Abstract 1112: PKCϵ: ERK Signaling Protects Cardiac Myocytes from Hyperglycemic Stress
Diabetes Mellitus (DM) is a risk factor for cardiovascular disease and is also associated with the development of a primary cardiomyopathy. Our laboratory has recently reported transgenic mouse hearts with intrinsically activated PKCϵ exhibit sustained cardioprotection following 12 weeks of experimental DM. We hypothesized that inducible PKCϵ-cardioprotection is mediated by PKCϵ: ERK signaling modules, which induce an oxidant resistant phenotype that promotes cardiac muscle cell (CMC) survival. Accordingly, adult rat ventricular myocytes (ARVM) were isolated by enzymatic dissociation, and plated in serum free medium (SFM), containing 5mM (C) or 25mM (H) glucose for 16 hours. PKCϵ-isozyme specific peptide translocation activator (ϵ agonist; 0. 75uM) was delivered to ARVM to promote PKCϵ translocation. To determine if ERK is required for PKCϵ-cardioprotection, the MEK inhibitor (UO126; 15uM) was employed. Mitochondria transmembrane potential (ΔΨμ) and apoptosis were used as indices of PKCϵ-cardioprotection. Apoptotic cell death was detected by ELISA cell death assay, which detects histone-associated DNA fragments within the cytoplasmic fraction of cells with high specificity. Immunoblot analyses of RACK II (receptor for activated C-kinase-II; PKCϵ-receptor) protein expressed in Arbitrary Units (AU) in lysates of ARVM maintained at H+ϵ agonist = 33AU and at C = 34AU were identical whereas RACK II in ARVM at H + 16AU and H+UO126+ϵ agonist peptide = 17AU were markedly reduced (n=3); indicative that PKCϵ : ERK restored RACK II in ARVM at H. Apoptosis was increased in ARVM in H (54%) and H+UO126+ϵ agonist (43%) when compared to C; the percentage of apoptotic cell death in H+ϵ agonist myocytes was similar to the baseline values detected in C (n=4). PKCϵ agonist dependent signals prevented the collapse of ΔΨm in H+ϵ agonist myocytes but this effect was diminished in the presence of MEK inhibitor (H+UO126+ϵ agonist peptide). In summary, in serum starved ARVM at H, ERK is essential for PKCϵ induced preservation of ΔΨm and attenuation of apoptosis.