Ca2+ cycling and particle image velocimetry (PIV). Results demonstrated that Wk-4 cardiomyocytes showed enhanced synchronization and maturation as compared to Wk-1 & Wk-2. Furthermore, ultrastructural morphology of Wk-4 cardiomyocytes closely mimicked the non-failing (NF) adult human heart. Additionally, modulation of cardiac genes, cell cycle genes, and pluripotency markers were analyzed by real-time PCR and compared with NF human heart. Increasing expression of fatty acid oxidation enzymes at Wk-4 supported the switching to lipid metabolism. Differential regulation of 12 miRNAs was observed in Wk-1 vs Wk-4 cardiomyocytes. Overall, this study demonstrated that Wk-4 hiPSC-CMs showed improved functional, metabolic and ultrastructural maturation, which could play a crucial role in optimizing timing for cell transplantation studies and drug screening.

Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease.

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.