Glycerol storage increases passive stiffness of muscle fibers through effects on titin extensibility

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.