Thursday, June 8, 2023
6/8/2023
ABSTRACT The scientific premise of this application regards the cardiac specific expression of the long ELC containing the N-terminal ELC extension (N-ELC) and its physiologically important role in regulating myosin motor function and force production in muscle. We hypothesize that cardiac N-ELC works as a molecular linker and/or energetic switch of the actin-myosin interaction by regulating the transition of myosin cross-bridges from a super- relaxed (SRX) to a disordered-relaxed (DRX) state, and the proportion of myosin heads that are available for the interaction with actin. In DRX, the myosin heads protrude into the interfilament space but are restricted from binding to actin, while in the SRX state, the heads are neatly ordered along the thick filament axis but cycle at a highly inhibited ATP turnover rate. We further hypothesize that hypertrophic cardiomyopathy mutations in myosin ELC promote the SRX-to-DRX transition and the deletion of the N-ELC counteracts this transition and stabilizes the energy conservation SRX state. We will test these hypotheses using two mouse models of hypertrophic HCM-A57G (Alanine57¿Glycine) and restrictive RCM-E143K (Glutamate143¿Lysine) cardiomyopathy. We will also use transgenic ¿43 mice expressing a 43-aa truncated ELC, and double transgenic mice: A57Gx¿43 and E143Kx¿43, expressing ¿43-ELC in the background of HCM or RCM mutations. The data will be compared to age and sex matched WT-ELC mice expressing the human ventricular ELC (MYL3 gene). SPECIFIC AIM 1: DEFINE THE ROLE OF MYOSIN ELC AND ITS N-TERMINUS (N-ELC) IN THE REGULATION OF MYOSIN MOTOR FUNCTION AND CARDIAC MUSCLE CONTRACTION. This aim will focus on the molecular triggers of ELC-induced heart remodeling studied at the level of myosin molecules, myofibers and the whole heart. The N-ELC-dependent regulation of actin-myosin interactions will be studied using: 1) mant-ATP-chase assays and the proportion of myosin heads in SRX vs. DRX states; 2) Quantum dot-labeled actin±Tm-Tn motility assays; 3) Skinned and intact muscle fibers and force-pCa as well as force and calcium transient measurements; and 4) echocardiography and invasive hemodynamics to assess contractile responses of the heart in vivo. SPECIFIC AIM 2: THE ROLE MYOSIN ELC IN ENERGETIC REMODELING OF THE HEART IN MOUSE MODELS OF CARDIOMYOPATHY. The analysis of the metabolic landscape in A57G, E143K, ¿43, A57Gx¿43 and E143Kx¿43 vs. WT hearts will be performed to identify the specific mechanisms that govern energy utilization in all ELC-mutant vs. WT mice. Mitochondrial function will be assessed by determination of mitochondrial respiration, enzymatic activity of respiratory complexes as well as ATP production/consumption. Steady-state levels of candidate proteins implicated in energy production such as Hexokinases I and II, various mitochondrial respiratory chain subunits (Complexes I to V) as well as peroxisome proliferator-activated receptor coactivator a (PGC-1a) will be assessed in all models to characterize the broader implications of cardiac muscle disease resulting from ELC-linked modifications.
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