Abstract 232: PKC-α Protection Against Simulated Ischemia/Reperfusion is Mediated by Induction of Heat Shock Protein 70i in Neonatal Rat Cardiomyocytes.
Recent research implies that PKCs and heat shock proteins (HSP) are involved in cardioprotection. We have investigated the possible interactions between two members of these families- the inducible heat shock protein 70 (hsp70i) and PKC-α. We find that adenoviral-mediated PKC-α overexpression in neonatal rat ventricular myocytes (NRVM) not only protects against simulated ischemia-reperfusion but also increases hsp70i expression. Electromobility shift assays (EMSA) show that overexpression of PKC-α, or other PKC isoforms, do not induce heat shock factor (HSF) activation. In addition, Western blots of PKC-α infected NRVM show that other HSPs are not induced. This confirms that PKC-α induced hsp70i is independent of HSF activation. We also measured cellular injury by assessing creatine kinase (CK) release following simulated ischemia to determine cardioprotection. NRVM infected with adenoviral construct AdPKC-α released 54% less CK than the control. Experiments using siRNA against hsp70i indicate that loss of PKC-α induced hsp70i expression results in increased CK release or loss of protection. In an effort to determine the protective mechanism, we created chimeric constructs that linked a luciferase reporter gene to either the 5′ promoter region or the 3′ untranslated region of hsp70i. Transfection of these constructs into rat myogenic H9c2 cells, followed by PKC-α overexpression, showed that the 5′ promoter region responded to PKC-α while the 3′end did not, although the 3′ end did respond to treatment by PMA. This indicates that PKC-α induction of hsp70i expression is through transcription activation and, potentially, mRNA stabilization is the result of the increased expression of other PKC isoforms. Our results show that there is a close interaction between PKC-α and hsp70i and that protection conferred by PKC-α overexpression is mediated at least partially by increased expression of hsp70. Experiments are n = 9, *p<0.01.