Heat stress (HS) induced-cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). Aims: The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) determine if myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. Methods: Following HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively using an adenoviral vector. Cell signaling was analyzed using Western blot analysis while oncosis/apoptosis was assayed by measuring trypan blue exclusion and/or TUNEL staining, respectively. Results: HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM prior to CI. HS increased association between FAK and phosphoinositol-3-kinase (PI3K) as well as caused a significant increase in AKT activity. Increased expression of wild type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time zero and after 10' of CI compared to myocytes expressing empty virus. Conclusions: Myocardial stress activates cytoskeletal-based signaling pathways that are associated with protection from lethal cell injury.