Time of day of skeletal muscle injury is a factor in short- and long-term regeneration outcomes

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 postregeneration outcomes in mice. C57BL/6NCrl mice (<i>n</i> = 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 postinjury (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 was performed to assess general fiber morphology and markers of regenerative state. Although the injury phase had largely no effect on most parameters, myofiber size distributions consistently displayed more small fibers in rest phase-injured mice, regardless of sex. Although 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 a persistent influence on the size distribution of regenerating myofibers, independent of functional recovery, and highlights the need to consider time-of-day and the contribution of postinjury activity as a biological variable in muscle regeneration research.<b>NEW & NOTEWORTHY</b> The timing of skeletal muscle injury influences myofiber morphology postregeneration. Although functional capacity was unaffected by injury timing, muscles injured in the circadian rest phase exhibited a greater quantity of smaller myofibers compared with their active phase counterparts. This was not accompanied by differences in regenerating cell morphology or nuclear centralization. This research suggests that circadian timing of injury exerts a lasting effect on muscle regeneration, and may reflect time-of-day-specific regulation of muscle remodeling.