Role of Ca[superscript 2+] in the Control of H[subscript 2]O[subscript 2-]Modulated Phosphorylation Pathways Leading to eNOS Activation in Cardiac Myocytes

Nitric oxide (NO) and hydrogen peroxide (H[subscript 2]O[subscript 2]) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H[subscript 2]O[subscript 2]–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H[subscript 2]O[subscript 2] treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca[superscript 2+] ([Ca[superscript 2+]][superscript i]), and document that activity of the L-type Ca[superscript 2+] channel is necessary for the H2O2-promoted increase in sarcomere shortening and of [Ca[superscript 2+]][superscript i]. Using the chemical NO sensor Cu[subscript 2](FL2E), we discovered that the H[subscript 2]O[subscript 2]-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca[superscript 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[subscript 2]O[subscript 2]-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca[superscript 2+]]i as well as the activation of protein kinase C (PKC). We also found that H[subscript 2]O[subscript 2]-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca[superscript 2+] channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H[subscript 2]O[subscript 2]-promoted eNOS phosphorylation. Here we present evidence documenting that H[subscript 2]O[subscript 2]-promoted Akt phosphorylation is dependent on activation of the L-type Ca[superscript 2+] channel, but is independent of PKC. These studies establish key roles for Ca[superscript 2+]- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H[subscript 2]O[subscript 2].National Science Foundation (U.S.) (Grant NSF CHE-0907905)National Institutes of Health (U.S.) (Grant K99GM092970

Effects of calphostin C on H₂O₂-promoted eNOS phosphorylation.

<p>In panel A, cardiac myocytes were incubated with nifedipine (100 µM, 30 min) or vehicle, then treated with H₂O₂ (25 µM) and analyzed in immunoblots probed with phospho-protein kinase C (PKC) phosphorylation (βII Ser660) or GAPDH antibodies. Panel B shows a representative experiment looking at the effects of calphostin C on H₂O₂-promoted eNOS phosphorylation. Freshly isolated adult murine cardiac myocytes were treated with calphostin C (1 µM, 30 min) or vehicle before treatment with H₂O₂ (25 µM, 15 min). Cell lysates were resolved by SDS-PAGE and probed using antibodies directed against phospho-eNOS Ser1177, phospho-eNOS Ser633, total eNOS, phospho-PKC, or GAPDH. Densitometric analyses from pooled data, plotting the fold increase of the degree of protein phosphorylation (in arbitrary units) relative to the signals present in unstimulated cardiac myocytes are also shown in this figure. Each data point represents the mean ± S.E. derived from three independent experiments, *indicates p<0.05 for respective phospho-protein versus unstimulated cells (<i>ANOVA</i>).</p

Effect of protein kinase A (PKA) inhibitor on H₂O₂-promoted eNOS phosphorylation.

<p>In panel A, cardiac myocytes were incubated with H89 (1 µM, 30 min) or vehicle, then treated with H₂O₂ (25 µM, 15 min) and analyzed in immunoblots probed with antibodies as shown. Below each representative immunoblot are shown the results of densitometric analyses from pooled data, documenting the changes in phospho-eNOS (Ser1177), and phospho-VASP (Ser157) plotted relative to the signals present in unstimulated cells. Each data point represents the mean ± S.E. derived from at least three independent experiments; *indicates p<0.05 (<i>ANOVA</i>). Panel B shows representative immunoblots from experiments documenting the effects of A23187 (40 µM, 5 min) on cardiac myocyte protein phosphorylation responses. Panel C shows the results of immunoblots analyzed in lysates prepared from cells treated with phorbol 12-myristate 13-acetate (10 µM, 15 min). Cell lysates were analyzed in immunoblots probed with antibodies as indicated. The immunoblot images shown are representative of three independent experiments that yielded similar results. Below each immunoblot panel are the results of densitometric analyses from pooled data, showing the fold increase in protein phosphorylation (in arbitrary units), *indicates p<0.05.</p

Nifedipine effects on H₂O₂-promoted NO synthesis, eNOS phosphorylation, and eNOS translocation.

<p>In Panel A, mouse cardiac myocytes were loaded with the NO chemical sensor Cu₂(FL2E), and then treated with nifedipine (100 µM) or vehicle followed by hydrogen peroxide (H₂O₂, 10 µM) treatment. Upper panel shows representative fluorescence images at 0, 2, and 5 minutes followed treatments as indicated. Middle panel shows representative fluorescence tracings of single cells treated with H₂O₂ or H₂O₂ in the presence of nifedipine. Lower panel shows the results of pooled data analyzed from at least three independent repetitions with a minimum of 4 cells analyzed per experiment that yielded equivalent results; *indicates p<0.05. In Panel B, cardiac myocytes were incubated with nifedipine (100 µM, 30 min) or vehicle, then treated with hydrogen peroxide (H₂O₂, 25 µM, 15 min) and analyzed in immunoblots probed with antibodies as shown. Panel C shows immunoblot analyses from cardiac myocytes incubated with the intracellular calcium chelator BAPTA AM (60 µM, 30 min) or vehicle, then treated with H₂O₂ (25 µM, 15 min). Below each representative immunoblot the results of densitometric analyses from pooled data are shown, documenting the changes in phospho-eNOS1177 and phospho-eNOS633 plotted relative to the signals present in unstimulated cells. Each data point represents the mean ± S.E. derived from at least three independent experiments; *indicates p<0.05 (<i>ANOVA</i>). Panel D shows confocal microscopic images of cardiac myocytes treated with nifedipine (100 µM, 30 min) or vehicle, then treated with H₂O₂ (10 µM) for the indicated times. The cells were fixed, permeabilized, and probed with antibodies against total caveolin-3 (Alexa Fluor-Red 568) or eNOS (Alexa Fluor-Green 488); overlapping signals are shown in yellow. The bar graph below shows pooled data from three experiments, quantitating the percent overlap between eNOS and caveolin-3 at different times after adding H₂O₂. *indicates p<0.05 compared to t = 0.</p

Pathways controlling H₂O₂-promoted phosphorylation of MEK/ERK 1/2 and Akt.

<p>In panel A, cardiac myocytes were incubated with nifedipine (100 µM, 30 min) or vehicle, then treated with hydrogen peroxide (H₂O₂, 25 µM, 15 min) and analyzed in immunoblots probed with antibodies as shown. Panel B shows immunoblot analyses from cardiac myocytes incubated with BAPTA AM (60 µM, 30 min) or vehicle, then treated with H₂O₂. Panel C shows cardiac myocytes treated with calphostin C (1 µM, 30 min) prior treatment with H₂O₂. Below each representative immunoblot are shown the results of densitometric analyses from pooled data, documenting the changes in phospho-MEK1/2 (Ser217/221), phospho-ERK1/2 (Thr202/Tyr204) (left panels), and phospho-Akt Ser473 (right panels) plotted relative to the signal present in unstimulated cells. Each data point represents the mean ± S.E. derived from at least three independent experiments (n = 4 for nifedipine, 3 for BAPTA and 6 for Calphostin C); *indicates p<0.05; **indicates p<0.01 (<i>ANOVA</i>).</p

Intersections of kinase pathways and [Ca²⁺]_i in control of H₂O₂-promoted eNOS responses.

<p>In panel A, adult mouse cardiac myocytes were loaded with the NO dye Cu₂(FL2E), and then treated with the AMPK inhibitor Compound C (1 µM) or vehicle followed by hydrogen peroxide (H₂O₂, 10 µM) treatment. Upper panel shows representative fluorescence images at 0, 2, and 5 minutes followed treatments as indicated. Middle panel shows representative fluorescence tracings of a cell treated with H₂O₂ or H₂O₂ in the presence of compound C. Lower panel shows the results of pooled data analyzed from at least three independent repetitions with a minimum of 4 cells analyzed per experiment that yielded equivalent results; *indicates p<0.05. Panels B, C, and D show representative experiments analyzing the effects of nifedipine (100 µM), BAPTA AM, (60 µM), or calphostin C (1 µM), on H₂O₂-promoted AMPK or ACC phosphorylation. Cardiac myocytes were pre-incubated with these compounds for 30 min, then treated with H₂O₂ (25 µM, 30 min) and analyzed in immunoblots probed with phospho-AMPK Thr172, phospho-ACC Ser79, AMPK, or ACC antibodies, as shown. Each data point represents the mean ± S.E. derived from at least three independent experiments; *indicates p<0.05 (<i>ANOVA</i>).</p

Scheme for H₂O₂-mediated regulation of eNOS signaling in cardiac myocytes.

<p>In this model, H₂O₂ activates the L-type Ca²⁺ channel (LTCC), causing an elevation in [Ca²⁺]_i. The increase in [Ca²⁺]_i promotes phosphorylation and activation of protein kinases PKC and Akt, which lead to an increase in eNOS phosphorylation. Activation of PKC is required for the phosphorylation of MEK1/2, ERK1/2, and AMPK, which in turn promote eNOS phosphorylation and an increase in NO synthesis. See the text for additional discussion.</p

Redox activation of Nox1 (NADPH Oxidase 1) involves an intermolecular disulfide bond between protein disulfide isomerase and p47phox in vascular smooth muscle cells

Objective— PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results— Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47phox and PDI and an intramolecular bond between Cys 196 and 378 in p47phox. PDI catalytic Cys 400 and p47phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47phox, whereas PDI mutant did not promote this effect. Mutation of p47phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47phox occurred only in PDI family members with the a′ redox active site. Conclusions— PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular diseas