Atrial SERCA2a Overexpression Has No Affect on Cardiac Alternans but Promotes Arrhythmogenic SR Ca2+ Triggers

Background

Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias.

Methods

Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca²⁺ was measured using Indo-1AM and Ca²⁺ alternans (Ca-ALT) was induced with a standard ramp pacing protocol.

Results

As predicted, SR Ca²⁺ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca²⁺ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20).

Conclusions

Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic.     

Atrial Fibrillation Complicated by Heart Failure Induces Distinct Remodeling of Calcium Cycling Proteins

Atrial fibrillation (AF) and heart failure (HF) are two of the most common cardiovascular diseases. They often coexist and account for significant morbidity and mortality. Alterations in cellular Ca²⁺ homeostasis play a critical role in AF initiation and maintenance. This study was designed to specifically elucidate AF-associated remodeling of atrial Ca²⁺ cycling in the presence of mild HF. AF was induced in domestic pigs by atrial burst pacing. The animals underwent electrophysiologic and echocardiographic examinations. Ca²⁺ handling proteins were analyzed in right atrial tissue obtained from pigs with AF (day 7; n = 5) and compared to sinus rhythm (SR) controls (n = 5). During AF, animals exhibited reduction of left ventricular ejection fraction (from 73% to 58%) and prolonged atrial refractory periods. AF and HF were associated with suppression of protein kinase A (PKA)_RII (-62%) and Ca²⁺-calmodulin-dependent kinase II (CaMKII) δ by 37%, without changes in CaMKIIδ autophosphorylation. We further detected downregulation of L-type calcium channel (LTCC) subunit α₂ (-75%), sarcoplasmic reticulum Ca²⁺-ATPase (Serca) 2a (-29%), phosphorylated phospholamban (Ser16, -92%; Thr17, -70%), and phospho-ryanodine receptor 2 (RyR2) (Ser2808, -62%). Na⁺-Ca²⁺ exchanger (NCX) levels were upregulated (+473%), whereas expression of Ser2814-phosphorylated RyR2 and LTCCα_1c subunits was not significantly altered. In conclusion, AF produced distinct arrhythmogenic remodeling of Ca²⁺ handling in the presence of tachycardia-induced mild HF that is different from AF without structural alterations. The changes may provide a starting point for personalized approaches to AF treatment.     

Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca²⁺-handling. We investigated the effects of ascending aortic banding (AoB) on Ca²⁺-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca²⁺-release at the cell edge whereas Ca²⁺ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na⁺/Ca²⁺ exchanger, SR Ca²⁺ ATPase or phospholamban. Mathematical modeling indicated that [Ca²⁺]_i transients at the cell center were accounted for by simple centripetal diffusion of Ca²⁺ released at the cell edge. In contrast, caffeine (10 mM) induced Ca²⁺ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca²⁺-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca²⁺ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca²⁺-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca²⁺-release.     

Decreased expression of small-conductance Ca2+-activated K+ channels SK1 and SK2 in human chronic atrial fibrillation

AimsSmall-conductance Ca^2 +-activated K⁺ (SK) channels are recognized as new ion channel candidates in atrial fibrillation (AF), with pivotal implications as novel drug targets due to their atrial-selective distribution in humans. The purpose of this study was to investigate whether SK channels and the Ca^2 +-activated K⁺ current (I_K,Ca) are involved in electrical remodeling of human chronic AF (cAF) and whether they display the differential distribution between the right (RA) and left atria (LA).Main methodsThe right (RAA) and left atrial appendage (LAA) myocytes were obtained from 29 sinus rhythm (SR) and 22 cAF patients. The I_K,Ca and action potential (AP) were recorded using the patch-clamp technique. Three SK channel subtypes (SK1–3) expressions were assayed by western blot and real-time quantitative PCR analysis.Key findingsThe I_K,Ca was decreased and its role in AP repolarization was attenuated in cAF, concomitant with a significant decrease in protein and mRNA levels of SK1 and SK2. In either SR or cAF, there was no difference in the I_K,Ca density and protein and mRNA expression levels of SK1–3 between RAA and LAA myocytes.SignificanceOur results demonstrated that SK1 and SK2 are involved in electrical remodeling of cAF. SK1–3 and I_K,Ca do not display the inter-atrial differential distribution in SR or cAF. These findings provide a new insight into mechanisms of electrical remodeling of human cAF.     

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca²⁺ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (P_o) are associated with Ca²⁺ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca²⁺]_o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca²⁺]_o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca²⁺ transient was normal, despite the reduced SR Ca²⁺ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca²⁺ transients because they are incapable of properly increasing the amount of Ca²⁺ stored in the SR because of a larger SR Ca²⁺ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca²⁺ channel current (I_Ca-L) and SR Ca²⁺ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 P_o and fractional Ca²⁺ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca²⁺ content, but cannot compensate for the inability to increase SR Ca²⁺ content during inotropic challenge.     

Inter-Subject Variability in Human Atrial Action Potential in Sinus Rhythm versus Chronic Atrial Fibrillation

Aims

Human atrial electrophysiology exhibits high inter-subject variability in both sinus rhythm (SR) and chronic atrial fibrillation (cAF) patients. Variability is however rarely investigated in experimental and theoretical electrophysiological studies, thus hampering the understanding of its underlying causes but also its implications in explaining differences in the response to disease and treatment. In our study, we aim at investigating the ability of populations of human atrial cell models to capture the inter-subject variability in action potential (AP) recorded in 363 patients both under SR and cAF conditions.

Methods and Results

Human AP recordings in atrial trabeculae (n = 469) from SR and cAF patients were used to calibrate populations of computational SR and cAF atrial AP models. Three populations of over 2000 sampled models were generated, based on three different human atrial AP models. Experimental calibration selected populations of AP models yielding AP with morphology and duration in range with experimental recordings. Populations using the three original models can mimic variability in experimental AP in both SR and cAF, with median conductance values in SR for most ionic currents deviating less than 30% from their original peak values. All cAF populations show similar variations in G_K1, G_Kur and G_to, consistent with AF-related remodeling as reported in experiments. In all SR and cAF model populations, inter-subject variability in I_K1 and I_NaK underlies variability in APD₉₀, variability in I_Kur, I_CaL and I_NaK modulates variability in APD₅₀ and combined variability in I_to and I_Kur determines variability in APD₂₀. The large variability in human atrial AP triangulation is mostly determined by I_K1 and either I_NaK or I_NaCa depending on the model.

Conclusion

Experimentally-calibrated human atrial AP models populations mimic AP variability in SR and cAF patient recordings, and identify potential ionic determinants of inter-subject variability in human atrial AP duration and morphology in SR versus cAF.     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

The Contribution of Ionic Currents to Rate-Dependent Action Potential Duration and Pattern of Reentry in a Mathematical Model of Human Atrial Fibrillation

Persistent atrial fibrillation (PeAF) in humans is characterized by shortening of action potential duration (APD) and attenuation of APD rate-adaptation. However, the quantitative influences of particular ionic current alterations on rate-dependent APD changes, and effects on patterns of reentry in atrial tissue, have not been systematically investigated. Using mathematical models of human atrial cells and tissue and performing parameter sensitivity analysis, we evaluated the quantitative contributions to action potential (AP) shortening and APD rate-adaptation of ionic current remodeling seen with PeAF. Ionic remodeling in PeAF was simulated by reducing L-type Ca²⁺ channel current (I_CaL), increasing inward rectifier K⁺ current (I_K1) and modulating five other ionic currents. Parameter sensitivity analysis, which quantified how each ionic current influenced APD in control and PeAF conditions, identified interesting results, including a negative effect of Na⁺/Ca²⁺ exchange on APD only in the PeAF condition. At high pacing rate (2 Hz), electrical remodeling in I_K1 alone accounts for the APD reduction of PeAF, but at slow pacing rate (0.5 Hz) both electrical remodeling in I_CaL alone (-70%) and I_K1 alone (+100%) contribute equally to the APD reduction. Furthermore, AP rate-adaptation was affected by I_Kur in control and by I_NaCa in the PeAF condition. In a 2D tissue model, a large reduction (-70%) of I_CaL becomes a dominant factor leading to a stable spiral wave in PeAF. Our study provides a quantitative and unifying understanding of the roles of ionic current remodeling in determining rate-dependent APD changes at the cellular level and spatial reentry patterns in tissue.     

Decreased sarcolipin protein expression and enhanced sarco(endo)plasmic reticulum Ca uptake in human atrial fibrillation

Sarcolipin (SLN), a key regulator of cardiac sarco(endo)plasmic reticulum (SR) Ca²⁺ ATPase, is predominantly expressed in atria and mediates β-adrenergic responses. Studies have shown that SLN mRNA expression is decreased in human chronic atrial fibrillation (AF) and in aortic banded mouse atria; however, SLN protein expression in human atrial pathology and its role in atrial SR Ca²⁺ uptake are not yet elucidated. In the present study, we determined the expression of major SR Ca²⁺ handling proteins in atria of human AF patients and in human and in a mouse model of heart failure (HF). We found that the expression of SR Ca²⁺ uptake and Ca²⁺ release channel proteins are significantly decreased in atria but not in the ventricles of pressure-overload induced HF in mice. In human AF and HF, the expression of SLN protein was significantly decreased; whereas the expressions of other major SR Ca²⁺ handling proteins were not altered. Further, we found that the SR Ca²⁺ uptake was significantly increased in human AF. The selective downregulation of SLN and enhanced SR Ca²⁺ uptake in human AF suggest that SLN downregulation could play an important role in abnormal intracellular Ca²⁺ cycling in atrial pathology.     

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

Background

Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive.

Methodology/Principal Findings

To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s.

Conclusions/Significance

We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.     

Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca²⁺ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca²⁺ handling, using a canine model of chronic HF. The cytosolic Ca²⁺ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca²⁺ content. Direct measurements of [Ca²⁺]_SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca²⁺] is markedly lowered in HF, suggesting that alterations in Ca²⁺ transport rather than fractional SR volume reduction accounts for the diminished Ca²⁺ release capacity of SR in HF. SR Ca²⁺ ATPase (SERCA2)-mediated SR Ca²⁺ uptake rate was not significantly altered, and Na⁺/Ca²⁺ exchange activity was accelerated in HF myocytes. At the same time, SR Ca²⁺ leak, measured directly as a loss of [Ca²⁺]_SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca²⁺]_SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca²⁺ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca²⁺ leak is a major factor in the abnormal intracellular Ca²⁺ handling that critically contributes to the reduced SR Ca²⁺ content of failing cardiomyocytes.     

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes

Abnormalities in cardiomyocyte Ca²⁺ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca²⁺ sparks (brief, localized increased in [Ca²⁺]_i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca²⁺]_i transient amplitude and sarcoplasmic reticulum (SR) Ca²⁺ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca²⁺ sparks were less frequent, whereas the fractional release and the frequency of Ca²⁺ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca²⁺ sparks were more frequent in PMI cells (under conditions of similar SR Ca²⁺ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca²⁺ sparks frequency normalized by the SR Ca²⁺ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca²⁺ spark frequency. Indeed, using an internal “failing solution” with less ATP (as found in HF), we observed a dramatic decrease in Ca²⁺ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca²⁺ load may mask these effects at the Ca²⁺ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca²⁺ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.     

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca²⁺) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca²⁺ from the sarcoplasmic reticulum (SR). The Ca²⁺ leak out of the SR is an important process for cellular Ca²⁺ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca²⁺ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca²⁺ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca²⁺ sparks and robust Ca²⁺ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca²⁺ spark and nonspark-based SR Ca²⁺ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca²⁺-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca²⁺ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca²⁺ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca²⁺ release properties due to variations in inter-RyR coupling via local subspace Ca²⁺ concentration ([Ca²⁺]_ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.     

Feedback-control induced pattern formation in cardiac myocytes: A mathematical modeling study

Cardiac alternans is a dangerous rhythm disturbance of the heart, in which rapid stimulation elicits a beat-to-beat alternation in the action potential duration (APD) and calcium (Ca) transient amplitude of individual myocytes. Recently, “subcellular alternans”, in which the Ca transients of adjacent regions within individual myocytes alternate out-of-phase, has been observed. A previous theoretical study suggested that subcellular alternans may result during static pacing from a Turing-type symmetry breaking instability, but this was only predicted in a subset of cardiac myocytes (with negative Ca to voltage (Ca→V_m) coupling) and has never been directly verified experimentally. A recent experimental study, however, showed that subcellular alternans is dynamically induced in the remaining subset of myocytes during pacing with a simple feedback control algorithm (“alternans control”). Here we show that alternans control pacing changes the effective coupling between the APD and the Ca transient (V_m→Ca coupling), such that subcellular alternans is predicted to occur by a Turing instability in cells with positive Ca→V_m coupling. In addition to strengthening the understanding of the proposed mechanism for subcellular alternans formation, this work (in concert with previous theoretical and experimental results) illuminates subcellular alternans as a striking example of a biological Turing instability in which the diffusing morphogens can be clearly identified.     

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

In vascular smooth muscle cells, Ca²⁺ release via IP₃ receptors (IP₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca²⁺ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca²⁺ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca²⁺ store is a single continuous interconnected network or multiple separate Ca²⁺ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca²⁺ available throughout the store was examined using localized photolysis of caged-IP₃ and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP₃R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP₃R and RyR was regulated by the Ca²⁺ concentration within the SR (luminal [Ca²⁺]). As the luminal [Ca²⁺] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP₃R. The SR Ca²⁺ store is a single luminally continuous entity which contains both IP₃R and RyR and within which Ca²⁺ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP₃R and RyR by luminal [Ca²⁺] explains the appearance of multiple stores in some functional studies.     

Cardiac action potential duration and calcium regulation in males and females

Adult women have longer QT intervals compared with men of a similar age, indicating differences in the speed of repolarisation of the ventricles. We investigate the influences of gender on ventricular electrophysiology and intracellular Ca²⁺ regulation of the guinea pig heart. Comparing sexually mature animals, females exhibited a significantly longer APD. Peak L-type Ca²⁺ current (I_CaL) was larger in females and when this current was inhibited with nifedipine the gender differences in APD were removed. APD differences also disappeared when the SR was depleted of Ca²⁺. Inactivation of I_CaL during a clamp step is faster in females but slower during an action potential and SR Ca²⁺ content is larger. We suggest that gender differences in APD result from variation in the kinetics of I_CaL stemming from alterations to Ca²⁺ release.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.     

Efficacy of RyR2 inhibitor EL20 in induced pluripotent stem cell-derived cardiomyocytes from a patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca²⁺) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca²⁺ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca²⁺ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the Stemdiff^TM kit. Confocal Ca²⁺ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca²⁺ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC₅₀ = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca²⁺ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca²⁺ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT.