Wednesday, February 23, 2022 515a contraction-induced force amplification and thick-filament disorder play a role in the pathophysiology of injury. These studies leveraged a selective fastskeletal myosin modulator, EDG-4131, to test the hypothesis that PTP is mediated by the population of myosin-heads available to form cross-bridges and the release of myosin-heads from the reserve super-relaxed-state (SRX). Enzymatic activity was evaluated via an NADH-coupled assay in myofibrils (pCa6. 0) and myosin-S1 (with 2.4 mM actin) isolated from rabbit psoas. SRX and disordered relaxed (DRX) populations/ATPase rates were derived via the labeled-ATP decay method in purified meromyosin (HMM). Ex vivo twitch tetanus force (at 200 Hz) as well as PTP were assessed (at 25 C) in intact Extensor Digitorum Longus (EDL) muscles from male C57BL/6 mice; data were collected before/after EDG-4131 incubation (2.5 mM). EDG-4131 selectively reduced fast-skeletal myofibrillar ATPase (IC50: 2.5 mM), by allosterically inhibiting myosin-S1 (IC50: 0.65 mM). EDG-4131 dose-dependently shifted the population of myosin-heads towards the SRX state, reducing the cross-bridge competent DRX pool (DRX/SRX from 7052/3052% to 6153/3953%@ 3.6 mM). EDL muscles showed a characteristic PTP force enhancement that was blunted by EDG-4131 (þ2% vs. þ25% in control, at 1 Hz), while pre-potentiated force was preserved; EDG-4131 attenuated both twitch and tetanic force (200 Hz). Pharmacological stabilization of the myosin’s SRX state with EDG-4131, a selective myosin-modulator, blunted PTP in EDL muscles, with negligible impact in pre-potentiated twitches.

2493-Pos The mechanism of cooperativity in thin filament activation studied in demembranated fibers of slow skeletal muscle Matteo Marcello, Cristina Gallart, Irene Pertici, Massimo Reconditi, Gabriella Piazzesi, Vincenzo Lombardi, Marco Linari, Marco Caremani. PhysioLab, University of Florence, Florence, Italy. Regulation of contraction in striated muscle is due to mechanisms on both thin and thick filaments. Thin filament is rapidly activated by Ca2þ binding to troponin C leading to a structural change in tropomyosin that makes the actin sites available for the interaction with the myosin motors. Biochemical and structural evidence suggests that the actin site availability is modulated by motor attachment to actin, which could also be responsible for the spread of activation along the thin filament, under a cooperative mechanism, still poorly defined, that determines the steepness of the sigmoidal isometric force-pCa relation (estimated by the Hill coefficient nH). Here the mechanism responsible for cooperativity in thin filament activation has been investigated in demembranated fibers of rabbit soleus muscle by determining the dependence of the characteristics of the force-pCa relation on the force per attached motor (F0), modulated by either temperature (range 12-35 C) or addition of the b/slow myosin activator omecamtiv mecarbil (OM). F0 was estimated by applying the half-sarcomere compliance analysis on the stiffness measured in fibers activated with Ca2þ-under the different conditions. Increase in temperature from 12 C to 35 C in control increases F0 from 1.8250. 08 pN to 2.7350. 07 pN and nH from 2.0050. 08 to 3.0250. 22. The same increase in temperature in 1 mM OM increases F0 from 0.9350. 07 pN to 1.9550. 13 pN, without significant change in nH (from 0. 7950.09 to 0.9950. 13). These results indicate that the cooperativity in thin filament activation depends on the stress induced along the thin filament by the attached, force-generating motors and that attachment of motors with force inhibited …