The transduction of signals from cell surface receptors to the nucleus is required for transcriptional activation of genes leading to the hypertrophic phenotype. A goal common to studies of signal transduction and of transcriptional regulation is to elucidate the pathways which connect extracellular signals to nuclear responses. One approach is to work from the nucleus outward, identifying transcription factors important for the regulation of the genes of interest. The other approach, which we have taken, is to begin outside the cell, defining the extracellular signals that induce nuclear responses and identifying the receptors, effectors, and kinase cascades which they activate. For the past 4 years of the previous program, my laboratory has utilized the in vitro cardiac myocyte cells assay system to map intracellular signaling pathways that lead to the activation of a hypertrophic response. Previous work on this project has demonstrated that a subset of G-protein linked receptors and a constitutively activated alpha subunit of G q, as well as the small G- protein Ras, participate in the induction of atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2) gene expression. These stimuli converge upon kinase cascades, some of which have only recently been discovered. The kinases that have received the most attention are protein kinase C and the extracellular-signal regulated kinase (ERK) subfamily of mitogen activated (MAP) kinases. More recent studies suggest that another member of the MAP kinase family, notably the c-Jun N-terminal kinase (JNK), may serve to transduce extracellular signals into the altered gene expression and morphological changes associated with hypertrophy. There is more limited evidence implicating tyrosine kinases and the cardiac (delta) isoform of the Ca++/calmodulin-dependent protein kinase II (CaM K II) in hypertrophic response. The first specific aim of the proposal is to compare the activation of MAP kinases (ERKs, JNKs, p38), CaM k II and tyrosine kinases by interventions that induce distinct hypertrophic phenotypes. Kinase activities will be measured in extracts from mouse hearts and mechanisms of activation studied in rat and louse cardiac myocytes. The second specific aim is to block kinase activation and determine whether there is functional blockade of specific features of hypertrophy seen in distinct animal models. Approaches will include the use of peptide inhibitors and dominant negative kinases, delivered in cell culture, by adenoviral infection, or in transgenic animals. The final aim is to disrupt kinase, delivered in cell culture, by adenoviral infection, or in transgenic animals. The final aim is to disrupt kinase function by generation of knockout mice, and to examine the effect of the kinase deficiency on development of specific features of hypertrophy. Cardiac-specific and conditional knockouts will be used to achieve spatial and temporal control. The proposed set of studies should allow us to establish the requirement for these kinases in the in vivo development of the genetic and morphological changes that characterize different forms of hypertrophy. Defining the critical kinase pathways utilized to transduce these signals has major implications for controlling the development and pathological consequences of cardiac hypertrophy.