Loss of ischaemic preconditioning in ovariectomized rat hearts: possible involvement of impaired protein kinase C   phosphorylation

AIMS: The aims of this study were to determine whether chronic oestrogen withdrawal influences the development of ischaemic preconditioning (IPC) in female hearts, to investigate the mechanism whereby IPC is impaired, and to assess whether direct activation of protein kinase C (PKC) can mimic IPC in female hearts with chronic oestrogen depletion. METHODS AND RESULTS: We performed Sham-operation (Sham) or bilateral ovariectomy on 16-week-old Sprague-Dawley female rats. Ovariectomized rats were randomized to subcutaneous implantation of 17beta-estradiol (OxE) or placebo (OxP) pellets. Four weeks later, isolated, perfused hearts were subjected to 30 min of ischaemia followed by 120 min of reperfusion with or without three cycles of 5 min ischaemia/5 min reperfusion. The cardioprotective effect of IPC was completely lost in the OxP group. Western immunoblots revealed that in the OxP group, IPC failed to translocate PKCepsilon to the membranous fraction and that phosphorylation of PKCepsilon (Ser(729)) and phosphoinositide-dependent kinase (PDK) 1 (Ser(241)) was impaired. Oestrogen replacement restored the IPC effect, the translocation and phosphorylation of PKCepsilon, and the phosphorylation of PDK1. In the OxP group, pre-treatment with a PKCepsilon selective activator peptide (Psi-epsilonRACK) mimicked the IPC effect. Pre-treatment with a phosphatidylinositol-3 kinase inhibitor before IPC abrogated the translocation and phosphorylation of PKCepsilon in the Sham group. CONCLUSIONS: The cardioprotective effect of IPC is lost in female hearts with chronic oestrogen withdrawal and this is due, at least in part, to impaired translocation and phosphorylation of PKCepsilon. Selective activation of PKCepsilon-mediated signalling can fully restore the IPC effect in a manner analogous to oestrogen replacement.