Calcium leak through ryanodine receptors leads to atrial fibrillation in 3 mouse models of catecholaminergic polymorphic ventricular tachycardia

Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca(2+)) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective: In the present study, we sought to determine whether intracellular diastolic SR Ca(2+) leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results: We generated 3 knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca(2+) leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3 CPVT mouse models (RyR2-R2474S(+/-), 70%; RyR2-N2386I(+/-), 60%; RyR2-L433P(+/-), 35.71%) but not in wild-type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca(2+) leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca(2+) leak through the channel. Atrial RyR2 from RyR2-R2474S(+/-) mice were oxidized, and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca(2+) leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. Conclusions: The present study demonstrates that RyR2-mediated diastolic SR Ca(2+) leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca(2+) leak through RyR2 and pacing-induced AF associated with CPVT mutations.     

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.     

FKBP12.6与RyR2的关系及其在心脏疾病中的意义

FKBP12.6从RyR2解离导致通道构象及功能改变。Ca2 从SR漏出使SR的Ca2 负荷减少,Ca2 瞬变减少,舒缩时RyR2耦联障碍,最终引起心肌功能异常。而且RyR2对CICR敏感性提高导致心肌迟后去极诱发心律失常。通过过表达或增加FKBP12.6对RyR2亲和力可以改善通道缺陷,从而使心脏功能及心律失常得到改善。     

Mechanisms of abnormal calcium homeostasis in mutations responsible for catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia is a heritable arrhythmia unmasked by exertion or stress and is characterized by triggered activity and sudden cardiac death. In this study, we simulated mutations in 2 genes linked to catecholaminergic polymorphic ventricular tachycardia, the first located in calsequestrin (CSQN2) and the second in the ryanodine receptor (RyR2). The aim of the study was to investigate the mechanistic basis for spontaneous Ca2+ release events that lead to delayed afterdepolarizations in affected patients. Sarcoplasmic reticulum (SR) luminal Ca2+ sensing was incorporated into a model of the human ventricular myocyte, and CSQN2 mutations were modeled by simulating disrupted RyR2 luminal Ca2+ sensing. In voltage-clamp mode, the mutant CSQN2 model recapitulated the smaller calcium transients, smaller time to peak calcium transient, and accelerated recovery from inactivation seen in experiments. In current clamp mode, in the presence of beta stimulation, we observed delayed afterdepolarizations, suggesting that accelerated recovery of RyR2 induced by impaired luminal Ca2+ sensing underlies the triggered activity observed in mutant CSQN2-expressing myocytes. In current-clamp mode, in a model of mutant RyR2 that is characterized by reduced FKBP12.6 binding to the RyR2 on beta stimulation, the impaired coupled gating characteristic of these mutations was modeled by reducing cooperativity of RyR2 activation. In current-clamp mode, the mutant RyR2 model exhibited increased diastolic RyR2 open probability that resulted in formation of delayed afterdepolarizations. In conclusion, these minimal order models of mutant CSQN2 and RyR2 provide plausible mechanisms by which defects in RyR2 gating may lead to the cellular triggers for arrhythmia, with implications for the development of targeted therapy.     

Ryanodine receptors and ventricular arrhythmias: emerging trends in mutations, mechanisms and therapies

It has been six years since the first reported link between mutations in the cardiac ryanodine receptor Ca(2+) release channel (RyR2) and catecholaminergic polymorphic ventricular tachycardia (CPVT), a malignant stress-induced arrhythmia. In this time, rapid advances have been made in identifying new mutations, and in understanding how these mutations disrupt normal channel function to cause VT that frequently degenerates into ventricular fibrillation (VF) and sudden death. Functional characterisation of these RyR2 Ca(2+) channelopathies suggests that mutations alter the ability of RyR2 to sense its intracellular environment, and that channel modulation via covalent modification, Ca(2+)- and Mg(2+)-dependent regulation and structural feedback mechanisms are catastrophically disturbed. This review reconciles the current status of RyR2 mutation-linked etiopathology, the significance of mutational clustering within the RyR2 polypeptide and the mechanisms underlying channel dysfunction. We will also review new data that explores the link between abnormal Ca(2+) release and the resultant cardiac electrical instability in VT and VF, and how these recent developments impact on novel anti-arrhythmic therapies. Finally, we evaluate the concept that mechanistic differences between CPVT and other arrhythmogenic disorders may preclude a common therapeutic strategy to normalise RyR2 function in cardiac disease.     

Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease characterized by adrenergically mediated polymorphic ventricular tachycardia leading to syncope and sudden cardiac death. The autosomal dominant form of CPVT is caused by mutations in the RyR2 gene encoding the cardiac isoform of the ryanodine receptor. In vitro functional characterization of mutant RyR2 channels showed altered behavior on adrenergic stimulation and caffeine administration with enhanced calcium release from the sarcoplasmic reticulum. As of today no experimental evidence is available to demonstrate that RyR2 mutations can reproduce the arrhythmias observed in CPVT patients. We developed a conditional knock-in mouse model carrier of the R4496C mutation, the mouse equivalent to the R4497C mutations identified in CPVT families, to evaluate if the animals would develop a CPVT phenotype and if beta blockers would prevent arrhythmias. Twenty-six mice (12 wild-type (WT) and 14RyR(R4496C)) underwent exercise stress testing followed by epinephrine administration: none of the WT developed ventricular tachycardia (VT) versus 5/14 RyR(R4496C) mice (P=0.02). Twenty-one mice (8 WT, 8 RyR(R4496C), and 5 RyR(R4496C) pretreated with beta-blockers) received epinephrine and caffeine: 4/8 (50%) RyR(R4496C) mice but none of the WT developed VT (P=0.02); 4/5 RyR(R4496C) mice pretreated with propranolol developed VT (P=0.56 nonsignificant versus RyR(R4496C) mice). These data provide the first experimental demonstration that the R4496C RyR2 mutation predisposes the murine heart to VT and VF in response caffeine and/or adrenergic stimulation. Furthermore, the results show that analogous to what is observed in patients, beta adrenergic stimulation seems ineffective in preventing life-threatening arrhythmias.     

Enhanced store overload-induced Ca2+ release and channel sensitivity to luminal Ca2+ activation are common defects of RyR2 mutations linked to ventricular tachycardia and sudden death

Ventricular tachycardia (VT) is the leading cause of sudden death, and the cardiac ryanodine receptor (RyR2) is emerging as an important focus in its pathogenesis. RyR2 mutations have been linked to VT and sudden death, but their precise impacts on channel function remain largely undefined and controversial. We have previously shown that several disease-linked RyR2 mutations in the C-terminal region enhance the sensitivity of the channel to activation by luminal Ca2+. Cells expressing these RyR2 mutants display an increased propensity for spontaneous Ca2+ release under conditions of store Ca2+ overload, a process we referred to as store overload-induced Ca2+ release (SOICR). To determine whether common defects exist in disease-linked RyR2 mutations, we characterized 6 more RyR2 mutations from different regions of the channel. Stable inducible HEK293 cell lines expressing Q4201R and I4867M from the C-terminal region, S2246L and R2474S from the central region, and R176Q(T2504M) and L433P from the N-terminal region were generated. All of these cell lines display an enhanced propensity for SOICR. HL-1 cardiac cells transfected with disease-linked RyR2 mutations also exhibit increased SOICR activity. Single channel analyses reveal that disease-linked RyR2 mutations primarily increase the channel sensitivity to luminal, but not to cytosolic, Ca2+ activation. Moreover, the Ca2+ dependence of [3H]ryanodine binding to RyR2 wild type and mutants is similar. In contrast to previous reports, we found no evidence that disease-linked RyR2 mutations alter the FKBP12.6-RyR2 interaction. Our data indicate that enhanced SOICR activity and luminal Ca2+ activation represent common defects of RyR2 mutations associated with VT and sudden death. A mechanistic model for CPVT/ARVD2 is proposed.     

RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR)

The cardiac ryanodine receptor (RyR2) governs the release of Ca2+ from the sarcoplasmic reticulum, which initiates muscle contraction. Mutations in RyR2 have been linked to ventricular tachycardia (VT) and sudden death, but the precise molecular mechanism is unclear. It is known that when the sarcoplasmic reticulum store Ca2+ content reaches a critical level, spontaneous Ca2+ release occurs, a process we refer to as store-overload-induced Ca2+ release (SOICR). In view of the well documented arrhythmogenic nature of SOICR, we characterized the effects of disease-causing RyR2 mutations on SOICR in human embryonic kidney (HEK)293 cells and found that, at elevated extracellular Ca2+ levels, HEK293 cells expressing RyR2 displayed SOICR in a manner virtually identical to that observed in cardiac cells. Using this cell model, we demonstrated that the RyR2 mutations linked to VT and sudden death, N4104K, R4496C, and N4895D, markedly increased the occurrence of SOICR. At the molecular level, we showed that these RyR2 mutations increased the sensitivity of single RyR2 channels to activation by luminal Ca2+ and enhanced the basal level of [3H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca2+ activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca2+ activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca2+ overload-associated VT.     

Loss of luminal Ca2+ activation in the cardiac ryanodine receptor is associated with ventricular fibrillation and sudden death

Different forms of ventricular arrhythmias have been linked to mutations in the cardiac ryanodine receptor (RyR)2, but the molecular basis for this phenotypic heterogeneity is unknown. We have recently demonstrated that an enhanced sensitivity to luminal Ca(2+) and an increased propensity for spontaneous Ca(2+) release or store-overload-induced Ca(2+) release (SOICR) are common defects of RyR2 mutations associated with catecholaminergic polymorphic or bidirectional ventricular tachycardia. Here, we investigated the properties of a unique RyR2 mutation associated with catecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2 mutations characterized previously, the A4860G mutation diminished the response of RyR2 to activation by luminal Ca(2+), but had little effect on the sensitivity of the channel to activation by cytosolic Ca(2+). This specific impact of the A4860G mutation indicates that the luminal Ca(2+) activation of RyR2 is distinct from its cytosolic Ca(2+) activation. Stable, inducible HEK293 cells expressing the A4860G mutant showed caffeine-induced Ca(2+) release but exhibited no SOICR. Importantly, HL-1 cardiac cells transfected with the A4860G mutant displayed attenuated SOICR activity compared with cells transfected with RyR2 WT. These observations provide the first evidence that a loss of luminal Ca(2+) activation and SOICR activity can cause ventricular fibrillation and sudden death. These findings also indicate that although suppressing enhanced SOICR is a promising antiarrhythmic strategy, its oversuppression can also lead to arrhythmias.     

Calmodulin kinase II inhibition prevents arrhythmias in RyR2(R4496C+/-) mice with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by life-threatening arrhythmias elicited by adrenergic activation. CPVT is caused by mutations in the cardiac ryanodine receptor gene (RyR2). In vitro studies demonstrated that RyR2 mutations respond to sympathetic activation with an abnormal diastolic Ca(2+) leak from the sarcoplasmic reticulum; however the pathways that mediate the response to adrenergic stimulation have not been defined. In our RyR2(R4496C+/-) knock-in mouse model of CPVT we tested the hypothesis that inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) counteracts the effects of adrenergic stimulation resulting in an antiarrhythmic activity. CaMKII inhibition with KN-93 completely prevented catecholamine-induced sustained ventricular tachyarrhythmia in RyR2(R4496C+/-) mice, while the inactive congener KN-92 had no effect. In ventricular myocytes isolated from the hearts of RyR2(R4496C+/-) mice, CaMKII inhibition with an autocamtide-2 related inhibitory peptide or with KN-93 blunted triggered activity and transient inward currents induced by isoproterenol. Isoproterenol also enhanced the activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), increased spontaneous Ca(2+) release and spark frequency. CaMKII inhibition blunted each of these parameters without having an effect on the SR Ca(2+) content. Our data therefore indicate that CaMKII inhibition is an effective intervention to prevent arrhythmogenesis (both in vivo and in vitro) in the RyR2(R4496C+/-) knock-in mouse model of CPVT. Mechanistically, CAMKII inhibition acts on several elements of the EC coupling cascade, including an attenuation of SR Ca(2+) leak and blunting catecholamine-mediated SERCA activation. CaMKII inhibition may therefore represent a novel therapeutic target for patients with CPVT.     

Distinct U wave changes in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT)

Although catecholaminergic polymorphic ventricular tachycardia (CPVT) is associated with fatal ventricular arrhythmias and sudden death, the ECG findings are not fully understood. In this paper, we report on alterations in the U-wave. Seven patients from 6 families with CPVT in which bidirectional tachycardia and polymorphic VT were induced by exercise or isoproterenol infusion visited our hospitals. VT was not inducible by programmed electrical stimulation. A novel gene mutation of the ryanodine receptor 2 (RyR2) was confirmed in 2 families. In one of these patients, U-wave alternans was observed following ventricular pacing at 160 beats/min. In the other patient, U-wave alternans was observed during the recovery phase after the exercise stress test, which was terminated because of polymorphic VT. In both cases, leads V3-V5 were the leads showing alternans most clearly. In the third patient, a negative U-wave became positive following a pause from sinus arrest and a change in T-wave was also noted. Since such findings were not found in the other subjects who underwent electrophysiologic study, isoproterenol infusion or exercise stress testing, the phenomenon seems to be relevant to the underlying pathogenesis of CPVT. The genesis and significance of U-wave alteration need to be determined.     

Arrhythmogenic mechanisms in a mouse model of catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (VT) is a lethal familial disease characterized by bidirectional VT, polymorphic VT, and ventricular fibrillation. Catecholaminergic polymorphic VT is caused by enhanced Ca2+ release through defective ryanodine receptor (RyR2) channels. We used epicardial and endocardial optical mapping, chemical subendocardial ablation with Lugol's solution, and patch clamping in a knockin (RyR2/RyR2(R4496C)) mouse model to investigate the arrhythmogenic mechanisms in catecholaminergic polymorphic VT. In isolated hearts, spontaneous ventricular arrhythmias occurred in 54% of 13 RyR2/RyR2(R4496C) and in 9% of 11 wild-type (P=0.03) littermates perfused with Ca2+and isoproterenol; 66% of 12 RyR2/RyR2(R4496C) and 20% of 10 wild-type hearts perfused with caffeine and epinephrine showed arrhythmias (P=0.04). Epicardial mapping showed that monomorphic VT, bidirectional VT, and polymorphic VT manifested as concentric epicardial breakthrough patterns, suggesting a focal origin in the His-Purkinje networks of either or both ventricles. Monomorphic VT was clearly unifocal, whereas bidirectional VT was bifocal. Polymorphic VT was initially multifocal but eventually became reentrant and degenerated into ventricular fibrillation. Endocardial mapping confirmed the Purkinje fiber origin of the focal arrhythmias. Chemical ablation of the right ventricular endocardial cavity with Lugol's solution induced complete right bundle branch block and converted the bidirectional VT into monomorphic VT in 4 anesthetized RyR2/RyR2(R4496C) mice. Under current clamp, single Purkinje cells from RyR2/RyR2(R4496C) mouse hearts generated delayed afterdepolarization-induced triggered activity at lower frequencies and level of adrenergic stimulation than wild-type. Overall, the data demonstrate that the His-Purkinje system is an important source of focal arrhythmias in catecholaminergic polymorphic VT.     

Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.     

A novel mutation in FKBP12.6 binding region of the human cardiac ryanodine receptor gene (R2401H) in a Japanese patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an autosomal dominant inherited disorder characterized by adrenergic induced polymorphic ventricular tachycardias and associated with sudden cardiac death. The human cardiac ryanodine receptor gene (RyR2) was linked to CPVT. A 20-year-old male was referred to our hospital because of recurrent syncope after physical and emotional stress. Routine cardiac examinations including catheterization revealed no structural abnormality. Exercise on treadmill induced premature ventricular contraction in bigeminy and bidirectional ventricular tachycardia was induced during isoproterenol infusion. Beta-blocking drug was effective in suppressing the arrhythmias. We performed genetic screening by PCR-SSCP method followed by DNA sequencing, and a novel missense mutation R2401H in RyR2 located in FKBP12.6 binding region was identified. This mutation was not detected in 190 healthy controls. Since FKBP12.6 plays a critical role in Ca channel gating, the R2401H mutation can be expected to alter Ca-induced Ca release and E-C coupling resulting in CPVT. This is the first report of RyR2 mutation in CPVT patient from Asia including Japan.     

Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB(+/-)) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na](i) and [Ca](i). Here we investigate how AnkB reduction affects cardiac [Na](i), [Ca](i) and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na](i) and diastolic [Ca](i) in myocytes from AnkB(+/-) vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB(+/-) myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB(+/-) vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB(+/-) mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB(+/-) mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB(+/-) myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB(+/-) mice.     

Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression

Defective regulation of the cardiac ryanodine receptor (RyR2)/calcium release channel, required for excitation-contraction coupling in the heart, has been linked to cardiac arrhythmias and heart failure. For example, diastolic calcium "leak" via RyR2 channels in the sarcoplasmic reticulum has been identified as an important factor contributing to impaired contractility in heart failure and ventricular arrhythmias that cause sudden cardiac death. In patients with heart failure, chronic activation of the "fight or flight" stress response leads to protein kinase A (PKA) hyperphosphorylation of RyR2 at Ser-2808. PKA phosphorylation of RyR2 Ser-2808 reduces the binding affinity of the channel-stabilizing subunit calstabin2, resulting in leaky RyR2 channels. We developed RyR2-S2808A mice to determine whether Ser-2808 is the functional PKA phosphorylation site on RyR2. Furthermore, mice in which the RyR2 channel cannot be PKA phosphorylated were relatively protected against the development of heart failure after myocardial infarction. Taken together, these data show that PKA phosphorylation of Ser-2808 on the RyR2 channel appears to be a critical mediator of progressive cardiac dysfunction after myocardial infarction.     

Ryanodine receptor as a new therapeutic target of heart failure and lethal arrhythmia.

Abnormal intracellular Ca(2+) handling by the sarcoplasmic reticulum (SR) is a critical factor in the development of heart failure (HF). Not only decreased Ca(2+) uptake, but also uncoordinated Ca(2+) release plays a significant role in contractile and relaxation dysfunction. Spontaneous Ca(2+) release through ryanodine receptor (RyR) 2, a huge tetrameric protein, during diastole leads to a decrease in the SR Ca(2+) content, and also triggers delayed after depolarization that is a substrate for lethal arrhythmia. Several disease-linked mutations of RyR have been reported in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) or arrhythmogenic right ventricular cardiomyopathy type 2 (ARVC2). The unique distribution of these mutation sites has lead to the concept that an interaction among the putative regulatory domains within RyR may play a key role in regulating channel opening, and that there seems to be a common abnormality in the channel disorder of HF and CPVT/ARVC2. Recent knowledge gained from pathological conditions may lead to the development of a new therapeutic strategy for the treatment of HF or cardiac arrhythmia.     

Mechanism underlying catecholaminergic polymorphic ventricular tachycardia and approaches to therapy

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by VT induced by adrenergic stress in the absence of structural heart disease and high incidence of sudden cardiac death. The diagnosis is made based on reproducible ventricular tachyarrhythmias including bidirectional VT and polymorphic VT during exercise testings. Two causative genes of CPVT have been identified: RYR2, encoding the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel, and CASQ2, encoding cardiac calsequestrin. A mutation in RYR2 or CASQ2 is identified in approximately 60% of patients with CPVT. Mutations in these two genes destabilize the RyR2 Ca²⁺ release channel complex in sarcoplasmic reticulum and result in spontaneous Ca²⁺ release through RyR2 channels leading to delayed after depolarization, triggered activity, and bidirectional/polymorphic VT. Implantable cardioverter defibrillators (ICDs) are recommended for prevention of sudden death in patients with CPVT.1. A.E. Epstein, J.P. DiMarco, K.A. Ellenbogen, et al., ACC/AHA/HRS 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices): developed in collaboration with the American Association for Thoracic Surgery and Society of Thoracic Surgeons. Circulation. 2008;117:e350 However, painful shocks can trigger further adrenergic stress and arrhythmias, and deaths have occurred despite appropriate ICD shocks. Treatment with β-adrenergic blockers reduces arrhythmia burden and mortality, but is not completely effective. The beneficial effects of Ca²⁺ channel blocker verapamil in combination with β-blocker have been reported, but the role of verapamil has not been well assessed. Because Ca²⁺ leakage through ryanodine channel is a common mechanism of CPVT, ryanodine channel block may have a therapeutic effect. We discovered that flecainide directly inhibits RyR2 channels and prevent CPVT. Left cardiac sympathetic denervation may be an effective alternative treatment in combination with ICD, especially for patients whose arrhythmias are not controlled by drug therapies.     

Catecholaminergic Polymorphic Ventricular Tachycardia from Bedside to Bench and Beyond

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a primary electrical myocardial disease characterized by exercise- and stress-related ventricular tachycardia manifested as syncope and sudden death. The disease has a heterogeneous genetic basis, with mutations in the cardiac Ryanodine Receptor channel (RyR2) gene accounting for an autosomal-dominant form (CPVT1) in approximately 50% and mutations in the cardiac calsequestrin gene (CASQ2) accounting for an autosomal-recessive form (CPVT2) in up to 2% of CPVT cases. Both RyR2 and calsequestrin are important participants in the cardiac cellular calcium homeostasis.We review the physiology of the cardiac calcium homeostasis, including the cardiac excitation contraction coupling and myocyte calcium cycling. The pathophysiology of cardiac arrhythmias related to myocyte calcium handling and the effects of different modulators are discussed.The putative derangements in myocyte calcium homeostasis responsible for CPVT, as well as the clinical manifestations and therapeutic options available, are described.