Influence of PKC- overexpression on HSP70 and cardioprotection

Coaxum SD, Griffin TM, Martin JL, Mestril R. Influence of PKCoverexpression on HSP70 and cardioprotection. Am J Physiol Heart Circ Physiol 292: H2220–H2226, 2007. First published January 5, 2007; doi:10.1152/ajpheart.01080.2006.—Recent research has indicated that the protein kinase C (PKC) isoforms and the heat shock proteins (HSPs) are involved in cardioprotection. We have investigated the possible interaction between these two protein families. We have found that adenoviral-mediated expression of PKCin neonatal rat ventricular myocytes (NRVM) not only increases the expression of HSP70 but also protects against simulated ischemia-reperfusion. In addition, Western blots of PKC-infected NRVM indicated that other HSPs are not induced in the same manner as HSP70. In an effort to determine the mechanism of induction of HSP70 by PKC, we tested a chimeric construct that linked the luciferase reporter gene to the 5 -promoter region of HSP70 in myogenic H9c2 cells. When PKCwas expressed, the 5 -promoter region of the HSP70 responded robustly, indicating that PKCinduction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay determined that overexpression of PKC, PKC, or PKCdid not induce activation of heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKCis independent of heat shock factor-1 activation. We also measured cellular injury by assessing creatine kinase (CK) release from NRVM after simulated ischemia to determine cardioprotection. NRVM infected with the wild-type adenoviral construct AdwtPKCreleased 54% less CK than control NRVM. Experiments using small interfering RNA against HSP70 indicate that loss of PKC-induced HSP70 expression results in increased CK release or a loss of protection. Our results show that there is a close interaction between PKCand HSP70, independent of heat shock factor-1 activation, and that the protection conferred by PKCoverexpression is mediated by the transcriptionally induced expression of HSP70.