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.

Regulation of myocyte contraction via neuronal nitric oxide synthase: role of ryanodine receptor S-nitrosylation.

The sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR2) has been proposed to be an end target of neuronal nitric oxide synthase (NOS1) signalling. The purpose of this study is to investigate the mechanism of NOS1 modulation of RyR2 activity and the corresponding effect on myocyte function. Myocytes were isolated from NOS1 knockout (NOS1(/)) and wild-type mice. NOS1(/) myocytes displayed a decreased fractional SR Ca(2+) release, NOS1 knockout also led to reduced RyR2 S-nitrosylation levels. RyR2 channels from NOS1(/) hearts had decreased RyR2 open probability. Additionally, knockout of NOS1 led to a decrease in [(3)H]ryanodine binding, Ca(2+) spark frequency (CaSpF) and a rightward shift in the SR Ca(2+) leak/load relationship. Similar effects were observed with acute inhibition of NOS1. These data are indicative of decreased RyR2 activity in myocytes with NOS1 knockout or acute inhibition. Interestingly, the NO donor and nitrosylating agent SNAP reversed the depressed RyR2 open probability, the reduced CaSpF, and caused a leftward shift in the leak/load relationship in NOS1(/) myocytes. SNAP also normalized Ca(2+) transient and cell shortening amplitudes and SR fractional release in myocytes with NOS1 knockout or acute inhibition. Furthermore, SNAP was able to normalize the RyR2 S-nitrosylation levels. These data suggest that NOS1 signalling increases RyR2 activity via S-nitrosylation, which contributes to the NOS1-induced positive inotropic effect. Thus, RyR2 is an important end target of NOS1.

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.

Protein protein interactions between triadin and calsequestrin are involved in modulation of sarcoplasmic reticulum calcium release in cardiac myocytes.

In cardiac muscle, intracellular Ca2+ release is controlled by a number of proteins including the ryanodine receptor (RyR2), calsequestrin (CASQ2), triadin-1 (Trd) and junctin (Jn) which form a complex in the junctional sarcoplasmic reticulum (SR) membrane. Within this complex, Trd appears to link CASQ2 to RyR2 although the functional significance of this interaction is unclear. In this study, we explored the functional importance of Trd-CASQ2 interactions for intracellular Ca2+ handling in rat ventricular myocytes. A peptide encompassing the homologous CASQ2 binding domain of Trd (residues 206-230 in the rat; TrdPt) was expressed in the lumen of the SR to disrupt Trd-CASQ2 interactions. Myocytes expressing TrdPt exhibited increased responsiveness of SR Ca2+ release to activation by ICa as manifested by flattened and broadened voltage dependency of the amplitude of cytosolic Ca2+ transients. Rhythmically paced, TrdPt-expressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca2+ and membrane potential that was not seen in control cells. In addition, the frequency of spontaneous Ca2+ sparks and Ca2+ waves was significantly increased in TrdPt-expressing, permeabilized myocytes. These alterations in SR Ca2+ release were accompanied by a significant decrease in basal free intra-SR[Ca2+] and total SR Ca2+ content in TrdPt-expressing cells. At the same time a synthetic peptide corresponding to the CASQ2 binding domain of Trd produced no direct effects on the activity of single RyR2 channels incorporated into lipid bilayers while interfering with the ability of CASQ2 to inhibit the RyR2 channel. These results suggest that CASQ2 stabilizes SR Ca2+ release by inhibiting the RyR2 channel through interaction with Trd. They also show that intracellular Ca2+ cycling in the heart relies on coordinated interactions between proteins of the RyR2 channel complex and that disruption of these interactions may represent a molecular mechanism for cardiac disease.

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 Ca(2+)-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 CASQ2(R33Q) 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 Ca(2+)-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 intrastore calcium signaling in chronic heart failure.

Diminished Ca release from the sarcoplasmic reticulum (SR) is an important contributor to the impaired contractility of the failing heart. Despite extensive effort, the underlying causes of abnormal SR Ca release in heart failure (HF) remain unknown. We used a combination of simultaneous imaging of cytosolic and SR intraluminal [Ca] in isolated cardiomyocytes and recordings from single-ryanodine receptor (RyR) channels reconstituted into lipid bilayers to investigate alterations in intracellular Ca handling in an experimental model of chronic HF. We found that diastolic free [Ca] inside the SR was dramatically reduced because of a Ca leak across the SR membrane, mediated by spontaneous local release events (Ca sparks), in HF myocytes. Additionally, the magnitudes of intrastore Ca depletion signals during global and focal Ca release events were blunted, and [Ca]SR recovery was slowed after global but not focal Ca release in HF myocytes. At the single-RyR level, the sensitivity of RyRs to activation by luminal Ca was greatly enhanced, providing a molecular mechanism for the maintained potentiation of Ca sparks (and increased Ca leak) at reduced intra-SR [Ca] in HF. This work shows that the diminished SR Ca release characteristic of failing myocardium could be explained by increased sensitivity of RyRs to luminal Ca, leading to enhanced spark-mediated SR Ca leak and reduced intra-SR [Ca].

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 (TRD(WT)) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRD(Del.200-224)) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRD(WT) reduced the amplitude of I(Ca)- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small I(Ca) at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRD(WT) myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRD(WT) myocytes. Periodic electrical stimulation of TRD(WT) myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRD(Del.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, TRD(WT) and TRD(Del.200-224) myocytes. Elevation of TRD(WT) 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.

Modulation of cytosolic and intra-sarcoplasmic reticulum calcium waves by calsequestrin in rat cardiac myocytes.

Waves of Ca2+-induced Ca2+ release occur in various cell types and are involved in the pathology of certain forms of cardiac arrhythmia. These arrhythmias include catecholaminergic polymorphic ventricular tachycardia (CPVT), certain cases of which are associated with mutations in the cardiac calsequestrin gene (CASQ2). To explore the mechanisms of Ca2+ wave generation and unravel the underlying causes of CPVT, we investigated the effects of adenoviral-mediated changes in CASQ2 protein levels on the properties of cytosolic and sarcoplasmic reticulum (SR) Ca2+ waves in permeabilized rat ventricular myocytes. The free [Ca2+] inside the sarcoplasmic reticulum ([Ca2+]SR) was monitored by fluo-5N entrapped into the SR, and cytosolic Ca2+ was imaged using fluo-3. Overexpression of CASQ2 resulted in significant increases in the amplitude of Ca2+ waves and interwave intervals, whereas reduced CASQ2 levels caused drastic reductions in the amplitude and period of Ca2+ waves. CASQ2 abundance had no impact on resting diastolic [Ca2+]SR or on the amplitude of the [Ca2+]SR depletion signal during the Ca2+ wave. However, the recovery dynamics of [Ca2+]SR following Ca2+ release were dramatically altered as the rate of [Ca2+]SR recovery increased approximately 3-fold in CASQ2-overexpressing myocytes and decreased to 30% of control in CASQ2-underexpressing myocytes. There was a direct linear relationship between Ca2+ wave period and the half-time of basal [Ca2+]SR recovery following Ca2+ release. Loading the SR with the low affinity exogenous Ca2+ buffer citrate exerted effects quantitatively similar to those observed on overexpressing CASQ2. We conclude that free intra-SR [Ca2+] is a critical determinant of cardiac Ca2+ wave generation. Our data indicate that reduced intra-SR Ca2+ binding activity promotes the generation of Ca2+ waves by accelerating the dynamics of attaining a threshold free [Ca2+]SR required for Ca2+ wave initiation, potentially accounting for arrhythmogenesis in CPVT linked to mutations in CASQ2.

The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium.

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.

Abnormal calcium signaling and sudden cardiac death associated with mutation of calsequestrin.

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 (CASQ2(D307H)) on Ca2+ signaling in adult rat myocytes. Total CASQ2 protein levels were consistently elevated approximately 4-fold in cells infected with adenoviruses expressing either wild-type CASQ2 (CASQ2(WT)) or CASQ2(D307H). Expression of CASQ2(D307H) reduced the Ca2+ storing capacity of the SR. In addition, the amplitude, duration, and rise time of macroscopic I(Ca)-induced Ca2+ transients and of spontaneous Ca2+ sparks were reduced significantly in myocytes expressing CASQ2(D307H). Myocytes expressing CASQ2(D307H) 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 CASQ2(D307H) 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.

Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes.

Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca(2+)-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.

Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes

Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca2+-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.

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.

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-mediated 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.

Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle.

The amount of Ca2+ released from the sarcoplasmic reticulum (SR) is a principal determinant of cardiac contractility. Normally, the SR Ca2+ stores are mobilized through the mechanism of Ca2+-induced Ca2+ release (CICR). In this process, Ca2+ enters the cell through plasmalemmal voltage-dependent Ca2+ channels to activate the Ca2+ release channels in the SR membrane. Consequently, the control of Ca2+ release by cytosolic Ca2+ has traditionally been the main focus of cardiac excitation-contraction (EC) coupling research. Evidence obtained recently suggests that SR Ca release is controlled not only by cytosolic Ca2+, but also by Ca2+ in the lumen of the SR. The presence of a luminal Ca2+ sensor regulating release of SR luminal Ca2+ potentially has profound implications for our understanding of EC coupling and intracellular Ca2+ cycling. Here we review evidence, obtained using in situ and in vitro approaches, in support of such a luminal Ca2+ sensor in cardiac muscle. We also discuss the role of control of Ca2+ release channels by luminal Ca2+ in termination and stabilization of CICR, as well as in shaping the response of cardiac myocytes to various inotropic influences and diseased states such as Ca2+ overload and heart failure.