A combined experimental and computational analysis of mantATP turnover in skinned muscle fibers

• 成果类型: Article
• 署名作者: Montesel, Mauro; De Napoli, Cosimo; Schmidt, Luisa; Germinario, Elena; Guzman, Ulises H.; V. Olsen, Jesper; Marcucci, Lorenzo; Nogara, Leonardo
• 署名单位: University of Padua; Veneto Institute Molecular Medicine; University of Copenhagen; University of Padua
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10347
• DOI: 10.1073/pnas.2502652122
• 发表日期: 2025-05-20
• 关键词: myosin subfragment-1; atp turnover; filament; myofibrils; heads; MODEL

摘要

Myosin is the primary motor protein in skeletal muscle, responsible for adenosine triphosphate (ATP) hydrolysis that drives muscle contraction. In addition to force production, resting myosin consumes ATP in futile cycles at two rates, the slower one being associated with the Super Relaxed State (SRX), in contrast to the less inhibited Disordered Relaxed State (DRX). The SRX is typically measured using the mantATP chasing technique, where the decay of a fluorescent ATP analogue is fitted using a multiexponential function. Recently, significant concerns have been raised regarding the use of this technique, particularly when applied to soluble myosin preparations. While skinned fibers offer the advantage of preserving the native thick filament structure and myosin cooperativity, limited diffusion and nonspecific mantATP binding pose challenges. In this study, we combine experimental data and in-silico modeling to dissect the contributions of different components in the mantATP chasing signal. We analyze control skinned fibers and fibers subjected to myosin extraction. Our analysis shows that the nonspecific component partially overlaps with the DRX timescale. In contrast, the slow component linked to myosin SRX nucleotide release is characterized by a time constant that significantly differs from those of the nonspecific signal and DRX, enabling its reliable estimation using this technique. Our findings indicate that evaluating nonspecific mantATP components is necessary to obtain a reliable estimation of both SRX and DRX. We validated our analysis by comparing populations and time constants obtained from chasing with mantATP to mantATPase rates in control conditions and upon piperine-induced SRX destabilization.