CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function - PubMed (original) (raw)

. 2010 Nov 2;107(44):19090-5.

doi: 10.1073/pnas.1014523107. Epub 2010 Oct 18.

Xiping Zhang, John J McCarthy, Erin L McDearmon, Troy A Hornberger, Brenda Russell, Kenneth S Campbell, Sandrine Arbogast, Michael B Reid, John R Walker, John B Hogenesch, Joseph S Takahashi, Karyn A Esser

Affiliations

CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function

Jessica L Andrews et al. Proc Natl Acad Sci U S A. 2010.

Abstract

MyoD, a master regulator of myogenesis, exhibits a circadian rhythm in its mRNA and protein levels, suggesting a possible role in the daily maintenance of muscle phenotype and function. We report that MyoD is a direct target of the circadian transcriptional activators CLOCK and BMAL1, which bind in a rhythmic manner to the core enhancer of the MyoD promoter. Skeletal muscle of Clock(Δ19) and Bmal1(-/-) mutant mice exhibited ∼30% reductions in normalized maximal force. A similar reduction in force was observed at the single-fiber level. Electron microscopy (EM) showed that the myofilament architecture was disrupted in skeletal muscle of Clock(Δ19), Bmal1(-/-), and MyoD(-/-) mice. The alteration in myofilament organization was associated with decreased expression of actin, myosins, titin, and several MyoD target genes. EM analysis also demonstrated that muscle from both Clock(Δ19) and Bmal1(-/-) mice had a 40% reduction in mitochondrial volume. The remaining mitochondria in these mutant mice displayed aberrant morphology and increased uncoupling of respiration. This mitochondrial pathology was not seen in muscle of MyoD(-/-) mice. We suggest that altered expression of both Pgc-1α and Pgc-1β in Clock(Δ19) and Bmal1(-/-) mice may underlie this pathology. Taken together, our results demonstrate that disruption of CLOCK or BMAL1 leads to structural and functional alterations at the cellular level in skeletal muscle. The identification of MyoD as a clock-controlled gene provides a mechanism by which the circadian clock may generate a muscle-specific circadian transcriptome in an adaptive role for the daily maintenance of adult skeletal muscle.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

MyoD is a clock-controlled gene in skeletal muscle. (A) Expression of MyoD in wild type (●) and ClockΔ19 (○) muscle was determined by quantitative PCR. Samples were collected every 4 h for 48 h starting at circadian time 18 (CT18) through CT62. Even though all collections were performed under total darkness, the dark and light stripes on the graph represent presumptive dark and light phases of the mice. (B) The diurnal expression (12:00 AM vs. 12:00 PM) of MyoD in wild-type (lanes 1–4) and Bmal1−/− (lanes 5–8) skeletal muscle was determined by semiquantitative PCR normalized to Rpl26 gene expression. Muscles (n = 4/group) were collected under DD at either 12:00 AM (lanes 1, 3, 5, and 7) or 12:00 PM (lanes 2, 4, 6, and 8). Histogram of densitometric quantification showed a significant (P < 0.05) diurnal expression of MyoD in wild-type muscle that is lost in Bmal1−/− muscle. (C) Western blots demonstrating circadian oscillation of MyoD levels in muscle of wild-type mice collected every 4 h for 28 h (CT18–46). (D) Illustration of MyoD reporter gene (CE+_MyoD_6.8) showing the position of the CE and DRR. The histogram summarizes results from transfection experiments using either a Per1 reporter gene or the MyoD reporter gene in C2C12 cells (n = 3/conditions). Over-expression of CLOCK and BMAL1 (black bar) significantly transactivated Per1 and CE-MyoD reporter genes by ∼2.5-fold and 6-fold, respectively, relative to control transfections (open bar). MyoD reporter was not activated by BMAL1:CLOCK, and activation of CE-MyoD reporter was significantly decreased by 50% when ClockΔ19 was over-expressed with BMAL1 (gray bar). Values are mean ± SEM with significance (P < 0.05) denoted by an asterisk or a pound sign (B+C vs. B+C_Δ_19). (E) Chromatin immunoprecipitation assays from muscles collected at CT26 and CT38 demonstrating CLOCK and BMAL1 binding to the CE at CT38 and no binding at the DRR of the MyoD promoter. The numbers under each lane represent the ratio of the intensity of the Ab band/No Ab band.

Fig. 2.

Fig. 2.

Decreased whole-muscle function, single-cell function, and myofilament structure in ClockΔ19, Bmal−/−, and MyoD−/− mice. (A) Representative force trace from the measurement of specific tension of whole-muscle (EDL) from wild-type mice. (B) Histogram of the average specific tensions of muscles for ClockΔ19, Bmal1−/−, and MyoD−/− mice (n = 3–6/strain). Significant difference (P < 0.05) from wild type is denoted by an asterisk. (C) Results from single-fiber mechanical analyses of wild-type (△) and Bmal1−/− (○) muscle fibers. Each point on the curve represents the average ± SEM for measures of 7–20 cells at each calcium concentration. (D) Data from C reported as tension relative to maximal tension for each calcium concentration. (E) Representative myofilament images obtained by electron microscopy (43,000×) from wild-type, ClockΔ19, Bmal1−/−, and MyoD−/− gastrocnemius muscles. The normal organization of thin and thick filaments is disrupted in muscle from the three different mutant animals.

Fig. 3.

Fig. 3.

Decreased mitochondrial volume and respiratory function in muscle of ClockΔ19 and Bmal1−/− mice. (A) Low-magnification EM images (4,000×) of skeletal muscle from wild-type, ClockΔ19, and Bmal1−/− mice. The white arrow in each image points to the region of the muscle under the sarcolemma where there are abundant mitochondria (wild type) or where mitochondria are lacking (ClockΔ19 and Bmal1−/−). (B) Histogram of mitochondrial volume measured using point-counting morphometry. The values are presented as a percentage of muscle-fiber volume from wild-type (black bar), ClockΔ19 (gray bar), and Bmal1−/− (open bar) mice. Values represent mean ± SEM (n = 5 muscles/strain) with significance (P < 0.05) denoted by an asterisk. (C) Representative high-magnification EM images (21,000×) of mitochondria within skeletal muscle of wild-type, ClockΔ19, and Bmal1−/− mice. Note swollen size and disrupted cristae of the mitochondria from muscle of ClockΔ19 and Bmal1−/− mice. (D) Histograms of biochemical measurements of respiratory control ratio (RCR) in gastrocnemius (GTN) and diaphragm (DIA) muscles of wild-type and Bmal1−/− mice (n = 6/strain). Values are means ± SEM with significance (P < 0.05) denoted by an asterisk. (E) Histograms showing significant reduction in state III respiration (ADP-stimulated, mmol O2/min/mg protein) in mitochondria isolated from GTN muscle of Bmal1−/− mice compared with wild type. Values are means ± SEM with significance (P < 0.05) indicated by an asterisk.

Fig. 4.

Fig. 4.

Altered expression of Pgc-1 coactivators in ClockΔ19 and Bmal1−/− mice. (A) Array data of Pgc-1β mRNA expression in skeletal muscle of wild-type mice (●) and ClockΔ19 mice (○); the light and dark stripes refer to the presumptive light and dark phases for the mice (7). (B) Quantitative PCR results for expression of Pgc-1β in wild-type muscle (●) and ClockΔ19 muscle (○). (C) Histogram of the mean expression level of PGC1α mRNA in muscle of wild-type, ClockΔ19, and Bmal1−/− mice as determined by quantitative PCR. A significant difference (P < 0.05) is denoted by an asterisk. (D) Proposed model of CLOCK:BMAL1 regulation of muscle phenotype and function via targeting of MyoD and Pgc-1 expression. Solid lines indicate known molecular links among components of the molecular clock, and dashed lines suggest potential links.

Similar articles

Cited by

References

    1. Bell-Pedersen D, et al. Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nat Rev Genet. 2005;6:544–556. - PMC - PubMed
    1. Green CB, Takahashi JS, Bass J. The meter of metabolism. Cell. 2008;134:728–742. - PMC - PubMed
    1. Emerson KJ, Bradshaw WE, Holzapfel CM. Concordance of the circadian clock with the environment is necessary to maximize fitness in natural populations. Evolution. 2008;62:979–983. - PMC - PubMed
    1. Woelfle MA, Ouyang Y, Phanvijhitsiri K, Johnson CH. The adaptive value of circadian clocks: An experimental assessment in cyanobacteria. Curr Biol. 2004;14:1481–1486. - PubMed
    1. Lowrey PL, Takahashi JS. Mammalian circadian biology: Elucidating genome-wide levels of temporal organization. Annu Rev Genomics Hum Genet. 2004;5:407–441. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources