Muscle growth is higher in the daytime, which is regulated by the biological clock - new study

The Hughes group from the Randall Centre for Cell & Molecular Biophysics, School of Basic & Medical Biosciences, has recently provided strong evidence to suggest that indeed circadian clock plays a crucial role in regulating muscle protein turnover and growth independent of physical activity and nutrition. The work has previously been published in Proceedings of the National Academy of Sciences of the United States of America (PNAS).

Animals exhibit robust circadian rhythms (~24 h oscillations due to Earth’s rotation) in behaviours and physiology. An obvious example is the day/night difference in the levels of physical activity, which is powered by muscle contractions. For more than 20 years, physiotherapists and sports scientists have observed that human muscle strength peaks in the late afternoon (~1600-2000 h).

A recent study investigating how the time of day affects Olympics swim times from 2004 to 2016 suggests that the physical performance of elite athletes is strongly affected by the time of day (i.e. highest in the afternoon hours). Thus, it appears that at least muscle functions are regulated by the biological clock. Whether the time of day affects muscle metabolism and hence growth, remains to be explored. Answers would help to identify the best time of day to exercise, build muscle, and prevent ageing- or disease-related sarcopenia (a syndrome characterized by progressive and generalised loss of skeletal muscle mass).

Physical activity such as exercise is long known to be a major intervention for improving population health. Beneficial effects of exercise are widespread, many of which are related to skeletal muscle homeostasis. Healthy functioning muscle may keep obesity, osteoporosis, heart and kidney disease, diabetes and associated disorders at bay. Muscle mass is maintained by both genetic and ‘environmental’ factors, such as exercise and nutrition, acting upon anabolic and catabolic processes that control muscle size. It is well-known that muscle growth-promoting factors such as hormone secretion, feeding and physical activity are all under biological clock control. Disrupted sleep and altered muscle metabolism are significant causes of obesity, and human studies suggest that disturbance of circadian rhythm via shift work or lack of sleep, disrupts skeletal muscle homeostasis. These studies, therefore, suggest a link between the biological clock, muscle physiology and population health. Nonetheless, a direct relationship between the dysregulation of the biological clock and loss of muscle and strength in people has yet to be established. We can now measure muscle growth in a simple vertebrate animal model system, zebrafish, and use their larvae to study the role of the biological clock in muscle growth and metabolism.

Zebrafish as a model to study muscle chronobiology

Zebrafish is an excellent model for the study of muscle chronobiology. First, zebrafish skeletal muscle is very similar to human muscle, and many human muscle genes are conserved in zebrafish, including those causing muscle disease. Second, zebrafish larvae are transparent, which facilitates direct microscopic observation, allowing the study of muscle growth and development in live animals. Third, zebrafish are diurnal like humans (i.e. active in the day and sleep at night) and they share the same sets of the core “clock genes” that oscillate over a 24 h period to maintain the functioning of the cellular clocks and their outputs. Last, larvae are transparent and thus cellular clocks can easily be set/reset by direct light exposure.

Day/night difference in muscle growth, irrespective of locomotor activity and feeding

By raising zebrafish at 12 h light/12 h dark (LD) cycles, we observed that the skeletal muscle of post-hatched larvae grows 50% more in the daytime than at night. This suggests there is a day/night difference in muscle growth. As said, feeding is a prominent contributing factor of muscle building, which is under circadian control such that diurnal animals feed in the active phase (day) and fast in the inactive phase (night). We therefore tested whether an altered supply of nutrients could account for day/night variation in muscle growth. At the stages examined, zebrafish larvae do not feed; nutrition and substrates for muscle anabolism are instead derived from the yolk sac via circulation. We showed that yolk consumption is constant over the diurnal cycle, arguing that nutrition does not account for day/night variation in muscle growth.

Diurnal zebrafish larvae spontaneously swim more during the day, raising the possibility that activity promotes muscle growth in the daytime. We therefore blocked all muscle contractions by growing fish larvae in anaesthetic. Strikingly, the day/night difference in muscle growth remained, such that the skeletal muscle of inactive fish grew during the day but failed to grow, and even showed a tendency to shrink, at night. We concluded that the zebrafish muscle grows more in the day than in the night, irrespective of feeding and muscle contractile activity.

Physical activity and daytime promote anabolism

Muscle growth is thought to be regulated by protein turnover, a balance between anabolism and catabolism. By assaying protein synthesis, we showed that in skeletal muscle, there is more synthesis in the day than at night, paralleling the observation of diurnal muscle growth. We further showed that the mechanistic target of rapamycin (mTOR) pathway, which is a central regulator of cell metabolism and growth, is more active in the daytime. Pharmacological inhibition of this pathway led to a reduction in muscle growth specifically in the day. Next, we looked at muscle catabolism by analysing the expression pattern of muscle RING Finger (MuRF) genes, prominent markers for muscle degradation. We found that the expression of MuRF genes peaks at night. Pharmacological inhibition of protein degradation led to an increase in muscle growth specifically at night. These results indicate that muscle anabolism and catabolism are more active in the day and at night, respectively. Thus, we uncovered a prominent role of the biological clock in regulating muscle growth, probably by increasing growth during the day and promoting atrophy at night. When the circadian rhythm of the fish was experimentally disrupted, muscle size was reduced and the day/night difference in growth was removed. This shows that the biological clock is crucial for optimal muscle growth.

Our findings, therefore, significantly advance understanding of the interaction between the circadian clock, physical activity, and the role of feeding on skeletal muscle growth. It remains to be seen whether the principles discerned in the current work apply more broadly to other species. If they do, deeper study may increase understanding of the effects on muscle growth and maintenance of aging, in which the circadian clock weakens and exercise diminishes.

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