Impaired Dynamic Sarcoplasmic Reticulum Ca Buffering in Autosomal Dominant CPVT2

Abstract

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene ( CASQ2 ), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2 -K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known. Methods: A K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators. Results: K180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness. Conclusions: CASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

The Structural–Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications

“…In those cases, we hypothesize that the extremely subtle defects, undetectable at the dimer level, might be amplified and, thus, exert their pathological effect only within the polymer structure(s), which could present altered Ca 2+ -binding/release properties and interactome. In this regard, it is indeed interesting to note that the only mouse model carrying a mutation from the second group (CASQ2-K180R) did not show any loss in the total intra-SR calcium buffering capacity (contrary to all CPVT2 recessive models), but did show a defect in the dynamic intra-SR calcium buffering [89]. Our hypothesis points to the CASQ dimerization of pathological mutants as a critical aspect in determining the intra-SR dominant/recessive pathological effect.…”

“…CASQ1 and CASQ2 missense mutations are scattered across all three thioredoxin domains of CASQ and the mutated proteins do not share any common defect in their Ca 2+ -dependent polymerization properties [31,36,85,86]. Moreover, most of the published biochemical analyses on either the polymerization and/or Ca 2+ -buffering capabilities of recombinant CASQ missense pathological mutants [15,28,31,[33][34][35][36][85][86][87][88][89][90] (Table 1) are sometimes difficult to interpret or in contrast with each other. Table 1.…”

“…The variable penetrance of inherited CASQ2 mutations [31,36], when combined with the 100% penetrance of the dominant K180R CASQ2 [91], has recently led to the hypothesis that two distinct molecular processes, triggered by two distinct groups of CASQ2 mutations, may underlie the recessive and dominant forms of CPVT2 [31,89].…”

“…In accordance with this hypothesis, the only recessive CASQ2 mutations that have been characterized in murine models, sharing a consistent loss of CASQ2 within the jSR [92,93], fall (R33Q [94], D307H [95]) or would fall (G112 + 5X [96]) within the first non-dimerizing group of proteins (on the left side of Table 1). The second group is that of dimerization-competent mutants (on the right side of Table 1), which includes many of the dominantly inherited CASQ1 variants, the only CASQ2 mutation whose dominance has been clinically and functionally validated to date [89,91], and other mutations clinically associated with CPVT2 in heterozygosis [36]. In those cases, we hypothesize that the extremely subtle defects, undetectable at the dimer level, might be amplified and, thus, exert their pathological effect only within the polymer structure(s), which could present altered Ca 2+ -binding/release properties and interactome.…”

Further exploration of cardiac channelopathy and cardiomyopathy genes in stillbirth

“…The study cohort consisted of 13 male and 6 female fetuses. The median fetal gestational age at stillbirth was 32 (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39) weeks. The median weight of stillborn fetuses was 1800 (410-3710) g with a median length of 42 (28 -54) cm (Table 2).…”