Scheduled exercise phase shifts the circadian clock in skeletal muscle

Abstract

Purpose: It has been well established in mammals that circadian behavior as well as the molecular clockwork can be synchronized to the light-dark cycle via the suprachiasmatic nucleus of the hypothalamus (SCN). In addition to light, it has been demonstrated that nonphotic time cues, such as restricting the time of food availability, can alter circadian behavior and clock gene expression in selected peripheral tissues such as the liver. Studies have also suggested that scheduled physical activity (exercise) can alter circadian rhythms in behavior and clock gene expression; however, currently, the effects of exercise alone are largely unknown and have not been explored in skeletal muscle.

Methods: Period2::Luciferase (Per2::Luc) mice were maintained under 12 h of light followed by 12 h of darkness then exposed to 2 h of voluntary or involuntary exercise during the light phase for 4 wk. Control mice were left in home cages or moved to the exercise environment (sham). A second group of mice had restricted access to food (4 h · d(-1) for 2 wk) to compare the effects of two nonphotic cues on PER2::LUC bioluminescence. Skeletal muscle, lung, and SCN tissue explants were cultured for 5-6 d to study molecular rhythms.

Results: In the exercised mice, the phase of peak PER2::LUC bioluminescence was shifted in the skeletal muscle and lung explants but not in the SCN suggesting a specific synchronizing effect of exercise on the molecular clockwork in peripheral tissues.

Conclusions: These data provide evidence that the molecular circadian clock in peripheral tissues can respond to the time of exercise suggesting that physical activity contributes important timing information for synchronization of circadian clocks throughout the body.

Figure 1. Restricted feeding alters the pattern of locomotor activity over the day without decreasing overall activity

(A) Double-plotted actograms of locomotor activity counts measured in the home cage averaged from free feeding (FF) or restricted feeding (RF), n=5 mice per group, dark bars indicate lights-off and gray shaded areas indicate time of restricted feeding in RF mice. (B) Activity profiles calculated during the two weeks of restricted feeding, averaged from n=5 per group. Gray shaded area indicates lights-off. (C) Mean activity counts over the entire day (Total), during lights-on, lights off, and anticipatory activity from ZT 1-4. Before refers to the averaged locomotor cage activity from all groups during time points indicated on each graph. * indicates significant from before (*p<0.05, mean ± SEM, ANOVA, post hoc Dunnett’s).

Figure 2. Restricted feeding phase shifts PER2∷LUC bioluminescence in peripheral tissues

(A) Representative raw bioluminescence data from FF in black or RF in gray. (B) Phase plot of cultured explants (mean ± SEM), black squares indicate FF, open squares RF. Mean phase data from Per2∷Luc explants was calculated using the time of peak bioluminescence following 24 hours in culture. In the RF group PER2∷LUC bioluminescence demonstrated a significant phase shift in both the lung and soleus explants (*p<0.05, mean ± SEM, Student’s t-test). (C) Representative sections from control soleus (fixed immediately) and one soleus that had been in the LumiCyle for 5 days, stained with H&E, imaged at 20X.

Figure 3. Alterations in locomotor cage activity following exercise in mice

(A) Double-plotted actograms of locomotor behavior throughout the experiment and (B) averaged activity profiles (calculated from the last two weeks) from control (n=3), sham treadmill (n=4) and treadmill (n=5) mice. (C) Mean activity counts throughout the day in control, sham treadmill (ShamT), treadmill, sham wheel (ShamW) and wheel groups. Before (black bars) is the locomotor cage activity in the two weeks prior to exercise averaged from all groups during time points indicated on each graph. During control, during ShamT, during treadmill, during ShamW, and during wheel were calculated from the final two weeks of exercise, * indicates significant from before (*p<0.05, mean ± SEM, ANOVA, post hoc Dunnett’s). Dark bars (A) and shaded area (B) represent time of lights-off, gray bars (A and B) indicate when animals were exposed to exercise.

Figure 4. Scheduled voluntary or involuntary exercise shifts PER2∷LUC bioluminescence in several skeletal muscles and lung

(A) Representative graphs of raw bioluminescence data (counts/sec) from Per2∷Luc tissue cultured explants over 5-6 days. For soleus and EDL representative raw data from control, ShamT, and treadmill groups. For FDB representative raw data from control, ShamW, and wheel. (B) The phase of PER2∷LUC bioluminescence was calculated using the time of peak bioluminescence following 24 hours in culture. Phase plot of cultured explants (mean ± SEM), black circles indicate control, black squares for sham groups, and red diamonds indicate both treadmill and wheel. In all three skeletal muscles from exercised mice the phase of PER2∷LUC bioluminescence was shifted to an earlier time (advanced) compared to control and in the lung compared to ShamW. There was no significant shift in the SCN. (*p<0.05, mean ± SEM, ANOVA within each tissue, post hoc Tukey, +p<0.05 compared with ShamW).