Familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DC) are diseases that affect the ability of the heart to effectively pump blood and can result in heart failure. Our main hypothesis is that cardiac muscle performance depends critically on cooperative activation of cardiac muscle thin filaments as they respond to Ca2+ binding to troponin and myosin binding to actin and tropomyosin (Tm); and that deficient or defective cooperative interactions due to mutations at these sites in the thin filament lead to cardiomyopathies. Here, using interdisciplinary approaches we test the hypothesis that cooperative interaction requires (1) specific and stable head-to-tail interactions between adjacent Tm molecules on thin filaments as well as (2) specific interactions between actin and Tm and myosin heads at rest and during the crossbridge cycle on actin.
Specific Aim 1 is to determine the structure and functional interactions required for proper assembly of thin filaments and test the hypothesis that cardiomyopathy-linked thin filament Tm mutations lead to faulty thin filament organization and responses to phosphorylation. We will: (i) determine the mode, and mutation- induced disruptions, of Tm binding to actin filaments, determine the effect of Tm mutations and phosphorylation on Tm-actin binding and Tm-Tm head-to-tail linkages using TIRFM and determine altered interactions between mutant tropomyosin and troponin using surface plasmon resonance (SPR); (ii) determine if specific sites in isolated mutant Tms show conformational alterations by electron microscopy (EM) and Molecular Dynamics (MD); (iii) compare head-to-tail Tm linkage of wild-type, mutant and phosphorylated Tm by EM and MD; and iv) define the thin filament interactome by zero-length cross-linking and hierarchical mass spectrometry (XL-MS) technology; and determine cardiomyopathy-mutation-induced alterations in the thin filament interactome.
Specific Aim 2 is to test the hypothesis that cardiomyopathy-linked thin filament mutations generate defective thin filament structures and perturb functional interactions between thin filament Tm with actin and myosin heads (and troponin) that, in turn, interfere with cooperative thin filament activation and relaxation. We will: (i) determine if the regulatory position of mutant Tm on thin filaments is affected by mutant Tms using 3D-EM and computational chemistry; (ii) determine if Tm mutation leads to defective or deficient thin filament cooperativity using in vitro motility assays and laser trapping methods; and (iii) Establish how alterations in thin filament molecular interactions translate to defective thin filament cooperativity in the more complex cellular and tissue environment. The studies are expected to yield a high resolution, residue specific, picture of normal and mutant cardiac muscle thin filaments that will facilitate the design of new therapies specific for particular mutations.
The human heart contracts to pump blood throughout the body when actin- and tropomyosin-containing thin filaments slide relative to myosin-containing thick filaments; and mutations in these proteins result in heart disease. The goal of this work is to determine the precise manner in which these proteins interact, which is expected to be a significant guide for future mutation-specific therapies and targeted drug design.
| Lehman, William; Li, Xiaochuan; Kiani, Farooq A et al. (2018) Precise Binding of Tropomyosin on Actin Involves Sequence-Dependent Variance in Coiled-Coil Twisting. Biophys J 115:1082-1092 |
| 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 |
| Schmidt, William M; Lehman, William; Moore, Jeffrey R (2015) Direct observation of tropomyosin binding to actin filaments. Cytoskeleton (Hoboken) 72:292-303 |
| 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 |
