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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 different concentrations of sildenafil (0.01–10 μM) at reperfusion on infarct size. Mouse hearts were subjected to 30 min global ischemia and 120 min of reperfusion. Bars represent means ± SE; n = 4–9 animals. *P < 0.001 vs. control (1-way ANOVA). B: the time dependence of protection by 0.1 μM sildenafil at reperfusion, assessing 10, 30, 60, or 120 min. Bars represent means ± SE; n = 5–11 animals. ***P < 0.001 vs. control; **P < 0.01 vs. control; *P < 0.05 vs. control (1-way ANOVA). Sildenafil administration beyond 10 min did not further attenuate infarction. C: the effect of 0.1 μM sildenafil administered at reperfusion on myocardial infarction in wild-type (WT) or natriuretic peptide receptor-A (NPR-A)  tissue following ischemia and reperfusion. Sildenafil (0.1 μM) was given at the onset of reperfusion for the first 10 min and protected both WT and NPR-A  hearts. Bars represent means ± SE; n = 5–13 animals. *P < 0.05 vs. control (1-way ANOVA). Infarction has been measured as infarction volume as a percentage of total myocardial volume. KO, knockout.
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Figure 2: A: the effect of different concentrations of sildenafil (0.01–10 μM) at reperfusion on infarct size. Mouse hearts were subjected to 30 min global ischemia and 120 min of reperfusion. Bars represent means ± SE; n = 4–9 animals. *P < 0.001 vs. control (1-way ANOVA). B: the time dependence of protection by 0.1 μM sildenafil at reperfusion, assessing 10, 30, 60, or 120 min. Bars represent means ± SE; n = 5–11 animals. ***P < 0.001 vs. control; **P < 0.01 vs. control; *P < 0.05 vs. control (1-way ANOVA). Sildenafil administration beyond 10 min did not further attenuate infarction. C: the effect of 0.1 μM sildenafil administered at reperfusion on myocardial infarction in wild-type (WT) or natriuretic peptide receptor-A (NPR-A) tissue following ischemia and reperfusion. Sildenafil (0.1 μM) was given at the onset of reperfusion for the first 10 min and protected both WT and NPR-A hearts. Bars represent means ± SE; n = 5–13 animals. *P < 0.05 vs. control (1-way ANOVA). Infarction has been measured as infarction volume as a percentage of total myocardial volume. KO, knockout.

Mentions: Figure 2A shows that infarct size (expressed as the percentage of area at risk) was 50.5 ± 2.5% under control conditions. Treatment with 0.1 μM sildenafil during the first 10 min of reperfusion significantly reduced infarct size (33.65 ± 3.61%; P < 0.001), but lower (0.01 μM) or higher concentrations (1 or 10 μM) during the first 10 min of reperfusion did not protect the heart (n = 4–9).


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 different concentrations of sildenafil (0.01–10 μM) at reperfusion on infarct size. Mouse hearts were subjected to 30 min global ischemia and 120 min of reperfusion. Bars represent means ± SE; n = 4–9 animals. *P < 0.001 vs. control (1-way ANOVA). B: the time dependence of protection by 0.1 μM sildenafil at reperfusion, assessing 10, 30, 60, or 120 min. Bars represent means ± SE; n = 5–11 animals. ***P < 0.001 vs. control; **P < 0.01 vs. control; *P < 0.05 vs. control (1-way ANOVA). Sildenafil administration beyond 10 min did not further attenuate infarction. C: the effect of 0.1 μM sildenafil administered at reperfusion on myocardial infarction in wild-type (WT) or natriuretic peptide receptor-A (NPR-A)  tissue following ischemia and reperfusion. Sildenafil (0.1 μM) was given at the onset of reperfusion for the first 10 min and protected both WT and NPR-A  hearts. Bars represent means ± SE; n = 5–13 animals. *P < 0.05 vs. control (1-way ANOVA). Infarction has been measured as infarction volume as a percentage of total myocardial volume. KO, knockout.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: A: the effect of different concentrations of sildenafil (0.01–10 μM) at reperfusion on infarct size. Mouse hearts were subjected to 30 min global ischemia and 120 min of reperfusion. Bars represent means ± SE; n = 4–9 animals. *P < 0.001 vs. control (1-way ANOVA). B: the time dependence of protection by 0.1 μM sildenafil at reperfusion, assessing 10, 30, 60, or 120 min. Bars represent means ± SE; n = 5–11 animals. ***P < 0.001 vs. control; **P < 0.01 vs. control; *P < 0.05 vs. control (1-way ANOVA). Sildenafil administration beyond 10 min did not further attenuate infarction. C: the effect of 0.1 μM sildenafil administered at reperfusion on myocardial infarction in wild-type (WT) or natriuretic peptide receptor-A (NPR-A) tissue following ischemia and reperfusion. Sildenafil (0.1 μM) was given at the onset of reperfusion for the first 10 min and protected both WT and NPR-A hearts. Bars represent means ± SE; n = 5–13 animals. *P < 0.05 vs. control (1-way ANOVA). Infarction has been measured as infarction volume as a percentage of total myocardial volume. KO, knockout.
Mentions: Figure 2A shows that infarct size (expressed as the percentage of area at risk) was 50.5 ± 2.5% under control conditions. Treatment with 0.1 μM sildenafil during the first 10 min of reperfusion significantly reduced infarct size (33.65 ± 3.61%; P < 0.001), but lower (0.01 μM) or higher concentrations (1 or 10 μM) during the first 10 min of reperfusion did not protect the heart (n = 4–9).

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