Skeletal muscle regeneration is a dynamic process diurnally regulated by circadian rhythms, which govern key myogenic factors. Previous studies have shown that the timing of muscle injury influences early regeneration outcomes, but it remains unclear whether these effects persist beyond early acute regeneration events. This study investigated whether the time of day at which muscle injury occurs alters post-regeneration outcomes in mice. C57BL/6NCrl mice (n = 80) received bilateral cardiotoxin (CTX) injury to the tibialis anterior (TA) during either the rest phase (ZT2-ZT4) or active phase (ZT14-ZT16), with tissues collected at 7- or 42- days post-injury (DPI). All mice were matched with uninjured controls. Gross functional performance, assessed via rotarod and grip strength testing, demonstrated no differences between rest and active phase injury groups across repeated testing. A series of immunohistological analyses were performed to assess general fiber morphology and markers of regenerative state. Although injury phase had largely no effect on most parameters, myofiber size distributions consistently displayed more smaller fibers in rest phase-injured mice, regardless of sex. While there were notable differences in myosin isoform expression, such trends were observed at both 7 and 42 DPI, suggesting a specific morphological effect of injury timing. These findings indicate that the circadian phase at which skeletal muscle injury occurs causes persistent influence on the size distribution of regenerating myofibers, independent of functional recovery, and highlights the need to consider time-of-day and contribution of post-injury activity as a biological variable in muscle regeneration research.
Scurto et al. (2026) studied this question.