Pharmacological Modulation of Mitochondrial Ca²⁺ Content Regulates Sarcoplasmic Reticulum Ca²⁺ Release via Oxidation of the Ryanodine Receptor by Mitochondria-Derived Reactive Oxygen Species

In a physiological setting, mitochondria increase oxidative phosphorylation during periods of stress to meet increased metabolic demand. This in part is mediated via enhanced mitochondrial Ca²⁺ uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca²⁺ uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca²⁺-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca²⁺ accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca²⁺ uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na⁺/Ca²⁺ exchanger, and MCU inhibitor Ru360 in rat ventricular myocytes (VMs) from control rats and rats with hypertrophy induced by thoracic aortic banding (TAB). In periodically paced VMs under β-adrenergic stimulation, treatment with kaempferol (10 μmol/L) or CGP-37157 (1 μmol/L) enhanced mitochondrial Ca²⁺ accumulation monitored by mitochondrial-targeted Ca²⁺ biosensor mtRCamp1h. Experiments with mitochondrial membrane potential-sensitive dye TMRM revealed this was accompanied by depolarization of the mitochondrial matrix. Using redox-sensitive OMM-HyPer and ERroGFP_iE biosensors, we found treatment with kaempferol or CGP-37157 increased the levels of reactive oxygen species (ROS) in mitochondria and the sarcoplasmic reticulum (SR), respectively. Confocal Ca²⁺ imaging showed that accelerated Ca²⁺ accumulation reduced Ca²⁺ transient amplitude and promoted generation of spontaneous Ca²⁺ waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca²⁺ release channel ryanodine receptor (RyR). Western blot analyses showed increased RyR oxidation after treatment with kaempferol or CGP-37157 vs. controls. Furthermore, in freshly isolated TAB VMs, confocal Ca²⁺ imaging demonstrated that enhancement of mitochondrial Ca²⁺ accumulation further perturbed global Ca²⁺ handling, increasing the number of cells exhibiting spontaneous Ca²⁺ waves, shortening RyR refractoriness and decreasing SR Ca²⁺ content. In ex vivo optically mapped TAB hearts, kaempferol exacerbated proarrhythmic phenotype. On the contrary, incubation of cells with MCU inhibitor Ru360 (2 μmol/L, 30 min) normalized RyR oxidation state, improved intracellular Ca²⁺ homeostasis and reduced triggered activity in ex vivo TAB hearts. These findings suggest facilitation of mitochondrial Ca²⁺ uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca²⁺ homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca²⁺ accumulation can be protective.