Thick and Thin Filament Mutations that cause Distal Arthrogryposis

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DESCRIPTION (provided by applicant): Arthrogryposis is a congenital contracture syndrome that affects 1 of 3000 live births. While the genetic causes of DA are in discovery, the biophysical mechanisms of how these mutations cause life-long contractures remains unknown. We have recently acquired and reported on contractile characterization for one DA patient, exhibiting an MYH3 R672C mutation. This mutation decreased steady state force production and increased relaxation time in single skinned myofibers from a human skeletal muscle biopsy, and our data suggest this may occur via altered crossbridge binding and cycling kinetics. The mechanism of how this occurs remains unknown. As such, there is much to be learned from contractile assessments from patient biopsies containing mutations in both thin and thick filament sarcomere proteins. I hypothesize that there are differences in how thin filament protein mutations and thick filament protein mutations result in altered contractures. Specifically, I expect thick filament mutations, such as MYH3 R672C and MYH8 R672H, to lead to direct changes in myosin (crossbridge) binding, detachment and interaction with actin. These mutations lead to a severe form of DA, Freeman-Sheldon Syndrome. In contrast, I expect thin filament mutations, such as TNNI2 R174Q andTNNT3 R63H, to lead to changes crossbridge binding via altered Ca2+ sensitivity and cooperativity of thin filament activation. These mutations lead to a slightly less severe form of DA, Sheldon-Hall syndrome. By determining functional changes and the causal mechanisms, we can begin to develop targeted therapies that would restore function on the molecular and cellular level. I plan to do so with three aims, focusing on muscle contractility of muscle fibers and muscle myofibrils. I will use muscle fibers to determine maximal force production, crossbridge binding, and Ca2+ sensitivity of force. I will use muscle myofibrils to determine thin filament activation and relaxation kinetics from single isolated myofibrils from human skeletal muscle biopsies. Finally, I will use muscle fibers and potentially myofibrils to determine if modulation of crossbridge binding or thin filament activation through use of an ATP analogue (2-deoxyATP) or TnC variants with altered Ca2+ binding, respectively, can restore function to human adult DA skeletal muscle.