Functional Calsequestrin-1 Is Expressed in the Heart and Its Deficiency Is Causally Related to Malignant Hyperthermia-Like Arrhythmia.

Abstract

Calsequestrins (Casqs), comprising the Casq1 and Casq2 isoforms, buffer Ca²⁺ and regulate its release in the sarcoplasmic reticulum of skeletal and cardiac muscle, respectively. Human inherited diseases associated with mutations in <i>CASQ1</i> or <i>CASQ2</i> include malignant hyperthermia/environmental heat stroke (MH/EHS) and catecholaminergic polymorphic ventricular tachycardia. However, patients with an MH/EHS event often experience arrhythmia for which the underlying mechanism remains unknown. Working hearts from conventional (<i>Casq1</i>-KO) and cardiac-specific (<i>Casq1</i>-CKO) <i>Casq1</i> knockout mice were monitored in vivo and ex vivo by ECG and electric mapping, respectively. MH was induced by 2% isoflurane and treated intraperitoneally with dantrolene. Time-lapse imaging was used to monitor intracellular Ca²⁺ activity in isolated mouse cardiomyocytes or neonatal rat ventricular myocytes with knockdown, overexpression, or truncation of the <i>Casq1</i> gene. Conformational change in both Casqs was determined by cross-linking Western blot analysis. Like patients with MH/EHS, <i>Casq1</i>-KO and <i>Casq1</i>-CKO mice had faster basal heart rate and ventricular tachycardia on exposure to 2% isoflurane, which could be relieved by dantrolene. Basal sinus tachycardia and ventricular ectopic electric triggering also occurred in <i>Casq1</i>-KO hearts ex vivo. Accordingly, the ventricular cardiomyocytes from <i>Casq1</i>-CKO mice displayed dantrolene-sensitive increased Ca²⁺ waves and diastole premature Ca²⁺ transients/oscillations on isoflurane. Neonatal rat ventricular myocytes with Casq1-knockdown had enhanced spontaneous Ca²⁺ sparks/transients on isoflurane, whereas cells overexpressing Casq1 exhibited decreased Ca²⁺ sparks/transients that were absent in cells with truncation of 9 amino acids at the C terminus of Casq1. Structural evaluation showed that most of the Casq1 protein was present as a polymer and physically interacted with ryanodine receptor-2 in the ventricular sarcoplasmic reticulum. The Casq1 isoform was also expressed in human myocardium. Mechanistically, exposure to 2% isoflurane or heating at 41 °C induced Casq1 oligomerization in mouse ventricular and skeletal muscle tissues, leading to a reduced Casq1/ryanodine receptor-2 interaction and increased ryanodine receptor-2 activity in the ventricle. Casq1 is expressed in the heart, where it regulates sarcoplasmic reticulum Ca²⁺ release and heart rate. Casq1 deficiency independently causes MH/EHS-like ventricular arrhythmia by trigger-induced Casq1 oligomerization and a relief of its inhibitory effect on ryanodine receptor-2-mediated Ca²⁺ release, thus revealing a new inherited arrhythmia and a novel mechanism for MH/EHS arrhythmogenesis.