Immunomodulatory Role of the Stem Cell Circadian Clock in Muscle Repair

The circadian clock orchestrates vital physiological processes such as metabolism, immune function, and tissue regeneration, aligning them with the optimal time of day. This study identifies an intricate interplay between the circadian clock within muscle stem cells (SCs) and their capacity to modulate the immune microenvironment during muscle regeneration. We uncover that the SC clock provokes time-of-day (TOD)-dependent induction in inflammatory response genes following injury, particularly those related to neutrophil activity and chemotaxis. These responses are driven by rhythms of cytosolic regeneration of the signaling metabolite NAD+. We demonstrate that genetically enhancing cytosolic NAD+ regeneration in SCs is sufficient to induce robust inflammatory responses that significantly influence muscle regeneration. Furthermore, using mononuclear single-cell sequencing of the regenerating muscle niche, we uncover a key role for the cytokine CCL2 in mediating SC-neutrophil crosstalk in a TOD-dependent manner. Our findings highlight a crucial intersection between SC metabolic shifts and immune responses within the muscle microenvironment, dictated by the circadian rhythms, and underscore the potential for targeting circadian and metabolic pathways to enhance tissue regeneration. Mononucleated cells from the tibialis anterior (TA) muscles of both hindlimbs were collected at ZT4 and ZT16 on day 0, 1, and 3 after injury. Single cells were collected through fluorescence-activated cell sorting (FACS), excluding dead cells identified by propidium iodide (PI) staining. Samples from each time point and day were captured in droplet emulsions using a 10x Chromium Controller (10x Genomics), aiming for 10,000 cells per sample, and then sequenced.