Abstract 150: FAK/Pyk2 Inhibitor Prevents Mitochondrial Ca 2 + Overload and Cardiac Injury during Adrenergic Stimulation.

Abstract

Introduction: Proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are abundantly expressed in cancer cells. In addition, these kinases are the potent therapeutic targets for cancer treatment and currently several selective FAK/Pyk2 inhibitors are in clinical trials. Recent study revealed that Pyk2 is also highly expressed in heart tissue and significantly activated during human heart failure. We have recently reported that α 1 -adrenoceptor (α 1 -AR) stimulation accelerates mitochondrial Ca 2+ uptake through Pyk2-dependent phospholylation of mitochondrial Ca 2+ uniporter (MCU). However, the roles of Pyk2 in cardiac mitochondrial physiology and pathophysiology have not been well established. Hypothesis: Persistent adrenergic signaling activates cell death signaling via Pyk2-dependent MCU activation and mitochondrial Ca 2+ overload. Methods: Using 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 measuring the amount of cytochrome c in cytosol by Western blotting or by monitoring the release of GFP-tagged mitochondrial protein, Smac-GFP using confocal microscopy. Results: Pyk2 was not only expressed in cytosol, but also in cardiac mitochondria. α 1 -AR agonist phenylephrine activated mitochondrial Pyk2 and enhanced mitochondrial Ca 2+ uptake via Pyk2-dependent MCU phosphorylation. In addition, persistent α 1 -AR stimulation increases ROS, activity of mPTP. These effects were abolished by co-expression of dominant-negative MCU or kinase-dead Pyk2, suggesting that Pyk2-dependent MCU activation followed by mitochondrial Ca 2+ overload are critical for this mechanism. Moreover, pretreatment of a potent FAK/Pyk2 inhibitor PF-431396 also effectively inhibited α 1 -AR-mediated ROS generation and mPTP activation. Conclusion: FAK/Pyk2 inhibitor prevents mitochondrial Ca 2+ overload, oxidative stress and mitochondrial injury under persistent adrenergic stimulation. Thus, Pyk2 may become a novel potent therapeutic target for preventing cardiac cell injury and death during heart failure.