Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation

Abstract

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca 2+ release channel/ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2 R420Q . Objective: To determine the arrhythmogenic mechanisms of this mutation. Methods and Results: Ventricular tachycardias under stress conditions were observed in both CPVT patients and KI mice. During action potential recording (by patch-clamp in KI mouse cardiomyocytes and by microelectrodes in mutant hiPSC-CM) we observed an increased occurrence of delayed after-depolarizations (DADs) under isoproterenol stimulation, associated with increased Ca 2+ waves during confocal Ca 2+ recording in both mouse and human RyR2 R420Q cardiomyocytes. In addition, Ca 2+ -induced Ca 2+ -release, as well as a rough indicator of fractional Ca 2+ release, were higher and Ca 2+ sparks longer in the RyR2 R420Q expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum (jSR) measured by g-STED super-resolution and electronic microscopy, respectively. The increase in jSR width might be due to the impairment of RyR2 R420Q binding to junctophilin-2, as there were less junctophilin-2 co-immunoprecipitated with RyR2 R420Q . At the single current level, the RyR2R420Q channel dwells longer in the open state at low [Ca 2+ ] i , but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N-terminus and the core solenoid, a RyR2 inter-domain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. Conclusions: The RyR2 R420Q CPVT mutation modifies the interdomain interaction of the channel and weaken its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction.

Local recovery of cardiac calcium‐induced calcium release interrogated by ultra‐effective, two‐photon uncaging of calcium

“…These observations are compatible with the notion that the CICR and RyRs in R420Q myocytes have a slightly elevated Ca 2+ sensitivity, consistent with the overall gain of function conferred by this genotype (Wang et al, 2017;Yin et al, 2021). Similarly, the activation of PKA in the presence of cAMP is expected to increase the RyR Ca 2+ sensitivity.…”

“…calsequestrin (Terentyev et al, 2002;Manno et al, 2017)). Impaired (shortened) refractoriness, as in case of mutation of ryanodine receptors R420Q (Figure 2A and Figure 3A), might lead to arrhythmogenic diastolic Ca 2+ releases (Lopez et al, 2020;Yin et al, 2021). PKA stimulation by cAMP increased the probability of triggering of Ca 2+ sparks (Figure 2A and Figure 3A), which agrees with results of spark recovery analysis using the "ryanodine method" (Sobie et al, 2005) in the presence of isoproterenol (Ramay et al, 2011;Poláková et al, 2015;Potenza et al, 2019).…”

“… 2017 ; Yin et al . 2021 ). Similarly, the activation of protein kinase A (PKA) in the presence of cAMP is expected to increase the RyR Ca 2+ sensitivity.…”

“… 2020 ; Yin et al . 2021 ). PKA stimulation by cAMP increased the probability of triggering of Ca 2+ sparks (Figs 2 A and 3 A ), which agrees with results of spark recovery analysis using the ‘ryanodine method’ (Sobie et al .…”

“…They are frequently used for classifications tasks (Noble, 2006). In this paper we introduce this procedure and validate the technique by using pharmacological modifiers for CICR and a transgenic mouse model harboring the arrhythmogenic RyR R420Q mutation (Domingo et al, 2015;Yin et al, 2021).…”