Divergent Regulation of Ryanodine Receptor 2 Calcium Release Channels by Arrhythmogenic Human Calmodulin Missense Mutants

Abstract

Rationale Calmodulin (CaM) mutations are associated with an autosomal-dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT)or long QT syndrome (LQTS).CaM binds to and inhibits RyR2 Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. Objective To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. Methods and Results We studied recombinant CaM mutants associated with CPVT (N54I, N98S) or LQTS (D96V, D130G, F142L). As a group, all LQTS-associated CaM mutants(LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants(CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared to wild-type (WT) CaM, CPVT-CaMs caused greater RyR2 single channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. By contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2 binding affinity (D130G, F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. Conclusions A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2-interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene

“…This mutation, as shown in Fig. 4A and in our previous work, increases RyR2 P o , the opposite effect to wild-type (wt)-CaM (Hwang et al, 2014). If dantrolene merely amplifies the action of CaM, then one would expect dantrolene to be an activator in the presence of N54I-CaM.…”

“…4C). These values are ∼2-fold lower than the binding affinities for these CaMs on RyR2 (Guo et al, 2011;Hwang et al, 2014).…”

“…Myocytes were first exposed to a Ca 21 -free relaxing solution and then permeabilized with saponin (40 mg/ml) for 60 seconds and placed in internal solution composed (in millimolar) of 120 K-aspartate, 15 KCl, 5 KH 2 PO 4 , 0.75 MgCl 2 , 4% dextran (40,000), 10 HEPES, 5 Mg 2 ATP, 10 glutathione (reduced), 0.025 Fluo-4, and 10 phosphocreatine (di-Na). These solutions also contained 10 U/ml creatine phosphokinase (Hwang et al, 2014) and had free [Ca 21 ] 5 120 nM. To allow complete removal of CaM binding to RyR2 in permeabilized myocytes , all Ca 21 wave recordings were done after 30-minute incubation with either dantrolene alone or dantrolene 1 CaM.…”

“…Ca 21 waves in myocytes were imaged with a confocal microscope (LSM 510; Zeiss, Thornwood, NY) in line scan mode. Ca 21 wave analysis was performed as described (Hwang et al, 2014). Given the variability between different experimental days, the Ca 21 wave frequency and amplitude data were normalized to the mean of vehicle group obtained on the same day.…”

Gene Transfer of Engineered Calmodulin Alleviates Ventricular Arrhythmias in a Calsequestrin‐Associated Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

“…CaM is associated with RyR2 at its cytosolic regulatory domain and inhibits RyR2 activity at elevated Ca 18. Recently several mutations of CaM have been linked to cardiac arrhythmia and specifically to CPVT 31. However, precisely how and through what molecular steps CaM regulates RyR2 mediated SR Ca release in cardiomyocytes and how these processes are altered to cause arrhythmia remains to be elucidated.…”