Person:

Michel, Thomas

Nitric oxide (NO) and hydrogen peroxide (H(_2)O(_2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(_2)O(_2)–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(_2)O(_2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(^{2+}) ([Ca(^{2+})](_i)), and document that activity of the L-type Ca(^{2+}) channel is necessary for the H(_2)O(_2)-promoted increase in sarcomere shortening and of [Ca(^{2+})](_i). Using the chemical NO sensor Cu(_2)(FL2E), we discovered that the H(_2)O(_2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(^{2+}) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(_2)O(_2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(^{2+})](_i) as well as the activation of protein kinase C (PKC). We also found that H(_2)O(_2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(^{2+}) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(_2)O(_2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(_2)O(_2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(^{2+})channel, but is independent of PKC. These studies establish key roles for Ca(^{2+})- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(_2)O(_2).