Investigating the role of the circadian clock and timed exercise on mouse skeletal muscle function

Abstract

An inactive, sedentary lifestyle directly impacts health and increases the risk of premature death. Likewise, circadian rhythm disruption (e.g., shift-work) is linked to various chronic diseases, including diabetes, cardiovascular disease, and cancer. Regular physical exercise is a potent regulator of skeletal muscle and whole-body metabolism and can prevent or even treat chronic medical disorders, including diabetes, cancer, and cardiovascular disease. Moreover, proper sleeping schedules, meal-timing, and chrono-medication all can ameliorate many of the aforementioned diseases. It is unclear, however, whether a chronotherapeutic approach of exercise can amplify the beneficial health effects of regular training, and whether these effects would be mediated by skeletal muscle. The first part of my Ph.D. work evaluated the transcriptomic, proteomic, and phosphoproteomic responses of mouse skeletal muscle to two widely used exercise modalities at distinct phases of the light-dark cycle. We found that maximal treadmill exercise capacity varies according to the time of day, and give evidence of subtle changes in systemic and muscle energy levels in response to exercise around the clock. Moreover, we reveal a timely activation of specific biological pathways within working muscles. For instance, mechanisms directing vesicular trafficking, phosphorylation of key regulators of glucose uptake and calcium metabolism, and putative secreted factors affecting hepatic glucose production were induced explicitly by early daytime treadmill exercise and could potentially enhance running exercise capacity. We furthermore established a completely new methodology to examine the effects of spontaneous wheel running. Using this method, we give evidence that exercise is a poor modifier of daily clock gene expression. On the other hand, we show that daytime wheel-running activity is a potent modifier of oxidative metabolism gene expression under constant environmental light conditions; however, foodintake has a more profound effect on the skeletal muscle clock. With our large-scale transcriptomic, proteomic, and phosphoproteomic data, we provide resources for future research projects and validation studies. We also hope that the use of scheduled wheel running at different times of the day could be useful in preclinical mouse models. Mouse models of whole-body clock gene deletion highlighted the potential role for circadian clock components in muscle cell development and repair, insulin sensitivity, glucose, and lipid metabolism. Yet, global gene deletion leads to multiple effects in other organs, which can be confounding factors for the evaluation of muscle-specific function. The second part of my Ph.D. work was to characterize the specific contribution of the core clock transcription factor RORα in skeletal muscle physiology and exercise behavior. We show that skeletal muscle-specific deletion of RORα significantly decreased spontaneous wheel-running activity in the absence of significant alteration in muscle structure, and systemic metabolism. Interestingly, in response to exercise, a decrease in the expression of key regulators of oxidative stress was observed. Furthermore, upon exposure to hypoxia, causing abnormal ROS elevation, or directly to H2O2, we reveal that RORα overexpression protects from cell death. Our data suggest that skeletal muscle RORα is important for modulating ROS metabolism and possibly the skeletal muscle adaptations to exercise. Taken together, our data provide new evidence that skeletal muscle molecular and cellular responses are dependent on the time of the day and systemic energy level. Notably, we propose that mechanisms promoting glucose delivery and uptake in muscle tissues, together with those critical for the response to inflammation and the secretion of myokines, are tied to a time period of the day. Our results, moreover, suggest that there is an optimal time to exercise to improve muscle adaptation and probably performance, as well as glucose homeostasis. Finally, the last part of my Ph.D. work gives evidence that the circadian component RORα is required to influence ROS metabolism and the cellular adaptations of skeletal muscle to a prolonged period of exercise training.