Protein kinase C and preconditioning: Role of the sarcoplasmic reticulum

Activation of protein kinase C (PKC) is cardioprotective, but the mechanism(s) by which PKC mediates protection is not fully understood. Inasmuch as PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca ²⁺ and because altered SR Ca²⁺ handling during ischemia is involved in cardioprotection, we examined the role of PKC-mediated alterations of SR Ca²⁺ in cardioprotection. Using isolated adult rat ventricular myocytes, we found that addition of 1,2-dioctanoyl-sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death associated with simulated ischemia and reperfusion, also reduced SR Ca²⁺. Cell death was 57.9 ± 2.9% and 47.3 ± 1.8% in untreated and DOG-treated myocytes, respectively (P < 0.05). Using fura 2 fluorescence to monitor Ca²⁺ transients and caffeine-releasable SR Ca²⁺. we examined the effect of DOG on SR Ca²⁺. Caffeine-releasable SR Ca ²⁺ was significantly reduced (by ∼65%) after 10 min of DOG treatment compared with untreated myocytes (P < 0.05). From our examination of the mechanism by which PKC alters SR Ca²⁺, we present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser¹⁶. This effect is mediated by PKC-ε, because a PKC-ε-selective inhibitory peptide blocked the DOG-mediated decrease in phosphorylation of PLB and abolished the DOG-induced reduction in caffeine-releasable SR Ca²⁺. Using immunoprecipitation, we further demonstrated that DOG increased the association between protein phosphatase 1 and PLB. These data suggest that activated PKC-ε reduces SR Ca²⁺ content through PLB dephosphorylation and that reduced SR Ca²⁺ may be important in cardioprotection.