MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca 2+ release in hypertrophic rat cardiomyocytes.

Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca ²⁺ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca ²⁺ ]). Pharmacological or genetic facilitation of mito-Ca ²⁺ uptake was shown to restore Ca ²⁺ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca ²⁺ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca ²⁺ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca ²⁺ ] restores systolic Ca ²⁺ release without adverse effects in diastole, we overexpressed mitochondrial Ca ²⁺ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca ²⁺ ] using genetic probe mtRCamp1h revealed that mito-[Ca ²⁺ ] in TAB VMs paced at 2 Hz under β-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca ²⁺ ]. However, it failed to improve cytosolic Ca ²⁺ handling and reduce proarrhythmic spontaneous Ca ²⁺ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca ²⁺ ], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca ²⁺ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca ²⁺ mishandling. These data establish nonlinear mito-[Ca ²⁺ ]/mito-ROS relationship, whereby partial restoration of mito-[Ca ²⁺ ] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca ²⁺ uptake exacerbates damaging mito-ROS emission. NEW & NOTEWORTHY Defective intracellular Ca ²⁺ homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca ²⁺ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca ²⁺ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca ²⁺ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.