Increased Intermembrane Space [Ca²⁺] Drives Mitochondrial Structural Damage in CPVT

Mitochondrial dysfunction caused by abnormally high RyR2 (ryanodine receptor) activity is a common finding in cardiovascular diseases. Mechanisms linking RyR2 gain of function with mitochondrial remodeling remain elusive. We hypothesized that RyR2 hyperactivity in cardiac disease increases [Ca²⁺] in the mitochondrial intermembrane space (IMS) and activates the Ca²⁺-sensitive protease calpain, driving remodeling of mitochondrial cristae architecture through cleavage of structural protein OPA1 (optic atrophy protein 1).

We generated a highly arrhythmogenic rat model of catecholaminergic polymorphic ventricular tachycardia, induced by RyR2 gain-of-function mutation S2236L(Ser2336Leu)^(+/-). We created a new biosensor to measure IMS-[Ca²⁺] in adult cardiomyocytes with intact Ca²⁺ cycling. We used ex vivo whole heart optical mapping, confocal and electron microscopy, as well as in vivo/in vitro gene editing techniques to test the effects of calpain in the IMS.

We found altered mitochondrial cristae structure, increased IMS-[Ca²⁺], reduced OPA1 expression, and augmented mito-reactive oxygen species emission in catecholaminergic polymorphic ventricular tachycardia myocytes. We show that calpain-mediated OPA1 cleavage led to disrupted cristae organization and, thereby, decreased electron transport chain supercomplex assembly, resulting in accelerated reactive oxygen species production. Genetic inhibition of calpain activity in IMS reversed mitochondria structural defects in catecholaminergic polymorphic ventricular tachycardia myocytes and reduced arrhythmic burden in ex vivo optically mapped hearts.

Our data suggest that RyR2 hyperactivity contributes to mitochondrial structural damage by promoting an increase in IMS-[Ca²⁺], sufficient to activate IMS-residing calpain. Calpain activation leads to proteolysis of OPA1 and cristae widening, thereby decreasing assembly of electron transport chain components into supercomplexes. Consequently, excessive mito-reactive oxygen species release critically contributes to RyR2 hyperactivation and ventricular tachyarrhythmia. Our new findings suggest that targeting IMS calpain may be beneficial in patients at risk for sudden cardiac death.