Abstract 18531: Adrenergic Stimulation Enhances Mitochondrial Ca 2 + Uptake and Cell Death Signaling Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca 2 + Uniporter

Abstract

Introduction: Mitochondrial Ca 2+ homeostasis is crucial for balancing cell survival and death. The recent breakthrough discovery of the molecular identity of the mitochondrial Ca 2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study the role of mitochondrial Ca 2+ regulation in cardiac cells. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca 2+ entry through post-translational modifications of MCU are completely unknown. Hypothesis: Persistent adrenergic signaling induces mitochondrial Ca 2+ overload and activates cell death signaling through MCU tyrosine phosphorylation. Methods: Tyrosine kinase activation and tyrosine phosphorylation of MCU were observed in rat neonatal cardiomyocytes and H9C2 cardiac myoblasts. Mitochondrial Ca 2+ and reactive oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca 2+ -sensitive inverse pericam and MitoSOX, respectively. Mitochondrial permeability transition pore (mPTP) activity was observed by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP, using confocal microscopy. Results: α 1 -adrenoceptor (α 1 -AR) signaling activates mitochondria-localized Ca 2+ -dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, persistent α 1 -AR stimulation increases ROS production, mPTP activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Moreover, pretreatment of a potent Pyk2 inhibitor PF-431396 or overexpression of dominant-negative mutant of MCU effectively inhibited α 1 -AR-mediated ROS generation and mPTP activation. Conclusion: The α 1 -AR-Pyk2 dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca 2+ entry and apoptosis in cardiac cells. Pyk2-MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca 2+ overload, oxidative stress and cardiac injury under pathophysiological states such as heart failure, where catecholamine levels highly increase.