Protein Kinase Cϵ Modulates Apoptosis Induced by β -Adrenergic Stimulation in Adult Rat Ventricular Myocytes via Extracellular Signal-regulated Kinase (ERK) Activity

Abstract

β -Adrenergic stimulation of ventricular myocytes has been shown to induce apoptosis; however, the cellular mechanisms involved in this pathway have not been completely characterized. This study examines the role of protein kinase C (PKC) in the signaling cascade that mediates β -adrenergic stimulation-induced apoptosis. Stimulation of β -adrenergic receptors using isoproterenol (ISO, 1–10 μ m, 24 h) induced apoptosis in cultured adult rat ventricular myocytes (ARVM) in a dose-dependent manner. Treatment with ISO significantly resulted in the membrane translocation of PKCϵ, but not of PKC α or δ in ARVM. The activation of PKCϵ by ISO was confirmed using an immune complex kinase assay. To address whether PKCϵ is involved in the mechanism of ISO-induced apoptosis, we used the PKCϵ-specific translocation inhibitor peptide,ϵV1-2. PeptideϵV1-2 significantly blocked the translocation of PKCϵ, as well as the enzymatic action of PKCϵ, resulting from ISO stimulation. The inhibition of PKCϵ attenuated ISO-induced apoptosis as measured by terminal deoxynucleotidyltransferase nick-end labeling (TUNEL) assay (18.2±3.8%v 49.0±2.4%P<0.05), while a PKC δ -specific peptide translocation inhibitor (δ V1-1) failed to do so (39.8±7.8%). In the presence of ISO, PKCϵ inhibition byϵV1-2 was found to significantly enhance activity of ERK, but not that of Akt/PKB. Inhibition of ERK activation by PD 98059 (10–50 μ m) attenuated theϵV1-2 peptide-mediated anti-apoptotic effect, thus suggesting that ERK activation is involved in this anti-apoptotic effect. Therefore, our results suggest that activation of PKCϵ downstream of β -adrenergic stimulation promotes apoptosis largely via inhibition of an ERK activation-dependent anti-apoptotic effect.