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

Juliano L Sartoretto,Simone M Sartoretto,Takashi Shiroto,Hermann Kalwa,Michael D Pluth,Thomas Michel,Stephen J Lippard,Gianfranco Pintus

doi:10.1371/journal.pone.0044627

Juliano L Sartoretto, Simone M Sartoretto + Show 6 more

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https://doi.org/10.1371/journal.pone.0044627

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Abstract

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.

Highlights

The endothelial isoform of nitric oxide synthase is robustly expressed in cardiac myocytes, and nitric oxide (NO) has been shown to play key roles in modulating cardiac function [1,2,3]. eNOS is a Ca2+/calmodulin-dependent enzyme that undergoes phosphorylation on multiple residues in response to extracellular stimuli, involving several protein kinases and phosphoprotein phosphatases
H2O2-promoted eNOS phosphorylation at ser1177 is blocked by this same protein kinase (PKA) inhibitor (Figure 7A). Both calcium ionophore A23187 and the protein kinase C (PKC) agonist phorbol 12myristate 13-acetate promote phosphorylation responses in these cells (Figure 7B and C). These studies have used a combination of cellular imaging and biochemical approaches to explore eNOS activation and phosphorylation pathways in isolated mouse cardiac myocytes treated with H2O2
It is unlikely that the short-term exposure to low concentrations of H2O2 used in the present study cause cardiac myocyte membrane damage (Figure S1F); instead, our findings suggest a physiological role for H2O2 in the modulation of myocyte L-type Ca2+ channels

Summary

Introduction

The endothelial isoform of nitric oxide synthase (eNOS) is robustly expressed in cardiac myocytes, and nitric oxide (NO) has been shown to play key roles in modulating cardiac function [1,2,3]. eNOS is a Ca2+/calmodulin-dependent enzyme that undergoes phosphorylation on multiple residues in response to extracellular stimuli, involving several protein kinases and phosphoprotein phosphatases. The role of H2O2 in modulation of cardiac myocyte Ca2+ metabolism is less well understood, and there are major gaps in our understanding of the pathways connecting H2O2–dependent phosphorylation pathways, intracellular Ca2+ signaling, and eNOS activation. Since abnormalities in PKC-modulated signaling pathways and alterations in intracellular Ca2+ metabolism have been implicated in cardiomyopathy and heart failure [4,5,6], we decided to explore the role of H2O2 in control of PKC activation, intracellular Ca2+ pathways, and eNOS phosphorylation responses in cardiac myocytes. We provide data that establish roles for Ca2+, PKC and PKA in modulating eNOS phosphorylation in response to H2O2, and identify the key protein kinase pathways that modulate H2O2–dependent NO synthesis in cardiac myocytes

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