The cardiac hypertrophy, myofibrillar disarray and sudden death caused by familial hypertrophic cardiomyopathy (FHC) results from autosomal dominant mutations in sarcomeric proteins. Myosin, the sarcomeric molecular motor that interacts with actin to power cardiac muscle contraction, is a hexameric protein consisting of two heavy chains. Each heavy chain binds two light chains, one essential (ELC) and one regulatory (RLC). The light chain binding (neck/lever) domain amplifies ATP dependent conformational changes originating in the myosin active site to generate force and motion. Given the importance of the light chain binding (neck/lever) domain of myosin in force production, it is not surprising that several FHC mutations have been identified in the RLC. The goal of this proposal is to provide a molecular basis for FHC in patients with mutations in the RLC. Since myosin molecule biochemistry is linked to force producing conformational changes, it is expected that several steps in the myosin biochemical cycle are strain dependent. Therefore, we will study the transmission of external forces to the myosin active site via the myosin neck region, . Since the clinical presentation depends on the specific mutation, these studies are a necessary precursor to development of therapeutic protocols. We will test the hypothesis that mutations in the myosin RLC decrease the ability of the myosin neck domain to act as a strain sensor, which alters the delivery of force to the active site, leading to altered strain dependent kinetics and power output. The mutations chosen for study are localized near the phosphorylatable serine and the EF-hand of the RLC molecule (A13T, N47K, R58Q and D166V). These regions have historically been shown to be important for myosin function;thus our experiments will not only provide a molecular basis for FHC but will also address fundamental aspects of RLC function and the molecular basis of myosin motion generation. Our approach will utilize in vitro motility assays to assess the effects of RLC mutations on power output (Aim 1) as well as strain dependent myosin kinetics at the ensemble (multiple molecule) level (Aim 2). Any alterations in ensemble strain dependence will be further pursued at the single myosin molecule level to determine the specific underlying strain dependent actomyosin kinetic transitions affected by the mutations (Aim 3). Furthermore, consistent with our preliminary data, RLC phosphorylation has been proposed to inhibit hypertrophy by contributing to enhanced contractile performance and efficiency. Therefore, we will determine if phosphorylation of the RLC rescues the RLC-FHC phenotypes (Aim 4). Our approach measures the mechanical properties of isolated contractile proteins. Therefore, we will determine the direct effects of the FHC mutations on actomyosin. Knowledge of how the RLC mutations affect myosin's inherent function will allow the degree of alteration of higher functional units, such as the cardiac muscle fiber, or the heart itself to be correlated with a primary contractile defect.
Familial hypertrophic cardiomyopathy (FHC) is a genetic heart disease that is caused by mutations in the molecular machinery that allows the heart to contract. This study will examine how FHC mutations in one of the proteins of the heart (the myosin regulatory light chain), alters the ability of the heart to generate force and power at the molecular level.
|Farman, Gerrie P; Rynkiewicz, Michael J; Orzechowski, Marek et al. (2018) HCM and DCM cardiomyopathy-linked ?-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments. Arch Biochem Biophys 647:84-92|
|Rynkiewicz, Michael J; Prum, Thavanareth; Hollenberg, Stephen et al. (2017) Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function. Biophys J 113:2444-2451|
|Moore, Jeffrey R; Campbell, Stuart G; Lehman, William (2016) Structural determinants of muscle thin filament cooperativity. Arch Biochem Biophys 594:8-17|
|Sewanan, Lorenzo R; Moore, Jeffrey R; Lehman, William et al. (2016) Predicting Effects of Tropomyosin Mutations on Cardiac Muscle Contraction through Myofilament Modeling. Front Physiol 7:473|
|Fischer, Stefan; Rynkiewicz, Michael J; Moore, Jeffrey R et al. (2016) Tropomyosin diffusion over actin subunits facilitates thin filament assembly. Struct Dyn 3:012002|
|Achal, Madhulika; Trujillo, Adriana S; Melkani, Girish C et al. (2016) A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila. J Mol Biol 428:2446-2461|
|Schmidt, William M; Lehman, William; Moore, Jeffrey R (2015) Direct observation of tropomyosin binding to actin filaments. Cytoskeleton (Hoboken) 72:292-303|
|Egan, Paul; Moore, Jeffrey; Schunn, Christian et al. (2015) Emergent systems energy laws for predicting myosin ensemble processivity. PLoS Comput Biol 11:e1004177|
|Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta et al. (2015) Myosin regulatory light chain phosphorylation enhances cardiac ?-myosin in vitro motility under load. Arch Biochem Biophys 580:14-21|
|Lehman, William; Medlock, Greg; Li, Xiaochuan Edward et al. (2015) Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain. Arch Biochem Biophys 571:10-5|
Showing the most recent 10 out of 28 publications