Role of Ca²⁺ in the Control of H₂O₂-Modulated Phosphorylation Pathways Leading to eNOS Activation in Cardiac Myocytes

Nitric oxide (NO) and hydrogen peroxide (H₂O₂) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H₂O₂-modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H₂O₂ treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca²⁺ ([Ca²⁺]_i), and document that activity of the L-type Ca²⁺ channel is necessary for the H₂O₂-promoted increase in sarcomere shortening and of [Ca²⁺]_i. Using the chemical NO sensor Cu₂(FL2E), we discovered that the H₂O₂-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca²⁺ channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H₂O₂-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca²⁺]_i as well as the activation of protein kinase C (PKC). We also found that H₂O₂-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca²⁺ channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H₂O₂-promoted eNOS phosphorylation. Here we present evidence documenting that H₂O₂-promoted Akt phosphorylation is dependent on activation of the L-type Ca²⁺ channel, but is independent of PKC. These studies establish key roles for Ca²⁺- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H₂O₂.