Slow myosin heavy chain expression in the absence of muscle activity.

Innervation has been generally accepted to be a major factor involved in both triggering and maintaining the expression of slow myosin heavy chain (MHC-1) in skeletal muscle. However, previous findings from our laboratory have suggested that, in the mouse, this is not always the case (30). Based on these results, we hypothesized that neurotomy would not markedly reduced the expression of MHC-1 protein in the mouse soleus muscles. In addition, other cellular, biochemical, and functional parameters were also studied in these denervated soleus muscles to complete our study. Our results show that denervation reduced neither the relative amount of MHC-1 protein, nor the percentage of muscle fibers expressing MHC-1 protein (P > 0.05). The fact that MHC-1 protein did not respond to muscle inactivity was confirmed in three different mouse strains (129/SV, C57BL/6, and CD1). In contrast, all of the other histological, biochemical, and functional muscle parameters were markedly altered by denervation. Cross-sectional area (CSA) of muscle fibers, maximal tetanic isometric force, maximal velocity of shortening, maximal power, and citrate synthase activity were all reduced in denervated muscles compared with innervated muscles (P < 0.05). Contraction and one-half relaxation times of the twitch were also increased by denervation (P < 0.05). Addition of tenotomy to denervation had no further effect on the relative expression of MHC-1 protein (P > 0.05), despite a greater reduction in CSA and citrate synthase activity (P < 0.05). In conclusion, a deficit in neural input leads to marked atrophy and reduction in performance in mouse soleus muscles. However, the maintenance of the relative expression of slow MHC protein is independent of neuromuscular activity in mice.

Genetic inactivation of acetylcholinesterase causes functional and structural impairment of mouse soleus muscles.

Acetylcholinesterase (AChE) plays an essential role in neuromuscular transmission. Not surprisingly, neuromuscular transmission during repetitive nerve stimulation is severely depressed in the AChE knockout mouse (KO). However, whether this deficit in AChE leads to skeletal muscle changes is not known. We have studied the in vitro contractile properties of the postural and locomotor soleus muscles of adult KO and normal (wildtype, WT) mice, and this was completed by histological and biochemical analyses. Our results show that muscle weight, cross-sectional area of muscle fibres and absolute maximal isometric force are all reduced in KO mice compared with WT mice. Of interest, the relative amount of slow myosin heavy chain (MHC-1) in muscle homogenates and the percentage of muscle fibres expressing MHC-1 are decreased in the KO mice. Surprisingly, AChE ablation does not modify twitch kinetics, absolute maximal power, fatigue resistance or citrate synthase activity, despite the reduced number of slow muscle fibres. Thus, a deficit in AChE leads to alterations in the structure and function of muscles but these changes are not simply related to the reduced body weight of KO mice. Our results also suggest that this murine model of congenital myasthenic syndrome with endplate AChE deficiency combines alterations in both neurotransmission and intrinsic muscle properties.

AAV-mediated delivery of a mutated myostatin propeptide ameliorates calpain 3 but not alpha-sarcoglycan deficiency.

Myostatin is a negative regulator of muscle mass whose inhibition has been proposed as a therapeutic strategy for muscle-wasting conditions. Indeed, blocking myostatin action through different strategies has proved beneficial for the pathophysiology of the dystrophin-deficient mdx mouse. In this report, we tested the inhibition of myostatin by AAV-mediated expression of a mutated propeptide in animal models of two limb-girdle muscular dystrophies: LGMD2A caused by mutations in the calpain 3 (CAPN3) gene and LGMD2D caused by mutations in the alpha-sarcoglycan gene (SGCA). In the highly regenerative Sgca-null mice, survival of the alpha-sarcoglycan-deficient muscle fibers did not improve after transfer of the myostatin propeptide. In calpain 3-deficient mice, a boost in muscle mass and an increase in absolute force were obtained, suggesting that myostatin inhibition could constitute a therapeutic strategy in this predominantly atrophic disorder.

Sequential expression of genes involved in muscular dystrophies during human development.

To elucidate the normal and pathophysiological roles of genes involved in the aetiology of muscular dystrophies, we studied the expression of dystrophin, four sarcoglycans, beta-dystroglycan and merosin during early human development. These proteins are expressed mainly in skeletal muscles while dystrophin, beta-dystroglycan, delta-sarcoglycan and merosin are in cardiac and smooth muscles. Dystrophin, beta-, delta-sarcoglycan and beta-dystroglycan are first expressed in the myotome at the 4th week of human embryogenesis, followed by gamma-sarcoglycan and merosin at the 6th week of development; alpha-sarcoglycan appears only at the level of the muscular fibre at the end of the embryonic period.

alpha11beta1 integrin is a receptor for interstitial collagens involved in cell migration and collagen reorganization on mesenchymal nonmuscle cells.

alpha11beta1 integrin constitutes a recent addition to the integrin family. Here, we present the first in vivo analysis of alpha11 protein and mRNA distribution during human embryonic development. alpha11 protein and mRNA were present in various mesenchymal cells around the cartilage anlage in the developing skeleton in a pattern similar to that described for the transcription factor scleraxis. alpha11 was also expressed by mesenchymal cells in intervertebral discs and in keratocytes in cornea, two sites with highly organized collagen networks. Neither alpha11 mRNA nor alpha11 protein could be detected in myogenic cells in human embryos. The described expression pattern is compatible with alpha11beta1 functioning as a receptor for interstitial collagens in vivo. To test this hypothesis in vitro, full-length human alpha11 cDNA was stably transfected into the mouse satellite cell line C2C12, lacking endogenous collagen receptors. alpha11beta1 mediated cell adhesion to collagens I and IV (with a preference for collagen I) and formed focal contacts on collagens. In addition, alpha11beta1 mediated contraction of fibrillar collagen gels in a manner similar to alpha2beta1, and supported migration on collagen I in response to chemotactic stimuli. Our data support a role for alpha11beta1 as a receptor for interstitial collagens on mesenchymally derived cells and suggest a multifunctional role of alpha11beta1 in the recognition and organization of interstitial collagen matrices during development.

Developmental expression of myotilin, a gene mutated in limb-girdle muscular dystrophy type 1A.

We analyzed developmental expression of myotilin, a novel sarcomeric component mutated in limb-girdle muscular dystrophy 1A (LGMD1A). In situ hybridization and immunostaining of embryonic mouse tissues revealed expression of myotilin initially (E9-10) in heart, somites and neuroepithelium. At E13 myotilin was expressed in a variety of tissues, including the nervous system, lung, liver and kidney, but upon organ differentiation expression became more restricted. The level of expression during early development is comparable between mouse and human, indicating that the mouse may provide a model for further studying the functions of myotilin and the pathogenesis of LGMD1A.

The muscle-specific enolase is an early marker of human myogenesis.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) is active as a dimer formed from three different subunits, alpha, beta and gamma, encoded by separate genes. The expression of these genes is developmentally regulated in a tissue-specific manner. A shift occurs during development, from the unique embryonic isoform alphaalpha, towards specific isoforms in two tissues with high energy demands: alphagamma and gammagamma in the nervous system, alphabeta and betabeta in striated muscles. The alphaalpha remains widely distributed in adult tissues. Here we report the results of the first extensive study of beta enolase expression during human development. Indeed, the beta subunit is specifically expressed at early stages of human myogenesis. Immunocytochemical analyses demonstrated that it is first detected in the heart of 3-week-old embryos and in the myotomal compartment of somites from 4-week-old embryos. At this stage, the muscle-specific sarcomeric protein titin is expressed in this structure, which will give rise to all body skeletal muscles, but embryonic myosin heavy chain is not yet present. Analyses at the protein level show that, during human ontogenesis, myogenesis is accompanied by an increase in beta enolase expression and by a decrease in the expression of the two other alpha and gamma subunits. Furthermore, beta enolase subunit is expressed in proliferating myoblasts from both embryonic and post-natal muscles. In addition, clonal analysis of primary cell cultures, obtained from the leg muscle of a 7-week-old human embryo, revealed that the beta subunit is present in the dividing myoblasts of all four types, according to the classification of Edom-Vovard et al. [(1999) J Cell Sci 112: 191-199], but not in cells of the non-myogenic lineage. Myoblast fusion is accompanied by a large increase in beta enolase expression. Our results demonstrate that this muscle-specific isoform of a glycolytic enzyme (beta enolase) is among the earliest markers of myogenic differentiation in humans.

Molecular tools for the study of titin's differential expression.

Although vertebrate genomes appear to contain only one titin gene, a large variety of quite distinct titin isoforms are expressed in striated muscle tissues. The isoforms appear to be generated by a series of complex, not yet fully characterized differential splicing mechanisms. Here, we provide an overview of the titin-specific antibodies that have been raised by our laboratory to study individual differentially expressed isoforms of titin. The staining patterns obtained in different tissues will contribute to the identification of both the particular titin isoforms that are expressed in the different tissues, as well as their intracellular distributions. In addition, antibodies to titin that are available are rapidly allowing for the refinement of our knowledge of titin's elastic spring properties. Knowledge of the nature and structure of vertebrate titins that may also be expressed in nonmuscle tissues may be broadened using these antibodies.

Calpain3 expression during human cardiogenesis.

Transcripts of calpain3, the gene involved in limb girdle muscular dystrophy type 2A, appear in organs other than the skeletal muscle during human development, the first of which being the early embryonic heart. We examined more precisely the spatio-temporal transcription pattern of calpain3 during human cardiogenesis and the appearance of its protein in fetal tissues, and correlated it to titin expression. Different events of the heart's maturation can be recognized: (i) the presence of titin RNA or protein constitute very precocious developmental cardiac markers appearing before the fusion of the two lateral endocardial tubes; (ii) the disappearance of calpain3 RNA from the ventricular compartment later in the embryonic heart. Finally, although calpain3 transcripts are present in the heart, the corresponding protein is not detected elsewhere than in skeletal muscle.

Series of exon-skipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity.

Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.

Human-mouse differences in the embryonic expression patterns of developmental control genes and disease genes.

Our understanding of early human development has been impeded by the general difficulty in obtaining suitable samples for study. As a result, and because of the extraordinarily high degree of evolutionary conservation of many developmentally important genes and developmental pathways, great reliance has been placed on extrapolation from animal models of development, principally the mouse. However, the strong evolutionary conservation of coding sequence for developmentally important genes does not necessarily mean that their expression patterns are as highly conserved. The very recent availability of human embryonic samples for gene expression studies has now permitted for the first time an assessment of the degree to which we can confidently extrapolate from studies of rodent gene expression patterns. We have found significant human-mouse differences in embryonic expression patterns for a variety of genes. We present detailed data for two illustrative examples. Wnt7a, a very highly conserved gene known to be important in early development, shows significant differences in spatial and temporal expression patterns in the developing brain (midbrain, telencephalon) of man and mice. CAPN3, the locus for LGMD2A limb girdle muscular dystrophy, and its mouse orthologue differ extensively in expression in embryonic heart, lens and smooth muscle. Our study also shows how molecular analyses, while providing explanations for the observed differences, can be important in providing insights into mammalian evolution.

A homeobox gene, vax2, controls the patterning of the eye dorsoventral axis.

We have identified a transcription factor specifically expressed in the developing vertebrate eye. We named this gene vax2 because of the high degree of sequence similarity to the recently described vax1. Both in the human and mouse genomes, vax2 is localized in the vicinity of the emx1 gene. This mapping assignment, together with the previously reported colocalization of Vax1 and Emx2 in mouse, indicates that the vax and the emx genes may be organized in clusters. vax2 has a remarkable expression domain confined to the ventral portion of the prospective neural retina in mouse, human, and Xenopus. The overexpression of either the frog Xvax2 or the human VAX2 in Xenopus embryos leads to an aberrant eye phenotype and, in particular, determines a ventralizing effect on the developing eye. The expression domain of the transcription factor Xpax2, normally confined to the ventral developing retina, extends to the dorsal region of the retina after overexpression of vax2. On the other hand, the expression of Xvent2, a molecular marker of the dorsal retina, is strongly reduced. Furthermore, vax2 overexpression induces a striking expansion of the optic stalk, a structure deriving from the ventralmost region of the eye vesicle. Altogether, these data indicate that vax2 plays a crucial role in eye development and, in particular, in the specification of the ventral optic vesicle.

Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events.

Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies.

Dysferlin is a plasma membrane protein and is expressed early in human development.

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

Expression of genes (CAPN3, SGCA, SGCB, and TTN) involved in progressive muscular dystrophies during early human development.

The developmental expression pattern of four human genes, three of which are involved in progressive muscular dystrophies, was investigated. The rationale for these experiments is that these patterns might provide useful information on the pathophysiology underlying these myopathies. Despite the presence of overlapping clinical signs, the spatiotemporal expression profiles of the corresponding genes differed widely. Transcripts of alpha-sarcoglycan (SGCA) were visible as soon as myotomes were formed, and constitute, together with titin transcripts, precocious muscular system landmarks. beta-sarcoglycan (SGCB) was initially transcribed in a ubiquitous manner, and, toward the second part of the embryonic period, became specific to striated muscle, heart, and the central nervous system. Whereas titin (TTN) transcription and translation seem to be coupled, for the sarcoglycans, translation seemed restricted to skeletal muscle. Calpain3 (CAPN3) RNA was found in only skeletal muscles during the fetal period. It was, however, present earlier in the whole heart, where it selectively disappeared. Finally, evidence for differentially spliced calpain3 variants in smooth muscles was also seen. The expression profiles of these genes is suggestive of their having a role during myogenesis, knowledge of which could be pertinent to the understanding of the pathophysiology of the associated diseases.

Cardiac myosin binding protein C gene is specifically expressed in heart during murine and human development.

Cardiac myosin binding protein C (MyBP-C) is a substantial component of the sarcomere, with both structural and regulatory roles. The gene encoding cardiac MyBP-C in humans is located on chromosome 11p11.2, and mutations that are most predicted to produce truncated proteins have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). To understand better the pathophysiology of FHC and with a view to the development of animal models for this disease, we have investigated by in situ hybridization the pattern of expression of the cardiac MyBP-C gene during human and mouse development using species-specific oligonucleotide probes. From 4 weeks of human development, a strong labeling of cardiac MyBP-C mRNAs was unambiguously detected in all heart compartments, and no signal could be visualized in somites. In murine embryos, from embryonic day 9.5 until birth, a strong signal was detected exclusively in the heart. Our results showed that during both human and murine development, in contrast to chicken development, the cardiac MyBP-C gene is abundantly and specifically expressed in the heart.

Dynamic molecular combing: stretching the whole human genome for high-resolution studies.

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

Identification of muscle-specific calpain and beta-sarcoglycan genes in progressive autosomal recessive muscular dystrophies.

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.