Influence of PKC-alpha overexpression on HSP70 and cardioprotection.
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 PKC-alpha 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-alpha-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-alpha, we tested a chimeric construct that linked the luciferase reporter gene to the 5'-promoter region of HSP70 in myogenic H9c2 cells. When PKC-alpha was expressed, the 5'-promoter region of the HSP70 responded robustly, indicating that PKC-alpha induction of HSP70 expression is through transcription activation. Electrophoretic mobility shift assay determined that overexpression of PKC-alpha, PKC-delta, or PKC-epsilon did not induce activation of heat shock factor-1 (HSF-1). Therefore, induction of HSP70 by PKC-alpha is 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 AdwtPKC-alpha released 54% less CK than control NRVM. Experiments using small interfering RNA against HSP70 indicate that loss of PKC-alpha-induced HSP70 expression results in increased CK release or a loss of protection. Our results show that there is a close interaction between PKC-alpha and HSP70, independent of heat shock factor-1 activation, and that the protection conferred by PKC-alpha overexpression is mediated by the transcriptionally induced expression of HSP70.
Coaxum
Sonya D
SD
Cardiovascular Institute, Loyola University Medical Center, 2160 S. First Avenue, Maywood, IL 60153, USA.
Griffin
Tina M
TM
Martin
Jody L
JL
Mestril
Ruben
R
D000818:Animals; D000831:Animals, Newborn; D002316:Cardiotonic Agents / Q000378:metabolism*; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism*; D015854:Up-Regulation
D002316:Cardiotonic Agents; D018840:HSP70 Heat-Shock Proteins; D051571:Protein Kinase C-alpha; D003402:Creatine Kinase