Abstract 13033: C-src Phosphorylates the Mitochondrial Ca 2+ Uniporter in Nonischemic Cardiomyopathy

Abstract

Introduction: We have described previously an arrhythmic mechanism in nonischemic heart failure (NI-HF) involving an increased mitochondrial Ca 2+ uptake that results in QT prolongation and lethal arrhythmias. This arrhythmic mechanism is associated with mitochondrial Ca 2+ uniporter (MCU) tyrosine phosphorylation (p-Tyr). Hypothesis: Here, we determined which kinase was responsible for MCU phosphorylation in cardiomyopathy. Methods: NI-HF was induced by hypertension in Wt C57BL6 mice by unilateral nephrectomy, deoxycorticosterone acetate (DOCA) treatment and substituting drinking water with 1% saline for six weeks. Western blot, immunoprecipitation, fluorescence resonance energy transfer (FRET) and patch-clamp techniques were employed in isolated mouse cardiomyocytes. Results: When compared to control hearts, the protein level of p-Src S17 increased significantly accompanied by significant enhancement of tyrosine but not serine/threonine phosphorylation of MCU in NI-HF mouse hearts. In a heterologous expression system, c-Src could bind MCU and tyrosine phosphorylate MCU. This phosphorylation was blocked by a c-Src inhibitor, PP1. Overexpression of constitutive active c-Src (Src-Y527F) significantly increased MCU inward current at -160 mV. It suggested that c-Src activation was directly related to mitochondrial-mediated arrhythmic risk. Downregulation of C-terminal Src kinase (CSK), a regulatory kinase inversely related to c-Src activity, increased p-Src, enhanced mitochondrial and SR contact, and facilitated mitochondrial Ca 2+ uptake in NI-HF mouse cardiomyocytes. Proline-rich tyrosine kinase 2 (Pyk2) did not contribute more phosphorylated MCU during cardiomyopathy as the protein expression of PyK2, p-PyK2 Y402 and p-PyK2 Y579/580 did not change during NI-HF. Correlation using human heart tissue showed that NI-HF patients had significantly increased p-Src S17 associated with an increased incidence of QTc elongation and arrhythmia. Conclusions: During cardiomyopathy, c-Src activation directly tyrosine phosphorylated MCU, increased MCU current, and increased mitochondrial/SR contact, suggesting this kinase is responsible for the increase in mitochondria-mediated arrhythmic risk seen in NI-HF.