Early Muscle Differentiation Research Articles

Mechanoresponsive regulation of myogenesis by the force-sensing transcriptional regulator Tono

Muscle morphogenesis is a multi-step program, starting with myoblast fusion, followed by myotube-tendon attachment and sarcomere assembly, with subsequent sarcomere maturation, mitochondrial amplification, and specialization. The correct chronological order of these steps requires precise control of the transcriptional regulators and their effectors. How this regulation is achieved during muscle development is not well understood. In a genome-wide RNAi screen in Drosophila, we identified the BTB-zinc-finger protein Tono (CG32121) as a muscle-specific transcriptional regulator. tono mutant flight muscles display severe deficits in mitochondria and sarcomere maturation, resulting in uncontrolled contractile forces causing muscle rupture and degeneration during development. Tono protein is expressed during sarcomere maturation and localizes in distinct condensates in flight muscle nuclei. Interestingly, internal pressure exerted by the maturing sarcomeres deforms the muscle nuclei into elongated shapes and changes the Tono condensates, suggesting that Tono senses the mechanical status of the muscle cells. Indeed, external mechanical pressure on the muscles triggers rapid liquid-liquid phase separation of Tono utilizing its BTB domain. Thus, we propose that Tono senses high mechanical pressure to adapt muscle transcription, specifically at the sarcomere maturation stages. Consistently, tono mutant muscles display specific defects in a transcriptional switch that represses early muscle differentiation genes and boosts late ones. We hypothesize that a similar mechano-responsive regulation mechanism may control the activity of related BTB-zinc-finger proteins that, if mutated, can result in uncontrolled force production in human muscle.

Filamin-A-interacting protein 1 (FILIP1) is a dual compartment protein linking myofibrils and microtubules during myogenic differentiation and upon mechanical stress

Variations in the gene encoding filamin-A-interacting protein 1 (FILIP1) were identified to be associated with a combination of neurological and muscular symptoms. While FILIP1 was shown to regulate motility of brain ventricular zone cells, a process important for corticogenesis, the function of the protein in muscle cells has been less well characterized. The expression of FILIP1 in regenerating muscle fibres predicted a role in early muscle differentiation. Here we analysed expression and localization of FILIP1 and its binding partners filamin-C (FLNc) and microtubule plus-end-binding protein EB3 in differentiating cultured myotubes and adult skeletal muscle. Prior to the development of cross-striated myofibrils, FILIP1 is associated with microtubules and colocalizes with EB3. During further myofibril maturation its localization changes, and FILIP1 localizes to myofibrillar Z-discs together with the actin-binding protein FLNc. Forced contractions of myotubes by electrical pulse stimulation (EPS) induce focal disruptions in myofibrils and translocation of both proteins from Z-discs to these lesions, suggesting a role in induction and/or repair of these structures. The immediate proximity of tyrosylated, dynamic microtubules and EB3 to lesions implies that also these play a role in these processes. This implication is supported by the fact that in nocodazole-treated myotubes that lack functional microtubules, the number of lesions induced by EPS is significantly reduced. In summary, we here show that FILIP1 is a cytolinker protein that is associated with both microtubules and actin filaments, and might play a role in the assembly of myofibrils and their stabilization upon mechanical stress to protect them from damage.

Abstract IA020: Reprogramming of myogenic transcription factors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma, a pediatric malignancy with partial resemblance to undifferentiated skeletal muscle, is characterized by high expression of myogenic-lineage transcription factors such as MYOD1 and MYOG. Despite high expression of these transcription factors, which in normal muscle result in differentiation, RMS cells fail to differentiate, suggesting the presence of factors that inhibit their normal differentiation-promoting functions. In this talk, I will present data that the key muscle transcriptional regulator, SIX1, which in development activates the myogenic regulatory factors (MRFs) and promotes muscle differentiation, in fact inhibits differentiation in fusion-negative (FN) RMS. SIX1 holds FN-RMS cells in a progenitor-like state by altering the chromatin landscape and causing MYOD1, a key MRF, to preferentially bind to regulatory regions of genes permissive to growth rather than differentiation. Loss of SIX1 results in re-localization of MYOD1 to promoters/enhancers of genes associated with differentiation, and further results in increased binding of MYOG at such loci. Altered binding of MYOD1 and MYOG in response to SIX1 loss results in marked inhibition of RMS growth in vivo, via induction of differentiation. These data suggest that SIX1 acts as a master regulatory factor controlling the fate of RMS cells. Data will be presented that suggest dynamic actions of SIX1 and its co-factors throughout normal muscle differentiation, whereby high levels of SIX1 expressed in early muscle differentiation may mimic a transcriptional state seen in RMS. We hypothesize that the specific levels of SIX1, combined with a unique combination of transcriptional co-factors, reprogram genome-wide binding of MRFs to different promoter/enhancer sites at specific developmental time points, and that RMS is trapped in an early developmental state where SIX1 represses differentiation via genome-wide alterations in MRF binding that favor growth. Understanding the co-factors that work with SIX1 to alter chromatin state and MRF binding may enable the discovery of novel targets whose inhibition could serve as a relatively non-toxic treatment to restore normal developmental processes and inhibit RMS progression. Citation Format: Heide L. Ford, Jessica Y. Hsu, Etienne P. Danis, Stephanie Nance, Jenean H. O’Brien, Annika L. Gustafson, Veronica M. Wessells, Andrew E. Goodspeed, Jared C. Talbot, Sharon L. Amacher, Paul Jedlicka, Joshua C. Black, James C. Costello, Adam D. Durbin, Kristin B. Artinger. Reprogramming of myogenic transcription factors in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA020.

Quaking RNA-Binding Proteins Control Early Myofibril Formation by Modulating Tropomyosin

Skeletal muscle contraction is mediated by myofibrils, complex multi-molecular scaffolds structured into repeated units, the sarcomeres. Myofibril structure and function have been extensively studied, but the molecular processes regulating its formation within the differentiating muscle cell remain largely unknown. Here we show in zebrafish that genetic interference with the Quaking RNA-binding proteins disrupts the initial steps of myofibril assembly without affecting early muscle differentiation. Using RNA sequencing, we demonstrate that Quaking is required for accumulation of the muscle-specific tropomyosin-3 transcript, tpm3.12. Further functional analyses reveal that Tpm3.12 mediates Quaking control of myofibril formation. Moreover, we identified a Quaking-binding site in the 3' UTR of tpm3.12 transcript, which is required invivo for tpm3.12 accumulation and myofibril formation. Our work uncovers a Quaking/Tpm3 pathway controlling de novo myofibril assembly. This unexpected developmental role for Tpm3 could be at the origin of muscle defects observed in human congenital myopathies associated with tpm3 mutation.

IRS4, a novel modulator of BMP/Smad and Akt signalling during early muscle differentiation

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.

Intestinal smooth muscle is required for patterning the enteric nervous system.

The development of the enteric nervous system (ENS) and intestinal smooth muscle occurs in a spatially and temporally correlated manner, but how they influence each other is unknown. In the developing mid-gut of the chick embryo, we find that α-smooth muscle actin expression, indicating early muscle differentiation, occurs after the arrival of migrating enteric neural crest-derived cells (ENCCs). In contrast, hindgut smooth muscle develops prior to ENCC arrival. Smooth muscle development is normal in experimentally aganglionic hindguts, suggesting that proper development and patterning of the muscle layers does not rely on the ENS. However, inhibiting early smooth muscle development severely disrupts ENS patterning without affecting ENCC proliferation or apoptosis. Our results demonstrate that early intestinal smooth muscle differentiation is required for patterning the developing ENS.

Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Cavβ1a NH2-terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation.

G.O.2: Mutations in LMOD3 cause severe nemaline myopathy by disrupting thin filament organisation in skeletal muscle

Nemaline myopathy (NM) is a disorder of the skeletal muscle thin filament characterised by muscle dysfunction and electron-dense protein accumulations (nemaline bodies). Pathogenic mutations have been described in nine genes to date, but the genetic basis remains unknown in many cases. We used whole exome sequencing (WES) in two families with NM and subsequent gene sequencing in over 540 additional genetically unresolved NM patients to identify and characterise a new genetic cause of NM. We developed a knock-down zebrafish model of this condition and used immunohistochemistry, western blotting, single-fibre contractility studies and recombinant protein studies to characterise the expression, localisation and biochemical functions of the new disease-related protein. We identified homozygous or compound heterozygous variants in LMOD3, which encodes leiomodin-3 (Lmod3) in 21 patients from 14 families. Affected individuals had severe generalised weakness and hypotonia, and most affected individuals died in the neonatal period. We demonstrated that Lmod3 is expressed from early muscle differentiation, localises to thin filaments with enrichment at the pointed ends, and has strong actin nucleating activity. Loss of Lmod3 in patient muscle results in shortening and disorganisation of thin filaments. Knockdown of lmod3 in the zebrafish replicates this phenotype. These findings define a new genetic subtype of congenital myopathy and demonstrate an essential, previously unrecognised role for Lmod3 in the regulation of sarcomeric thin filaments in skeletal muscle.

POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations.

Dystroglycan is a transmembrane glycoprotein whose interactions with the extracellular matrix (ECM) are necessary for normal muscle and brain development, and disruptions of its function lead to dystroglycanopathies, a group of congenital muscular dystrophies showing extreme genetic and clinical heterogeneity. Specific glycans bound to the extracellular portion of dystroglycan, α-dystroglycan, mediate ECM interactions and most known dystroglycanopathy genes encode glycosyltransferases involved in glycan synthesis. POMK, which was found mutated in two dystroglycanopathy cases, is instead involved in a glycan phosphorylation reaction critical for ECM binding, but little is known about the clinical presentation of POMK mutations or of the function of this protein in the muscle. Here, we describe two families carrying different truncating alleles, both removing the kinase domain in POMK, with different clinical manifestations ranging from Walker-Warburg syndrome, the most severe form of dystroglycanopathy, to limb-girdle muscular dystrophy with cognitive defects. We explored POMK expression in fetal and adult human muscle and identified widespread expression primarily during fetal development in myocytes and interstitial cells suggesting a role for this protein during early muscle differentiation. Analysis of loss of function in the zebrafish embryo and larva showed that pomk function is necessary for normal muscle development, leading to locomotor dysfuction in the embryo and signs of muscular dystrophy in the larva. In summary, we defined diverse clinical presentations following POMK mutations and showed that this gene is necessary for early muscle development.

Cell Aspect Ratio Alters Stem Cell Traction Stresses and Lineage

Adult mesenchymal stem cells (MSCs) “feel” the stiffness of their environment and differentiate in response to it; thus aberrant stiffness resulting from fibrosis in muscle dystrophies could misdirect MSCs into the wrong lineage. Conversely, MSCs have been shown to respond in a myosin-dependent manner to adipogenic and osteogenic media when cell spread area changes from 103 to 104 μm2 or when cultured in specific shapes, e.g. circles versus rectangles, and polygons, respectively. When both cues are present in a disparate fashion, e.g. highly elongated cells similar to muscle despite the presence of an abnormally stiff microenvironment, we hypothesized that a myosin contraction-dependent balance could induce a subset of MSCs to differentiate in to a muscle-like phenotype despite residing in a dystrophic-like stiffness. To regulate MSC morphology, we patterned fibronectin in shapes of varying aspect ratios but common area on polyacrylamide substrates of known stiffness. MSCs spread to the patterns and localized their focal adhesion in a stiffness and shape-dependent manner. Using traction force microscopy, we found that strain energy from cell-generated forces scaled with stiffness, but decreased as a function of cell elongation with isotropic cell patterns producing the highest contractile energy in contrast to our hypothesis. Muscle-specific myosin heavy chain (mMHC), an indicator of early muscle differentiation, also was expressed in a stiffness and elongation dependent manner. On muscle-like stiffness of 11 kiloPascals (kPa), cells with only minimal elongation, i.e. 1:1 and 3:1 patterns, expressed mMHC most strongly. In contrast on osteogenic-like matrices of 34 kPa, highest MHC expression corresponded to the most elongated patterns. These shape- and stiffness-dependent lineage changes with muscle markers correlated to contractility-based observations suggest that muscle induction may be possible in non-permissive stiffer environments and could prove beneficial to treat fibrotic muscle diseases.

Abstract 5343: Aberrant activation of hedgehog signaling confers a poor prognosis in embryonal and fusion gene negative alveolar rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). The hedgehog (Hh) pathway has recently been implied in tumor formation and progression of various cancers. In the current study we investigated whether Hh pathway activation presents a general feature also of RMS and whether it bears prognostic impact. Here, we report that marker genes of active Hh signaling, i.e. Patched1 (Ptch1), Gli1, Gli3 and Myf5, are elevated in two distinct cohorts of RMS patients with a total number of 235 primary samples. Interestingly, all these marker genes of active Hh signaling were expressed at significantly higher levels in specific subtypes of RMS, i.e. ERMS and fusion gene negative ARMS, compared to fusion gene positive ARMS. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene negative ARMS, but not in fusion gene positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation in primary RMS samples. In support of this notion, the addition of Shh inhibits muscle differentiation in parallel experiments in muscle satellite cells and myoblasts. Moreover, we identify Myf5 is as a novel excellent class predictor for RMS by receiver operating characteristic (ROC) analysis. Most importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene negative RMS (P< 0.004) underscoring the clinical relevance of these findings. By identifying aberrant Hh activation in specific subgroups of RMS, our study has important implications for the development of molecular targeted therapies, such as small molecule Hh inhibitors, in RMS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2011-5343

Alterations of excitation-contraction coupling and excitation coupled Ca2+ entry in human myotubes carrying CAV3 mutations linked to rippling muscle

Rippling muscle disease is caused by mutations in the gene encoding caveolin-3 (CAV3), the muscle-specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin-3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca2+ homeostasis. In the present study we examined Ca2+ homeostasis and excitation–contraction (E-C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin-3 expression; one patient harboured the heterozygous c.84C>A mutation while the other patient harbored a homozygous splice-site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca2+]i and 4-chloro-m-cresol-induced Ca2+ release but reduced KCl-induced Ca2+ influx. Detailed analysis of the voltage-dependence of Ca2+ transients revealed a significant shift of Ca2+ release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin-3 leads to microscopic disarrays in the colocalization of the voltage-sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation–contraction coupling. Hum Mutat 32:309–317, 2011. © 2011 Wiley-Liss, Inc.

Activation of the hedgehog pathway confers a poor prognosis in embryonal and fusion gene-negative alveolar rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and comprises two major histological subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Seventy-five percent of ARMS harbor reciprocal chromosomal translocations leading to fusion genes of the forkhead transcription factor FOXO1 and PAX3 or PAX7. The hedgehog (Hh) pathway has been implied in tumor formation and progression of various cancers including RMS. However, whether Hh pathway activation presents a general feature of RMS or whether it is restricted to specific subgroups has not yet been addressed. Here, we report that marker genes of active Hh signaling, that is, Patched1 (Ptch1), Gli1, Gli3 and Myf5, are expressed at significantly higher levels in ERMS and fusion gene-negative ARMS compared with fusion gene-positive ARMS in two distinct cohorts of RMS patients. Consistently, Gli1 expression correlates with Ptch1 expression in ERMS and fusion gene-negative ARMS, but not in fusion gene-positive ARMS. In addition, expression levels of MyoD1 are significantly lower in ERMS and fusion gene-negative ARMS, pointing to an inverse association of Hh activation and early muscle differentiation. Moreover, Myf5 is identified as a novel excellent class predictor for RMS by receiver operating characteristic analysis. Importantly, high expression of Ptch1 or low MyoD1 expression significantly correlate with reduced cumulative survival in fusion gene-negative RMS underscoring the clinical relevance of these findings. By showing that Hh signaling is preferentially activated in specific subgroups of RMS, our study has important implications for molecular targeted therapies, such as small molecule Hh inhibitors, in RMS.

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose–derived Stem Cells With Forced Expression of MyoD

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose-derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2(-/-)gammaC(-/-) mice resulted in a substantial increase in the number of hMADS cell-derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.

From T‐tubule to sarcolemma: damage‐induced dysferlin translocation in early myogenesis

The dysferlin gene is mutated in limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. In mature skeletal muscle, dysferlin is located predominantly at the sarcolemma, where it plays a role in membrane fusion and repair. To investigate the role of dysferlin during early muscle differentiation, its localization was studied at high resolution in a muscle cell line. This demonstrated that dysferlin is not expressed at the plasmalemma of myotubes but mostly localizes to the T-tubule network. However, dysferlin translocated to the site of injury and toward the plasma membrane in a Ca2+-dependent fashion in response to a newly designed in vitro wounding assay. This reaction was specific to the full-length protein, as heterologously expressed deletion mutants of distinct C2 domains of dysferlin did not show this response. These results shed light on the dynamics of muscle membrane repair and are highly indicative of a specific role of dysferlin in this process in early myogenesis.

In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21(waf1/cip1) and p27(kip1) cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.

Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes.

Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen-activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N-terminal transactivation domain, resulting in reduced MRF4-mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and alpha-actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and alpha-actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation.

Myogenin induces higher oxidative capacity in pre-existing mouse muscle fibres after somatic DNA transfer

Muscle is a permanent tissue, and in the adult pronounced changes can occur in pre-existing fibres without the formation of new fibres. Thus, the mechanisms responsible for phenotype transformation in the adult might be distinct from mechanisms regulating muscle differentiation during muscle formation and growth. Myogenin is a muscle-specific, basic helix-loop-helix transcription factor that is important during early muscle differentiation. It is also expressed in the adult, where its role is unknown. In this study we have overexpressed myogenin in glycolytic fibres of normal adult mice by electroporation and single-cell intracellular injection of expression vectors. Myogenin had no effects on myosin heavy chain fibre type, but induced a considerable increase in succinate dehydrogenase and NADH dehydrogenase activity, with some type IIb fibres reaching the levels observed histochemically in normal type IIx and IIa fibres. mRNA levels for malate dehydrogenase were similarly altered. The size of the fibres overexpressing myogenin was reduced by 30–50 %. Thus, the transfected fibres acquired a phenotype reminiscent of the phenotype obtained by endurance training in man and other animals, with a higher oxidative capacity and smaller size. We conclude that myogenin can alter pre-existing glycolytic fibres in the intact adult animal.

Myogenin induces higher oxidative capacity in pre-existing mouse muscle fibres after somatic DNA transfer.

Muscle is a permanent tissue, and in the adult pronounced changes can occur in pre-existing fibres without the formation of new fibres. Thus, the mechanisms responsible for phenotype transformation in the adult might be distinct from mechanisms regulating muscle differentiation during muscle formation and growth. Myogenin is a muscle-specific, basic helix-loop-helix transcription factor that is important during early muscle differentiation. It is also expressed in the adult, where its role is unknown. In this study we have overexpressed myogenin in glycolytic fibres of normal adult mice by electroporation and single-cell intracellular injection of expression vectors. Myogenin had no effects on myosin heavy chain fibre type, but induced a considerable increase in succinate dehydrogenase and NADH dehydrogenase activity, with some type IIb fibres reaching the levels observed histochemically in normal type IIx and IIa fibres. mRNA levels for malate dehydrogenase were similarly altered. The size of the fibres overexpressing myogenin was reduced by 30-50 %. Thus, the transfected fibres acquired a phenotype reminiscent of the phenotype obtained by endurance training in man and other animals, with a higher oxidative capacity and smaller size. We conclude that myogenin can alter pre-existing glycolytic fibres in the intact adult animal.

Myne-1, a spectrin repeat transmembrane protein of the myocyte inner nuclear membrane, interacts with lamin A/C

Mutations in the genes encoding the inner nuclear membrane proteins lamin A/C and emerin produce cardiomyopathy and muscular dystrophy in humans and mice. The mechanism by which these broadly expressed gene products result in tissue-specific dysfunction is not known. We have identified a protein of the inner nuclear membrane that is highly expressed in striated and smooth muscle. This protein, myne-1 (myocyte nuclear envelope), is predicted to have seven spectrin repeats, an interrupted LEM domain and a single transmembrane domain at its C-terminus. We found that myne-1 is expressed upon early muscle differentiation in multiple intranuclear foci concomitant with lamin A/C expression. In mature muscle, myne-1 and lamin A/C are perfectly colocalized, although colocalization with emerin is only partial. Moreover, we show that myne-1 and lamin A/C coimmunoprecipitate from differentiated muscle in vitro. The muscle-specific inner nuclear envelope expression of myne-1, along with its interaction with lamin A/C, indicates that this gene is a potential mediator of cardiomyopathy and muscular dystrophy.