SchroderExerc Sport
SchroderExerc Sport
SchroderExerc Sport
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Abstract
Skeletal muscle comprises approximately 40 % of total body mass and, as such, contributes to
maintenance of human health. In this review we will discuss the current state of knowledge
regarding the role of molecular clocks in skeletal muscle. In addition we discuss a new function
for exercise as a time setting cue for muscle and other peripheral tissues.
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Keywords
circadian; skeletal muscle; exercise; peripheral tissues; zeitgeber
Introduction
Circadian rhythms are the approximate 24-hour biological cycles that function to prepare the
organism for daily environmental changes. Almost all organisms ranging from single cell
bacteria to plants and animals, exhibit behavioral, physiological and biochemical rhythms
termed circadian rhythms.(7, 9) Underlying circadian rhythms is a molecular clock
mechanism found in most, if not all, cell types including skeletal muscle. At the cellular
level, the presence of a molecular clock is argued to be a necessary timekeeping mechanism
to prepare the cell for daily changes in environmental conditions. The ability to synchronize
the molecular clock and intracellular physiology with external day-night cycles denotes an
evolutionarily conserved adaptation to environmental conditions.(7, 9)
Environmental stimuli capable of shifting the timing of molecular rhythms are critically
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Additional components to the core molecular clock family include the orphan nuclear
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receptors Rora (RAR-related orphan receptor-α) and Rev-erb α/β. These gene products
function to link the feedback loops by activating (Rora) or repressing (Rev-erb) Bmal1
transcription.(21, 24) Most recently, studies have added new elements, known as E3 ligases
(e.g. Fbxl3), to the core molecular clock and these elements function by changing stability of
the PER and CRY proteins.(33) In addition to their role in timekeeping, components of the
core clock (Bmal1 and Clock) have also been shown to transcriptionally regulate the
expression of genes that do not function in timekeeping and these genes are designated as
clock-controlled genes (CCGs).
While the identity of all the direct clock controlled genes in a specific tissue, like skeletal
muscle, have not been defined, they often encode transcription factors (e.g. MyoD1) or
proteins that control rate-limiting steps in cell physiology (e.g. PBEF, the rate-limiting
enzyme in the NAD+ salvage pathway).(17, 18) For more detailed reviews of the molecular
clock mechanism there are several recent reviews by other groups.(16)
suprachiasmatic nucleus (SCN) came when it was discovered that surgical ablation of the
SCN resulted in arrhythmic behavior patterns.(27) SCN ablation in hamsters creates an
actively arrhythmic animal and transplantation of a healthy SCN (from either a hamster or
mouse) back into the lesioned hamsters restores activity rhythms with rhythms matching that
of the donor animal (26) demonstrating the role of the SCN as a system-wide circadian
synchronizer. Cell autonomy of the molecular clock in peripheral tissue was first established
almost 15 years ago.(3) The development of a mouse model in which the luciferase cDNA
was knocked into the Per2 coding region to generate a chimeric protein provided the
powerful resource (PER2:LUC mice) to directly test the cell autonomy of the clock
mechanism.(32) For these studies, tissue explants from the SCN, liver and lung from these
PER2:LUC mice were placed in cell culture in the presence of luciferin and real time light
emission was monitored for up to two weeks. While oscillations in luminescence were
expected from the central clock (SCN slice) these findings demonstrated that the clock
mechanism from peripheral tissue explants maintained normal 24hr circadian periodicity in
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the absence of systemic neural or humoral factors.(32) These studies also validated the use
of the PER2::LUC mouse as a valuable resource to study molecular clock function in
multiple tissues including skeletal muscle. (31, 35)
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While studies have established that the molecular clock mechanism is intrinsic to each cell
and that it can run in a cell autonomous manner, a critical feature is that the phase of the
molecular clock can be set or reset by cues from the environment. The ability to reset the
circadian clock is a critical function to be able to adapt to environmental changes. The
timing or phase of the central clock is primarily entrained by cues from light.(29) Recent
studies have demonstrated that the molecular clock in many peripheral tissues, including
skeletal muscle can be dissociated from the rhythm of the SCN by restricting time of
feeding.(4) In addition, Zambon et al.(34) reported that there is an interaction between time
of day and contraction on expression of clock genes in human muscle, suggesting contractile
activity might be a zeitgeber for the molecular clock mechanism in skeletal muscle. In this
study, microarray analysis was used to determine the effects of resistance exercise on gene
expression in the quadriceps at 6 and 18 hours after an acute bout exercise. Gene expression
was also examined in the non-exercised leg at the same time points for comparison. These
results indicate that feeding, and potentially contractile activity, can act as dominant
zeitgebers for setting clocks in peripheral tissues. (Figure 2) They also demonstrate that
circadian rhythms in peripheral tissues can be dissociated from the SCN through non-photic
environmental signals.
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The timing of the molecular clock can also be modulated by post-translational mechanisms
using phosphorylation, acetylation and/or ubiquitination pathways. These modifications
impact the stability and/or translocation of core molecular clock components with the
greatest impact on period length (time for completion of 1 cycle (24h)). The most well-
known regulator of clock function through changing phosphorylation status is casein
kinase1ε (CK1ε). This serine/threonine kinase has been the subject of studies in both
hamster and humans in which the mutations of either CK1ε or PER2 affect period length.
The tau mutant hamster, has a point mutation (Arg178Cys) in the catalytic region of CK1ε,
termed the tau mutation, which results in a hamster that exhibits short circadian periods.(23)
In these hamsters, CK1ε is unable to phosphorylate PER proteins. With diminished levels of
phosphorylation, PER translocates to the nucleus leading to a more rapid repression of the
CLOCK: BMAL1 mediated transcription, effectively shortening the circadian period to 20
hours. (10) In humans, a mutation in PER2 that affects a phosphorylation site (target of
CK1ε) results in the clinical condition, familial advance sleep phase syndrome (FASPS).
Studies of these individuals determined that they have a shortened period length of
approximately 20 hours compared to the normal 24 hours observed in most humans and is
associated with early awake and early to sleep.(30)
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direct or permissive mechanism for controlling clock gene expression and propose roles for
acetylation/deacetylation in the initiation, duration and termination of both the activating
and repressing phases of the circadian cycle.
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behavioral rhythms of 27 hours.(2) We found that several core clock genes including Bmal1
and Per2, no longer oscillated in the muscle of Clock mutant mice. Oscillation was also lost
in several known clock controlled genes in the skeletal muscle such as Tef, Dbp, Myod1 and
Pgc1β. In addition to analysis of the circadian genes, McCarthy et al, found that
approximately 35% of all the expressed genes were differentially expressed in the muscle of
Clock mutant mice demonstrating the substantial impact disruption of the core clock
machinery has on gene expression with implications for normal cellular function and health.
With continued development of analysis tools for time course gene expression studies, the
list of genes expressed in a circadian manner in skeletal muscle has grown to greater than
800. Pizarro et al (2013)(20) have established a valuable website to allow for queries into
circadian expression from time course expression experiments in tissues and cell lines
(http://circadb.org). These new data will open many exciting avenues of research in the
study of skeletal muscle circadian physiology. However, one area that is still understudied is
whether the circadian transcriptome differs among individual skeletal muscles of different
developmental origin (e.g. limb vs. facial muscle) or whether they differ among muscles
with markedly different fiber type, metabolic function and mechanical function.
Wheel running or cage activity has long been utilized as a read-out of circadian behavior but
the idea that physical activity/exercise and more importantly the timing of exercise could
serve as an entrainment cue is relatively new. (25, 31) Studies in the late 1980’s and early
90’s were the first to show that novel wheel access at different times of day was sufficient to
shift the phase of circadian activity rhythms in mice and hamsters. (5) Further studies
demonstrated that exercise is a sufficient environmental cue to effect clock gene expression
in the SCN (central clock) located in the hypothalamus of the brain. (12) These studies
established that activity, in the form of access to a novel running wheel, during light
conditions decreased peak expression of the clock genes Per1 and Per2 in the SCN.
Schroeder et al took this idea further examining both timing of exercise as well as multiple
bouts of exercise. They explored effects on the central clock utilizing a scheduled exercise
paradigm in control and mutant mice in which the central molecular clock mechanism was
weakened.(25) In attempts to better mimic the timing of exercise in the human population,
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mice were allowed free access to a wheel, no access to a wheel, or access limited to 6 hour
time frames at the beginning or end of the dark or active phase for a minimum of 16 days.
Similar to previous studies examining single bouts of exercise, they observed changes in the
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properties of the molecular clock in the SCN after 16 days in the control mice suggesting the
phase shifts observed in previous studies were not solely an acute phase response. Moreover,
PER2:LUC amplitude was damped in mice with wheel access scheduled early in the dark
phase but unaffected with scheduled activity late in the dark phase or with free access to
wheel running suggesting that the timing of exercise may be critical for the maintenance of
molecular rhythms in the SCN. Utilizing the vasoactive intestinal polypeptide (VIP) knock-
out mouse, shown to have an unstable clock mechanism, they found that scheduled exercise
functioned to enhance the stability of both activity and heart rate rhythms.
The core molecular clock gene Clock has also been demonstrated to be critical for healthy
skeletal muscle as Clock mutant mice exhibit ~30% reductions in normalized maximal force
at both the muscle and single-fiber level. (1) In addition, myofiber architecture is disrupted
and mitochondrial volume is diminished. Recent work from Pastore et al supports these
results. (19) They demonstrated that CLOCK protein is critical for mitochondrial
maintenance in skeletal muscle. Taking this a step further, they examined the ability of clock
mutant mice to adapt to chronic exercise and found that despite the pathology as a result of
the mutant Clock gene the ability of these mice to adapt to chronic exercise was not
changed. Utilizing endurance training they were able to partially rescue the metabolic
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Most studies of exercise and shifting of circadian rhythms have relied on endurance exercise
paradigms. Less is known about the potential for resistance exercise but Zambon et al.,
reported that one bout of 60 contractions was associated with changes in molecular clock
gene expression in skeletal muscle of humans.(34) Experiments in neonatal cardiomyocytes
have shown that contractile activity may modulate the molecular clock through the actions
of the CLOCK protein. Histological and biochemical analysis demonstrated that CLOCK
localizes to the Z-disk in neonatal cardiomyocytes and translocates to the nucleus to
influence gene expression in response to contractile activity. CLOCK localization at the Z-
disk puts CLOCK in an appropriate location to sense mechanical function associated with
contractile activity. (22) While these studies are suggestive that resistance exercise can also
modulate molecular clock function in muscle, there is still much to be determined.
With over 600 different muscles in the human body, comprising approximately 40 percent
total body mass, understanding the effects of exercise on the molecular rhythms in
individual skeletal muscles may provide critical insight into systemic mechanisms
contributing to daily rhythms. At this stage, there is only one study that has examined more
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than one muscle in mice exposed to scheduled bouts of either voluntary or involuntary
endurance exercise for 2 hours/day in the light phase four hours after lights on. In this study,
the authors found a significant shift in clock gene expression (PER2:LUC bioluminescence)
in three different skeletal muscles and the lung from exercised mice, whereas the molecular
clock in the SCN remained unshifted demonstrating that scheduled exercise can alter the
molecular clock in peripheral tissues. In addition, one of the muscles examined, the flexor
digitorum brevis (FDB) was phase advanced more than the other two muscles (soleus and
extensor digitorum longus) suggesting the potential for differential regulation of the
molecular clock in individual muscles.(31) Lumicycle data in combination with data
demonstrating rescue of phenotype resulting from exercise,(19, 25, 31) implicates exercise
as a non-photic time cue in peripheral tissues and suggests that the molecular clock in all
muscle tissues may not respond in a similar manner to non-photic cues. Since muscle is such
a large contributor to systems physiology, these data have broad implications for human
health and disease suggesting the power of exercise, and more specifically the interaction of
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Summary
Circadian rhythms and molecular clocks in skeletal muscle is a new and rapidly emerging
area of research. Studies in the last 15 years have demonstrated that molecular clocks exist
in skeletal muscle and more importantly, studies have demonstrated that the phase of the
clocks in skeletal muscle can be reset by altering time of exercise or time of feeding
independent of the central clock in the brain. In parallel studies it has been shown that
skeletal muscles from mice in which the core clock genes have been disrupted are weaker
and exhibit decreased mitochondrial content and function. These findings suggest a potential
new role for exercise in human health through its role in providing timing information to
skeletal muscle and other peripheral tissue clocks. Although difficult to extrapolate to
humans, exercise studies in mice do suggest an optimal time of day for exercise to enhance
the robust nature of circadian rhythms at the molecular level. Until an optimal time is
determined, consistently timed daily exercise may be a viable alternative. These studies
highlight the recognition that time of day matters for maintenance of proper molecular clock
function and is important for issues of muscle strength and endurance.
Acknowledgments
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This work was supported by the following NIH grants RC1ES018636 and R01AR55246 (KAE).
The authors would like to thank Jonathan England for his help with the figures in the preparation of this manuscript.
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Summary
This review discusses the relationship between the molecular clock, skeletal muscle, and
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exercise.
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Figure 1.
The core circadian clock is an autoregulatory transcriptional feedback loop comprised of a
transcription factors, CLOCK and BMAL1 and their target genes; Per1, Per2, Cry1, and
Cry2. The CLOCK:BMAL1 heterodimer also functions to drive transcription of Rev-erbα
and Rorα which in turn either represses or activates Bmal1 transcription respectively. The
CLOCK:BMAL1 heterodimer also regulates downstream targets known as clock-controlled
genes (CCG). The kinases, casein kinase 1ε (CK1ε) and AMP kinase (AMPK),
phosphorylate PER(CK1ε) and CRY(AMPK) proteins to promote polyubiquitination by E3
ubiquitin ligase complexes. PER and CRY proteins are then degraded by the 26S
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proteasome complex.
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Figure 2.
Central and peripheral clocks can respond to both photic (light) and non-photic (feeding and
exercise) zeitgebers. This schematic demonstrates the interplay between the different
zeitgebers and central and peripheral clocks. It also suggests that central and peripheral
clocks may have differential responses depending upon the zeitgeber present.
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