Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca²⁺-induced ATP production in cardiac H9c2 myoblasts

Ca²⁺ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca2 influx is mainly mediated by the mitochondrial Ca²⁺ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca²⁺ influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca²⁺ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca²⁺ influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca²⁺ transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca²⁺ elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca²⁺-induced ATP production in cardiac H9c2 myoblasts.