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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2005 Jan 12;102(4):1082–1087. doi: 10.1073/pnas.0409103102

Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice

Shijie Li *, Michael P Czubryt *, John McAnally *, Rhonda Bassel-Duby *, James A Richardson †, Franziska F Wiebel ‡, Alfred Nordheim ‡, Eric N Olson *,§
PMCID: PMC545866  PMID: 15647354

Abstract

Serum response factor (SRF) controls the transcription of muscle genes by recruiting a variety of partner proteins, including members of the myocardin family of transcriptional coactivators. Mice lacking SRF fail to form mesoderm and die before gastrulation, precluding an analysis of the roles of SRF in muscle tissues. To investigate the functions of SRF in skeletal muscle development, we conditionally deleted the Srf gene in mice by skeletal muscle-specific expression of Cre recombinase. In mice lacking skeletal muscle SRF expression, muscle fibers formed, but failed to undergo hypertrophic growth after birth. Consequently, mutant mice died during the perinatal period from severe skeletal muscle hypoplasia. The myopathic phenotype of these mutant mice resembled that of mice expressing a dominant negative mutant of a myocardin family member in skeletal muscle. These findings reveal an essential role for the partnership of SRF and myocardin-related transcription factors in the control of skeletal muscle growth and maturation in vivo.

Keywords: hypertrophy, myocardin-related transcription factor, myofiber, myopathy

Skeletal muscle development involves a precisely orchestrated series of steps that begins when mesodermal precursor cells become committed to the skeletal muscle lineage, giving rise to proliferating myoblasts. In response to extracellular cues, myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes that express an array of muscle-specific genes encoding proteins that mediate the specialized contractile, metabolic, and structural functions of the muscle fiber. Subsequent hypertrophic growth of the muscle fiber through the assembly of sarcomeres and increased diameter of the fiber is required to enhance contractile force to meet the functional demands associated with postnatal life.

The early steps in skeletal muscle development are controlled by combinatorial interactions between members of the MyoD family of basic helix–loop–helix transcription factors (MyoD, myogenin, Myf5, and MRF4) and the myocyte enhancer factor-2 (MEF2) family of MADS (MCM1, Agamous, Deficiens, serum response factor) box transcription factors (1). MyoD and Myf5 play redundant roles in specification of muscle cell fate, whereas myogenin and MRF4 act together with MEF2 factors to activate and sustain the muscle differentiation program (2, 3).

Serum response factor (SRF), a MADS box transcription factor related to MEF2, also regulates skeletal, as well as cardiac and smooth muscle genes by binding a DNA sequence known as a CArG box (4–6). Like MEF2 and other MADS box transcription factors, SRF activates transcription by associating with a variety of signal-responsive and cell type-restricted cofactors (7). Myocardin, an especially powerful SRF coactivator expressed specifically in cardiac and smooth muscle cells, has been shown to be necessary and sufficient for cardiac and smooth muscle gene expression (8–16). The myocardin-related transcription factors (MRTFs) MRTF-A (also called MAL/MKL1/BASC) and MRTF-B (also called MKL2) also are expressed in skeletal, cardiac, and smooth muscle cells, as well as other cell types (17–21). Their potential contributions to muscle development in vivo have not yet been investigated.

A requisite role for SRF in skeletal muscle development has been inferred from experiments in cultured muscle cells in which injection with anti-SRF antibody or expression of a dominant negative SRF mutant blocks myoblast fusion and differentiation (22–24). However, knockout mice lacking SRF die before gastrulation, precluding the analysis of potential functions of SRF in muscle development in vivo (25). Several groups recently have generated conditional Srf null alleles allowing for temporal and spatial specificity of gene deletion in the mouse (26–28). Cardiac-specific deletion of Srf results in embryonic lethality from cardiac defects (27), and deletion of the gene in smooth muscle results in embryonic lethality from a deficiency of differentiated smooth muscle cells (28).

To determine the function of SRF in developing skeletal muscle, we conditionally deleted the Srf gene in mice by using skeletal muscle-specific transgenes encoding Cre recombinase. Mice lacking skeletal muscle expression of SRF died during the first few days after birth with a severe skeletal muscle myopathy characterized by a deficiency in muscle growth. The muscle abnormalities in these mice were similar to the myopathic phenotype of mice expressing a dominant negative mutant of MRTF-A (dnMRTF-A) in skeletal muscle. These findings reveal an essential role for SRF and MRTFs in the control of muscle fiber growth and maturation.

Materials and Methods

Transgenic Mice. To create a muscle-specific Cre recombinase transgene, a Cre recombinase expression cassette was placed under the control of the 1.5-kb mouse myogenin promoter (29) and the 1-kb mouse MEF2C enhancer (30), yielding a transgene called Myo-Cre. A skeletal muscle-specific transgene encoding dnMRTF-A was created by placing a cDNA encoding the N-terminal 712 aa of mouse MRTF-A (17), lacking the transcription activation domain, under the control of a 4.8-kb muscle creatine kinase (MCK) promoter (31). Transgenic mice were generated by oocyte injection according to standard procedures. The MCK-Cre transgenic line and the ROSA26-lacZ indicator line have been described (32, 33). All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Texas Southwestern Medical Center.

Skeletal Muscle-Specific Deletion of Srf. The conditional Srf allele (Srfflex1), which contains loxP sites in the 5′ UTR and first intron of the gene, has been described (26). Mice homozygous for this allele are viable and fertile. Cre:loxP recombination results in deletion of part of exon 1, which encodes the start codon and the DNA binding domain of SRF. Breeding of Cre transgenic mice heterozygous for the floxed Srf allele (Cre:Srfflex1/+) with Srfflex1/flex1 mice yielded Cre:Srfflex1/flex1 mice. Breedings were performed in the 129SvEv and C57BL/6 mixed backgrounds. DNA prepared from tail biopsies was used for genotyping by PCR, using two primers (SRF-L and SRF-R) as described (26). This process allowed amplification of a 1.34-kb fragment from the undeleted Srfflex1 allele and a 380-bp DNA fragment from the Srflx allele obtained when floxed Srfflex1 alleles had been recombined by Cre recombinase.

RT-PCR. Total RNA was purified from tissues with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. One microgram of RNA from each sample was used to generate cDNA by using a SuperScript II First-Strand Synthesis kit (Invitrogen). The cDNA was used for PCR under conditions of linearity with respect to input DNA. Primer sequences are available on request.

β-Galactosidase Staining and Histology. Staining of embryos for β-galactosidase was performed as described (29). Skeletal muscle was dissected from the hind limbs of WT and mutant mice. Embedding of tissues, histological sectioning, and staining with hematoxylin and eosin (H&E) were performed by standard procedures.

Electron Microscopy. For electron microscopy, skeletal muscle was fixed overnight in 2% glutaraldehyde in PBS at 4°C, then postfixed in 1% OsO4, and dehydrated in an ethanol series. Samples were then embedded in Spurr resin (Ted Pella, Inc., Redding, CA), stained with uranyl acetate and lead citrate, and sectioned at 80 nm.

Western Blot Analysis. Skeletal muscle extracts were prepared and used for Western blotting with anti-FLAG antibodies and horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biosciences). Signal was detected with Western blotting Luminol Reagent (Santa Cruz Biotechnology), followed by exposure of blots to BioMax film (Kodak).

Results

Creation of a Skeletal Muscle-Specific Cre Transgene. To enable the skeletal muscle-specific deletion of a floxed Srf gene, we created a transgene in which Cre recombinase expression was controlled by the mouse myogenin promoter and the skeletal muscle-specific enhancer of the mouse MEF2C gene. Both of these regulatory elements are active only in the skeletal muscle lineage from embryonic day (E) 8.5 to adulthood (29, 30). The expression pattern of this transgene, referred to as Myo-Cre, was determined by crossing mice harboring the transgene into the ROSA26R heterozygous background, which contains a “floxed” lacZ allele that is activated in the presence of Cre (33). As shown in Fig. 1A, the Myo-Cre transgene directed the expression of lacZ specifically in skeletal muscle cells within the somite myotome at E9.5. Expression was initiated in the anterior somites at ≈E9.0 (data not shown), and strong expression throughout skeletal muscle was maintained throughout embryogenesis (Fig. 1 A). Serial histological sections through stained embryos at multiple stages confirmed the skeletal muscle specificity of lacZ expression and showed that all skeletal muscle cells were stained for lacZ (data not shown).

Fig. 1.

Fig. 1.

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Deletion of Srf with a skeletal muscle-specific Cre transgene. (A) Myo-Cre transgenic mice were bred with ROSA26R indicator mice to determine the temporal and tissue specificity of Cre expression. Whole-mount photographs of β-galactosidase-stained embryos of the indicated embryonic ages are shown. The lacZ reporter gene is activated specifically in the skeletal muscle lineage. (B) WT and Srfflex1/flex1/Myo-Cre (KO) mice immediately after birth are shown. The mutant is cyanotic and displays curvature of the spine. (C) The structure of the Srfflex1 allele before (Upper) and after (Lower) Cre-mediated recombination is shown. Triangles represent loxP sites. Exons 1 and 2 are shown in black boxes with the 5′ UTR as a white box. Primers used by PCR are designated L and R, and sizes of PCR fragments are indicated. (D) PCR of genomic DNA from skeletal muscle of mice of the indicated genotypes. Primers L and R yield a product of 1,340 bp with the Srfflex1 allele and 380 bp with the Srf/x1 allele in the presence of the Myo-Cre transgene.

Skeletal Muscle-Specific Deletion of Srf with the Myo-Cre Transgene. To delete Srf specifically in skeletal muscle, we used a conditional Srf allele (called Srfflex1) harboring loxP sites flanking exon 1 of the gene, which encodes the DNA binding domain of SRF. Cre-mediated recombination of this locus generates the Srflx deletion allele essentially identical to that in the previously described null allele (25, 26). Mice heterozygous for the Srfflex1 allele and heterozygous for the Myo-Cre transgene were bred with homozygous Srfflex1/flex1 mice to yield mice homozygous for the Srfflex1 allele and the Myo-Cre transgene. Genotyping of litters from these crosses revealed that offspring with the Myo-Cre;Srfflex1/flex1 genotype were born at Mendelian ratios. At birth, the hearts of these mutant mice were beating, but the animals were immobile and died from an inability to breathe. These mutant mice were recognizable by their cyanotic appearance and kyphosis (Fig. 1B).

Efficient deletion of Srf from skeletal muscle was confirmed by PCR with genomic DNA (Fig. 1 C and D). There was an ≈80% reduction in the PCR product from the floxed Srf gene in skeletal muscle at E19.5 in the presence of the Myo-Cre transgene. Given that cells other than muscle (e.g., neurons and fibroblasts), in which the Cre transgene is not expressed, also are contained in these tissue samples, we estimate that the efficiency of Srf gene deletion was at least 90%.

Perinatal Lethality and Skeletal Muscle Hypoplasia Resulting from Skeletal Muscle Deletion of Srf. Histological analysis of skeletal muscle from mice at E19.5 or birth lacking skeletal muscle expression of Srf showed the presence of multinucleated muscle fibers that were thinner than normal and were separated by prominent interstitial space (Fig. 2A). The diameters of fibers in the mutant were also much more variable than in WT controls. All skeletal muscle groups appeared to be affected comparably in Srf mutant animals. Perinatal lethality is likely caused by abnormalities in the diaphragm muscle, which prevent breathing. The mean body weights (±SD) of WT and mutant mice at birth were 1.28 ± 0.05 g (n = 9) versus 1.06 ± 0.11 g (n = 6) (P < 0.005). Because the deletion of Srf is specific for skeletal muscle, this difference in body weight reflects the lack of muscle mass in the mutants.

Fig. 2.

Fig. 2.

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Histology of skeletal muscle of Srfflex1/flex1/Myo-Cre mice. (A) Histological sections of representative muscle groups of WT and Srfflex1/flex1/Myo-Cre mice at E19.5 were stained with H&E. The muscle fibers in the mutant are thinner than those of WT. (Bar: 20 μm.) (B) Hindlimb muscle of WT and Srfflex1/flex1/Myo-Cre mice was analyzed by electron microscopy at E19.5. The muscle fibers in the mutant are disorganized and less developed than those of WT. Magnifications are shown at left. (Bar: 2 μm, Upper; 0.5 μm Lower.)

Given that the MEF2C enhancer and myogenin promoter are activated at the onset of myogenesis, we considered it unlikely that the deficiency of skeletal muscle fibers in Myo-Cre;Srfflex1/flex1 mutants reflected a deficiency in myoblasts. Indeed, the number of nuclei in muscle fibers of WT and mutant mice was comparable, suggesting that the skeletal muscle hypoplasia of the mutant was caused by a failure in growth of muscle fibers rather than a deficiency of muscle cells or a partial block of myoblast fusion.

Ultrastructural analysis of skeletal muscle fibers by electron microscopy showed the presence of sarcomeres in Myo-Cre;Srfflex1/flex1 mutants (Fig. 2B). However, the sarcomere units were smaller, and the fibers were narrowed and disorganized. Electron-dense material, likely glycogen, filled the interstitial spaces between the hypoplastic muscle fibers in the mutant.

Analysis of representative skeletal muscle transcripts at birth by semiquantitative RT-PCR showed a 70% decrease in SRF mRNA in Myo-Cre;Srfflex1/flex1 mutants compared with WT littermates (Fig. 3). Skeletal α-actin and cardiac α-actin transcripts also were down-regulated ≈30% in the mutants, whereas other transcripts for smooth muscle α-actin, neonatal skeletal myosin heavy chain, MCK, and myogenic basic helix–loop–helix and MEF2 factors were unaffected (Fig. 3).

Fig. 3.

Fig. 3.

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Analysis of muscle markers in Srfflex1/flex1/Myo-Cre mice. RNA was isolated from hindlimb muscles of WT and Srfflex1/flex1/Myo-Cre (KO) mice at birth and analyzed by semiquantitative RT-PCR for the indicated transcripts. Samples from two animals of each genotype are shown.

Skeletal Muscle Deletion of Srf Using MCK-Cre. Because the myogenin and MEF2C regulatory elements used to direct Cre recombinase are active early in the pathway of skeletal muscle development, we wondered whether deletion of Srf from skeletal muscle at a later time might result in a different phenotype. To explore this possibility, we additionally used a MCK-Cre transgene to delete the Srfflex1 allele. Prior studies have shown that this transgene is activated in skeletal muscle cells during late embryonic development (32). This transgene also is expressed in the developing heart and smooth muscle cells of the large arteries (32, 34). Deletion of Srf with the MCK-Cre transgene resulted in perinatal lethality with complete penetrance. These mutant mice were mobile, nursed, and appeared normal at birth (data not shown). However, by postnatal day (P) 3, these animals were lethargic and began to display growth retardation (Fig. 4A). No viable offspring with skeletal muscle deletion were observed beyond P7.

Fig. 4.

Fig. 4.

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Deletion of Srf with a MCK-Cre transgene. (A) WT and Srfflex1/flex1/MCK-Cre (KO) mice at P3 are shown. The mutant is severely runted. (B) PCR of genomic DNA from mice of the indicated genotypes. Primers L and R yield a product of 1,340 bp with the floxed Srfflex1 allele and 380 bp with the deleted Srflx allele, generated in the presence of the MCK-Cre transgene. (C) Histological sections of representative muscle groups of WT and Srfflex1/flex1/MCK-Cre mice were stained with H&E. The cross-sectional area of the muscle fibers in the mutant is smaller than that of WT. (Bar: 20 μm.) (D) RNA was isolated from hindlimb muscles of WT and Srfflex1/flex1/MCK-Cre (KO) mice at P3 and analyzed by semiquantitative RT-PCR for the indicated transcripts.

Based on PCR of genomic DNA of mutant mice at P3, we estimate that the MCK-Cre transgene directed at least 90% deletion of Srf in skeletal muscle (Fig. 4B). Histological analysis of skeletal muscle from MCK-Cre;Srfflex1/flex1 mutants at P3 showed thinner myofibers than normal, although the phenotype appeared less severe than that of Myo-Cre;Srfflex1/flex1 mutants (Fig. 4C). The delayed phenotype of these animals compared with those using the Myo-Cre transgene for Srf deletion is likely to reflect the later activation of the MCK-Cre transgene. We detected no abnormalities in the hearts of MCK-Cre;Srfflex1/flex1 mutants, leading us to conclude that skeletal muscle abnormalities were the cause of death. Expression of Srf transcripts in skeletal muscle from MCK-Cre;Srfflex1/flex1 mutants at P3 was reduced by 80% compared with controls. However, we detected only a modest (<50%) decrease in expression of α-skeletal and α-cardiac actin and no decrease in other muscle genes in these animals.

Skeletal Muscle Hypoplasia Resulting from Expression of dnMRTF-A. SRF cooperates with members of the myocardin family to activate specific programs of gene expression (8–10). To determine whether MRTFs might participate in the control of skeletal muscle genes in vivo, we expressed dnMRTF-A in skeletal muscle of transgenic mice by using the MCK promoter. The mutant form of MRTF-A, which contained the SRF binding domain, but lacked the transcription activation domain, can dimerize with WT MRTF proteins and suppress SRF activity in vitro (10, 12). Expression of FLAG-tagged dnMRTF-A in skeletal muscle of transgenic mice was confirmed by Western blot (Fig. 5A).

Fig. 5.

Fig. 5.

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Skeletal muscle abnormalities resulting from expression of dnMRTF-A. (A) Western blot analysis of skeletal muscle from MCK-dnMRTF-A transgenic mice. Extracts from skeletal muscle of WT and MCK-dnMRTF-A transgenic mice were analyzed by Western blot with anti-FLAG antibody to detect FLAG-tagged dnMRTF-A. Two transgenic lines are shown. (B) Hindlimb muscles of WT and MCK-dnMRTF-A transgenic (line 1) mice at 4 weeks of age are shown. The transgenic animal shows severe skeletal myopathy. (C) Histological sections of hindlimb muscles of WT and MCK-dnMRTF-A transgenic mice at 4 weeks of age were stained with H&E. The muscle fibers in the transgenic animals are thinner than those of WT. Transgenic line 1 shows the most severe phenotype with extensive fibrosis and centrally located nuclei. (Bar: 20 μm.) (D) RNA was isolated from hindlimb muscles of WT and MCK-dnMRTF-A transgenic (Tg) mice line 1 at 4 weeks of age and analyzed by semiquantitative RT-PCR for the indicated transcripts.

Mice expressing dnMRTF-A were viable, but failed to thrive and showed skeletal myopathy and hypoplasia reminiscent of, although less severe than, the phenotype resulting from skeletal muscle-specific Srf deletion (Fig. 5 B and C). The severity of the muscle phenotype depended on the level of dnMRTF-A expression (Fig. 5B). Transgenic line 1, which expressed FLAG-dnMRTF-A at a level ≈4-fold higher than line 2, showed a more severe myopathic phenotype (Fig. 5 A and C and data not shown). In contrast with mice lacking skeletal muscle expression of Srf, these transgenic mice survived to adulthood, likely because dnMRTF-A is unable to completely silence SRF activity. Myofibers from MCK-dnMRTF-A transgenic mice also showed extensive fibrosis and centrally located nuclei, indicative of muscle damage and regeneration (Fig. 5C). Transgenic animals also were runted, reflecting the failure in skeletal muscle growth. The mean body weights (±SD) of WT and transgenic mice at 8 weeks of age were 21.4 ± 1.6 g (n = 12) versus 18.9 ± 1.0 g (n = 9) (P < 0.005). We detected no abnormalities in cardiac structure in MCK-dnMRTF-A transgenic mice. RNA analysis showed a decline in expression of skeletal and cardiac α-actin genes, as well as the MCK gene, in these transgenic mice (Fig. 5D). Based on the intensity of MRTF-A transcripts in WT and transgenic mice (line 1), we estimate the transcript encoding dnMRTF-A to be expressed at a level ≈4-fold higher than the endogenous MRTF-A transcript.

Discussion

To determine the role of SRF in skeletal muscle development, we deleted a conditional Srf gene specifically in the skeletal muscle lineage by using two Cre transgenes with different temporal patterns of expression in the muscle developmental pathway. The phenotypes of these mutant mice reveal an essential role for SRF in the control of skeletal muscle growth. The resemblance of myopathic phenotypes of mice lacking skeletal muscle expression of SRF and expressing dnMRTF-A in skeletal muscle suggests that the partnership of SRF and MRTFs plays a critical role in skeletal muscle growth and maturation in vivo.

Control of Myofiber Growth and Maturation by SRF. We believe the failure of skeletal muscle to grow and mature properly in mice lacking skeletal muscle expression of Srf results in lethality caused by skeletal muscle weakness, which disrupts breathing and/or nursing. The early onset of lethal muscle deficits in these mutant mice is distinct from most myopathic phenotypes in mice, which do not manifest until adulthood reflecting, at least in part, the regenerative capacity of skeletal muscle. The myopathic phenotype resulting from skeletal muscle-specific deletion of Srf is also distinct from those of mice lacking myogenic basic helix–loop–helix genes. MyoD and Myf5 play redundant roles in specification of the skeletal muscle cell lineage such that deletion of one gene or the other does not substantially affect muscle development, whereas deletion of both genes eliminates all traces of the skeletal muscle lineage (35, 36). In contrast, deletion of the myogenin gene results in perinatal lethality from a block in myoblast fusion and differentiation (37, 38).

The skeletal muscle phenotype of Srf mutant mice could, in principle, reflect an early or late developmental function of SRF. The Myo-Cre transgene is activated by E9.5 before the first round of myoblast fusion, whereas the MCK-Cre transgene is activated later during muscle fiber differentiation. The finding that two skeletal muscle Cre transgenes activated at different times in development lead to similar phenotypes, albeit with differing severity, suggests that the Srf mutant phenotype reflects a late function of SRF in hypertrophic growth rather than an early developmental role, for example, in myoblast fusion.

Sarcomeric actin genes, as well as other contractile protein genes, require CArG boxes for expression (5, 6). Thus, it is intriguing that some CArG box-dependent genes were expressed normally in Srf-deficient skeletal muscle. We suggest two possible explanations for this finding. (i) Residual SRF caused by incomplete or delayed gene deletion might be adequate to activate certain SRF-dependent genes that are more sensitive to SRF levels than others. (ii) SRF-independent mechanisms might bypass a requirement of SRF for activation of some CArG box-dependent genes.

Given the evidence for the involvement of SRF in myoblast differentiation in vitro (22–24), why does myogenesis appear to proceed normally in mice lacking skeletal muscle expression of SRF? We suggest three possibilities, which are not mutually exclusive. (i) Myogenesis in vitro might have a more stringent dependency on SRF. (ii) The kinetics of Srf gene deletion in vivo might be delayed such that an initial requirement for SRF in activation of the differentiation program is bypassed. (iii) Residual, low-level expression of SRF in the Srf lx1 animals might be sufficient to support the initial steps in myogenesis, whereas later steps in muscle growth and maturation might require higher SRF levels.

The apparent block to myofiber growth after skeletal muscle deletion of Srf is reminiscent of the cardiac phenotype resulting from cardiac expression of dominant negative SRF (39) or cardiac deletion of Srf, which results in embryonic lethality from a defect in ventricular growth and maturation (27, 28). Conversely, overexpression of SRF results in lethal cardiomyopathy with associated myocyte hypertrophy in adult cardiac muscle (40). Thus, SRF might play comparable roles in regulating growth of skeletal and cardiac muscle in vivo.

A Role for MRTFs in Muscle Growth. Members of the myocardin family stimulate SRF activity and have been implicated in differentiation of cardiac and smooth muscle (8–17). Similarly, a dominant negative mutant of MRTF-B/MKL2 inhibits differentiation of skeletal muscle cells in vitro (41). MRTFs form homodimers and heterodimers through a leucine zipper (12). The dominant negative mutant used in these studies contains the leucine zipper domain and the SRF-binding region, but lacks the transcription activation domain. This mutant can compete with WT MRTFs for association with SRF and can form heterodimers with WT MRTFs with diminished transcriptional activity. Although we favor the interpretation that dnMRTF-A blocks muscle growth by disrupting the functions of MRTF-A or MRTF-B, it is also possible that it displaces other transcription factors from SRF or even interferes with the activities of transcriptional partners that function independently of SRF.

Mechanisms for Skeletal Muscle Hypertrophy. Growth of skeletal muscle during late fetal and postnatal development involves the assembly of sarcomeres and an increase in volume of individual myofibers. Several signaling pathways have been shown to control skeletal muscle hypertrophy (reviewed in ref. 42). Signaling by insulin-like growth factor-1 to Akt and its downstream effectors promotes hypertrophy, and SRF has been shown to be a target of Akt signaling (43). The secreted bone morphogenetic protein myostatin also suppresses muscle hypertrophy, and follistatin, its antagonist, promotes hypertrophy (44, 45). SRF could be a critical component of these hypertrophic signaling pathways. Alternatively, the reduction in expression of one or more SRF target genes, α-actin, for example, could perturb myofiber growth through secondary mechanisms. In this regard, α-skeletal actin knockout mice die during the perinatal period from abnormalities in skeletal muscle growth and force generation (46).

Consistent with the notion that SRF plays a role in hypertrophic growth of skeletal muscle, SRF expression is up-regulated during load-induced hypertrophy of skeletal muscle (47), and the CArG box in the α-skeletal actin promoter is a target for hypertrophic signaling (48). The recognition that SRF plays a role in skeletal muscle growth and maturation suggests strategies for enhancing SRF activity in the settings of muscle-wasting disorders, possibly by modulating the signaling pathways that stimulate the activity of SRF or its cofactors.

Acknowledgments

We thank C. R. Kahn (Harvard Medical School, Cambridge, MA) for MCK-Cre transgenic mice, B. A. Rothermel and W. H. Klein for comments on the manuscript, J. Page for editorial assistance, and A. Tizenor for graphics. E.N.O.'s laboratory was supported by the National Institutes of Health, the Donald W. Reynolds Cardiovascular Clinical Research Center, the Muscular Dystrophy Association, and the Robert A. Welch Foundation. A.N. was supported by Deutsche Forschungsgemeinschaft Grant SFB446/B7. M.P.C. was supported by a fellowship grant from the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research.

Author contributions: C.L. designed research; M.C., J.M., R.B.-D., J.A.R., F.F.W., A.N., and E.N.O. performed research; and E.N.O. wrote the paper.

Abbreviations: MEF2, myocyte enhancer factor 2; SRF, serum response factor; MRTF, myocardin-related transcription factor; dnMRTF-A, dominant negative mutant of MRTF-A; MCK, muscle creatine kinase; H&E, hematoxylin and eosin; E(n), embryonic day n; P(n), postnatal day n.

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