MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca2+ release in hypertrophic rat cardiomyocytes

Abstract

Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca²⁺ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca²⁺]). Pharmacological or genetic facilitation of mito-Ca²⁺ uptake was shown to restore Ca²⁺ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca²⁺ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca²⁺] restores systolic Ca²⁺ release without adverse effects in diastole, we overexpressed mitochondrial Ca²⁺ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca²⁺] using genetic probe mtRCamp1h revealed that mito-[Ca²⁺] 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-[Ca²⁺]. However, it failed to improve cytosolic Ca²⁺ handling and reduce proarrhythmic spontaneous Ca²⁺ 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-[Ca²⁺], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca²⁺ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca²⁺ mishandling. These data establish nonlinear mito-[Ca²⁺]/mito-ROS relationship, whereby partial restoration of mito-[Ca²⁺] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca²⁺ uptake exacerbates damaging mito-ROS emission.NEW & NOTEWORTHY Defective intracellular Ca²⁺ 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-Ca²⁺ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca²⁺ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca²⁺ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.

Keywords: calcium-dependent ventricular arrhythmia; mitochondrial calcium uniporter; mitochondrial calcium uptake; mitochondrial reactive oxygen species; ryanodine receptor.