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Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.

Madhani M, Hall AR, Cuello F, Charles RL, Burgoyne JR, Fuller W, Hobbs AJ, Shattock MJ, Eaton P - Am. J. Physiol. Heart Circ. Physiol. (2010)

Bottom Line: PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated.The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive.Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.

ABSTRACT
The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na(+)/K(+)-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 muM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na(+)/K(+)-ATPase activation during reperfusion would attenuate Na(+) overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na(+)/K(+)-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

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A: the effect of sildenafil on Na+/K+-ATPase pump current (Ip). Isolated rat ventricular myocytes were superfused with solutions containing control vehicle or 0.1 μM sildenafil. The sildenafil treatment robustly activated the Na+/K+-ATPase. Bars represent means ± SE; n = 8 animals. *P < 0.05 vs. control (2-tailed t-test). The effect of sildenafil on phospholemman (PLM) phosphorylation at Ser63 (B), Ser68 (C), and Ser69 (D). Isolated mouse hearts were subjected to 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil at the onset of reperfusion. Heart samples were collected at the end of 10-min treatment for immunoblotting analysis. Representative blots are shown with quantitative analysis of repeat experiments. The dotted line on the immunoblots indicates that they were edited using computer software to present the control and sildenafil treatment samples directly adjacent to each other. The band intensities are, however, directly comparable as the sample pairs are from the same immunoblots and the data are normalized to the control levels. Sildenafil treatment selectively induced the phosphorylation of Ser69, whereas the two other sites remain unchanged. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA).
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Figure 4: A: the effect of sildenafil on Na+/K+-ATPase pump current (Ip). Isolated rat ventricular myocytes were superfused with solutions containing control vehicle or 0.1 μM sildenafil. The sildenafil treatment robustly activated the Na+/K+-ATPase. Bars represent means ± SE; n = 8 animals. *P < 0.05 vs. control (2-tailed t-test). The effect of sildenafil on phospholemman (PLM) phosphorylation at Ser63 (B), Ser68 (C), and Ser69 (D). Isolated mouse hearts were subjected to 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil at the onset of reperfusion. Heart samples were collected at the end of 10-min treatment for immunoblotting analysis. Representative blots are shown with quantitative analysis of repeat experiments. The dotted line on the immunoblots indicates that they were edited using computer software to present the control and sildenafil treatment samples directly adjacent to each other. The band intensities are, however, directly comparable as the sample pairs are from the same immunoblots and the data are normalized to the control levels. Sildenafil treatment selectively induced the phosphorylation of Ser69, whereas the two other sites remain unchanged. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA).

Mentions: Figure 4A shows that in isolated rat ventricular myocytes, 0.1 μM sildenafil (1.72 ± 0.08 pA/pF) significantly increased the Na+/K+-ATPase pump activity compared with controls (1.51 ± 0.09 pA/pF, P < 0.05, n = 8 preparations).


Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.

Madhani M, Hall AR, Cuello F, Charles RL, Burgoyne JR, Fuller W, Hobbs AJ, Shattock MJ, Eaton P - Am. J. Physiol. Heart Circ. Physiol. (2010)

A: the effect of sildenafil on Na+/K+-ATPase pump current (Ip). Isolated rat ventricular myocytes were superfused with solutions containing control vehicle or 0.1 μM sildenafil. The sildenafil treatment robustly activated the Na+/K+-ATPase. Bars represent means ± SE; n = 8 animals. *P < 0.05 vs. control (2-tailed t-test). The effect of sildenafil on phospholemman (PLM) phosphorylation at Ser63 (B), Ser68 (C), and Ser69 (D). Isolated mouse hearts were subjected to 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil at the onset of reperfusion. Heart samples were collected at the end of 10-min treatment for immunoblotting analysis. Representative blots are shown with quantitative analysis of repeat experiments. The dotted line on the immunoblots indicates that they were edited using computer software to present the control and sildenafil treatment samples directly adjacent to each other. The band intensities are, however, directly comparable as the sample pairs are from the same immunoblots and the data are normalized to the control levels. Sildenafil treatment selectively induced the phosphorylation of Ser69, whereas the two other sites remain unchanged. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944484&req=5

Figure 4: A: the effect of sildenafil on Na+/K+-ATPase pump current (Ip). Isolated rat ventricular myocytes were superfused with solutions containing control vehicle or 0.1 μM sildenafil. The sildenafil treatment robustly activated the Na+/K+-ATPase. Bars represent means ± SE; n = 8 animals. *P < 0.05 vs. control (2-tailed t-test). The effect of sildenafil on phospholemman (PLM) phosphorylation at Ser63 (B), Ser68 (C), and Ser69 (D). Isolated mouse hearts were subjected to 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil at the onset of reperfusion. Heart samples were collected at the end of 10-min treatment for immunoblotting analysis. Representative blots are shown with quantitative analysis of repeat experiments. The dotted line on the immunoblots indicates that they were edited using computer software to present the control and sildenafil treatment samples directly adjacent to each other. The band intensities are, however, directly comparable as the sample pairs are from the same immunoblots and the data are normalized to the control levels. Sildenafil treatment selectively induced the phosphorylation of Ser69, whereas the two other sites remain unchanged. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA).
Mentions: Figure 4A shows that in isolated rat ventricular myocytes, 0.1 μM sildenafil (1.72 ± 0.08 pA/pF) significantly increased the Na+/K+-ATPase pump activity compared with controls (1.51 ± 0.09 pA/pF, P < 0.05, n = 8 preparations).

Bottom Line: PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated.The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive.Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.

ABSTRACT
The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na(+)/K(+)-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 muM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na(+)/K(+)-ATPase activation during reperfusion would attenuate Na(+) overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na(+)/K(+)-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

Show MeSH
Related in: MedlinePlus