The overall goal of this proposal is to elucidate the signaling molecules involved in transducing hypertrophic stimuli assembly of newly synthesized contractile proteins. An essential step in the assembly of contractile proteins into sarcomeres is focal adhesion formation (FAF). Recent studies indicate that FAF in cardiomyocytes is dependent on phosphorylation of FAK and paxillin, which is dependent upon upstream activation of one or more isoenzymes of PKC. Preliminary data demonstrates that inhibition of PKCs results in the disappearance of decorated actin filaments and adenovirally-mediated overexpression of constitutively active PKC-epsilon (caPKCe) alters myocyte shape and enhances sarcomeric assembly (SA). Therefore, experiments will be performed to determine the effects of overexpression of mutant forms of PKCe on FAF and SA in cultured neonatal rat myocytes. Preliminary data also shows that caPKCe increases basal ERK1/2 activation and FRNK greatly reduces endothelin-dependent ERK1/2 activation. The signaling pathway linking activation of PKCe to FAK-dependent ERK1/2 phosphorylation is hypothesized to involve GTP-loading of Rho and/or Ras. Experiments are designed to examine overexpression of mutant forms of PKCe, Rho, Ras, and FRNK on FA-dependent signaling, FAF and SA. As an in vivo correlate, GFP-dnPKCe and GFP-caPKCe will be generated and injected directly into the hearts of neonatal rats. Their effects on FAF and SA will be investigated. A better understanding of the signaling pathways which contribute to cardiac hypertrophy would enable novel, therapeutic agents of disease prevention to be discovered.
