Recent research has indicated that the protein kinase C isoforms (PKC) and the heat shock proteins (HSP) are involved in cardioprotection. We have investigated the possible interaction between these two protein families. We find that adenoviral-mediated expression of PKC- in 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 PKC- was expressed the 5' promoter region of the HSP70 responded robustly, indicating that PKC- induction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay (EMSA) analysis determined that over-expression of PKC-, PKC- or PKC- isoforms, did not induce activation of the heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKC- is independent of HSF-1 activation. We also measured cellular injury by assessing creatine kinase (CK) release from NRVM following simulated ischemia to determine cardioprotection. NRVM infected with adenoviral construct AdwtPKC- released 54% less CK than control NRVM. Experiments using siRNA 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 PKC- and HSP70, independent of HSF-1 activation and that the protection conferred by PKC- over-expression is mediated by the transcriptionally induced expression of HSP70.