Abstract 18531: Adrenergic Stimulation Enhances Mitochondrial Ca2+ Uptake and Cell Death Signaling Through Pyk2-Dependent Tyrosine Phosphorylation of the Mitochondrial Ca2+ Uniporter
Introduction: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent breakthrough discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study the role of mitochondrial Ca2+ 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 Ca2+ entry through post-translational modifications of MCU are completely unknown.
Hypothesis: Persistent adrenergic signaling induces mitochondrial Ca2+ 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 Ca2+ and reactive oxygen species (ROS) were measured using mitochondrial matrix-targeted Ca2+-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: α1-adrenoceptor (α1-AR) signaling activates mitochondria-localized Ca2+-dependent tyrosine kinase named proline-rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, persistent α1-AR stimulation increases ROS production, mPTP activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Moreover, pretreatment of a potent Pyk2 inhibitor PF-431396 or overexpression of dominant-negative mutant of MCU effectively inhibited α1-AR-mediated ROS generation and mPTP activation.
Conclusion: The α1-AR-Pyk2 dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Pyk2-MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca2+ overload, oxidative stress and cardiac injury under pathophysiological states such as heart failure, where catecholamine levels highly increase.
- © 2013 by American Heart Association, Inc.