Store Overload-induced Ca Release Research Articles

Loss of luminal Ca 2+ 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.

Removal of FKBP12.6 Does Not Alter the Conductance and Activation of the Cardiac Ryanodine Receptor or the Susceptibility to Stress-induced Ventricular Arrhythmias

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for spontaneous Ca(2+) release and the occurrence of ventricular arrhythmias. Single channel recordings in lipid bilayers showed that FK506 treatment of recombinant RyR2 co-expressed with or without FKBP12.6 or native canine RyR2 did not induce long-lived subconductance states. [(3)H]Ryanodine binding studies revealed that coexpression with or without FKBP12.6 or treatment with or without FK506 did not alter the sensitivity of RyR2 to activation by Ca(2+) or caffeine. Furthermore, single cell Ca(2+) imaging analyses demonstrated that HEK293 cells co-expressing RyR2 and FKBP12.6 or expressing RyR2 alone displayed the same propensity for spontaneous Ca(2+) release or store overload-induced Ca(2+) release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca(2+) release and stress-induced ventricular arrhythmias.

Similarities and Contrasts in Ryanodine Receptor Localization and Function in Osteoclasts and Striated Muscle Cells

This review compares ryanodine receptor (RyR)-mediated Ca(2+) signaling processes in muscle and osteoclast cells. In muscle, RyR-mediated release of an intracellularly stored, sarcoplasmic reticular (SR), Ca(2+) is triggered by voltage-sensitive dihydropyridine receptor (DHPR)-L-type Ca(2+) channels either through an allosteric coupling with the RyR in skeletal muscle or a Ca(2+)-induced Ca(2+) release initiated by extracellular Ca(2+) entry in cardiac muscle. Both cell subtypes are nevertheless capable of Ca(2+)-induced SR Ca(2+) release with cardiac muscle additionally showing a store overload-induced Ca(2+) release (SOICR) driven by SR luminal Ca(2+) under some pathological conditions. Osteoclasts similarly show cytosolic Ca(2+) elevations driven by release of intracellular Ca(2+) stores that culminate in motile activity in turn modifying bone resorptive activity. However, such triggering is controlled by ambient Ca(2+) rather than membrane potential with features strongly suggestive of control by a surface membrane Ca(2+) receptor. Yet common actions of the RyR-specific agents perchlorate, dantrolene Na, ryanodine, caffeine, adenosine 3',5'-cyclic diphosphate ribose (cADPr) and ruthenium red implicate RyR in signaling in all these cell types. These findings were reconciled by reports confirming and uniquely localizing a cell surface rather than microsomal osteoclastic RyR that might itself detect ambient Ca(2+) possibly through its otherwise intraluminal positioned low-affinity Ca(2+)-binding site in parallel with the SOICR mechanism in cardiac muscle. Such a mechanism could interact with other osteoclast processes transferring Ca(2+) between cytosol, intracellular stores and extracellular space and be integrated with systemic processes regulating Ca(2+) homeostasis.

Functional Consequence of Protein Kinase A-dependent Phosphorylation of the Cardiac Ryanodine Receptor

The phosphorylation of the cardiac Ca(2+)-release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.

Skeletal and Cardiac Ryanodine Receptors Exhibit Different Responses to Ca 2+ Overload and Luminal Ca 2+

Spontaneous Ca 2+ release occurs in cardiac cells during sarcoplasmic reticulum Ca 2+ overload, a process we refer to as store-overload-induced Ca 2+ release (SOICR). Unlike cardiac cells, skeletal muscle cells exhibit little SOICR activity. The molecular basis of this difference is not well defined. In this study, we investigated the SOICR properties of HEK293 cells expressing RyR1 or RyR2. We found that HEK293 cells expressing RyR2 exhibited robust SOICR activity, whereas no SOICR activity was observed in HEK293 cells expressing RyR1. However, in the presence of low concentrations of caffeine, SOICR could be triggered in these RyR1-expressing cells. At the single-channel level, we showed that RyR2 is much more sensitive to luminal Ca 2+ than RyR1. To identify the molecular determinants responsible for these differences, we constructed two chimeras between RyR1 and RyR2, N-RyR1(1–4006)/C-RyR2(3962–4968) and N-RyR2(1–3961)/C-RyR1(4007–5037). We found that replacing the C-terminal region of RyR1 with the corresponding region of RyR2 (N-RyR1/C-RyR2) dramatically enhanced the propensity for SOICR and the response to luminal Ca 2+, whereas replacing the C-terminal region of RyR2 with the corresponding region of RyR1 (N-RyR2/C-RyR1) reduced the propensity for SOICR and the luminal Ca 2+ response. These observations indicate that the C-terminal region of RyR is a critical determinant of both SOICR and the response to luminal Ca 2+. These chimeric studies also reveal that the N-terminal region of RyR plays an important role in regulating SOICR and luminal Ca 2+ response. Taken together, our results demonstrate that RyR1 differs markedly from RyR2 with respect to their responses to Ca 2+ overload and luminal Ca 2+, and suggest that the lack of spontaneous Ca 2+ release in skeletal muscle cells is, in part, attributable to the unique intrinsic properties of RyR1.

Diagnosis and treatment of catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (VT) was first described by Reid et al 1 Reid D.S. Tynan M. Braidwood L. Fitzgerald G.R. Bidirectional tachycardia in a child A study using His bundle electrography. Br Heart J. 1975; 37: 339-344 Crossref PubMed Scopus (138) Google Scholar in 1975 and by Coumel et al in 1978. The condition was described as a familial cardiac arrhythmia that occurs in patients with structurally normal heart and causes exercise-/emotion-triggered syncope and sudden death with a distinctive pattern of ventricular and supraventricular arrhythmias. Since the first ryanodine receptor mutations were identified in 2001, 2 Priori S.G. Napolitano C. Tiso N. Memmi M. Vignati G. Bloise R. Sorrentino V. Danieli G.A. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation. 2001; 103: 196-200 Crossref PubMed Scopus (1135) Google Scholar it appeared evident that catecholaminergic polymorphic VT was caused by uncontrolled Ca2+ release from the sarcoplasmic reticulum. 3 Jiang D. Xiao B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR). Proc Natl Acad Sci U S A. 2004; 101: 13062-13067 Crossref PubMed Scopus (350) Google Scholar Subsequent experimental studies demonstrated that such abnormal calcium handling caused arrhythmias mediated by delayed afterdepolarizations and triggered activity. 4 Liu N. Colombi B. Memmi M. Zissimopoulos S. Rizzi N. Negri S. Imbriani M. Napolitano C. Lai F.A. Priori S.G. Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia Insights from a RyR2 R4496C knock-in mouse model. Circ Res. 2006; 99: 292-298 Crossref PubMed Scopus (261) Google Scholar This article reviews the current knowledge of the diagnosis and therapy for catecholaminergic polymorphic VT and outlines the open issues to be addressed in order to reduce the burden of life-threatening cardiac events in patients with this lethal disorder.

Enhanced Store Overload–Induced Ca 2+ Release and Channel Sensitivity to Luminal Ca 2+ 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.

Can Novel Therapies for Arrhythmias Caused by Spontaneous Sarcoplasmic Reticulum Ca 2+ Release be Developed Using Mouse Models?

See related article, pages e77–e82 Ventricular arrhythmias are a significant cause of premature death in Western society and occur both in the absence and presence of heart disease. For example, in heart failure close to 50% of patients die of lethal forms of ventricular arrhythmias. In spite of a large amount of basic and clinical investigations, current pharmacotherapy for ventricular arrhythmias is inadequate, demonstrating the need for novel therapeutic approaches. Abnormal automaticity is responsible for induction and maintenance of different types of ventricular tachycardias. The form of abnormal automaticity that results from abnormal release of Ca2+ from a Ca2+ overloaded sarcoplasmic reticulum (SR), referred to as a “triggered arrhythmia”, is the topic of this editorial. In vitro studies have shown that increasing the SR Ca2+ load beyond a critical level causes spontaneous opening of the SR Ca2+ release channel (ryanodine receptor; RYR) and results in spontaneous SR Ca2+ release.1 The subsequent increase in cytosolic [Ca] activates an inward current through the sarcolemmal Na+/ Ca2+ exchanger which causes membrane depolarization.1,2 Spontaneous SR Ca2+ release during the diastolic interval causes depolarization (delayed after depolarizations; DADs) which has been shown to result in action potentials in single cells and in the intact heart when the aberrant release is adequately synchronized within and among cardiac myocytes.3,4 Factors that lead to DADs include increased heart rate and catecholamine stress, which both induce triggered arrhythmias by increasing SR Ca2+ loading.3 Whereas many molecules are involved in SR Ca2+ overload (DAD) related arrhythmias, abnormal opening …

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.