To define better the subcellular mechanism of heat shock-induced cardioprotection, we examined the effect of heat shock (HS) as well as selective expression of individual heat shock proteins (HSP) on cellular injury in neonatal rat ventricular myocytes (NRVM). Methods: HS was induced in NRVM by rapidly raising the temperature to 42°C. for 20 min followed by 20-24 hrs of recovery at 37°C. Other NRVM were infected with a replication-deficient adenovirus encoding either HSP27 or HSP70. All groups were subjected to metabolic inhibition (MI). Cell injury was assayed by measuring the percent of total LDH released, the percentage of cells staining with trypan blue, or TUNEL staining while cell signaling was assayed using immunoblot analysis and co-immunoprecipitation. Results: Prior to MI, the viability of treated groups did not differ significantly from control (C) NRVM. HS significantly increased both HSP70 and HSP27 expression. Infection with either virus caused a significant increase in selective HSP content. HS protected NRVM from injury. NRVM selectively expressing HSP27 or HSP70 alone was not protective but dual infection with both viral vectors (HSP27 and HSP70) was protective. Both HS and HSP27/70 expression increased paxillin in the membrane fraction, which persisted during MI. HS increased the interaction of integrin-paxillin-FAK while targeted inhibition of FAK activity abolished integrin-paxillin association and increased cell death. Conclusions: HS and dual HSP27/70 expression increases the association of members of the focal adhesion complex as well as protect NRVM against irreversible injury. Cytoskeletal-based signaling pathways may represent a unique pathway of cardioprotection.