The long term objective of research in our laboratory is to understand the functional significance of regulation of cardiac function downstream of altered Ca- fluxes at the level of the sarcomere. The present proposal emphasizes tropomyosin phosphorylation (Tm-P) in molecular signaling in thin filament activation with focus on potential synergistic and antagonistic inter- and intra-molecular alterations that modulate its effects on myofilament response to Ca. The rationale for this hypothesis is related to our nearly complete lack of understanding of the functional significance of Tm-P and to compelling pilot data demonstrating novel changes in Tm-P associated with altered cardiac and sarcomeric function. The objectives are:
Aim #1 : Do effects of modifications in cTnT and cTnl promote or diminish the functional effects of Tm phosphorylation (Tm-P) on sarcomeric response to Ca and/or cooperative activation of the myofilaments by strong crossbridges? Aim #2: Do intrinsic stresses, especially Tm mutations linked to cardio-myopathies, induce intra-molecular alterations that modulate the effect of Tm-P in sarcomeric function or alter it as a substrate for kinases/phosphatases? Aim #3: Do extrinsic stresses (hypertension, p38 MAPK activation) on the myocardium induce a change in Tm-P that correlates with altered function? The approach employs transgenic models expressing mutant forms of Tm, Tm(S283D) and Tm(S283A), viral transfer of cDNA expressing various forms of Tm into cardiac myocytes, and exchange of various forms of cTnl and cTnT in skinned fibers with and without Tm(S283D) or Tm(S283A). We also study a cTnl mutation that generates loss of function"""""""" with regard to cooperative activation of the myofilaments by strong crossbridges. Measurements are made of Ca2+ and shortening in myocytes, and mechanical and biochemical activity of detergent extracted fibers and reconstituted systems with the use of NEM-S1 as a probe of cooperative activation.. Phospho-proteomic approaches are applied to determination of altered sarcomeric protein phosphorylation in these experiments and in long term responses of the myocardium to p38 MAPK and hypertension. Results of these studies will reveal the functional significance of Tm phosphorylation in cardiac function and add novel and important new dimensions to our understanding of modulation of cooperative activation of cardiac thin filaments in physiological and patho-physiological states.
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