Pharmacological Modulation of Mitochondrial Ca Content Regulates Sarcoplasmic Reticulum Ca 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 Ca uptake, an important regulator of cellular ATP homeostasis. In a pathophysiological setting pharmacological modulation of mitochondrial Ca uptake or retention has been suggested as a therapeutic strategy to improve metabolic homeostasis or attenuate Ca-dependent arrhythmias in cardiac disease states. To explore the consequences of mitochondrial Ca accumulation, we tested the effects of kaempferol, an activator of mitochondrial Ca uniporter (MCU), CGP-37157, an inhibitor of mitochondrial Na/Ca 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 Ca accumulation monitored by mitochondrial-targeted Ca 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 Ca imaging showed that accelerated Ca accumulation reduced Ca transient amplitude and promoted generation of spontaneous Ca waves in VMs paced under ISO, suggestive of abnormally high activity of the SR Ca 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 Ca imaging demonstrated that enhancement of mitochondrial Ca accumulation further perturbed global Ca handling, increasing the number of cells exhibiting spontaneous Ca waves, shortening RyR refractoriness and decreasing SR Ca content. In 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 Ca homeostasis and reduced triggered activity in TAB hearts. These findings suggest facilitation of mitochondrial Ca uptake in cardiac disease can exacerbate proarrhythmic disturbances in Ca homeostasis via ROS and enhanced activity of oxidized RyRs, while strategies to reduce mitochondrial Ca accumulation can be protective.