Normal Skeletal Muscle Development Research Articles

MiR-205 Regulates the Fusion of Porcine Myoblast by Targeting the Myomaker Gene.

Skeletal muscle formation is an extremely important step in animal growth and development. Recent studies have found that TMEM8c (also known as Myomaker, MYMK), a muscle-specific transmembrane protein, can promote myoblast fusion and plays a key role in the normal development of skeletal muscle. However, the effect of Myomaker on porcine (Sus scrofa) myoblast fusion and the underlying regulatory mechanisms remain largely unknown. Therefore, in this study, we focused on the role and corresponding regulatory mechanism of the Myomaker gene during skeletal muscle development, cell differentiation, and muscle injury repair in pigs. We obtained the entire 3' UTR sequence of porcine Myomaker using the 3' RACE approach and found that miR-205 inhibited porcine myoblast fusion by targeting the 3' UTR of Myomaker. In addition, based on a constructed porcine acute muscle injury model, we discovered that both the mRNA and protein expression of Myomaker were activated in the injured muscle, while miR-205 expression was significantly inhibited during skeletal muscle regeneration. The negative regulatory relationship between miR-205 and Myomaker was further confirmed in vivo. Taken together, the present study reveals that Myomaker plays a role during porcine myoblast fusion and skeletal muscle regeneration and demonstrates that miR-205 inhibits myoblast fusion through targeted regulation of the expression of Myomaker.

Abstract 6736: Suppression of antigen presentation is a hallmark of pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children and young adults, accounting for approximately 350-400 new cases annually in the US. These tumors are diagnosed based on their expression of skeletal muscle lineage markers. Clinical trials testing conventional immunotherapies such as cancer vaccines or immune checkpoint blockade (ICB) therapy have found no activity in RMS, although the root causes of failure are largely unexplored. A recent study by our group found that RMS tumors have a lack of immune infiltrate, consistent with previous reports. However, we made an unexpected observation that RMS tumors had a near absence of antigen processing and presentation (APP) gene expression, a key requisite for anti-tumor CD8+ T lymphocytes. In this study, we investigate the root causes of the lack of APP in RMS and evaluate implications for immunotherapy. Methods: Publicly available DNA sequencing datasets were used to evaluate APP gene mutations in RMS tumor samples. RNAseq from reporter iPSC cell lines demarcating specific stages of skeletal muscle differentiation were used to evaluate APP gene expression during myogenesis. Surface MHC class I expression was evaluated using flow cytometry against a panel of pediatric sarcoma cell lines. RMS PDX and cell line APP protein levels were measured by western blot. Stimulation of APP in RMS cell lines was tested using IFNγ or sub-cytotoxic doses of different epigenetic therapeutics (DNMTi, HDACi, EZH2i) before analyzing surface MHC class I expression by flow cytometry. Results: RNAseq analysis of the differentiation states across the normal human skeletal muscle lineage revealed a myoblastic cell state which transiently downregulates the APP pathway. RNA expression of HLA-A,B,C genes were lower in RMS than any other pediatric extracranial solid tumor histology studied. RMS cell lines and PDX models also displayed low APP pathway gene expression by western blot and flow cytometry, with FOXO1 fusion positive RMS having a near absence of expression. RMS tumor exome sequencing data from RMS patients showed a lack of enrichment of APP gene mutations. Instead, we found that APP could be induced in nearly all samples tested using IFNγ or epigenetic stimulation with Decitabine, Entinostat, or Tazemetostat. Conclusions: We uncovered a previously unappreciated lack of antigen presentation in RMS which phenocopies myoblasts in normal skeletal muscle development. The downregulation of MHC class I antigen presentation is not due to APP pathway mutations but rather a partially reversible downregulation mirroring this specific stage of normal skeletal muscle development. Our findings suggest that pharmacological reversal of the APP pathway downregulation may enhance immunotherapies targeting MHC class I antigens in RMS. Citation Format: David Milewski, Meijie Tian, Yong Kim, Jun Wei, Javed Khan. Suppression of antigen presentation is a hallmark of pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6736.

Med12 is Essential for Skeletal Muscle Development

Tight regulation of tissue‐ and cell‐specific gene expression is essential for healthy physiological development. Mediator is a multisubunit protein complex that links basal transcriptional machinery, transcription factors, and epigenetic modifiers to gene targets to coordinate transcriptional regulation during development. One subunit, MED12, controls tissue‐ and cell‐specific gene expression. Some mutations in MED12 cause neurological developmental disorders, while others are associated with uterine leiomyomas. In addition to these diseases, human MED12 mutations have been linked to muscle disorders such as skeletal muscle hypotonia and congenital heart defects, but the function of MED12 in muscle and muscle cell development has not been elucidated. The objective in this study was to discover a molecular role of MED12 in transcriptional control of muscle development. We hypothesized that MED12 is required for healthy muscle development, and that MED12 helps recruit myogenic transcription factors (TFs) to Mediator at the Pre‐Initiation Complex (PIC) to achieve transcriptional control. Our lab generated a conditional skeletal muscle‐specific Med12 knockout mouse model (Med12mKO) driven by a Myogenin‐Cre transgene that is expressed during embryonic muscle development. Med12mKOmice have a runted phenotype, smaller muscles, disorganized muscle architecture, and suffer premature death, compared to littermate controls. Gene expression analysis by RNA‐sequencing (RNA‐seq) of muscle from 1‐day old Med12mKO and control mice revealed that Med12 deletion dysregulates metabolic and structural genes, including several myosin genes. MED12 is a transcriptional regulator, but does not directly bind DNA. To identify putative candidate TFs that regulate muscle gene expression in a MED12‐dependent manner we performed Upstream Regulator Analysis (URA) of dysregulated genes in Med12mKO muscle. Multiple TFs that regulate muscle gene expression were predicted to do so via MED12, including SRF, which is known to regulate expression of many myosin genes. Using differentiating C2C12 mouse myoblasts as a cell model of myogenesis, knockdown of Med12by shRNA resulted in diminished myotube size and reduced expression of myosin genes, recapitulating the gene expression and muscle phenotype observed in the Med12mKOmodel. However, no interaction was detected between SRF and MED12 in C2C12 myoblasts by Co‐Immunoprecipitation (Co‐IP), which did not support our hypothesis of muscle gene expression regulation through a MED12‐SRF mechanism. To further investigate how loss of MED12 dysregulates muscle gene expression, we used an unbiased approach to identify TFs that interact with MED12. We immunoprecipitated MED12 from C2C12 myoblasts and identified interacting TFs using mass spectrometry. We are currently investigating other TFs through which MED12 could be regulating gene expression during muscle development. We conclude that Med12 is required for normal skeletal muscle development, including normal gene expression of structural and metabolic genes during myogenesis.

Chchd10 is dispensable for myogenesis but critical for adipose browning

The Chchd10 gene encodes a coiled-coil-helix-coiled-coil-helix-domain containing protein predicted to function in the mitochondrion and nucleus. Mutations of Chchd10 are associated with ALS, dementia and myopathy in humans and animal models, but how knockout of Chchd10 (Chchd10KO) affects various tissues especially skeletal muscle and adipose tissues remains unclear. Here we show that Chchd10 expression increases as myoblasts and preadipocytes differentiate. During myogenesis, CHCHD10 interacts with TAR DNA binding protein 43 (TDP-43) in regenerating myofibers in vivo and in newly differentiated myotubes ex vivo. Surprisingly, Chchd10KO mice had normal skeletal muscle development, growth and regeneration, with moderate defects in grip strength and motor performance. Chchd10KO similarly had no effects on development of brown and white adipose tissues (WAT). However, Chchd10KO mice had blunted response to acute cold and attenuated cold-induced browning of WAT, with markedly reduced UCP1 levels. Together, these results demonstrate that Chchd10 is dispensable for normal myogenesis and adipogenesis but is required for normal motility and cold-induced, mitochondrion-dependent browning of adipocytes. The data also suggest that human CHCHD10 mutations cause myopathy through a gain-of-function mechanism.

Loss of splicing factor IK impairs normal skeletal muscle development

BackgroundIK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo.ResultsThe ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells.ConclusionThis study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

Miswired Enhancer Logic Drives a Cancer of the Muscle Lineage.

SummaryCore regulatory transcription factors (CR TFs) establish enhancers with logical ordering during embryogenesis and development. Here we report that in fusion-positive rhabdomyosarcoma, a cancer of the muscle lineage, the chief oncogene PAX3-FOXO1 is driven by a translocated FOXO1 super enhancer (SE) restricted to a late stage of myogenesis. Using chromatin conformation capture techniques, we demonstrate that the extensive FOXO1 cis-regulatory domain interacts with PAX3. Furthermore, RNA sequencing and chromatin immunoprecipitation sequencing data in tumors bearing rare PAX translocations implicate enhancer miswiring across all fusion-positive tumors. HiChIP of H3K27ac showed connectivity between the FOXO1 SE, additional intra-domain enhancers, and the PAX3 promoter. We show that PAX3-FOXO1 transcription is diminished when this network of enhancers is ablated by CRISPR. Our data reveal a hijacked enhancer network that disrupts the stepwise CR TF logic of normal skeletal muscle development (PAX3 to MYOD to MYOG), replacing it with an “infinite loop” enhancer logic that locks rhabdomyosarcoma in an undifferentiated stage.

Fndc5 loss-of-function attenuates exercise-induced browning of white adipose tissue in mice.

Fibronectin type III domain containing 5 (Fndc5) is a transmembrane protein highly expressed in the skeletal muscle. It was reported that exercise promotes the shedding of the extracellular domain of Fndc5, generating a circulating peptide (irisin) that cross-talks to adipose tissues to convert lipid-storing white adipocytes to energy-catabolizing beige adipocytes. However, the requirement of Fndc5 in mediating the beneficial effect of exercise remains to be determined. Here, we created a mouse model of Fndc5 mutation through transcription activator-like effector nuclease-mediated DNA targeting. The Fndc5 mutant mice have normal skeletal muscle development, growth, regeneration, as well as glucose and lipid metabolism at resting state, even when fed a high-fat diet. In response to running exercise, however, the Fndc5 mutant mice exhibit reduced glucose tolerance and insulin sensitivity and have lower maximal oxygen consumption compared with the exercised wild-type mice. Mechanistically, Fndc5 mutation attenuates exercise-induced browning of white adipose tissue that is crucial for the metabolic benefits of physical activities. These data provide genetic evidence that Fndc5 is dispensable for muscle development and basal metabolism but essential for exercise-induced browning of white adipose tissues in mice.-Xiong, Y., Wu, Z., Zhang, B., Wang, C., Mao, F., Liu, X., Hu, K., Sun, X., Jin, W., Kuang, S. Fndc5 loss-of-function attenuates exercise-induced browning of white adipose tissue in mice.

NEXT GENERATION SEQUENCING AND EXPERIMENTAL MYOLOGY: P.383Cullin-3 is required for normal skeletal muscle development

E3-ubiquitin ligases are protein complexes that are essential for the recognition and the degradation of specific substrates by the ubiquitin-proteasome pathway. Cullin-RING ligases represent the largest E3-ubiquitin ligase family in mammals. The Cullins constitute the backbone of the E3-ubiquitin ligase complex, and interact with specific substrate adaptors that are necessary for the recruitment of their individual substrates for degradation. While roles for Cullin-RING ligases were intensively studied in the context of cancer, very little is known about their functions in muscle biology. Recently, several genes of the BTB-domain family (KBTBD13, KLHL40 and KLHL41) were found mutated in severe and early onset forms of nemaline myopathy. Due to their BTB-domains, these proteins are thought to be substrate adaptors for Cullin-3. These findings argue in favor of important and yet largely uncharacterized roles for Cullin-3 mediated protein turnover for muscle development, maintenance and function. We demonstrated that Cullin-RING ligase function is essential for myogenesis in vitro through the use of MLN4924, an inhibitor of neddylation. However, the role of Cullin-3 in muscle development remains poorly understood. Using conditional knockout mice for Cullin-3 in skeletal muscles, we demonstrate the pivotal role of this protein for postnatal life. Our data reveal that absence of Cullin-3 leads to respiratory defects at birth, associated with a strong skeletal muscle atrophy and neuromuscular junction disorganization. Large-scale proteome analysis of embryonic muscles revealed that lack of Cullin-3 results in the accumulation of muscle specific and cytoskeletal proteins, presumably representing novel substrates of this E3-ligase. Our results demonstrate that Cullin-3 mediated protein turnover is required for proper early skeletal muscle development. We are currently investigating the molecular mechanisms that contribute to the severe myopathy observed in these animals.

Delayed muscle development in small pig fetuses around birth cannot be rectified by maternal early feed restriction and subsequent overfeeding during gestation

Intrauterine variations in nutrient allowance can alter body composition and tissue features of the porcine offspring around birth. This study aimed to determine the effects of fetal weight variations between littermates and of maternal dietary regimen during gestation on fetal muscle traits just before birth. Fourteen pregnant gilts were reared under a conventional (control, CTL; n=7) or an experimental (treatment, TRT; n=7) dietary regimen during gestation. The dietary treatment provided 70% of the protein and digestible energy contents of the CTL diet during the first 70 days of gestation and then, 115% of the protein and digestible energy contents up to farrowing. At 110 days of gestation, sows were sacrificed and one fetus having a low (824±140 g) and one having a normal (1218±192 g) BW per litter were sampled. Irrespective of maternal dietary regimen, the longissimus muscle of the small fetuses exhibited higher expression levels of DLK1/Pref1 and NCAM1/CD56, two genes known to be downregulated during normal skeletal muscle development. Expression levels of the embryonic isoform of the myosin heavy chain (MyHC), both at the mRNA and at the protein levels, were also higher in small fetuses. In addition, the ratios of perinatal to embryonic and of adult fast to developmental MyHC isoforms were generally lower in light fetuses compared with their medium-weight littermates. These modifications suggest a delayed myofiber development in spontaneous growth-retarded fetuses. Finally, GLUT1 was expressed to a lesser extent in the muscle of small v. normal fetuses, suggesting decreased ability for glucose uptake in muscle. Initial feed restriction and subsequent overfeeding of sows during gestation led to a lower expression of the myogenic factor MYOD1, a prerequisite for myogenic initiation in skeletal muscle. This maternal strategy was also associated with a lower expression level of insulin-like growth factor 1 receptor (IGFR) but an upregulation of IGF2. This suggests an altered susceptibility of muscle cells to IGFs’ signal in fetuses from treated sows. Altogether, intrauterine growth restriction impaired fetal muscle development, and restricted feeding followed by overfeeding of gestating sows did not allow small fetuses to recover normal contractile and metabolic characteristics.

Alterations in thin filament length during postnatal skeletal muscle development and aging in mice.

The lengths of the sarcomeric thin filaments vary in a skeletal muscle-specific manner and help specify the physiological properties of skeletal muscle. Since the extent of overlap between the thin and thick filaments determines the amount of contractile force that a sarcomere can actively produce, thin filament lengths are accurate predictors of muscle-specific sarcomere length-tension relationships and sarcomere operating length ranges. However, the striking uniformity of thin filament lengths within sarcomeres, specified during myofibril assembly, has led to the widely held assumption that thin filament lengths remain constant throughout an organism's lifespan. Here, we rigorously tested this assumption by using computational super-resolution image analysis of confocal fluorescence images to explore the effects of postnatal development and aging on thin filament length in mice. We found that thin filaments shorten in postnatal tibialis anterior (TA) and gastrocnemius muscles between postnatal days 7 and 21, consistent with the developmental program of myosin heavy chain (MHC) gene expression in this interval. By contrast, thin filament lengths in TA and extensor digitorum longus (EDL) muscles remained constant between 2 mo and 2 yr of age, while thin filament lengths in soleus muscle became shorter, suggestive of a slow-muscle-specific mechanism of thin filament destabilization associated with aging. Collectively, these data are the first to show that thin filament lengths change as part of normal skeletal muscle development and aging, motivating future investigations into the cellular and molecular mechanisms underlying thin filament adaptation across the lifespan.

Stac3 inhibits myoblast differentiation into myotubes.

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.

P.4.1 PIP kinases, muscle development, and the pathogenesis of myotubular myopathy

Myotubular myopathy (MTM) is a severe congenital myopathy with no currently identified treatment. MTM is caused by mutations in MTM1, a phosphatase that dephosphorylates 3-position phosphoinositides. Through the use of vertebrate model systems, the consequences (s) of MTM1 mutation in skeletal muscle in vivo are beginning to be unraveled. In particular, work from several laboratories (including our own) has demonstrated that loss of MTM1 (1) increases the levels of PI3P in skeletal muscle and (2) results in the disruption of the structure and function of the EC coupling apparatus. Based on these data, one hypothesis to explain MTM is that pathologic elevation of PI3P results in aberrant EC coupling, which in turn causes muscle weakness and severe neurologic disability. A correlate hypothesis is that lowering PI3P levels in MTM will enable normal muscle structure and will thus ameliorate the disease. These hypotheses lead to 2 critical questions: What is the consequence of reducing PI3P levels on normal skeletal muscle development? What is the impact of reducing PI3P levels on MTM pathogenesis? The goal of this study is to address these questions by studying models with reduced PI3P. We have created knockout mice that lack either Pik3c2b or Pik3c3, the kinases responsible for PI3P production in skeletal muscle. Using these animals, we have identified unique and non-overlapping functions for these kinases, including the observation that loss of Pik3c3 results in a muscular dystrophy phenotype. Furthermore, we have tested the concept of PI3P reduction as a therapeutic strategy for MTM by crossbreeding individual kinase knockouts with Mtm1 knockouts. Preliminary data shows that reduced expression of one of the kinases significantly improves the Mtm1 phenotype. In all, our data reveals for the first time the role of 3-PI kinases in skeletal muscle, and identifies a potential therapeutic target for the treatment of MTM.

Laminin-111 Protein Therapy Reduces Muscle Pathology and Improves Viability of a Mouse Model of Merosin-Deficient Congenital Muscular Dystrophy

Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a lethal muscle-wasting disease that is caused by mutations in the LAMA2 gene, resulting in the loss of laminin-α2 protein. MDC1A patients exhibit severe muscle weakness from birth, are confined to a wheelchair, require ventilator assistance, and have reduced life expectancy. There are currently no effective treatments or cures for MDC1A. Laminin-α2 is required for the formation of heterotrimeric laminin-211 (ie, α2, β1, and γ1) and laminin-221 (ie, α2, β2, and γ1), which are major constituents of skeletal muscle basal lamina. Laminin-111 (ie, α1, β1, and γ1) is the predominant laminin isoform in embryonic skeletal muscle and supports normal skeletal muscle development in laminin-α2-deficient muscle but is absent from adult skeletal muscle. In this study, we determined whether treatment with Engelbreth-Holm-Swarm-derived mouse laminin-111 protein could rescue MDC1A in the dy(W-/-) mouse model. We demonstrate that laminin-111 protein systemically delivered to the muscles of laminin-α2-deficient mice prevents muscle pathology, improves muscle strength, and dramatically increases life expectancy. Laminin-111 also prevented apoptosis in laminin-α2-deficient mouse muscle and primary human MDC1A myogenic cells, which indicates a conserved mechanism of action and cross-reactivity between species. Our results demonstrate that laminin-111 can serve as an effective protein substitution therapy for the treatment of muscular dystrophy in the dy(W-/-) mouse model and establish the potential for its use in the treatment of MDC1A.

The Role of Fibroblasts in Self-Assembled Skeletal Muscle

Small facial skeletal muscles often have no autologous donor source to effect surgical reconstruction. Autologously derived muscles could be engineered for replacement tissue, but must be vascularized and innervated to be functional. As a critical step, engineered muscle must mimic the morphology, protein and gene expression, and function of native muscle. This study utilized a self-assembly process to engineer three-dimensional (3D) muscle from a statically strained muscle cell monolayer. Primary mouse myoblasts (PMMs) and mouse embryonic fibroblasts (MEFs) were separately proliferated and coseeded on a fibrin sheet with anchored sutures. Within 10 days of initiating PMM differentiation, the cell-gel layer contracted, lifted, and rolled into a cylindrical 3D structure around the tendon-like suture anchors; the myotubes longitudinally aligned along the lines of tensile force. The objectives of this study were to characterize these engineered muscles and to elucidate the role of the fibroblasts in the self-assembly process. Fibroblasts maintained myotube viability, mediated fibrin degradation, and assisted in muscle self-assembly. The optimal 1:1 PMM:MEF ratio resulted in tissue morphology remarkably similar to native muscle. Through gene and protein expression assays, the development and maturation of the engineered muscle tissue was demonstrated to recapitulate normal skeletal muscle development.

Regulation of Skeletal Muscle Sarcomere Integrity and Postnatal Muscle Function by Mef2c

Myocyte enhancer factor 2 (MEF2) transcription factors cooperate with the MyoD family of basic helix-loop-helix (bHLH) transcription factors to drive skeletal muscle development during embryogenesis, but little is known about the potential functions of MEF2 factors in postnatal skeletal muscle. Here we show that skeletal muscle-specific deletion of Mef2c in mice results in disorganized myofibers and perinatal lethality. In contrast, neither Mef2a nor Mef2d is required for normal skeletal muscle development in vivo. Skeletal muscle deficient in Mef2c differentiates and forms normal myofibers during embryogenesis, but myofibers rapidly deteriorate after birth due to disorganized sarcomeres and a loss of integrity of the M line. Microarray analysis of Mef2c null muscles identified several muscle structural genes that depend on MEF2C, including those encoding the M-line-specific proteins myomesin and M protein. We show that MEF2C directly regulates myomesin gene transcription and that loss of Mef2c in skeletal muscle results in improper sarcomere organization. These results reveal a key role for Mef2c in maintenance of sarcomere integrity and postnatal maturation of skeletal muscle.

Insulin-Like Growth Factor (IGF) Binding Protein-5 Blocks Skeletal Muscle Differentiation by Inhibiting IGF Actions

Signaling through the IGF-I receptor by locally produced IGF-I or -II is critical for normal skeletal muscle development and repair after injury. In most tissues, IGF action is modulated by IGF binding proteins (IGFBPs). IGFBP-5 is produced by muscle cells, and previous studies have suggested that when overexpressed it may either facilitate or inhibit IGF actions, and thus potentially enhance or diminish IGF-mediated myoblast differentiation or survival. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5, we studied its effects in cultured muscle cells. Purified wild-type (WT) mouse IGFBP-5 or a variant with diminished extracellular matrix binding (C domain mutant) each prevented differentiation at final concentrations as low as 3.5 nm, whereas analogs with reduced IGF binding (N domain mutant) were ineffective even at 100 nm. None of the IGFBP-5 variants altered cell number. An IGF-I analog (R(3)IGF-I) with diminished affinity for IGFBPs promoted full muscle differentiation in the presence of IGFBP-5(WT), showing that IGFBP-5 interferes with IGF-dependent signaling pathways in myoblasts. When IGFBP-5(WT) or variants were overexpressed by adenovirus-mediated gene transfer, concentrations in muscle culture medium reached 500 nm, and differentiation was inhibited, even by IGFBP-5(N). As 200 nm of purified IGFBP-5(N) prevented activation of the IGF-I receptor by 10 nm IGF-II as effectively as 2 nm of IGFBP-5(WT), our results not only demonstrate that IGFBP-5 variants with reduced IGF binding affinity impair muscle differentiation by blocking IGF actions, but underscore the need for caution when labeling effects of IGFBPs as IGF independent because even low-affinity analogs may potently inhibit IGF-I or -II if present at high enough concentrations in biological fluids.

Regulation of chloride ion conductance during skeletal muscle development and in disease. Focus on “Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1”

productive investigations of disease mechanisms that also reveal new information about normal regulation elicit a particularly satisfying sense of a two-for-one deal. In the case of Lueck et al. (Ref. [13][1]; see page 1291 of this issue), a detailed study of the molecular basis for the myotonia (

Muscle cell survival mediated by the transcriptional coactivators p300 and PCAF displays different requirements for acetyltransferase activity

Normal skeletal muscle development requires the proper orchestration of genetic programs by myogenic regulatory factors (MRFs). The actions of the MRF protein MyoD are enhanced by the transcriptional coactivators p300 and the p300/CBP-associated factor (PCAF). We previously described C2 skeletal myoblasts lacking expression of insulin-like growth factor-II (IGF-II) that underwent progressive apoptotic death when incubated in differentiation-promoting medium. Viability of these cells was sustained by addition of IGF analogs or unrelated peptide growth factors. We now show that p300 or PCAF maintains myoblast viability as effectively as added growth factors through mechanisms requiring the acetyltransferase activity of PCAF but not of p300. The actions of p300 to promote cell survival were not secondary to increased expression of known MyoD targets, as evidenced by results of gene microarray experiments, but rather appeared to be mediated by induction of other genes, including fibroblast growth factor-1 (FGF-1). Conditioned culture medium from cells expressing p300 increased myoblast viability, and this was blocked by pharmacological inhibition of FGF receptors. Our results define a role for p300 in promoting cell survival, which is independent of its acetyltransferase activity and acts at least in part through FGF-1.

Thyroid hormones and the mRNA of the GH receptor and IGFs in skeletal muscle of fetal sheep.

Thyroid hormones are required for the normal development of skeletal muscle in utero, although their mechanism of action is poorly understood. The present study examined the effects of the thyroid hormones on the gene expression of the growth hormone receptor (GHR) and the insulin-like growth factors (IGFs) IGF-I and IGF-II, in skeletal muscle of fetal sheep during late gestation (term 145 +/- 2 days) and after manipulation of plasma thyroid hormone concentration. Thyroidectomy at 105-110 days of gestation suppressed muscle GHR and IGF-I gene expression in fetuses studied at 127-130 and 142-145 days. Muscle GHR mRNA abundance remained unchanged with increasing gestational age in intact and thyroidectomized fetuses. In the intact fetuses, a decrease in muscle IGF-I gene expression was observed between 127-130 and 142-145 days, which coincided with the normal prepartum surges in plasma cortisol and triiodothyronine (T3). At 127-130 days, downregulation of muscle IGF-I mRNA abundance was induced prematurely in intact fetuses by an infusion of cortisol for 5 days (2-3 mg x kg(-1) x day(-1) iv), which increased plasma cortisol and T3 concentrations to values seen near term. However, increasing plasma T3 alone by an infusion of T3 for 5 days (8-12 microg x kg(-1) x day(-1) iv) in intact fetuses at this age had no effect on GHR or IGF-I gene expression in skeletal muscle. In the thyroidectomized fetuses, no additional change in the low level of muscle IGF-I mRNA abundance was seen with increasing gestational age, but at 127-130 days, IGF-I gene expression was reduced further when plasma cortisol and T3 concentrations were increased by exogenous cortisol infusion. Muscle IGF-II mRNA abundance was not affected by thyroidectomy, gestational age, or exogenous hormone infusion. These findings show, in the sheep fetus, that thyroid hormones may influence the growth and development of skeletal muscle via changes in the local activity of the somatotrophic axis.

Acetylcholine receptor epsilon-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice.

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the gamma-subunit of the fetal acetylcholine receptor (AChR) by the epsilon-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the epsilon-subunit gene caused in homozygous mutant mice the persistence of gamma-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of epsilon-subunit containing AChRs is essential for normal development of skeletal muscle.