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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: whole heart cGMP levels. Isolated mouse hearts were subjected to 30 min global ischemia followed by 10 min reperfusion with control vehicle or the cardioprotective 0.1 μM sildenafil treatment. cGMP analysis showed that the cyclic nucleotide levels were similar in both groups. Bars represent means ± SE; n = 6 animals; not significantly different when compared with controls. B: the effect of 0.1 μM sildenafil at reperfusion in the absence or presence of the PKG inhibitor KT-5823 on infarct size. Infarction has been measured as a percentage of total myocardial volume. PKG inhibition blocked the sildenafil-dependent protection from infarct. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA); **P < 0.01.
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Figure 3: A: whole heart cGMP levels. Isolated mouse hearts were subjected to 30 min global ischemia followed by 10 min reperfusion with control vehicle or the cardioprotective 0.1 μM sildenafil treatment. cGMP analysis showed that the cyclic nucleotide levels were similar in both groups. Bars represent means ± SE; n = 6 animals; not significantly different when compared with controls. B: the effect of 0.1 μM sildenafil at reperfusion in the absence or presence of the PKG inhibitor KT-5823 on infarct size. Infarction has been measured as a percentage of total myocardial volume. PKG inhibition blocked the sildenafil-dependent protection from infarct. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA); **P < 0.01.

Mentions: Hearts treated with control vehicle or sildenafil (0.1 μM) for 10 min at the onset of reperfusion had been snap frozen for the assessment of myocardial cGMP. Figure 3A shows that sildenafil treatment did not alter myocardial cGMP levels. The hearts receiving vehicle contained 31.17 pmol/mg tissue cGMP, whereas those treated with sildenafil contained 32.44 pmol/mg tissue (n = 6; P = not significant).


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: whole heart cGMP levels. Isolated mouse hearts were subjected to 30 min global ischemia followed by 10 min reperfusion with control vehicle or the cardioprotective 0.1 μM sildenafil treatment. cGMP analysis showed that the cyclic nucleotide levels were similar in both groups. Bars represent means ± SE; n = 6 animals; not significantly different when compared with controls. B: the effect of 0.1 μM sildenafil at reperfusion in the absence or presence of the PKG inhibitor KT-5823 on infarct size. Infarction has been measured as a percentage of total myocardial volume. PKG inhibition blocked the sildenafil-dependent protection from infarct. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA); **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: A: whole heart cGMP levels. Isolated mouse hearts were subjected to 30 min global ischemia followed by 10 min reperfusion with control vehicle or the cardioprotective 0.1 μM sildenafil treatment. cGMP analysis showed that the cyclic nucleotide levels were similar in both groups. Bars represent means ± SE; n = 6 animals; not significantly different when compared with controls. B: the effect of 0.1 μM sildenafil at reperfusion in the absence or presence of the PKG inhibitor KT-5823 on infarct size. Infarction has been measured as a percentage of total myocardial volume. PKG inhibition blocked the sildenafil-dependent protection from infarct. Bars represent means ± SE; n = 3–5 animals. *P < 0.05 vs. control (1-way ANOVA); **P < 0.01.
Mentions: Hearts treated with control vehicle or sildenafil (0.1 μM) for 10 min at the onset of reperfusion had been snap frozen for the assessment of myocardial cGMP. Figure 3A shows that sildenafil treatment did not alter myocardial cGMP levels. The hearts receiving vehicle contained 31.17 pmol/mg tissue cGMP, whereas those treated with sildenafil contained 32.44 pmol/mg tissue (n = 6; P = not significant).

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