Molecular signaling in cardiac myofilaments

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(Adapted from the Applicant's Abstract) Experiments proposed here test the hypothesis that isoform switching and phosphorylation of cardiac TnI (cTnI) and cTnT are important elements in the intrinsic (Starling's Law) and extrinsic (neurohumoral) control of cardiac output. The long term objective is to identify: 1) unique molecular mechanisms by which Ca2+- and cross-bridge binding to cardiac thin filaments control contraction and relaxation, and 2) the impact of modulation of these mechanisms on activation and relaxation. The specific aims are: Aim #1: To identify functional effects of isoform specific regions of interaction of cTnT and cTnI and of cTnT with cTnC. Aim #2: To test the hypothesis that functional effects of protein kinase C (PKC) dependent phosphorylation of cTnI and cTnT are site specific and synergistic. Aim #3: To test the hypothesis that isoform switching of actin, cTnT and cTnT and/or phosphorylation of cTnI and cTnT and of cTnT with cTnC. Aim #2: To test the hypothesis that functional effects of protein kinase C (PKC) dependent phosphorylation of cTnI and cTnT are site specific and synergistic. Aim #3: To test the hypothesis that isoform switching of actin, cTnI and cTnT and/or phosphorylation of cTnI and cTnT modulate effects of sarcomere length and cross-bridge binding on myofilament activation. Aim #4: To determine steady- and pre-steady state binding between the regulatory (N domain) and structural (C- domain) of cTnC with cTnI and cTnT. This objective tests the hypothesis that modulation of association/dissociation rates between Tn components affects the rates of cardiac contraction and/or relaxation. This hypotheses are approached using mutagenesis, reconstitution, and transgenic models combined with structure-function analysis of myofilament proteins. The experiments include determination of mechanics and force/ATPase rate in skinned fiber bundles, and surface plasmon resonance spectroscopy to determine rates of interaction between Tn components. Results of these experiments provide information crucial to the understanding of normal events that signal contraction and relaxation of the heart. The results are also essential in understanding the mechanism for changes in the thin filament signaling mechanism associated with ischemia, heart failure, and genetically linked hypertrophic myopathies involving the thin filament proteins.