Biophysical Society Thematic Meeting | Canterbury 2023

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

Monday Speaker Abstracts

SUPER-RESOLUTION SINGLE-MOLECULE IMAGING OF ATP USAGE IN MYOFIBRILS TO STUDY THICK FILAMENT REGULATION Matvey Pilagov 1 ; Sonette Steczina 2 ; Ateeqa Niam 1 ; Michael Regnier 2 ; Michael A Geeves 1 ; Neil M Kad 1 ; 1 University of Kent, School of Biological Sciences, Canterbury, United Kingdom 2 University of Washington, Department of Bioengineering, Seattle, WA, USA Controlling access of myosin to actin in muscle modulates how energy is used. This control occurs at both the thin and thick filaments levels; with the latter involving two states of myosin: disordered relaxed (DRX – able to interact with actin), and super-relaxed (SRX – does not interact with actin). The amount of DRX defines the number of myosins available to interact with actin. However, it is still uncertain how this SRX-DRX equilibrium is distributed within the sarcomere. By fluorescently labelling ATP, we are able to measure the activity of individual myosins within myofibrils. Super-resolution deconvolution provides ~30 nm localization of these activities, enabling assignment to the three zones of the thick filament P, C and D. For fast skeletal muscle (rabbit psoas), we observed 53% of all heads in the C-zone were SRX, compared with 35% and 44% in the P- and D-zones, respectively. This suggests myosin binding protein C (MyBP-C) influences the SRX-DRX equilibrium. To investigate if phosphorylation of MyBP-C affects the population of SRX, we treated myofibrils with protein kinase A (PKA). We found that PKA decreased SRX to 34% in the C-zone, whereas the P- and D-zones were not affected. Nowhere is the control of myosin head accessibility more relevant than in the heart, therefore we have studied the localization of DRX and SRX in porcine cardiac myofibrils, which possess human equivalent beta-cardiac myosin. We will report on the effects of PKA phosphorylation and the recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten, in these myofibrils. In summary, our results directly show that PKA treatment of muscle releases some but not all myosin heads, equally mavacamten increases SRX but does not completely eradicate DRX. Therefore, the activation and repression of myosins within the sarcomere is a combination of multiple competing factors that require an in-depth understanding, especially relevant as we begin to develop drugs to modulate this equilibrium.

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