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

Abstract

ϩ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca 2ϩ influx is mainly mediated by the mitochondrial Ca 2ϩ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca 2ϩ 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 Ca 2ϩ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ -induced ATP production in cardiac H9c2 myoblasts. fluorescence resonance energy transfer; mitochondrial Ca 2ϩ uniporter; mitochondria; mitochondrial morphology; ryanodine receptor type 1 MITOCHONDRIAL Ca 2ϩ is critical for the regulation of various cellular functions, including energy metabolism, ROS generation, spatiotemporal dynamics of Ca 2ϩ signaling, and cell growth/development and death (11,18,23). Historically, Ca 2ϩ was found to be accumulated by mitochondria over 5 decades ago (for reviews, see Refs. 24, 64, and 69), and, shortly thereafter, it was also recognized that Ca 2ϩ stimulates the oxidative phosphorylation [tricarboxylic acid (TCA) cycle] and electron transport chain activity, which results in the stimulation of ATP synthesis (for a review, see Ref. 23). Additionally, the coexistence of mitochondrial dysfunction and loss of cellular Ca 2ϩ homeostasis are frequently observed in various cardiovascular diseases, but it is still not clear how altered mitochondrial Ca 2ϩ handing and/or mitochondrial dysfunction are involved in the pathogenesis of each different disease setting (23,33,56).Although the basic functional and pharmacological properties of mitochondrial ...

Adrenergic Signaling Regulates Mitochondrial Ca2+ Uptake Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter

“…This study is the first to show the modulation of MCU by adrenergic signaling and the regulation of ROS generation and cell death signaling via a posttranslational modification of MCU in cardiac cell. (39,53), in intact cardiac H9c2 myoblasts ( Fig. 1).…”

“…H9c2 cells and HEK293T cells were maintained, transfected with plasmids, and used for experiments (37,53,54). HEK293T-MCU-Flag cells were generated by transecting with pcDNA3.1(+ )-MCU-Flag and maintained in the presence of G-418 (53).…”

“…HEK293T-MCU-Flag cells were generated by transecting with pcDNA3.1(+ )-MCU-Flag and maintained in the presence of G-418 (53). NCMs and ACMs were isolated from Sprague-Dawley rats, plated, cultured, and used for experiments (37).…”

“…Mitochondrial protein isolation, Western immunoblot analysis, in vitro kinase, and binding assay Whole cell lysates and mitochondria-enriched and cytosolic proteins were prepared (53). For electrophoresis under nondissociating conditions, mitochondrial proteins or whole cell lysates were separated by 10% polyacrylamide gel (Native-PAGE) (63).…”

Molecular and functional identification of a mitochondrial ryanodine receptor in neurons

“…Since cardiac mRyR exhibits a bell-shaped Ca 2+ -dependent activation (bimodal activation) in the physiological range of [Ca 2+ ] c , this unique property places mRyR as an ideal candidate for sequestering Ca 2+ quickly and transiently during physiological [Ca 2+ ] c oscillation in excitable cells. In addition, using not only native cardiomyocytes, but also RyR overexpression/knock-out in cultured cardiac myoblasts and knock-out mouse hearts, we showed that the molecular identity of mRyR is possibly a skeletal-muscle type-isoform RyR type 1 (RyR1) and is required for Ca 2+ -dependent acceleration of ATP production in cardiomyocytes even though the expression level is much lower than RyR2 which is the main RyR isoform expressed in cardiac sarcoplasmic reticulum (SR) /endoplasmic reticulum (ER) [3,23]. …”