title: Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.
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.
authors.affiliation: Cardiovascular Division, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.
authors.forename: Melanie
authors.initials: M
authors.lastname: Madhani
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Hall
authors.forename: Friederike
authors.initials: F
authors.lastname: Cuello
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Charles
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Burgoyne
authors.forename: William
authors.initials: W
authors.lastname: Fuller
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Hobbs
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shattock
authors.forename: Philip
authors.initials: P
authors.lastname: Eaton
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002316:Cardiotonic Agents / Q000494:pharmacology; D002478:Cells, Cultured; D006321:Heart / Q000187:drug effects*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology*; D010879:Piperazines / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology; D015427:Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control*; D015398:Signal Transduction / Q000187:drug effects; D000068677:Sildenafil Citrate; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology*
chemical_list: D002316:Cardiotonic Agents; D008565:Membrane Proteins; D010750:Phosphoproteins; D010879:Piperazines; D011687:Purines; D013450:Sulfones; C069141:phospholemman; D000068677:Sildenafil Citrate; D000254:Sodium-Potassium-Exchanging ATPase