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. 2011 Jun 21;108(25):10196-201.
doi: 10.1073/pnas.1107413108. Epub 2011 Jun 1.

Concerted regulation of myofiber-specific gene expression and muscle performance by the transcriptional repressor Sox6

Daniel Quiat  1 , Kevin A Voelker, Jimin Pei, Nick V Grishin, Robert W Grange, Rhonda Bassel-Duby, Eric N Olson
Affiliations

Concerted regulation of myofiber-specific gene expression and muscle performance by the transcriptional repressor Sox6

Daniel Quiat et al. Proc Natl Acad Sci U S A. .

Abstract

In response to physiological stimuli, skeletal muscle alters its myofiber composition to significantly affect muscle performance and metabolism. This process requires concerted regulation of myofiber-specific isoforms of sarcomeric and calcium regulatory proteins that couple action potentials to the generation of contractile force. Here, we identify Sox6 as a fast myofiber-enriched repressor of slow muscle gene expression in vivo. Mice lacking Sox6 specifically in skeletal muscle have an increased number of slow myofibers, elevated mitochondrial activity, and exhibit down-regulation of the fast myofiber gene program, resulting in enhanced muscular endurance. In addition, microarray profiling of Sox6 knockout muscle revealed extensive muscle fiber-type remodeling, and identified numerous genes that display distinctive fiber-type enrichment. Sox6 directly represses the transcription of slow myofiber-enriched genes by binding to conserved cis-regulatory elements. These results identify Sox6 as a robust regulator of muscle contractile phenotype and metabolism, and elucidate a mechanism by which functionally related muscle fiber-type specific gene isoforms are collectively controlled.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Sox6 is enriched in fast muscle fibers and effects gross muscle fiber morphology. (A) Sox6 mRNA is enriched in muscle groups that predominantly contain fast myofibers (TA and EDL) compared with slow myofiber-enriched muscle (soleus), as measured by qPCR. (B) Sox6 expression levels are reduced by qPCR in slow fiber containing MCK-CnA transgenic TA muscle compared with WT fast TA muscle. (C) Gross examination of adult Sox6 cKO hindlimb muscles demonstrates increased redness and a reduction in muscle size compared with WT control. (D) Staining of myofiber cell membranes with wheat germ agglutinin indicates a reduction in myofiber size in Sox6 cKO TA muscle. (Scale bars, 200 μm.) (E) Quantification of myofiber size following wheat germ agglutinin staining reveals an increase in the proportion of smaller myofibers and a reduction in the proportion of larger myofibers in Sox6 cKO TA muscle. (F) Sox6 cKO and WT TA muscle contain a similar number of myofibers.
Fig. 2.
Fig. 2.
Altered physiological performance of Sox6 cKO muscle. (A) A stress-frequency plot of WT and Sox6 cKO EDL and soleus muscle reveals no difference in stress generation at various frequencies of stimulation. (B) Measurement of the shortening velocity in WT and Sox6 cKO EDL and soleus muscle reveals a reduced maximal shortening velocity (y-intercept) in Sox6 cKO EDL, but not soleus. Lines represent curve fit of datapoints. (C) Measurement of time-to-fatigue (i.e., to 50% of initial force; horizontal dashed line) for EDL muscle reveals an increase by 50% in Sox6 cKO EDL. Time-to-fatigue (i.e., 75% of initial force; horizontal dashed line) measurements for soleus muscle demonstrate an increase by 100%. These results indicate a significant improvement in Sox6 cKO muscle endurance. Vertical dashed lines indicate extrapolation of time-to-fatigue. WT (n = 4), Sox6 cKO (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Fig. 3.
Fig. 3.
Increased slow myofibers and mitochondrial activity in Sox6 cKO mice. (A) Immunohistochemistry directed against type I slow myosin indicates an increase in slow myofibers in all muscle groups from Sox6 cKO mice at 8 wk of age. Slow myofibers are stained brown. (Scale bar, 400 μm.) (B) Measurement of Myh7, encoding type I slow myosin, by qPCR reveals significantly increased expression in Sox6 cKO muscle. (C) Separation of myosin isoforms by gel electrophoresis reveals increased type I myosin in soleus, TA, and EDL muscle from Sox6 cKO mice. In addition, a switch from type IIb to type IIa/x fibers is evident in cKO TA and EDL. (D) Sox6 cKO mice display altered expression of fast myosin isoforms compared with WT by qPCR. (E) Western blots for slow skeletal troponin (Tnni1) and myoglobin demonstrate robust up-regulation of these proteins in TA and gastrocnemius/plantaris (GP) muscle from Sox6 cKO mice. (F) Sox6 cKO TA muscle demonstrates increased SDH and NADH-TR enzymatic staining, consistent with an increase in mitochondrial activity in Sox6 cKO myofibers. (Scale bar, 200 μm.)
Fig. 4.
Fig. 4.
Sox6 is a direct repressor of slow myofiber genes. (A) Schematic representation of highly conserved Sox6 consensus binding motifs found near loci encoding slow fiber-enriched genes. (B) Sox6 ChIP reveals that all five loci are bound significantly more by Sox6 in WT TA muscle compared with Sox6 cKO TA negative-control tissue. (C) Direct Sox6 target genes are up-regulated in TA muscle from Sox6 cKO mice as measured by qPCR. (D) Sox6 target gene transcripts are reduced in hindlimb muscle from MCK-Sox6 transgenic mice as measured by qPCR. (E) Gel-shift analysis of the palindromic Sox6 binding site upstream of the Myh7 locus. Sox6 binds WT (W), but not mutant (M) consensus binding sequence. The Sox6 binding complex is supershifted with addition of α-FLAG antibody. (F) Gel-shift analysis of single Sox6 binding sites near loci for Myh1, Myl2, Myl3, and ATP2a2 reveals that Sox6 can bind to consensus binding sites in vitro.
Fig. 5.
Fig. 5.
Our data suggest a model by which Sox6 represses multiple contractile genes that collectively contribute to slow muscle fiber contractile properties.
See this image and copyright information in PMC

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