Concerted Regulation of Myofiber-Specific Gene Expression

Concerted regulation of myofiber-specific gene expression and muscle performance by the transcriptional repressor Sox6 Daniel Quiata, Kevin A. Voelkerb, Jimin Peic,d,NickV.Grishinc,d, Robert W. Grangeb, Rhonda Bassel-Dubya, and Eric N. Olsona,1 aDepartment of Molecular Biology, cDepartment of Biochemistry, and dThe Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; and bDepartment of Human Nutrition, Foods, and Exercise, Virginia Tech University, Blacksburg, VA 24061 Contributed by Eric N. Olson, May 10, 2011 (sent for review April 25, 2011) In response to physiological stimuli, skeletal muscle alters its Recently, we identified a microRNA (miRNA)-mediated myofiber composition to significantly affect muscle performance transcriptional regulatory network that reinforces slow myofiber and metabolism. This process requires concerted regulation of gene expression in skeletal muscle via a reciprocal negative- myofiber-specific isoforms of sarcomeric and calcium regulatory feedback loop (18). In this pathway, miRNAs miR-499 and miR- proteins that couple action potentials to the generation of contrac- 208b, intronically encoded within slow myosin heavy-chain genes, tile force. Here, we identify Sox6 as a fast myofiber-enriched re- target a collection of transcriptional repressors, and promote a pressor of slow muscle gene expression in vivo. Mice lacking Sox6 fast-to-slow myofiber-type switch. One transcriptional repressor specifically in skeletal muscle have an increased number of slow integral to this regulatory pathway is Sox6, a member of the SoxD myofibers, elevated mitochondrial activity, and exhibit down- family of transcription factors that exhibits dual functions as a regulation of the fast myofiber gene program, resulting in enhanced transcriptional activator or repressor (19, 20). Sox6-null mice muscular endurance. In addition, microarray profiling of Sox6 knock- exhibit failure to thrive and expire within 2 wk following birth (21); out muscle revealed extensive muscle fiber-type remodeling, and however, studies in these mice, as well as in zebrafish, have identified numerous genes that display distinctive fiber-type enrich- identified an important role for Sox6 in embryonic muscle de- ment. Sox6 directly represses the transcription of slow myofiber- velopment (22–24). enriched genes by binding to conserved cis-regulatory elements. In the present study, we investigate the function of Sox6 in adult These results identify Sox6 as a robust regulator of muscle contrac- skeletal muscle. We show that Sox6 is required for fast myofiber tile phenotype and metabolism, and elucidate a mechanism by maintenance in adult muscle, as conditional loss of Sox6 leads to fi fi which functionally related muscle ber-type speci c gene isoforms conversion of muscle to a slow myofiber phenotype, resulting in are collectively controlled. significant changes in skeletal muscle mechanics. We demonstrate that Sox6 regulation of muscle phenotype occurs independent fi calcium handling | myosin heavy-chain isoforms | slow-twitch ber of changes in other regulatory factors implicated in fiber-type switching, such as PGC-1α and AMP-kinase (AMPK), and instead he striated appearance of skeletal muscle arises from the tan- results from direct repression of a constellation of slow isoforms Tdem arrangement of individual sarcomeric contractile units. of sarcomeric and calcium regulatory proteins. Together, these Sarcomere contraction occurs when thick filament myosins bind data reveal an important role for Sox6 in the coordinated regu- to actin thin filaments, and hydrolyze ATP to generate a power lation of multiple properties of myofiber phenotype that pro- stroke (1). This process is tightly regulated by the calcium-sensitive foundly effect muscle performance. troponin-tropomyosin complex, which alters its conformation to permit actin–myosin interactions in response to increased intra- Results cellular calcium (2). Multiple isoforms of contractile apparatus Sox6 Is Enriched in Fast Myofibers. Previous work suggests a role proteins are regulated in a fiber-type specific manner, and can in- for Sox6 in embryonic muscle differentiation, but the potential fluence overall muscle function (3). For example, differential ex- functions of Sox6 in adult muscle are unknown; therefore, we pression of myosin heavy chain (Myh) isoforms alters force-velocity asked if Sox6 displays differential expression between muscle fiber parameters of individual muscle fibers, and alternate combinations types in adult mice. Comparison of slow myofiber-enriched soleus 2+ of troponin T, I, and C isoforms influence myofiber Ca sensitivity muscle to fast myofiber-enriched tibialis anterior (TA) and ex- (4, 5). The mechanism by which contractile and calcium regulatory tensor digitorum longus (EDL) by qPCR revealed that Sox6 genes are concordantly regulated to produce a unified effect on mRNA is expressed approximately threefold higher in fast TA muscle contraction is poorly understood. and EDL muscle groups (Fig. 1A). To more thoroughly study the Skeletal muscle is composed of a heterogeneous population of influence of fiber type on the expression of Sox6 within an in- fi slow and fast myo bers that display contrasting contractile and dividual muscle group, we utilized transgenic mice that express an fi metabolic properties. Slow type I bers exhibit oxidative metab- active form of the phosphatase calcineurin (CnA) under control olism, express slow isoforms of sarcomeric proteins, and are clas- of muscle creatine kinase (MCK) regulatory elements, which fi sically identi ed by their expression of type I myosin heavy chain drives slow myofiber formation in skeletal muscle (10). Expression β (Myh7 or -Myh). In contrast, fast type IIa, IIx/d, and IIb myo- of Sox6 in slow myofiber-enriched MCK-CnA TA muscle was fi bers use glycolytic metabolism, express fast isoforms of contrac- reduced by ∼50% compared to WT TA by qPCR (Fig. 1B). Based tile proteins, and are designated by their expression of fast myosin heavy chain isoforms (Myh2, Myh1, and Myh4, respectively) (6). Chronic muscle stimulation induces a reversible transition in Author contributions: D.Q. and E.N.O. designed research; D.Q., K.V., J.P., and R.W.G. myofiber phenotype from fast to slow, which occurs in a sequential performed research; N.V.G. contributed new reagents/analytic tools; D.Q., K.V., N.V.G., manner through several fast myofiber intermediates (type IIb → R.W.G., R.B.-D., and E.N.O. analyzed data; and D.Q., R.B.-D., and E.N.O. wrote the paper. type IIx/d → type IIa → type I) (7). Previous studies have im- The authors declare no conflict of interest. plicated multiple transcriptional pathways in the regulation of 1To whom correspondence should be addressed. E-mail: [email protected]. fi fi basal-muscle ber-type speci c gene expression, stress-induced This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fiber-type remodeling, and myofiber metabolism (8–17). 1073/pnas.1107413108/-/DCSupplemental. 10196–10201 | PNAS | June 21, 2011 | vol. 108 | no. 25 www.pnas.org/cgi/doi/10.1073/pnas.1107413108 Downloaded by guest on September 26, 2021 Sox6 in WT Sox6 expression controls. Analysis of Sox6 expression in 8-wk-old cKO mice by skeletal muscle in MCK-CnA TA A 5 B 1.5 qPCR revealed a reduction in Sox6 transcript levels by 70% in TA and EDL muscles, and by 25% in the soleus muscle (Fig. 4 A 1.0 S1 ). By gross examination, hindlimb musculature of adult Sox6 3 cKO mice was markedly darker and more red in color than that 2 C 0.5 of WT littermates (Fig. 1 ). Furthermore, cKO muscle weighed 1 less than WT controls (Fig. S1B). Examination of myofiber cross- Relative expression Relative expression fi 0 0 sectional area by histochemical staining revealed that ber size Soleus EDLTA WT MCK-CnA distribution was altered in Sox6 cKO TA muscle, with a shift toward a reduced cross-sectional area relative to WT (Fig. 1 D C and E). Quantification of total myofiber number revealed that Sox6 cKO TA muscle contained a comparable number of individual fibers to WT controls (Fig. 1F), indicating that the de- crease in Sox6 cKO muscle mass results solely from a reduction WT cKO in myofiber size. Importantly, gross morphological changes in Sox6 cKO muscle were absent in young mice at stage P10 (Figs. D S1C and S2 B, and C), suggesting that the observed phenotype in Sox6 cKO adult mice is not caused by adverse effects on normal muscle development or early fiber-type patterning. Enhanced Muscle Performance in Sox6 cKO Mice. The altered ap- WT cKO pearance of Sox6 cKO muscle is consistent with changes observed in endurance trained muscle (27), leading us to ask if muscle from Myofiber size distribution E 200 Sox6 cKO mice displayed altered mechanical performance. EDL WT cKO and soleus muscles were isolated from WT and Sox6 cKO mice, 150 and physiological performance was assessed ex vivo in a muscle 100 bath. Sox6 cKO soleus and EDL muscle generated stress comparable to WT controls over a range of stimulation frequencies 50 (Fig. 2A). Measurement of shortening velocity revealed that Sox6 Number of fibers 0 cKO EDL, but not cKO soleus, had a reduced maximal shortening 0 velocity compared with WT (Fig. 2B). 4000 8000 1200016000200002400028000320003600040000 We assayed muscle endurance by calculating the time-to- Myofiber cross-sectional area (arbitrary units) fatigue: measured as the length of time for a muscle to fall to a F defined percentage of its initial force under constant stimulating TA 2500 frequency. Remarkably, the time-to-fatigue for cKO EDL muscle (50% of initial force) was 50% greater than WT muscle, indi- 2000 cating a significant increase in cKO muscle endurance (Fig. 2C). 1500 This pronounced improvement in physiological performance 1000 was also evident in the cKO soleus, which displayed a 100% increase in the time-to-fatigue (75% of initial force) (Fig. 2C). Total myofibers 500 Sox6 cKO EDL and soleus muscle also demonstrated improved 0 WT cKO recovery following fatigue (Fig.

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