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bioRxiv · 10.1101/2025.02.17.638391

INVESTIGATING THE INHIBITORY EFFECTS OF BLEBBISTATIN ON ACTOMYOSIN INTERACTIONS IN MYOFIBRILS AND ISOLATED MYOFILAMENTS

Elkrief, D.·Cheng, Y.-S.·Rassier, D.
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Abstract

Myosin II is the molecular motor responsible for muscle contraction. Myosin II hydrolyses ATP into Pi and ADP, to convert chemical energy into mechanical work while attached to actin filaments. The relation between force generation and Pi release remains unclear. Many studies use chemical substances, such as blebbistatin, to study the transitions during actomyosin interactions. Blebbistatin and its derivatives selectively inhibit the actin-activated ATPase of myosin II, accumulating myosin cross-bridges in a pre-power-stroke state. Although the effects of blebbistatin have been explored, it is still unclear how blebbistatin affects force generation and the velocity of contraction. In this study, we used individual myofibrils, myosin and actin filaments, and isolated heavy meromyosin (HMM) and actin filaments to characterize the effects of blebbistatin. We observed that increasing concentrations of blebbistatin (i) decreased the force produced by myofibrils and isolated myosin filaments, (ii) decreased the maximum velocity of shortening produced by myofibrils and the myosin-induced actin sliding velocity, (iii) decreased the curvature of the force-velocity relation in a dose-dependent manner. Furthermore, UV radiation reduced the effect of blebbistatin, which was partially reversed if blebbistatin was bound to myosin before exposure to UV light. These results show that blebbistatin alters force and velocity generation at the molecular, myofilamentous and myofibrillar levels. This study has interesting implications in fields which rely on using blebbistatin to study cellular processes and confirms several results published in different experimental arrangements. Thus, this study is exploratory and confirmatory, and the findings have utility surrounding cell migration, muscle biophysics, cellular reproduction, or any processes that rely on the action of myosin II.

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Elkrief, D., Cheng, Y.-S., Rassier, D.. 2025-02-21. INVESTIGATING THE INHIBITORY EFFECTS OF BLEBBISTATIN ON ACTOMYOSIN INTERACTIONS IN MYOFIBRILS AND ISOLATED MYOFILAMENTS. https://doi.org/10.1101/2025.02.17.638391

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