Han Seong-Won, Kolb Justin, Farman Gerrie P, Gohlke Jochen, Granzier Henk L
Department of Cellular and Molecular Medicine, Molecular Cardiovascular Research Program, University of Arizona, Tucson, AZ, USA.
J Gen Physiol. 2025 Jul 7;157(4). doi: 10.1085/jgp.202413729. Epub 2025 May 9.
To study the physiological and pathological mechanisms of muscle, it is crucial to store muscle samples in ways that preserve their properties. Glycerol is commonly used for storage, as it stabilizes muscle proteins, slows enzymatic activity, and minimizes degradation. However, previous studies validating glycerol storage have not examined its effects on passive properties. In this study, mouse extensor digitorum longus (EDL) muscles were stored in 50% glycerol in relaxing solution with protease inhibitors for various durations, then rehydrated in physiological solutions to assess mechanical properties. Active properties remained unchanged, but passive stress was sensitive to glycerol storage, showing a 56.5 ± 13.6% increase after 4 days, and this effect was permanent. The increase was most pronounced at sarcomere lengths, where titin's PEVK segment extension dominates. Using gelsolin, we determined whether the passive stress increase requires the thin filament, which is known to interact with titin's PEVK region. Both glycerol-stored fibers with and without thin filament extraction exhibited increased passive stress, suggesting that the underlying mechanism is intrinsic to titin. Finally, fibers treated with methylglyoxal, a reactive carbonyl and glycating agent that forms cross-links on lysine residues, showed a significant increase in passive stress in fibers stored in relaxing solution but not in glycerol. Thus, glycerol storage elevates passive stress in a titin-specific manner, likely involving lysine residues in the PEVK. Therefore, glycerol storage should be avoided when assessing passive stiffness. We further showed that, for long-term preservation, storage of rapidly frozen muscle at -80°C is a viable option.
为了研究肌肉的生理和病理机制，以能够保持其特性的方式储存肌肉样本至关重要。甘油常用于储存，因为它能稳定肌肉蛋白质、减缓酶活性并使降解最小化。然而，先前验证甘油储存效果的研究并未考察其对被动特性的影响。在本研究中，将小鼠趾长伸肌（EDL）肌肉置于含有蛋白酶抑制剂的松弛溶液中的50%甘油中储存不同时长，然后在生理溶液中复水以评估力学特性。主动特性保持不变，但被动张力对甘油储存敏感，在4天后增加了56.5±13.6%，且这种效应是永久性的。在肌节长度处这种增加最为明显，此时肌联蛋白的PEVK区段伸展起主导作用。使用凝溶胶蛋白，我们确定被动张力增加是否需要细肌丝，已知细肌丝与肌联蛋白的PEVK区域相互作用。无论有无提取细肌丝，经甘油储存的纤维均表现出被动张力增加，这表明潜在机制是肌联蛋白固有的。最后，用甲基乙二醛处理的纤维，甲基乙二醛是一种活性羰基和糖基化剂，可在赖氨酸残基上形成交联，结果显示在松弛溶液中储存的纤维被动张力显著增加，但在甘油中储存的纤维未增加。因此，甘油储存以肌联蛋白特异性方式提高被动张力，可能涉及PEVK中的赖氨酸残基。所以，在评估被动僵硬度时应避免使用甘油储存。我们进一步表明，对于长期保存，将肌肉快速冷冻在-80°C是一种可行的选择。
