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Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes.

ABSTRACT: 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).

SUBMITTER: Sartoretto JL

PROVIDER: S-EPMC3435284 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes.

Sartoretto Juliano L JL Kalwa Hermann H Shiroto Takashi T Sartoretto Simone M SM Pluth Michael D MD Lippard Stephen J SJ Michel Thomas T

PloS one 20120906 9

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 necessa ...[more]

PMID: 22970272

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Project description:RATIONALE:Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE:To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS:Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS:Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Dataset Information

Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes.

Publications

Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes.

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