Biophysical Society Thematic Meeting | Canterbury 2023

Towards a More Perfect Union: Multi-Scale Models of Muscle and Their Experimental Validation

Poster Abstracts

15-POS Board 15 EMBRYONIC MYOSIN MUTATIONS ASSOCIATED WITH DISTAL ARTHROGRYPOSIS ALTER THE MECHANICS AND MATURATION OF HIPSC DERIVED SKELETAL MUSCLE Christian Mandrycky 1 ; Saffie Mohran 1 ; Matthew Childers 1 ; Shawn Luttrell 3 ; Elizabeth Choi 1 ; Kati Buckingham 2 ; Michael Bamshad 2 ; David Mack 3 ; Michael Regnier 1 ; 1 University of Washington, Bioengineering, Seattle, WA, USA 2 University of Washington, Pediatrics, Seattle, WA, USA 3 University of Washington, Rehabilitation Medicine, Seattle, WA, USA Distal arthrogryposis (DA) is a skeletal muscle disorder characterized by joint contractures predominantly localized in the distal extremities. DA associated syndromes like Freeman-Sheldon Syndrome (FSS) are linked to mutations in the MYH3 gene that encodes the embryonic skeletal muscle myosin. To study the mutation and its effect on developing muscle, we generated human induced pluripotent stem cell (hiPSC) lines bearing T178I or R672C MYH3 mutations. hiPSC were differentiated into skeletal muscle and evaluated for differences in the maturation of the contractile unit and its functional performance. R672C mutations in MYH3 were associated with alterations in myosin isoform content, with homozygous R672C having reduced MYH3 levels in day 7 cells and elevated levels of MYH7 relative to isogenic control. Preliminary myofibril mechanics measurements showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to control myofibrils. Myofibril preparations were also used to compare ATP binding rates across conditions. Preliminary results showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the B sheet and SH-Helix than in controls. Mutation also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

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