Molecular Signaling in Cardiac Sarcomeres

University Of Illinois At Chicago, Chicago IL

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Abstract

Our hypothesis is that altered signaling at the level of sarcomere proteins occurs in the course of compensation to hemodynamic stressors leading to hypertrophy and is a significant factor in the decompensation leading to pump failure. Preliminary and published data indicate that altered phosphorylation of cTnl and alterations at the Z-disc are among these significant factors. In the aims we ask the following: Aim #1 Do PKA or PKC dependent phosphorylations induce or modify an interaction of the unique N-terminus of cTnl with itself at the inhibitory region (Ip) or other actin-binding sites or with cTnT, and thus modulate the actin-crossbridge reaction? Aim #2. What are functional consequences of transgenic expression in a mouse model of a DCM-linked mutant form of cTnl with a Val/Ala substitution at position 2 in the unique N-terminal extension? Are intra-molecular or intermolecular interactions of cTnl with other thin filament proteins altered by the mutation? Aim #3. Does reduction in the amounts of the Z-disk actin capping protein, CapZ, alter function of ejecting mouse hearts beating under basal conditions, during receptor-mediated activation of the adrenergic signaling pathways, or during the anti-adrenergic effects of adenosine and/or bradykinin? Aim #4. Does depletion of the Z-disk actin capping protein (CapZ) from thin filaments act directly to alter myofilament activation or indirectly via alterations in PKC signaling? Does extraction of CapZ from sarcomeres induce differences in docking of signaling proteins at the Z-disc. The approach to these aims involves studies of intra- and inter-molecular protein interactions, and mutagenesis to reveal mechanisms of critical interactions. The approaches include FRET, cross-linking, and surface plasmon resonance spectroscopy. Mechanical studies are carried out at the level of the single cell, bundles of cells, and trabeculae undergoing contractions that simulate pressure volume changes in the heart. Studies of hemodynamics of in situ beating hearts including echo-cardiography are carried out in conjunction with Core B. Core C serves this project in the analysis of cross-linked peptides and in determination of altered phosphorylation of sarcomeric proteins employing gel electrophoresis, in gel digestion, and mass spectrometry. Results of these experiments will add new understanding of the role of sarcomeric proteins in control of power of the myocardium, and provide information critical to the prevention, diagnosis, and therapy of heart failure.