Abstract 1606: Cytoskeletal-Based Survival Signaling in Myocardium
Heat shock (HS) induced-cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). In this report, we further characterize the effect of HS on cytoskeletal-based cell survival pathways and extend our findings to an in vivo model of heat shock.
Methods: In vitro HS was induced in NRVM by raising the temperature of myocyte cultures to 43°C. for 20 min followed by 24 hrs of recovery at 37°C. Following HS, myocytes were subjected to metabolic inhibition (MI) at 37°C. Akt expression was increased using an adenoviral vector. Whole animal HS was induced by raising the core temperature of retired breeder rats to 42° C. for 20 min followed by 24 hours of recovery at room temperature. Cell signaling was analyzed from myocyte cultures or heart tissue using Western blot analysis and subsequent quantified scan analysis. Cell death was assayed by manual counting trypan blue (TB) and/or TUNEL staining.
Results: HS did not adversely affect the viability of NRVM prior to MI. HS as well as increased expression of Akt protected myocytes from both oncotic and apoptotic cell death (HS: 33.6 ± 4.4% vs. 26.9 ± 3.4% TB positive; p<0.005; 46.4 ± 1.8% vs. 17.6 ± 4.6% TUNEL positive; p<0.05; control vs. HS, respectively; Akt: 41.6 ± 7.0% vs. 22.6 ± 2.8% TB positive; p<0.02; 16.6 ± 3.1% vs. 7.1 ± 1.1% TUNEL positive; p<0.005; empty virus vs. Akt expressing, respectively). HS caused a shift of FAK, the adapter proteins paxillin and p130cas, and phosphoinositol-3-kinase (PI3K) from the soluble to the membrane fraction. HS also increased association between FAK and PI3K as well as causing significant (p<0.05) activation of FAK and Akt (scanned blot analysis). Importantly, whole animal HS resulted in a significant activation of both FAK and Akt and movement of PI3K to the membrane fraction in intact hearts.
Conclusions: Myocardial stress activates cytoskeletal-based signaling pathways in both cultured NRVM and intact adult rat myocardium that are associated with protection from lethal ischemic injury.