Phosphorylation and function of the contractile proteins

Children'S Hospital Med Ctr (Cincinnati), Cincinnati OH

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Abstract

The long-term objective of subproject 1 is to understand how the contractile apparatus is tuned to match prevailing conditions and to understand the functional consequences of PKA and PKC pathway convergence at the level of the contractile proteins. The PKA and PKC mediated events that converge via MyBP-C and cTnI phosphorylation, proteins that represent potential convergence points for adrenergic signaling in the cardiomyocyte, will be studied via the following Specific Aims: (1) The PKA, PKC and PKA/PKC sites in TnI will be ablated and the constructs expressed in transgenic mice. The hypothesis is that calcium sensitivity will be increased and the resultant alterations in calcium sensitivity will lead to pathophysiological changes particularly under stress. Specific Aim 2: The PKA, PKC and PKA/PKC sites in cardiac MyBP-C will be ablated and the constructs expressed in transgenic mice. The hypothesis is that phosphorylation of this protein at the PKA and/or PKC sites modulates cardiac contractility either by structural alterations or by directly affecting the myofibril's kinetics or mechanics. Transgenic expression will be driven by the MLC2v promoter in order to restrict expression to the ventricle. As changes in the protein's role may be dependent upon MyHC isoform content, the resultant mice will be bred into a model in which an alpha- MyHC->beta-MyHC in the heart was effected. The functional consequences of dephosphorylated C protein will be explored at the structural, biochemical, cellular, organ and whole animal levels. Specific Aim 3: Mice mimicking a constant state of complete PKA and PKC-mediated phosphorylation of TnI will be made. The hypothesis is that constant myofibrillar protein phosphorylation may in fact be detrimental to a healthy animal. To achieve a state of constant phosphorylation, the relevant residues will be mutated to aspartic acid, which mimics the biochemical effects of phosphorylation. Animals will be generated in which ~ 50% and ~ 100% of the endogenous cardiac protein is replaced by the "phosphorylation-plus" species. The effects of these proteins will be explored at the molecular, biochemical, cellular, whole organ and whole animal levels throughout the animals' lifetimes. These experiments will determine the shore and long term effects of the final post-translational modifications of the contractile proteins brought about as the result of adrenergic stimulation at the molecular, biochemical, cellular, whole organ and whole animal levels.