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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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Related in: MedlinePlus

Experimental protocols implemented to assess infarction and signaling events that contribute to sildenafil-mediated protection via reduced Na+ loading during myocardial reperfusion. In all experiments, hearts were allowed to stabilize for 40 min and in relevant protocols were subjected to 30 min global ischemia and 120 min reperfusion. A: hearts were perfused with control vehicle or 0.01, 0.1, 1, or 10 μM sildenafil at the onset of reperfusion for the first 10 min, followed by perfusion with Krebs. B: hearts were perfused with control vehicle or 0.1 μM sildenafil for 10, 30, 60, 90, or 120 min. C: hearts were perfused with control vehicle or 0.1 μM sildenafil in the absence or presence of PKG inhibitor, KT-5823 (1 μM). D: hearts samples were collected following 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil in the absence or presence of 1 μM KT-5823 for protein analysis. E: protocols used to assess myocardial Na+/K+-ATPase activity. F: hearts were perfused with rubidium (Rb)-Krebs-Henseleit buffer (KHB) during aerobic perfusion or at reperfusion (with or without sildenafil) before a 0.5-min washout period with standard KHB to remove extracellular Rb. Each heart was then analyzed for its Rb content using flame photometry. TTC, triphenyltetrazolium chloride.
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Figure 1: Experimental protocols implemented to assess infarction and signaling events that contribute to sildenafil-mediated protection via reduced Na+ loading during myocardial reperfusion. In all experiments, hearts were allowed to stabilize for 40 min and in relevant protocols were subjected to 30 min global ischemia and 120 min reperfusion. A: hearts were perfused with control vehicle or 0.01, 0.1, 1, or 10 μM sildenafil at the onset of reperfusion for the first 10 min, followed by perfusion with Krebs. B: hearts were perfused with control vehicle or 0.1 μM sildenafil for 10, 30, 60, 90, or 120 min. C: hearts were perfused with control vehicle or 0.1 μM sildenafil in the absence or presence of PKG inhibitor, KT-5823 (1 μM). D: hearts samples were collected following 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil in the absence or presence of 1 μM KT-5823 for protein analysis. E: protocols used to assess myocardial Na+/K+-ATPase activity. F: hearts were perfused with rubidium (Rb)-Krebs-Henseleit buffer (KHB) during aerobic perfusion or at reperfusion (with or without sildenafil) before a 0.5-min washout period with standard KHB to remove extracellular Rb. Each heart was then analyzed for its Rb content using flame photometry. TTC, triphenyltetrazolium chloride.

Mentions: Specific isolated heart perfusion protocols were used to determine whether sildenafil is cardioprotective at reperfusion. Detailed protocol information is described below and in Fig. 1 (protocols A and B). Additional protocols were designed to investigate the potential role of the PKG activation (protocol C), natriuretic peptide receptor-A (NPR-A, protocol D), and Na+/K+-ATPase activity (protocol E) in sildenafil-induced protection (see Fig. 1).


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)

Experimental protocols implemented to assess infarction and signaling events that contribute to sildenafil-mediated protection via reduced Na+ loading during myocardial reperfusion. In all experiments, hearts were allowed to stabilize for 40 min and in relevant protocols were subjected to 30 min global ischemia and 120 min reperfusion. A: hearts were perfused with control vehicle or 0.01, 0.1, 1, or 10 μM sildenafil at the onset of reperfusion for the first 10 min, followed by perfusion with Krebs. B: hearts were perfused with control vehicle or 0.1 μM sildenafil for 10, 30, 60, 90, or 120 min. C: hearts were perfused with control vehicle or 0.1 μM sildenafil in the absence or presence of PKG inhibitor, KT-5823 (1 μM). D: hearts samples were collected following 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil in the absence or presence of 1 μM KT-5823 for protein analysis. E: protocols used to assess myocardial Na+/K+-ATPase activity. F: hearts were perfused with rubidium (Rb)-Krebs-Henseleit buffer (KHB) during aerobic perfusion or at reperfusion (with or without sildenafil) before a 0.5-min washout period with standard KHB to remove extracellular Rb. Each heart was then analyzed for its Rb content using flame photometry. TTC, triphenyltetrazolium chloride.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Experimental protocols implemented to assess infarction and signaling events that contribute to sildenafil-mediated protection via reduced Na+ loading during myocardial reperfusion. In all experiments, hearts were allowed to stabilize for 40 min and in relevant protocols were subjected to 30 min global ischemia and 120 min reperfusion. A: hearts were perfused with control vehicle or 0.01, 0.1, 1, or 10 μM sildenafil at the onset of reperfusion for the first 10 min, followed by perfusion with Krebs. B: hearts were perfused with control vehicle or 0.1 μM sildenafil for 10, 30, 60, 90, or 120 min. C: hearts were perfused with control vehicle or 0.1 μM sildenafil in the absence or presence of PKG inhibitor, KT-5823 (1 μM). D: hearts samples were collected following 30 min global ischemia and 10 min perfusion with control vehicle or 0.1 μM sildenafil in the absence or presence of 1 μM KT-5823 for protein analysis. E: protocols used to assess myocardial Na+/K+-ATPase activity. F: hearts were perfused with rubidium (Rb)-Krebs-Henseleit buffer (KHB) during aerobic perfusion or at reperfusion (with or without sildenafil) before a 0.5-min washout period with standard KHB to remove extracellular Rb. Each heart was then analyzed for its Rb content using flame photometry. TTC, triphenyltetrazolium chloride.
Mentions: Specific isolated heart perfusion protocols were used to determine whether sildenafil is cardioprotective at reperfusion. Detailed protocol information is described below and in Fig. 1 (protocols A and B). Additional protocols were designed to investigate the potential role of the PKG activation (protocol C), natriuretic peptide receptor-A (NPR-A, protocol D), and Na+/K+-ATPase activity (protocol E) in sildenafil-induced protection (see Fig. 1).

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