Satellite cell molecular clock regulation on muscle mitochondria, contractile function, and muscle repair

Background: Circadian rhythms are an evolutionary conserved mechanism that underpin diurnal variance in biological and physiological processes. These rhythms are produced by a central molecular clock located in the suprachiasmatic nucleus (SCN) and are communicated to “peripheral” clocks located in peripheral tissues and organs. Of the peripheral clocks, several studies have demonstrated skeletal muscle houses some of the most robust rhythmicity in molecular clock oscillations throughout a 24 h day. A wide range of muscle biological/physiological processes are under molecular clock control including exercise-capacity, mitochondrial metabolism, contractile-function, and muscle repair/regeneration. However, there are two myogenic molecular clock sources within muscle: myonuclei and satellite cells (SC). Recent evidence has demonstrated that SCs house a functioning molecular clock with the transcription of several genes related to mitochondrial metabolism, muscle contractile function, and myogenesis exhibiting circadian expression patterns. Additionally, as other works have demonstrated that mitochondrial function, contractility, and muscle repair are all molecular clock regulated aspects of muscle physiology, this suggests the SC molecular clock may also play a role in such regulations. Systematic evaluation of mitochondrial function, contractile function, and muscle repair across different times of the day and in the presence/absence of SC-molecular clocks will provide novel information on the role that SC-molecular clocks may have in these processes.

Methods: Using the Pax7DTA (Pax7CRE-ERT2/+; Rosa26DTA/+) mouse model capable of inducible depletion of SC’s, muscle mitochondrial function was assessed in the tibialis anterior (TA) (glycolytic muscle) and soleus (oxidative muscle) in the morning, afternoon, and evening (0700h, 1500h, 1900h). Mitochondrial citrate synthase activity, ETC-I activity, copy number, mitochondrial and molecular clock gene expression were undertaken at the same timepoints/SC-conditions in the TA and quadriceps. At time points demonstrating oscillatory clock gene expression, ex vivo submaximal muscle fatigue in the EDL was assessed to determine if fatiguing contractions reliant on mitochondrial-energy also demonstrate time-dependent variance. Next, utilizing the Pax7DTA mouse model, ex vivo maximal contractile function and eccentric injury of the EDL were assessed in the morning and afternoon (0700h and 1500h) in the presence and absence of SCs. Immunohistochemical methods were used to quantify dystrophinnegative fibers, cross sectional area, and SC abundance in these muscles. Gene expression was performed in contralateral, uninjured TA muscle. Finally, utilizing a mouse model capable of inducible depletion of molecular clock gene, Bmal1, in SCs (SC-Bmal1iKO) muscle damage, SC progression, and muscle repair following in vivo eccentric contractile injury were compared to control animals (SC- Bmal1Cntrl). Baseline in vivo torque and ex vivo specific force were quantified in both groups who underwent in vivo contractile injury consisting of 200 eccentric contractions. All mechanical experiments were performed at the same time of day (1000 h). Following injury, animals were sacrificed at 24 h, 72 h, and 7 days. Muscles were frozen, sectioned, and histologically and/or immunohistologically labeled for markers of muscle damage, neutrophil content, muscle repair, and satellite cell myogenic progression.

Results/Discussion: The results from these studies demonstrate that SC presence/absence does not affect time-of-day mitochondrial respiration. In line with peak, trough Bmal1 and CLOCK gene expression, mitochondrial-dependent submaximal fatigue showed ~35% greater fatigue-resistance in the morning versus afternoon. Collectively, SCs are not a factor that influence time-of-day mitochondrial function and therefore time-of-day differences in submaximal fatigue I (and others) observe may not be due to time-of-day regulations (SC, muscle, or otherwise) on mitochondrial respiration. With notable diurnal differences observed in submaximal contractile-fatigue, maximal contractile function with respect to time-of-day and SC presence/absence was evaluated in a separate study at the same timepoints. Morning-SC+ animals demonstrated reduced maximal tetanic and eccentric specific forces compared to SC- counterparts. However, no such differences were observed between Afternoon-SC+/SC- groups. Consequently, Morning-SC+ animals experienced reduced extents of contractile injury (less force-loss and dystrophinnegataive fibers) compared to SC- counterparts, whereas no differences were noted between afternoon groups. These findings demonstrate that the regulatory role of SCs over contractility is time-of-day specific. Evaluations of ex vivo caffeine-contracture force, a surrogate for maximal Ca++ availability to contractile units, revealed similar patterns of lower force in Morning-SC+ versus SC- counterparts indicating lower volumes of Ca++ may be underpinning the lower forces observed in these animals. These observations suggest SCs influence maximal force-production in the morning but, not in the afternoon and thus the extent of injury is concordant with the level of maximal force produced at specific times of day and the prevailing status of SCs. To further unravel SC-molecular clock regulation on contractile injury and how that may impact repair, a mouse model allowing for inducible depletion of SC-specific clock gene, Bmal1, was utilized for the last experiments. Following in vivo contractile injury, these animals demonstrated lesser extents of fiber-necrosis (24 h, 72 h), neutrophil content (24 h, 72 h), eMHC+ fibers (7 dpi), and centralized nuclei (7 dpi) compared to control animals. Of note, as SC-Bmal1iKO animals produced lower torque and specific forces, these animals may have sustained less damage/repair in-line with the established notion that higher forces lead to higher damage. As necrosis delays SC kinetics, the lesser necrosis noted in SC-Bmal1iKO animals may have led to the earlier peaks in SC activation/proliferation (24 h versus 72 h in control animals). The extent of SC activation (Pax7+/MyoD+) was approximately two-fold higher in SC-Bmal1iKO animals, suggesting that SC-Bmal1 has an additional role on the temporal and volumetric regulation of MyoD during SC-mediated repair.

Conclusion: The results of the experiments performed provide evidence that SC-molecular clocks play a regulatory role in contractile function and muscle repair. While past works have shown muscle molecular clocks regulate these events, the results from the experiments undertaken for this thesis provide novel insights to demonstrate that satellite cell specific molecular clocks also regulate these processes. Collectively, the results from these studies provide preliminary insight suggesting that SC-molecular clocks, in part, regulate maximal force-production, contractile injury-induced muscle damage/repair, and SC-mediated myogenic progression. Future work will be able to use these initial studies as foundational knowledge to further explore the mechanisms of how SC-molecular clocks regulate muscle physiology according to time-of-day.

This work © 2025, Ryan Kahn.