Biophysical Society Thematic Meeting | Canterbury 2023

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

Poster Abstracts

17-POS Board 17 HYPERTROPHIC CARDIOMYOPATHY MUTATION R403Q DEVELOPS ACTIN MEDIATED MECHANICAL AND STRUCTURAL DYSFUNCTION IN PORCINE VENTRICLE TISSUE Saffie Mohran 1,3 ; Matthew Childers 1,3 ; Kristina Kooiker 2,3 ; Jing Zhao 4 ; Timothy McMillen 3 ; Christian Mandrycky 1,3 ; Stephanie Neys 2 ; Jingyuan Yu 4 ; Farid Moussavi-Harami 2,3 ; Michael Geeves 5 ; Thomas Irving 6 ; Weikang Ma 6 ; Michael Regnier 1,3 ; 1 University of Washington, Bioengineering, Seattle, WA, USA 2 University of Washington, Cardiology, Seattle, WA, USA 3 University of Washington, Center for Translational Muscle Research, Seattle, WA, USA 4 Dalian Medical University, College of Basic Medical Sciences, Dalian, China 5 University of Kent, School of Biosciences, Canterbury, United Kingdom 6 Illinois Institute of Technology, Biology, Chicago, IL, USA Missense mutations in human β -cardiac myosin were first reported to cause malignant hypertrophic cardiomyopathy with the discovery of R403Q. Located in the upper 50KDa cardiomyopathy loop of myosin, there are several reports on the impact of R403Q on myosin structure, sarcomere contractile properties, and ATPase activity. Conflicting findings in contractile kinetics between human-patient samples and a transgenic rabbit model motivated us to study a novel MYH7 R403Q porcine model. Preliminary contractile experiments comparing control and R403Q demembranated ventricle strips showed R403Q expresses significantly faster k TR (1.18s-1 vs 1.89s-1), greater Ca 2+ sensitivity (pCa 50 = 5.72 vs 5.89), and a decreased Hill coefficient (4.96 vs 2.57). Small angle x-ray diffraction on R403Q tissues demonstrated greater myosin disorder with decreased intensities in the myosin-based reflections (I MLL1 = 6.30 vs 0.69, I M3 = 9.35 vs 3.56) compared to WT cardiac muscle. Interestingly, R403Q tissue had blunted structural changes in response to dATP, a myosin activator, while the structural changes in response to mavacamten, a myosin inhibitor, were increased. These structural changes suggest that R403Q increases the population of ON myosin heads, a potential mechanism of hyper contractility. To understand the direct impact of R403Q on myosin head structure in the absence of actin, we performed molecular dynamic (MD) simulations of the pre-power stroke state (M.ADP.Pi). Simulations showed the R403Q mutation primarily altered the surface area and electrostatic potential of myosin’s actin binding surface. To complement the MD simulations, we isolated full-length myosin and performed stop-flow measurements of ATP-binding and single nucleotide turnover utilizing Mant-ATP. These actin-absent experiments showed no difference in ATP-binding and single nucleotide turnover kinetics between control and R403Q myosin. Future experiments will utilize HMM in the absence and presence of actin to assess ADP dissociation and actin-mediated ATP-binding.

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