The cardiac ryanodine receptor (RyR2) governs the release of Ca 2؉ 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 Ca 2؉ content reaches a critical level, spontaneous Ca 2؉ release occurs, a process we refer to as store-overload-induced Ca 2؉ 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 Ca 2؉ 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 Ca 2؉ and enhanced the basal level of [ 3 H]ryanodine binding. We conclude that disease-causing RyR2 mutations, by enhancing RyR2 luminal Ca 2؉ activation, reduce the threshold for SOICR, which in turn increases the propensity for triggered arrhythmia. Abnormal RyR2 luminal Ca 2؉ activation likely contributes to the enhanced SOICR commonly observed in various cardiac conditions, including heart failure, and may represent a unifying mechanism for Ca 2؉ overload-associated VT. V entricular tachycardia (VT) is the leading cause of sudden death in patients with heart failure (HF). Delayed afterdepolarizations (DAD) frequently occur in failing hearts and are a major cause of VT, but the reason for the increased incidence of DAD-associated VT in patients with HF is not completely clear. Abnormal Ca 2ϩ handling is believed to be involved in the pathogenesis of VT (1-4). In keeping with this view, mutations in Ca 2ϩ handling proteins, including the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2), have been linked to catecholaminergic polymorphic VT (CPVT), which is also thought to be DAD-based (5-10). These similarities suggest that CPVT and DAD-associated VT in patients with HF may share a common arrhythmogenic mechanism. Thus, knowledge gained from investigation of inherited CPVT should lead to a better understanding of the molecular basis of the more commonly occurring VT in patients with HF and other cardiac diseases.RyR2 is an intracellular Ca 2ϩ release channel located in the sarcoplasmic reticulum (SR) (11). It is a key component of excitation contraction (EC) coupling in cardiac muscle, which is believed to take place via a mechanism known as Ca A number of conditions, such as physical and emotional stresses, digitalis toxicity, elevated extracellular Ca 2ϩ , ischemia͞ reperfusion, etc., can lead to SR Ca 2ϩ overload and subsequent SOICR in cardiac cells (19), and SOICR can activate inward currents. These Ca...
Abstract-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 Ca 2ϩ . Cells expressing these RyR2 mutants display an increased propensity for spontaneous Ca 2ϩ release under conditions of store Ca 2ϩ overload, a process we referred to as store overload-induced Ca 2ϩ 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, Ca
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