Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation.

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H O resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca influx phosphorylated Ca -calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H O -treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.