Person:

Kalwa, Hermann

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).

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1null mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1null mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.