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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 ability of PKG or PKC to phosphorylate a recombinant PLM peptide in vitro was assessed using WT or a T69A (which cannot be phosphorylated at that site) recombinant peptide. Dot blots were probed with an anti-PLM CP69 antibody to index phosphorylation. The T69A serves as a control that shows the phosphospecific antibody only recognizes modification of Ser/Thr69, and does not cross-react with other sites. Whilst PKC efficiently phosphorylated the PLM peptide, PKG did not. From previous HPLC studies (not shown), we know that the PKC-dependent signal represents a stoichiometric phosphorylation of the PLM peptide. Arb units, arbitrary units. B: the effect of PKC inhibition on PLM Thr69 phosphorylation in cultured adult rat ventricular myocytes. Sildenafil or 8-bromo-cGMP each increased PLM Thr69 phosphorylation, and this was robustly blocked by the PKC inhibitor bisindolylmaleimide (Bis). OD, optical density; Ab units, arbitrary units. Representative blots are shown with quantitative analysis of repeat experiments. Bars represent means ± SE; n = 3–5 experiments. *P < 0.05 vs. control (1-way ANOVA).
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Figure 6: A: the ability of PKG or PKC to phosphorylate a recombinant PLM peptide in vitro was assessed using WT or a T69A (which cannot be phosphorylated at that site) recombinant peptide. Dot blots were probed with an anti-PLM CP69 antibody to index phosphorylation. The T69A serves as a control that shows the phosphospecific antibody only recognizes modification of Ser/Thr69, and does not cross-react with other sites. Whilst PKC efficiently phosphorylated the PLM peptide, PKG did not. From previous HPLC studies (not shown), we know that the PKC-dependent signal represents a stoichiometric phosphorylation of the PLM peptide. Arb units, arbitrary units. B: the effect of PKC inhibition on PLM Thr69 phosphorylation in cultured adult rat ventricular myocytes. Sildenafil or 8-bromo-cGMP each increased PLM Thr69 phosphorylation, and this was robustly blocked by the PKC inhibitor bisindolylmaleimide (Bis). OD, optical density; Ab units, arbitrary units. Representative blots are shown with quantitative analysis of repeat experiments. Bars represent means ± SE; n = 3–5 experiments. *P < 0.05 vs. control (1-way ANOVA).

Mentions: To investigate the signaling pathways involved in sildenafil-dependent cardioprotection, we assessed the ability of PKG and PKCε to directly phosphorylate PLM Thr69 using an in vitro kinase assay. Figure 6A shows that PKCε efficiently phosphorylated WT (but not mutant T69A) PLM at Thr69, as detected in dot blots probed with an anti-phospho-Thr/Ser69 antibody. Indeed, in separate experiments (not shown) described previously (13), this level of phosphorylation represents a complete stoichiometric modification of PLM. This allows an absolute quantitative comparison of the ability of PKG to phosphorylate PLM compared with PKC. Indeed, while PKG does not phosphorylate the T69A mutant peptide at all, it similarly does not phosphorylate the WT protein efficiently (n = 3). This suggests that PKG, despite the evidence presented from the Langendorff and ARVM studies for an upstream role in cardioprotection induced by sildenafil, is unlikely to be the kinase that directly modifies PLM during sildenafil or other stimuli that culminate in Thr/Ser69 phosphorylation.


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 ability of PKG or PKC to phosphorylate a recombinant PLM peptide in vitro was assessed using WT or a T69A (which cannot be phosphorylated at that site) recombinant peptide. Dot blots were probed with an anti-PLM CP69 antibody to index phosphorylation. The T69A serves as a control that shows the phosphospecific antibody only recognizes modification of Ser/Thr69, and does not cross-react with other sites. Whilst PKC efficiently phosphorylated the PLM peptide, PKG did not. From previous HPLC studies (not shown), we know that the PKC-dependent signal represents a stoichiometric phosphorylation of the PLM peptide. Arb units, arbitrary units. B: the effect of PKC inhibition on PLM Thr69 phosphorylation in cultured adult rat ventricular myocytes. Sildenafil or 8-bromo-cGMP each increased PLM Thr69 phosphorylation, and this was robustly blocked by the PKC inhibitor bisindolylmaleimide (Bis). OD, optical density; Ab units, arbitrary units. Representative blots are shown with quantitative analysis of repeat experiments. Bars represent means ± SE; n = 3–5 experiments. *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 6: A: the ability of PKG or PKC to phosphorylate a recombinant PLM peptide in vitro was assessed using WT or a T69A (which cannot be phosphorylated at that site) recombinant peptide. Dot blots were probed with an anti-PLM CP69 antibody to index phosphorylation. The T69A serves as a control that shows the phosphospecific antibody only recognizes modification of Ser/Thr69, and does not cross-react with other sites. Whilst PKC efficiently phosphorylated the PLM peptide, PKG did not. From previous HPLC studies (not shown), we know that the PKC-dependent signal represents a stoichiometric phosphorylation of the PLM peptide. Arb units, arbitrary units. B: the effect of PKC inhibition on PLM Thr69 phosphorylation in cultured adult rat ventricular myocytes. Sildenafil or 8-bromo-cGMP each increased PLM Thr69 phosphorylation, and this was robustly blocked by the PKC inhibitor bisindolylmaleimide (Bis). OD, optical density; Ab units, arbitrary units. Representative blots are shown with quantitative analysis of repeat experiments. Bars represent means ± SE; n = 3–5 experiments. *P < 0.05 vs. control (1-way ANOVA).
Mentions: To investigate the signaling pathways involved in sildenafil-dependent cardioprotection, we assessed the ability of PKG and PKCε to directly phosphorylate PLM Thr69 using an in vitro kinase assay. Figure 6A shows that PKCε efficiently phosphorylated WT (but not mutant T69A) PLM at Thr69, as detected in dot blots probed with an anti-phospho-Thr/Ser69 antibody. Indeed, in separate experiments (not shown) described previously (13), this level of phosphorylation represents a complete stoichiometric modification of PLM. This allows an absolute quantitative comparison of the ability of PKG to phosphorylate PLM compared with PKC. Indeed, while PKG does not phosphorylate the T69A mutant peptide at all, it similarly does not phosphorylate the WT protein efficiently (n = 3). This suggests that PKG, despite the evidence presented from the Langendorff and ARVM studies for an upstream role in cardioprotection induced by sildenafil, is unlikely to be the kinase that directly modifies PLM during sildenafil or other stimuli that culminate in Thr/Ser69 phosphorylation.

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