Characterizing the functional impact of embryonic myosin mutations in stem cell-derived skeletal muscle

Divergent contractile properties of different myosins suggest that each isoform performs distinct functions in embryonic and adult skeletal muscles. To better understand the role of embryonic myosin heavy chain 3 (MYH3) in regulating skeletal muscle pattern formation, growth and regeneration, our group investigated the functional defects caused by multiple mutations. Our team generated human induced pluripotent stem cell (iPSC) lines bearing T178I or R672C mutations in MYH3. Human iPSCs bearing these mutations were differentiated into skeletal muscle and evaluated for differences in the structure, maturation and functional performance of the sarcomere. Isolated myofibril mechanics 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 controls. Myofibril preparations were also used to compare ATP binding rates and showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. When incorporated into 3D engineered tissues, these changes in MYH3 mechanics resulted in slower relaxation kinetics and an incomplete relaxation of the tissue in response to repetitive tetanic stimulation, which may explain the emergence of developmental contractures in patients possessing MYH3 mutations. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the β-sheet and SH-Helix than in controls. Mutations 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.