Mitochondrial calpain inhibition restores defective SR-mitochondrial crosstalk in CPVT rat myocytes: Calcium Signaling and Excitation–Contraction in Cardiac, Skeletal and Smooth Muscle

Cardiac RYR2-mediated sarcoplasmic Ca²⁺ (SR) release is essential for matching increased energy demand during fight-or-flight response with mitochondrial metabolic output by delivering Ca²⁺ into the mitochondrial matrix to activate Ca²⁺-dependent Krebs cycle dehydrogenases. RYR2 complex gain-of-function mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) have been linked to mitochondrial structural damage and enhanced production of reactive oxygen species (ROS). Despite being critical for arrhythmogenesis in CPVT, the exact causes of these phenomena remain undetermined. Taking advantage of a new rat model of CPVT induced by heterozygous RYR2 gain-of-function mutation S2222L, we tested how RYR2 overactivity alters mitochondrial Ca²⁺ and ROS handling, and how these changes cause mitochondrial structural defects. Injection of epinephrine (1 mg/kg) and caffeine (120 mg/kg) induced bigamy and bidirectional VT in vivo in 100% of CPVT rats. Simultaneous whole-cell patch clamp and confocal Ca²⁺-imaging demonstrated that under β-adrenergic stimulation with isoproterenol (50 nM), CPVT ventricular myocytes (VMs) exhibited severe Ca²⁺ mishandling and high propensity for generation of spontaneous Ca²⁺ waves (SCWs) that cause arrhythmogenic afterdepolarizations. Diminished Ca²⁺ transient amplitude in CPVT VMs resulted in a significant reduction in mitochondrial matrix–[Ca²⁺], and thereby a mito-ROS surge, visualized using matrix-targeted biosensors mtRCaMP1h and MLS-HyPer, respectively. Importantly, using novel Ca²⁺-biosensors targeted to intermembrane space (IMS-GECO), we uncovered that [Ca²⁺] in this compartment reaches 1 µM, sufficient for activation of Ca²⁺-dependent protease μ-calpain. Adenoviral overexpression of IMS-targeted calpastatin, an endogenous calpain inhibitor, reduced mito-ROS, restored cytosolic Ca²⁺ transient amplitude and SR Ca²⁺ content, and reduced RYR2-mediated SCWs in CPVT VMs. These changes were paralleled by restored expression levels of OPA1, a mitochondrial structural protein responsible for tight cristae organization. Our data suggest that enhanced mito-ROS due to matrix-[Ca²⁺] reduction in CPVT VMs and unexpectedly high IMS-[Ca²⁺] promotes IMS-calpain–mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling.