Mutations In Calsequestrin Research Articles

The Structural-Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications.

Calsequestrin (CASQ) is a key intra-sarcoplasmic reticulum Ca2+-handling protein that plays a pivotal role in the contraction of cardiac and skeletal muscles. Its Ca2+-dependent polymerization dynamics shape the translation of electric excitation signals to the Ca2+-induced contraction of the actin-myosin architecture. Mutations in CASQ are linked to life-threatening pathological conditions, including tubular aggregate myopathy, malignant hyperthermia, and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The variability in the penetrance of these phenotypes and the lack of a clear understanding of the disease mechanisms associated with CASQ mutations pose a major challenge to the development of effective therapeutic strategies. In vitro studies have mainly focused on the polymerization and Ca2+-buffering properties of CASQ but have provided little insight into the complex interplay of structural and functional changes that underlie disease. In this review, the biochemical and structural natures of CASQ are explored in-depth, while emphasizing their direct and indirect consequences for muscle Ca2+ physiology. We propose a novel functional classification of CASQ pathological missense mutations based on the structural stability of the monomer, dimer, or linear polymer conformation. We also highlight emerging similarities between polymeric CASQ and polyelectrolyte systems, emphasizing the potential for the use of this paradigm to guide further research.

NEW GENES AND DISEASES: EP.304 Early onset lower limp assymmetry: a new clinical phenotype of calsequestrin mutation

Calsequestrin is a protein that handles calcium in sarcoplasmic reticulum (SR),Ca2+ transients are crucial in skeletal muscle excitation-contraction coupling. Synchronous Ca2+ release from the SR terminal cisternae into the cytoplasm and its reuptake into the SR store are granted by a structural and functional unit and their defect causes myopathy. In an 11-year-old girl, affected by lower limb asymmetry (R limb shorter of 3 cm), upper limb hypotrophy and winging scapulae were found; she had high CK:2900 U/l, her sister presented myalgias and CK:1500 U/l, the mother CK 500 U/l and grandmother had both congenital club foot. We investigated the causes of the high CK by EMG that was myopathic, muscle biopsy that showed few atrophic fibres, in ATPase rare 2C fibres, some increased NADH-TR stain at the periphery, increased central nuclei and rare basophilic fibres. We performed immunohistochemistry for dystrophin, caveolin-3, alpha-sarcoglycan, western blot for dysferlin: all normal. The girl was operated at 15 years for elongation of right femur and by epiphysiodesis left femur and the limb asymmetry was reduced to only half a centimeter: however she complained of fatiguability, especially after physical education at school, and high CK (2186 U). She presented with scoliosis, difficulty walking with calf hypertrophy. A search for the causes for hyperCKemia was done with WGS at TIGEM and resulted in detection of a pathogenetic variant of CASQ1 gene (p.ASP244Gly), coding for calsequestrin, which allowed the solution of this puzzling familial case. In 7 members of 4 Italian families with vacuolar myopathy with CASQ1 mutation calsequestrin aggregates and a similar variant was found. In CASQ1 myopathy in several cases hyperCKemia, myalgia or other vague symptoms are present: in 22 CASQ1-mutated patients from 12 families 21 sharing the same variant of our case presented symptoms in the sixth decade with exercise intolerance and myalgias and later developed mild to moderate, slowly progressive proximal weakness with quadriceps atrophy and scapular winging. In the present family, clubfoot, early onset, limb asymmetry were the predominant clinical symptoms, they could be the effect of somatic mosaicism. In this respect our patient presents an early onset and a new clinical phenotype of lower limb asymmetry that is unique. The present familial case expands the clinical phenotype of CASQ1-myopathy.

The structure of a calsequestrin filament reveals mechanisms of familial arrhythmia.

Mutations in the calcium-binding protein calsequestrin cause the highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but an atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a human cardiac calsequestrin filament with supporting mutational analysis and in vitro filamentation assays. We identify and characterize a novel disease-associated calsequestrin mutation, S173I, that is located at the filament-forming interface, and further show that a previously reported dominant disease mutation, K180R, maps to the same surface. Both mutations disrupt filamentation, suggesting that disease pathology is due to defects in multimer formation. An ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. Our study thus provides a unifying molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias.

SK4 K+ channels are therapeutic targets for the treatment of cardiac arrhythmias

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress‐provoked ventricular arrhythmia, which also manifests sinoatrial node (SAN) dysfunction. We recently showed that SK4 calcium‐activated potassium channels are important for automaticity of cardiomyocytes derived from human embryonic stem cells. Here SK4 channels were identified in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) from healthy and CPVT2 patients bearing a mutation in calsequestrin 2 (CASQ2‐D307H) and in SAN cells from WT and CASQ2‐D307H knock‐in (KI) mice. TRAM‐34, a selective blocker of SK4 channels, prominently reduced delayed afterdepolarizations and arrhythmic Ca2+ transients observed following application of the β‐adrenergic agonist isoproterenol in CPVT2‐derived hiPSC‐CMs and in SAN cells from KI mice. Strikingly, in vivo ECG recording showed that intraperitoneal injection of the SK4 channel blockers, TRAM‐34 or clotrimazole, greatly reduced the arrhythmic features of CASQ2‐D307H KI and CASQ2 knockout mice at rest and following exercise. This work demonstrates the critical role of SK4 Ca2+‐activated K+ channels in adult pacemaker function, making them promising therapeutic targets for the treatment of cardiac ventricular arrhythmias such as CPVT.

Ryanodine Receptor Sensitivity Governs the Stability and Synchrony of Local Calcium Release during Cardiac Excitation-Contraction Coupling

Synchronous coordination of calcium-induced calcium release (CICR) across thousands of calcium release units (CRUs) within the cardiomyocyte is critical for normal excitation-contraction coupling (ECC). Here we utilize a novel “local control” mathematical model of ECC, designed to simulate a mouse cardiomyocyte, to investigate physiological and pathophysiological calcium (Ca2+) signaling. The model contains 20,000 independent CRUs each composed of 6 stochastically gated L-type Ca2+ channels (LCCs) in the transverse tubule membrane that are positioned across a local “dyadic subspace” from a junctional sarcoplasmic reticulum (JSR) compartment containing a cluster of 50 stochastically gated ryanodine receptors (RyR2s). Action potential depolarization of the sarcolemmal membrane activates LCC openings which elevate subspace [Ca2+] ([Ca2+]ds) across each of the cell's 20,000 CRUs. This rise in [Ca2+]ds promotes RyR2 opening to initiate the fundamental element of triggered Ca2+ release, the Ca2+ spark. The mechanistic design and true SR Ca2+ pump/leak balance displayed by our model allows us to quantify ECC fidelity and Ca2+ spark fidelity (the probability that a LCC opening or RyR2 opening induces a Ca2+ spark, respectively). We study the effect of excess SR Ca2+ leak in the context of “catecholaminergic polymorphic ventricular tachycardia (CPVT)” caused by the R33Q mutation in calsequestrin (CASQ2). CPVT is simulated by increasing RyR2 sensitivity to [Ca2+]i and reducing JSR Ca2+ buffering capacity to levels identified by prior studies of the CASQ2-R33Q “knock-in” mice. Our model effectively reproduces both normal and arrhythmogenic CPVT RyR2 [Ca2+]i sensitivity, local Ca2+ spark activity, and global Ca2+ signals. Under CPVT conditions enhanced RyR2 open probability is critical for the development of “unstable”, quiescent Ca2+ sparks that fail to terminate robustly as well as asynchronous systolic Ca2+ spark activity and increased diastolic SR Ca2+ leak.

Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca2+ handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation-contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²+ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2R420Q and CASQ2D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca2+]i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca2+]i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.

EP News: Basic and Translational

Arrhythmias caused by abnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum. Surprisingly, these patients often respond to treatment with Na+ channel blockers. The relationship between Na+ influx and disturbances in Ca2+ handling preceding CPVT arrhythmias remains poorly understood. Radwański et al (Cardiovasc Res 2014; PMID 25538156) performed optical Ca2+ and membrane potential imaging in myocytes and intact cardiac muscle as well as electrocardiograms on a CPVT mouse model with a mutation in calsequestrin. They demonstrated that a subpopulation of Na+ channels (neuronal Nav [nNav]) colocalize with ryanodine receptor channels. Disruption of the cross-talk between nNav and ryanodine receptor channels by nNav blockade with riluzole reduced and desynchronized DCR in both myocytes and intact muscle. Such desynchronization of DCR on cellular and tissue levels translated into decreased arrhythmias in CPVT mice. The authors conclude that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy.

Flecainide: Current status and perspectives in arrhythmia management.

Flecainide acetate is a class IC antiarrhythmic agent and its clinical efficacy has been confirmed by the results of several clinical trials. Nowadays, flecainide is recommended as one of the first line therapies for pharmacological conversion as well as maintenance of sinus rhythm in patients with atrial fibrillation and/or supraventricular tachycardias. Based on the Cardiac Arrhythmia Suppression Trial study results, flecainide is not recommended in patients with structural heart disease due to high proarrhythmic risk. Recent data support the role of flecainide in preventing ventricular tachyarrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia associated both with ryanodine receptor and calsequestrin mutations. We herein review the current clinical data related to flecainide use in clinical practice and some concerns about its role in the management of patients with coronary artery disease.

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

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

Decreased RyR2 refractoriness determines myocardial synchronization of aberrant Ca 2+ release in a genetic model of arrhythmia

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

Dantrolene rescues arrhythmogenic RYR2 defect in a patient‐specific stem cell model of catecholaminergic polymorphic ventricular tachycardia

Coordinated release of calcium (Ca2+) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptor (RYR2) channels is essential for cardiomyocyte function. In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disease characterized by stress-induced ventricular arrhythmias in young patients with structurally normal hearts, autosomal dominant mutations in RYR2 or recessive mutations in calsequestrin lead to aberrant diastolic Ca2+ release from the SR causing arrhythmogenic delayed after depolarizations (DADs). Here, we report the generation of induced pluripotent stem cells (iPSCs) from a CPVT patient carrying a novel RYR2 S406L mutation. In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca2+ concentrations, a reduced SR Ca2+ content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes. This was due to increased frequency and duration of elementary Ca2+ release events (Ca2+ sparks). Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca2+ spark properties and rescued the arrhythmogenic phenotype. This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation. Our work provides a new in vitro model to study the pathogenesis of human cardiac arrhythmias and develop novel therapies for CPVT.

The Genetics of Malignant Hyperthermia and Related Muscular Syndromes

To the Editor: The review by Capacchione and Muldoon1 discusses relationships between malignant hyperthermia (MH) and other muscular diseases presenting with some of the clinical features of MH. The task is made more difficult because of the uncertain relationships between a positive in vitro contracture test for halothane and caffeine, specific expression of the triggering mechanism of MH, and the presence of a genetic defect in the muscular ryanodine receptor type 1 (RYR1) gene. Capacchione and Muldoon state that “a negative RYR1 screen with a positive contracture test does not necessarily mean that the patient is not MH susceptible as there are likely yet unidentified non-RYR1 mutations that confer MHS (MH susceptibility).” Indeed, in the 20% to 30% of individuals lacking an RYR1 mutation, genetic alterations of other proteins involved in calcium release or its regulation could be present.2 Monnier et al.3 described such a mutation in the α-1 subunit of the human dihydropyridine receptor responsible for MH in a French family. Protasi et al.2 and Dainese et al.,4 studying knockout mice lacking skeletal muscle calsequestrin, a calcium-binding and buffering protein involved in the modulation of calcium release from RYR1, suggested that this mutation could be involved in MH and environmental heat stroke. On the basis of analogies between MH and catecholaminergic polymorphic ventricular tachycardia, a cardiac disorder caused by mutation in the cardiac RYR2 gene, MacLennan and Chen5 suggested that both diseases may have a common mechanism. In fact, a mutation in RyR can decrease the threshold for calcium release from sarcoplasmic reticulum, and a mutation in calsequestrin can increase the free calcium concentration over the same threshold, giving rise, in the presence of halothane and catecholamines, to contracture (MH) or arrhythmias (catecholaminergic polymorphic ventricular tachycardia), respectively. It is also possible that a mutation in different genes involved in the production and regulation of different calcium handling proteins could explain not only MH and similar muscular diseases but also some cardiac-related diseases sharing the same genetic anomaly. Luca Siracusano, MD Viviana Girasole, MD Department of Neuroscience, Psychiatric and Anesthesiological Sciences University of Messina Messina, Italy [email protected] or [email protected]

The Calsequestrin Mutation CASQ2D307H Does Not Affect Protein Stability and Targeting to the Junctional Sarcoplasmic Reticulum but Compromises Its Dynamic Regulation of Calcium Buffering

Mutations in cardiac ryanodine receptor (RYR2) and cardiac calsequestrin (CASQ2) genes are linked to catecholaminergic polymorphic ventricular tachycardia, a life-threatening genetic disease. They predispose young individuals to cardiac arrhythmia in the absence of structural abnormalities. One such mutation that changes an aspartic residue to histidine at position 307 in CASQ2 has been linked to catecholaminergic polymorphic ventricular tachycardia. In this study we made a transgenic mouse model expressing the mutant CASQ2(D307H) protein in a CASQ2 null background and investigated if the disease is caused by accelerated degradation of the mutant protein. Our data suggest that the mutant protein can be expressed, is relatively stable, and targets appropriately to the junctional sarcoplasmic reticulum. Moreover, it partially normalizes the ultrastructure of the sarcoplasmic reticulum, which was altered in the CASQ2 null background. In addition, overexpression of the mutant protein does not cause any pathology and/or structural changes in the myocardium. We further demonstrate, using purified protein, that the mutant protein is very stable under chemical and thermal denaturation but shows abnormal Ca(2+) buffering characteristics at high calcium concentrations. In addition, trypsin digestion studies reveal that the mutant protein is more susceptible to protease activity only in the presence of high Ca(2+). These studies collectively suggest that the D307H mutation can compromise the dynamic behavior of CASQ2 including supramolecular rearrangement upon Ca(2+) activation.

Junctin – the quiet achiever

Recent advances in understanding skeletal and cardiac muscle function have evolved with recognition of the active role played by the intracellular sarcoplasmic reticulum (SR) Ca2+ store in contraction. The key proteins in this store are the Ca2+ binding protein calsequestrin (CSQ), the ryanodine receptor (RyR) Ca2+ release channel and triadin and junctin (Beard et al. 2004). The CSQ–triadin–junctin–RyR complex (Fig. 1) in the SR lumen forms a ‘Ca2+ transduction machine’ that is central to EC coupling and to normal muscle development. Other proteins in the lumen of the SR, including the histidine rich calcium binding protein (HRC) (Suk et al. 1999), JP-45 (Anderson et al. 2003) and SRP-27 (Bleunven et al. 2008), must also contribute to control of SR intraluminal Ca2+ load, but the precise nature of their role remains undetermined. JP-45 in particular is ideally placed to communicate store load to the excitation–contraction (EC) coupling process as it binds to both CSQ and the dihydropyridine receptor (DHPR) in the surface/transverse tubule membrane. The importance of the luminal proteins has been underlined by the recent discovery that changes in Ca2+ signalling due to mutations in CSQ or to lack of its expression can result in sudden cardiac death (Viatchenko-Karpinski et al. 2004). Furthermore, studies in animal models show that changes in CSQ, junctin, triadin and HRC expression can lead to defective Ca2+ signalling. The review by Pritchard & Kranius (2009) examines the roles of junctin and HRC in Ca2+ cycling and their potential significance in heart failure. This commentary focuses on protein-protein interactions between CSQ, triadin, junctin and RyR proteins that may underlie their roles in Ca2+ cycling. Figure 1 Illustration of the possible interactions between triadin, junctin, CSQ1 and RyR1 in the lumen of the SR

Ca2+ signaling in striated muscle: the elusive roles of triadin, junctin, and calsequestrin

This review focuses on molecular interactions between calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca(2+) release in response to changes in the Ca(2+) load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca(2+) homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in calsequestrin lead to functional changes which can be fatal. We find that calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca(2+) release in the heart, but depressing Ca(2+) release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation-contraction coupling, while junctin alone supports functional interactions between calsequestrin and the ryanodine receptor.

Catecholaminergic polymorphic ventricular tachycardia with structural heart disease is caused by a mutation in calsequestrin 2 that leads to increased spark-mediated calcium leak in cardiomyocytes

CAR-2 Catecholaminergic polymorphic ventricular tachycardia with structural heart disease is caused by a mutation in calsequestrin 2 that leads to increased spark-mediated calcium leak in cardiomyocytes Aygun Dindar, Oya Uyguner, ES Sahin, D Terentyev, Kemal Nisli, A Uzumcu, R Terentyeva, E Yekeler, S Vedamoorthyrao, A Belevych, S Gyorke, B Wollnik, K Adalet Istanbul University, Istanbul Medical Faculty, Department of Pediatrics, Division of Pediatric Cardiology, Istanbul, Turkey Istanbul University, Istanbul Medical Faculty, Department of Medical Genetics, Istanbul, Turkey Dorothy Davis Heart and Lung Research Institute, Ohio State University, Columbus USA, Ohio, United States Istanbul University, Istanbul, Medical Faculty, Department of Radiology, Istanbul, Turkey Center for Molecular Medicine Cologne (CMMC), Institute of Human Genetics, University of Cologne, Cologne, Germany Istanbul University, Istanbul Medical Faculty, Department of Cardiology, Istanbul, Turkey

Calsequestrin mutation and catecholaminergic polymorphic ventricular tachycardia: A simulation study of cellular mechanism

Patients with a missense mutation of the calsequestrin 2 gene (CASQ2) are at risk for catecholaminergic polymorphic ventricular tachycardia. This mutation (CASQ2(D307H)) results in decreased ability of CASQ2 to bind Ca2+ in the sarcoplasmic reticulum (SR). In this theoretical study, we investigate a potential mechanism by which CASQ2(D307H) manifests its pro-arrhythmic consequences in patients. Using simulations in a model of the guinea pig ventricular myocyte, we investigate the mutation's effect on SR Ca2+ storage, the Ca2+ transient (CaT), and its indirect effect on ionic currents and membrane potential. We model the effects of isoproterenol (ISO) on Ca(V)1.2 (the L-type Ca2+ current, I(Ca(L))) and other targets of beta-adrenergic stimulation. ISO increases I(Ca(L)), prolonging action potential (AP) duration (Control: 172 ms, +ISO: 207 ms, at cycle length of 1500 ms) and increasing CaT (Control: 0.79 microM, +ISO: 1.61 microM). ISO increases I(Ca(L)) by reducing the fraction of channels which undergo voltage-dependent inactivation and increasing transitions from a non-conducting to conducting mode of channel gating. CASQ2(D307H) reduces SR storage capacity, thereby reducing the magnitude of CaT (Control: 0.79 microM, CASQ2(D307H): 0.52 microM, at cycle length of 1500 ms). The combined effect of CASQ2(D307H) and ISO elevates SR free Ca2+ at a rapid rate, leading to store-overload-induced Ca2+ release and delayed afterdepolarization (DAD). If resting membrane potential is sufficiently elevated, the Na+-Ca2+ exchange-driven DAD can trigger I(Na) and I(Ca(L)) activation, generating a triggered arrhythmogenic AP. The CASQ2(D307H) mutation manifests its pro-arrhythmic consequences due to store-overload-induced Ca2+ release and DAD formation due to excess free SR Ca2+ following rapid pacing and beta-adrenergic stimulation.

A mutation in calsequestrin, CASQ2D307H, impairs Sarcoplasmic Reticulum Ca2+ handling and causes complex ventricular arrhythmias in mice

A naturally-occurring mutation in cardiac calsequestrin (CASQ2) at amino acid 307 was discovered in a highly inbred family and hypothesized to cause Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). The goal of this study was to establish a causal link between CASQ2(D307H) and the CPVT phenotype using an in vivo model. Cardiac-specific expression of the CASQ2(D307H) transgene was achieved using the alpha-MHC promoter. Multiple transgenic (TG) mouse lines expressing CASQ2(D307H) from 2- to 6-fold possess structurally normal hearts without any sign of hypertrophy. The hearts displayed normal ventricular function. Myocytes isolated from TG mice had diminished I(Ca)-induced Ca2+ transient amplitude and duration, as well as increased Ca2+ spark frequency. These myocytes, when exposed to isoproterenol and caffeine, displayed disturbances in their rhythmic Ca2+ oscillations and membrane potential, and delayed afterdepolarizations. ECG monitoring revealed that TG mice challenged with isoproterenol and caffeine developed complex ventricular arrhythmias, including non-sustained polymorphic ventricular tachycardia. The findings of the present study demonstrate that expression of mutant CASQ2(D307H) in the mouse heart results in abnormal myocyte Ca2+ handling and predisposes to complex ventricular arrhythmias similar to the CPVT phenotype observed in human patients.

The molecular basis of catecholaminergic polymorphic ventricular tachycardia: What are the different hypotheses regarding mechanisms?

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disorder characterized by exercise- and stress-induced ventricular tachycardias associated with syncope and sudden cardiac death.1 Two genetic variants of this disease have been described: an autosomal-dominant form caused by mutations in the cardiac ryanodine receptor gene (RyR2),2–4 and a recessive form associated with homozygous mutations in calsequestrin (CASQ2).5 RyR2 and CASQ2 are both critically involved in the regulation of cardiac excitation-contraction coupling, during which opening of plasmalemmal Ca2+ channels triggers a much greater release of Ca2+ from the sarcoplasmic reticulum (SR) via RyR2 channels. CASQ2 associates with the luminal side of RyR2, and represents a high capacity, low affinity Ca2+-binding protein that plays an important role in buffering Ca2+ within the SR and preventing diastolic Ca2+ release via RyR2.6 Accordingly, abnormalities in the control of SR Ca2+ release constitute the central pathogenic abnormality in CPVT, although considerable controversy exists about the molecular mechanisms causing these defects. This viewpoint will discuss some of these hypotheses, mechanisms, and controversies.

Unzipping RyR2 in adult cardiomyocytes: Getting closer to mechanisms of inherited ventricular arrhythmias?

See article by Yang et al. [19] (pages 475–485) in this issue . Malignant ventricular arrhythmias (VA) are a major cause of mortality and morbidity worldwide. Causative mechanisms are multiple and complex. VA can be generated by abnormal or instable repolarization of the action potential (AP) in myocytes. Perverted repolarization results mainly from congenital channelopathies, acquired diseases, including hypertrophy and heart failure (HF), or therapeutic intervention. Inherited arrhythmias are due to mutations, mostly in transmembrane ionic channels (Na+, K+, Ca2+) [1,2]. However, because Ca2+ regulates several ionic currents involved in AP repolarization [3], it was not counterintuitive to find that mutations in proteins regulating intracellular Ca2+ homeostasis can also generate VA and sudden cardiac death (SCD). For example, mutations in the cardiac Ca2+ release channel (ryanodine receptor; RyR2) of the sarcoplasmic reticulum (SR) are involved in type 2 arrhythmogenic right ventricular cardiomyopathy and catecholaminergic polymorphic ventricular tachycardia (CPVT) characterized by effort-induced VA in structurally normal hearts [2,4,5]. CPVT are also caused by mutations in calsequestrin, a protein binding Ca2+ and associated to the RyR2 in the lumenal side of the SR [2,4]. The intrinsic mechanisms by which changes in [Ca2+]i handling promote arrhythmias are not completely elucidated. Obviously, spontaneous SR Ca2+ release can activate a transient inward current ( I ti) mediated mainly by the electrogenic Na+/Ca2+ exchanger (NCX) in the forward mode [6] … * Corresponding author. Tel.: +33 467 41 52 41; fax: +33 467 41 52 42. Email address: srichard{at}montp.inserm.fr