Serge Viatchenko-Karpinski

Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia.

Calsequestrin is a high-capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR), an intracellular Ca release and storage organelle in muscle. Mutations in the cardiac calsequestrin gene (CSQ2) have been linked to arrhythmias and sudden death. We have used Ca-imaging and patch-clamp methods in combination with adenoviral gene transfer strategies to explore the function of CSQ2 in adult rat heart cells. By increasing or decreasing CSQ2 levels, we showed that CSQ2 not only determines the Ca storage capacity of the SR but also positively controls the amount of Ca released from this organelle during excitation–contraction coupling. CSQ2 controls Ca release by prolonging the duration of Ca fluxes through the SR Ca-release sites. In addition, the dynamics of functional restitution of Ca-release sites after Ca discharge were prolonged when CSQ2 levels were elevated and accelerated in the presence of lowered CSQ2 protein levels. Furthermore, profound disturbances in rhythmic Ca transients in myocytes undergoing periodic electrical stimulation were observed when CSQ2 levels were reduced. We conclude that CSQ2 is a key determinant of the functional size and stability of SR Ca stores in cardiac muscle. CSQ2 appears to exert its effects by influencing the local luminal Ca concentration-dependent gating of the Ca-release channels and by acting as both a reservoir and a sink for Ca in SR. The abnormal restitution of Ca-release channels in the presence of reduced CSQ2 levels provides a plausible explanation for ventricular arrhythmia associated with mutations of CSQ2.

Luminal Ca2+ Controls Termination and Refractory Behavior of Ca2+-Induced Ca2+ Release in Cardiac Myocytes

Abstract— Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivtion of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to “internal” solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by ICa and deranged the smoothly graded ICa-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.

Clinical Phenotype and Functional Characterization of CASQ2 Mutations Associated With Catecholaminergic Polymorphic Ventricular Tachycardia

Background— Four distinct mutations in the human cardiac calsequestrin gene (CASQ2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. Methods and Results— We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2G112+5X) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2L167H) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2G112+5X did not bind Ca2+, whereas CASQ2L167H had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of ICa-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2G112+5X. Conclusions— CASQ2L167H and CASQ2G112+5X alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca2+ release and calcium content. In addition, CASQ2G112+5X displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.

Abnormal Interactions of Calsequestrin With the Ryanodine Receptor Calcium Release Channel Complex Linked to Exercise-Induced Sudden Cardiac Death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca2+-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2R33Q in adult rat myocytes led to an increase in excitation–contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca2+-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.

Abnormal Calcium Signaling and Sudden Cardiac Death Associated With Mutation of Calsequestrin

Abstract— Mutations in human cardiac calsequestrin (CASQ2), a high-capacity calcium-binding protein located in the sarcoplasmic reticulum (SR), have recently been linked to effort-induced ventricular arrhythmia and sudden death (catecholaminergic polymorphic ventricular tachycardia). However, the precise mechanisms through which these mutations affect SR function and lead to arrhythmia are presently unknown. In this study, we explored the effect of adenoviral-directed expression of a canine CASQ2 protein carrying the catecholaminergic polymorphic ventricular tachycardia–linked mutation D307H (CASQ2D307H) on Ca2+ signaling in adult rat myocytes. Total CASQ2 protein levels were consistently elevated ≈4-fold in cells infected with adenoviruses expressing either wild-type CASQ2 (CASQ2WT) or CASQ2D307H. Expression of CASQ2D307H reduced the Ca2+ storing capacity of the SR. In addition, the amplitude, duration, and rise time of macroscopic ICa-induced Ca2+ transients and of spontaneous Ca2+ sparks were reduced significantly in myocytes expressing CASQ2D307H. Myocytes expressing CASQ2D307H also displayed drastic disturbances of rhythmic oscillations in [Ca2+]i and membrane potential, with signs of delayed afterdepolarizations when undergoing periodic pacing and exposed to isoproterenol. Importantly, normal rhythmic activity was restored by loading the SR with the low-affinity Ca2+ buffer, citrate. Our data suggest that the arrhythmogenic CASQ2D307H mutation impairs SR Ca2+ storing and release functions and destabilizes the Ca2+-induced Ca2+ release mechanism by reducing the effective Ca2+ buffering inside the SR and/or by altering the responsiveness of the Ca2+ release channel complex to luminal Ca2+. These results establish at the cellular level the pathological link between CASQ2 mutations and the predisposition to adrenergically mediated arrhythmias observed in patients carrying CASQ2 defects.

Redox modification of ryanodine receptors underlies calcium alternans in a canine model of sudden cardiac death

AIMS Although cardiac alternans is a known predictor of lethal arrhythmias, its underlying causes remain largely undefined in disease settings. The potential role of, and mechanisms responsible for, beat-to-beat alternations in the amplitude of systolic Ca(2+) transients (Ca(2+) alternans) was investigated in a canine post-myocardial infarction (MI) model of sudden cardiac death (SCD). METHODS AND RESULTS Post-MI dogs had preserved left ventricular (LV) function and susceptibility to ventricular fibrillation (VF) during exercise. LV wedge preparations from VF dogs were more susceptible to action potential (AP) alternans and the frequency-dependence of Ca(2+) alternans was shifted towards slower rates in myocytes isolated from VF dogs relative to controls. In both groups of cells, cytosolic Ca(2+) transients ([Ca(2+)](c)) alternated in phase with changes in diastolic Ca(2+) in sarcoplasmic reticulum ([Ca(2+)](SR)), but the dependence of [Ca(2+)](c) amplitude on [Ca(2+)](SR) was steeper in VF cells. Abnormal ryanodine receptor (RyR) function in VF cells was indicated by increased fractional Ca(2+) release for a given amplitude of Ca(2+) current and elevated diastolic RyR-mediated SR Ca(2+) leak. SR Ca(2+) uptake activity did not differ between VF and control cells. VF myocytes had an increased rate of reactive oxygen species production and increased RyR oxidation. Treatment of VF myocytes with reducing agents normalized parameters of Ca(2+) handling and shifted the threshold of Ca(2+) alternans to higher frequencies. CONCLUSION Redox modulation of RyRs promotes generation of Ca(2+) alternans by enhancing the steepness of the Ca(2+) release-load relationship and thereby providing a substrate for post-MI arrhythmias.

Modulation of SR Ca Release by Luminal Ca and Calsequestrin in Cardiac Myocytes: Effects of CASQ2 Mutations Linked to Sudden Cardiac Death

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2(DEL)) or CASQ2 containing a point mutation (CASQ2(R33Q)), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2(DEL) or CASQ2(R33Q) altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2(DEL) disrupted the CASQ2 polymerization required for high capacity Ca binding, whereas CASQ2(R33Q) compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild-type CASQ2 and CASQ2(DEL)-expressing myocytes, suggesting that a decline in [Ca](SR) is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2(R33Q) markedly lowered the [Ca](SR) threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.

Protein Phosphatases Decrease Sarcoplasmic Reticulum Calcium Content by Stimulating Calcium Release in Cardiac Myocytes

Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation−contraction coupling; furthermore abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca2+ sparks and SR Ca2+ load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca2+ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca2+ stores, as determined by application of caffeine. These changes in Ca2+ release and SR Ca2+ load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1‐medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti‐RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR‐mediated SR Ca2+ release leading to depleted SR Ca2+ stores in cardiac myocytes.

Modulation of the Ca2+‐induced Ca2+ release cascade by β‐adrenergic stimulation in rat ventricular myocytes

1 To define the sub‐cellular mechanisms of modulation of cardiac excitation‐contraction (E‐C) coupling by the β‐adrenergic pathway, we carried out confocal Ca2+ imaging in conjunction with recordings of inward Ca2+ current in fluo‐3‐loaded patch‐clamped rat ventricular myocytes. 2 Isoproterenol (isoprenaline; ISO) increased the amplitude of the inward Ca2+ current and the globally measured intracellular Ca2+ transients. The gain of calcium‐induced calcium release (CICR) was increased at all membrane potentials but especially at positive membrane potentials (> +30 mV). ISO dramatically broadened the bell‐shaped voltage dependence of intracellular Ca2+ transients by shifting the descending portion of the curve to very high membrane potentials. 3 The number of local release events (solitary sparks and conglomerates of overlapping sparks) induced by depolarizing steps to +30 mV was increased significantly by ISO. This potentiation of events was due to increased trigger calcium current (ICa) as well the enhanced ability of ICa to induce release. The amplitude and duration of solitary sparks were increased in the presence of ISO. In addition, ISO dramatically increased the proportion and the size (‘mass’) of clustered events. 4 Exclusion of Na+ from the intra‐ and extracellular solutions prevented ISO from enhancing the ability of ICa to trigger sparks. 5 We conclude that β‐adrenergic stimulation enhances the gain of the CICR cascade by increasing the fidelity of dihydropyridine receptor (DHPR)‐ryanodine receptor (RyR) coupling and by promoting cross‐activation of RyRs in neighbouring release sites. Reverse Na+‐Ca2+ exchange (NCX) appears to play a role in the β‐adrenergic enhancement of CICR by effectively contributing to the Ca2+ trigger.

Triadin Overexpression Stimulates Excitation-Contraction Coupling and Increases Predisposition to Cellular Arrhythmia in Cardiac Myocytes

Triadin 1 (TRD) is an integral membrane protein that associates with the ryanodine receptor (RyR2), calsequestrin (CASQ2) and junctin to form a macromolecular Ca signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). To define the functional role of TRD, we examined the effects of adenoviral-mediated overexpression of the wild-type protein (TRDWT) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRDDel.200–224) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRDWT reduced the amplitude of ICa- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small ICa at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRDWT myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRDWT myocytes. Periodic electrical stimulation of TRDWT myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRDDel.200–224 failed to produce any of the effects of the wild-type protein. The lipid bilayer technique was used to record the activity of single RyR2 channels using microsome samples obtained from control, TRDWT and TRDDel.200–224 myocytes. Elevation of TRDWT levels increased the open probability of RyR2 channels, whereas expression of the mutant protein did not affect RyR2 activity. We conclude that TRD enhances cardiac excitation-contraction coupling by directly stimulating the RyR2. Interaction of TRD with RyR2 may involve amino acids 200 to 224 in C-terminal domain of TRD.