Abstract 093: Adrenergic Stimulation Induces Mitochondrial Fragmentation and Cell Injury through PKD1-dependent Phosphorylation of DLP1 in H9c2 Cardiac Myoblasts.

Abstract

Introduction: Regulation of mitochondrial morphology and dynamics is crucial for the maintenance of various cellular functions in cardiac myocytes. Abnormal mitochondrial morphologies concomitant with mitochondrial dysfunction are frequently observed in various pathophysiological states of human heart such as heart failure, where the catecholamine level is elevated. However, it is still unclear what kinds of cardiac signaling pathways regulate mitochondrial morphology and function under pathophysiological conditions. Hypothesis: Adrenergic signaling induces cardiac mitochondrial morphology changes and mitochondrial dysfunction, which simultaneously contribute to cardiac injury. Methods: H9c2 cardiac myoblasts were stimulated by α 1 -adrenoceptor (α 1 -AR) agonist phenylephrine and mitochondrial morphology was monitored by confocal microscopy. Translocation and phosphorylation of a mitochondrial fission protein, dynamin-like protein 1 (DLP1) was observed from whole cell lysates, cytosolic proteins and mitochondrial proteins by western blotting. Results: We found that persistent α 1 -AR stimulation induced mitochondrial fragmentation, followed by an increase in the production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria to the cytosol in H9c2 cardiac myoblasts. These effects were abolished by the treatment of α 1 -AR antagonist, prazosin. Further, mitochondrial fragmentation by α 1 -AR stimulation was inhibited by expression of the dominant-negative fission mutant DLP1-K38A, suggesting that the mitochondrial fission is required for mitochondrial fragmentation observed in α 1 -AR stimulation. We also found that DLP1 was translocated from cytosol to mitochondria under α 1 -AR stimulation. In addition, activation of protein kinase D1 (PKD1), a protein kinase downstream of α 1 -AR signaling, led to the phosphorylation of DLP1 at serine 637 which lies within a putative PKD phosphorylation consensus motif. Conclusion: α 1 -AR signaling induces mitochondrial fragmentation and cell injury, possibly through PKD1-dependent phosphorylation of DLP1.