Store overload‐induced Ca2+ release as a triggering mechanism for CPVT and MH episodes caused by mutations in RYR and CASQ genes

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic cardiac disorder caused, in part, by dominant mutations in RYR2, the cardiac ryanodine receptor (RyR2) gene (Liu et al. 2009). The disorder manifests as syncopal events and sudden cardiac death, usually in response to intensive exercise; elevated catecholamines induce arrhythmogenic episodes that respond to β-blockers. In other CPVT families arrhythmia is caused by recessive mutations in CASQ2 that cause truncation and/or misfolding of cardiac calsequestrin (CASQ2). In a mouse model, casq2-null or partially null mice also exhibit arrhythmias (Knollmann et al. 2006; Chopra et al. 2007; Knollmann, 2009, this symposium). Malignant hyperthermia (MH) is an acute response to volatile anaesthetics, such as halothane, and depolarizing muscle relaxants, characterized by hypermetabolism, skeletal muscle contracture and fever (Loke Protasi et al. 2009, this symposium). CASQ1 mutations have not yet been reported to cause human MH. It is accepted that CPVT arrhythmias result from repetitive, premature activation of RyR2. It has long been known that RyR is activated by elevation of luminal Ca2+ beyond a certain threshold (Palade et al. 1983; Venetucci et al. 2008). Here, we refer to this phenomenon as store overload-induced Ca2+ release (SOICR). RyR2 CPVT mutant channels expressed in the human embryonic kidney cell line HEK-293 were activated by much lower luminal Ca2+ levels than wild-type (wt) (Jiang et al. 2004, 2005). Critically, the frequency of Ca2+ oscillations was markedly increased and the size of the Ca2+ store was decreased. When single channel activities of RyR2 CPVT mutants were measured in planar lipid bilayers with 45 nm cytoplasmic Ca2+, incremental elevation of luminal Ca2+ from 0 to 1000 μm increased the probability of opening to 0.3–0.4 for CPVT mutant RyR2 vs. 0.02 for wt RyR2. These results demonstrate that RyR2 CPVT mutant channels, in isolation from other sarcoplasmic reticulum (SR) proteins, manifest a lower SOICR threshold than wt RyR2. In comparable studies, RyR1 MH mutant channels expressed in HEK-293 cells are activated by much lower luminal Ca2+ levels than wt (Jiang et al. 2008). Furthermore, in single channel measurements, the MH-triggering agent, halothane, enhanced the frequency of activation of RyR1 MH mutant channels in the presence of luminal Ca2+, but had no effect in the absence of luminal Ca2+. Appropriately, dantrolene suppressed SOICR in HEK-293 cells expressing an RyR1 MH mutant. These results demonstrate that RyR1 MH mutations lower the threshold for luminal Ca2+ activation, that volatile anaesthetics further lower the SOICR threshold, and that dantrolene suppresses SOICR. Calsequestrin plays at least three prominent roles: (1) it increases the SR luminal total Ca2+ content through its highly cooperative, high capacity, low affinity Ca2+ binding; (2) it buffers free Ca2+ levels in the SR lumen; and (3) it may convey bound Ca2+ into the RyR pore through its strategic localization in relation to triadin (Tri) and RyR (Wang et al. 1998; MacLennan Park et al. 2003; Royer Györke et al. 2009, this symposium) and Qin et al. (2008) lead them to postulate that a CASQ2–Tri–RyR2 complex regulates RyR2 function and to assign a 4th and even a 5th role to CASQ in this complex: (4) the deactivation of RyR in low luminal Ca2+; and (5) the activation of RyR by elevated luminal Ca2+. Their theory is that mutations in CASQ2 (and, by extension, mutations in CASQ1) alter protein–protein interactions within the complex, resulting in the abnormal openings of RyR2 (or RyR1) that cause episodes of CPVT (or MH). There is general agreement in both theories and for both disorders, whether caused by mutations in RyR or CASQ, that manifestations of disease are ultimately caused by the abnormal response of RyR channels to elevations in luminal Ca2+. To evaluate the relative merits of the hypotheses that SOICR acts directly through RyR or through a RyR–Tri–CASQ complex, let us consider critical features of the diseases. First, the dominant inheritance of RyR mutations in CPVT and MH is expected, since openings of abnormal channels in the presence of normal closed channels can cause full disease manifestations. Why then are all cases of CPVT caused by CASQ2 mutations inherited recessively? Surely if dysregulation of a CASQ2–Tri–RyR2 complex were causal, then mutation of one CASQ2 allele would be sufficient to trigger abnormal activation in abnormal complexes, manifesting as an arrhythmia – but this is not the case. Second, CASQ2 CPVT causal mutations have diverse properties: R33Q activates wt RYR2 channels in response to elevated luminal Ca2+, whereas L167H did not (Qin et al. 2008); R33Q is an unstable molecule and may be degraded (Rizzi et al. 2008); R33Q causes a compensatory elevation of the expression of RyR2 and calreticulin (Song et al. 2007); R33Q, but not CASQdel lowers both basal and nadir SR Ca2+ levels (Terentyev et al. 2008). In contrast to those papers that relate differences among CASQ2 mutants to CPVT, one paper pinpoints the common feature of all CASQ2 CPVT mutants – their loss of the ability to oligomerize properly and therefore to bring about cooperative high capacity Ca2+ binding (Kim et al. 2007). Thus, the critical common feature in CASQ2-associated CPVT is a decrease in luminal Ca2+ binding and Ca2+ buffering that would permit an overshoot of luminal Ca2+ when SERCA pumps are stimulated. Final questions are: why, if CASQ is critical to the regulation of normal RyR channels, do CASQ2-null humans or casq2-null mice, susceptible to CPVT, only display arrhythmias under stressful conditions that elevate luminal Ca2+ loading; and why do casq1-null mice only display MH symptoms in the presence of MH-triggering anaesthetics or heat? The answers cannot lie in CASQ–Tri–RyR interactions, since CASQ is absent. Therefore, the answer is most likely to lie in normal RyR itself, which, we propose, is intrinsically programmed to open in response to a tsunami of Ca2+ in a luminal space depleted of its capacity to buffer a surge of incoming Ca2+ (CPVT), or to open in response to halothane, which lowers its SOICR threshold (MH). This work was supported by Grants to D.H.M. from the Canadian Institutes of Health Research (CIHR) and to S.R.W.C. from the National Institutes of Health (HL75210) and CIHR and the Alberta Heritage Foundation for Medical Research.