Diagnosis and etiology of congenital muscular dystrophy.

OBJECTIVE: We aimed to determine the frequency of all known forms of congenital muscular dystrophy (CMD) in a large Australasian cohort. METHODS: We screened 101 patients with CMD with a combination of immunofluorescence, Western blotting, and DNA sequencing to identify disease-associated abnormalities in glycosylated alpha-dystroglycan, collagen VI, laminin alpha2, alpha7-integrin, and selenoprotein. RESULTS: A total of 45% of the CMD cohort were assigned to an immunofluorescent subgroup based on their abnormal staining pattern. Abnormal staining for glycosylated alpha-dystroglycan was present in 25% of patients, and approximately half of these had reduced glycosylated alpha-dystroglycan by Western blot. Sequencing of the FKRP, fukutin, POMGnT1, and POMT1 genes in all patients with abnormal alpha-dystroglycan immunofluorescence identified mutations in one patient for each of these genes and two patients had mutations in POMT2. Twelve percent of patients had abnormalities in collagen VI immunofluorescence, and we identified disease-causing COL6 mutations in eight of nine patients in whom the genes were sequenced. Laminin alpha2 deficiency accounted for only 8% of CMD. alpha7-Integrin staining was absent in 12 of 45 patients studied, and ITGA7 gene mutations were excluded in all of these patients. CONCLUSIONS: We define the distribution of different forms of congenital muscular dystrophy in a large cohort of mixed ethnicity and demonstrate the utility and limitations of current diagnostic techniques.

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice.

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.

Laminin and alpha7beta1 integrin regulate agrin-induced clustering of acetylcholine receptors.

The clustering of acetylcholine receptors (AChRs) in the post-synaptic membrane of skeletal muscle is an early developmental event in the formation of the neuromuscular junction. Several studies show that laminin, as well as neural agrin, can induce AChR clustering in C2C12 myofibers. We recently showed that specific isoforms of the alpha7beta1 integrin (a receptor normally found at neuromuscular junctions) colocalize and physically interact with AChR clusters in a laminin-dependent fashion. In contrast, induction with agrin alone fails to promote localization of the integrin with AChR clusters. Together both agrin and laminin enhance the interaction of the integrin with AChRs and their aggregation into clusters. To further understand this mechanism we investigated cluster formation and the association of the alpha7beta1 integrin and AChR over time following induction with laminin and/or agrin. Our results show that the alpha7beta1 integrin associates with AChRs early during the formation of the post-synaptic membrane and that laminin modulates this recruitment. Laminin induces a rapid stable association of the integrin and AChRs and this association is independent of clustering. In addition to laminin-1, merosin (laminin-2/4) is present both before and after formation of neuromuscular junctions and also promotes AChR clustering and colocalization with the integrin as well as synergism with agrin. Using site directed mutagenesis we demonstrate that a tyrosine residue in the cytoplasmic domain of both (&agr;)7A and (&agr;)7B chains regulates the localization of the integrin with AChR clusters. We also provide evidence that laminin, through its association with the alpha7beta1 integrin, reduces by 20-fold the concentration of agrin required to promote AChR clustering and accelerates the formation of clusters. Thus laminin, agrin and the alpha7beta1 integrin act in a concerted manner early in the development of the post-synaptic membrane, with laminin priming newly formed myofibers to rapidly and vigorously respond to low concentrations of neural agrin produced by innervating motor neurons.

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling.

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.

The alpha7beta1 integrin in muscle development and disease.

The alpha7beta1 integrin is a laminin receptor on the surface of skeletal myoblasts and myofibers. Alternative forms of both the alpha7 and beta1 chains are expressed in a developmentally regulated fashion during myogenesis. These different alpha7beta1 isoforms localize at specific sites on myofibers and appear to have distinct functions in skeletal muscle. These functions include the migration and proliferation of developing myoblasts, the formation and integrity of neuromuscular and myotendinous junctions, and the "gluing" together of muscle fibers that is essential to the generation of contractile force. The alpha7beta1 integrin appears to be both directly and indirectly causally related to several muscle diseases. Enhanced expression of alpha7beta1-mediated linkage of the extracellular matrix is seen in Duchenne muscular dystrophy and may compensate for the absence of the dystrophin-mediated linkage. Downregulation of expression of the integrin may contribute to the development of pathology in congenital laminin deficiencies. Mutations in the alpha7 integrin gene underlie additional congenital muscle diseases. The functional roles of this integrin in the formation and stability of the neuromuscular and myotendinous junctions and its localization between fibers suggest that altered expression or function of this integrin may have widespread involvement in other myopathies. The localization of the alpha7 gene at human chromosome 12q13 is a useful clue for focusing such studies.

A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering.

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

Mutations in the integrin alpha7 gene cause congenital myopathy.

The basal lamina of muscle fibers plays a crucial role in the development and function of skeletal muscle. An important laminin receptor in muscle is integrin alpha7beta1D. Integrin beta1 is expressed throughout the body, while integrin alpha7 is more muscle-specific. To address the role of integrin alpha7 in human muscle disease, we determined alpha7 protein expression in muscle biopsies from 117 patients with unclassified congenital myopathy and congenital muscular dystrophy by immunocytochemistry. We found three unrelated patients with integrin alpha7 deficiency and normal laminin alpha2 chain expression. To determine if any of these three patients had mutations of the integrin alpha7 gene, ITGA7, we cloned and sequenced the full-length human ITGA7 cDNA, and screened the patients for mutations. One patient had splice mutations on both alleles; one causing a 21-bp insertion in the conserved cysteine-rich region, and the other causing a 98-bp deletion. A second patient was a compound heterozygote for the same 98-bp deletion, and had a 1-bp frame-shift deletion on the other allele. A third showed marked deficiency of ITGA7 mRNA. Clinically, these patients showed congenital myopathy with delayed motor milestones. Our results demonstrate that mutations in ITGA7 are involved in a form of congenital myopathy.

The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin.

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.

Altered expression of the alpha7beta1 integrin in human and murine muscular dystrophies.

The alpha7beta1 integrin is the primary laminin receptor on skeletal myoblasts and adult myofibers. It has distinct functions during muscle development and contributes to muscle structural integrity. We have studied this integrin in cases where expression of dystrophin or laminin are compromised. Immunofluorescence demonstrates an increase in alpha7beta1 in patients with Duchenne muscular dystrophy and in mdx mice that lack dystrophin. Analysis of RNA from mdx mice and from patients with Duchenne and Becker muscular dystrophies indicates that the increase in the alpha7beta1 integrin is regulated at the level of alpha7 gene transcription. In contrast, the levels of alpha7beta1 integrin are severely diminished in patients with laminin alpha2 chain congenital dystrophy muscular dystrophy and in dy/dy mice that also do not make the alpha2 laminin chain. Analysis of RNA from the hindlimbs of dy/dy mice demonstrated that in the absence of laminin alpha7 gene transcription is inhibited and limited to specific alternatively spliced isoforms. We suggest that the increased expression of alpha7beta1 integrin in the absence of dystrophin compensates for the reduced dystrophin-mediated linkage of fibers with the basal lamina and modulates the development of pathology associated with these diseases. The decrease in alpha7beta1 integrin and its transcripts in the absence of laminin likely contributes to the severe myopathy that results from laminin alpha2 chain deficiency and suggests that laminin-2 regulates expression of the alpha7 integrin gene. The role of the alpha7beta1 integrin in muscle integrity also suggests that compromised expression of this receptor may underlie as yet undefined myopathies.

Synaptic integrins in developing, adult, and mutant muscle: selective association of alpha1, alpha7A, and alpha7B integrins with the neuromuscular junction.

Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As beta1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin alpha subunits known to form dimers with beta1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin alpha1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while alpha7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus alpha1 and alpha7 integrins are present in synaptic membranes. Expression of the alpha7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the alpha7 gene (A, B, and C), all of which were expressed in muscle. The alpha7A and alpha7B isoforms were confined to synaptic sites in adult muscle, while alpha7C was present both synaptically and extrasynaptically. In developing muscle, alpha7A appeared postnatally and specifically at the synapse; alpha7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and alpha7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the alpha7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the alpha7 integrins. In normal mice, laminin beta2 is concentrated in synaptic basal lamina. In beta2-null mutant mice, alpha7A was still present at synaptic sites, but alpha7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution.

Localization of the alpha 7 integrin gene (ITGA7) on human chromosome 12q13: clustering of integrin and Hox genes implies parallel evolution of these gene families.

Expression of the alpha 7 integrin gene (ITGA7) is developmentally regulated during the formation of skeletal muscle. Increased levels of expression and production of isoforms containing different cytoplasmic and extracellular domains accompany myogenesis. To determine whether a single or multiple alpha 7 genes underlie the structural diversity in this alpha chain that accompanies development, we have examined the rat and human genomes by Southern blotting and in situ hybridization. Our results demonstrate that there is only one alpha 7 gene in both the rat and the human genomes. In the human, ITGA7 is present on chromosome 12q13. Phylogenetic analysis of the integrin alpha chain sequences suggests that the early integrin genes evolved in two pathways to form the I-integrins and the non-I-integrins. The I-integrin alpha chains contain an additional sequence of approximately 180 amino acids and arose as a result of an early insertion into the non-I-gene. The I-chain subfamily further evolved by duplications within the same chromosome. The non-I-integrin alpha chain genes are localized in clusters on chromosomes 2, 12, and 17, and this closely coincides with the localization of the human homeobox gene clusters. Non-I-integrin alpha chain genes appear to have evolved in parallel and in proximity to the Hox clusters. Thus, the Hox genes that underlie the design of body structure and the Integrin genes that underlie informed cell-cell and cell-matrix interactions appear to have evolved in parallel and coordinate fashions.

SV40 T antigen inhibits expression of MyoD and myogenin, up-regulates Myf-5, but does not affect early expression of desmin or alpha 7 integrin during muscle development.

The terminal stage of myogenic development is marked by the cessation of replication, fusion, and expression of genes which encode the myofibrillar proteins. Prior to terminal differentiation one or more stages of myogenic development take place. Expression of alpha 7 integrin and desmin have been used as markers for these earlier stages of myogenesis. Both proteins are expressed in replicating secondary myoblasts prior to terminal differentiation and when these cells differentiate further, the expression of the alpha 7 integrin and desmin genes is up-regulated. To determine whether the stages of myogenesis which precede terminal development and the factors which regulate them are distinct, the expression of alpha 7 integrin and desmin was assayed in a variety of myogenic cell lines in which terminal differentiation was inhibited. L8E63 and C2 myoblasts in which terminal differentiation was inhibited by SV40 large T antigen, adenovirus E1A protein, or ras and an L6 mutant whose terminal differentiation is sensitive to alpha-amanitin were studied. In all cases, when terminal myogenic differentiation is inhibited the basal levels of desmin and alpha 7 expression are not altered. Under these same conditions expression of the myogenic regulatory genes myogenin and MyoD also were inhibited whereas Myf-5 persisted. These results indicate that expression of the early myogenic phenotype and terminal differentiation are discrete and independent stages of myogenesis and that different transcription factors likely regulate the expression of each stage. In contrast with myoblasts in cultures of newborn rat hindlimb cells and the C2 cell line, myogenic cells derived from C3H10T1/2 cells by treatment with 5-azacytidine or by transfection with MyoD, Myf-5, or MRF4 do not express desmin as replicating myoblasts but do so upon terminal differentiation. This indicates that in vitro, terminal differentiation can proceed in the absence of the phenotypes that normally develop earlier and that the conversion of 10T1/2 cells to myogenic cells can bypass developmental stages which normally occur in vivo. These results are discussed in the context of a model of the myogenic lineage that is based on the expression of desmin.

Selective modulation of the interaction of alpha 7 beta 1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation.

The alpha 7 beta 1 integrin was originally identified and isolated from differentiating skeletal muscle and shown to be a laminin-binding protein (Song et al. (1992) J. Cell Biol. 117, 643-657). Expression of the alpha 7 gene and protein are developmentally regulated during skeletal muscle differentiation and have been used to identify cells at distinct stages of the myogenic lineage (George-Weinstein et al. (1993) Dev. Biol. 156, 209-229). The lactoside-binding protein L-14 exists as a dimer and has been localized on a variety of cells, in association with extracellular matrix. During myogenesis in vitro, L-14 is synthesized within replicating myoblasts but it is not secreted until these cells commence terminal differentiation and fusion into multinucleate fibers (Cooper and Barondes, J. Cell Biol. (1990) 110, 1681-1691). Addition of purified L-14 to myogenic cells plated on laminin inhibits myoblast spreading and fusion, suggesting that the L-14 lectin regulates muscle cell interactions with the extracellular matrix that are germane to myogenic development (Cooper et al. (1991) J. Cell Biol. 115, 1437-1448). We demonstrate here, using affinity chromatography and immunoblots, that alpha 7 beta 1 also binds to fibronectin and to the L-14 lectin. L-14 binds to both laminin and to the alpha 7 beta 1 integrin, and it can effectively inhibit the association of laminin and this integrin. Modulation of alpha 7 beta 1 interaction with its ligands by L-14 is selective: L-14 does not bind to fibronectin, nor does it interfere with the binding of fibronectin to alpha 7 beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of alpha 7 integrin cytoplasmic domains during skeletal muscle development: alternate forms, conformational change, and homologies with serine/threonine kinases and tyrosine phosphatases.

We recently reported the cloning and sequencing of the alpha 7 integrin chain and its regulated expression during the development of skeletal muscle (Song et al. (1992) J. Cell Biol. 117, 643-657). The alpha 7 chain is expressed during the development of the myogenic lineage and on adult muscle fibers and this suggests that it participates in multiple and diverse functions at different times during muscle development. One interesting portion of this isoform is its cytoplasmic domain; comprised of 77 amino acids it is the largest in the alpha chains thus reported. In these experiments we begin to study the potential functions of the alpha 7 cytoplasmic domain by analyzing homologies between the rat and human sequences, by immunologic studies using an anti-cytoplasmic domain antiserum, and by identifying two alternate forms. In keeping with the nomenclature used to describe the alpha 3 and alpha 6 alternate cytoplasmic domains, we refer to the originally reported species as alpha 7B and the two additional forms as alpha 7A and alpha 7C. These three cytoplasmic domains likely arise as a consequence of alternate splicing. A splice site at the junctions of the transmembrane and cytoplasmic domains is used to generate the alpha 3, alpha 6 and alpha 7 A and B forms. The alpha 7A form RNA contains an additional 113 nucleotides compared to the B form, and a common coding region in the A and B form RNAs is used in alternate reading frames. Part of the coding region of alpha 7B appears to be used as the 3'-untranslated region of the alpha 7A form. The alpha 7C mRNA is 595 nucleotides smaller than the alpha 7B RNA and part of the 3'-untranslated region of the alpha 7B isoform is used as coding sequence in alpha 7C. There is developmental specificity in expression of these alternate mRNAs: alpha 7A and alpha 7C transcripts are found upon terminal myogenic differentiation whereas alpha 7B is present earlier in replicating cells and diminishes upon differentiation. We suggest this selective expression of the alpha 7 cytoplasmic domains underlies the diversity in function of the alpha 7 beta 1 integrin at different stages of muscle development. Immunochemical analyses indicate that the alpha 7B cytoplasmic domain undergoes a change in conformation in response to binding laminin or upon crosslinking initiated with antibody reactive with the integrin extracellular domain. Crosslinking also promotes association of the integrin with the cell cytoskeleton.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro and in vivo expression of alpha 7 integrin and desmin define the primary and secondary myogenic lineages.

Skeletal muscle fibers form during two periods of development and differ biochemically, functionally and in their morphology. Primary fibers develop in the rat hindlimb during Days 14 to 16 of embryogenesis. These fibers are subsequently surrounded by secondary fibers that eventually constitute the bulk of muscle mass in the limbs. We have used the expression of the alpha 7 muscle laminin binding integrin (Song et al., J. Cell Biol. 117, 643-657, 1992) and the intermediate filament protein desmin to identify myogenic cells at distinct stages of development both in vitro and in vivo. The phenotypes of these cells, determined by immunofluorescence microscopy, discriminate two lineages and indicate that the development of primary and secondary muscle fibers is regulated by multiple mechanisms. The cells which compose the primary myogenic lineage are derived from a population of precursor cells that is in part present in the Day 12 embryo limb bud and which do not express either alpha 7 integrin or desmin. These precursor cells develop into cells that express desmin, but not alpha 7, and which subsequently mature into replicating myoblasts that are competent to undergo terminal differentiation. This maturation process requires the in vivo environment of the Day 13 embryo limb. The alpha 7 integrin and slow myosin heavy chain are first expressed in primary muscle cells well after the onset of terminal differentiation. Some cells that give rise to secondary muscle fibers also are present in the Day 12 embryo hindlimb. The precursors of secondary fibers will develop into cells which express either alpha 7 integrin or desmin and subsequently into replicating myoblasts that express both proteins. Upon terminal differentiation of secondary myoblasts there is an increase in the expression of both alpha 7 integrin and desmin. The temporal regulation of expression of these proteins indicates that the environment of the limb plays a role in the maturation of precursors of both lineages. At least two roles of alpha 7 integrin during myogenesis are related to its association with beta 1 integrin and its function as a laminin receptor. Laminin selectively maintains the proliferation of secondary myoblasts and modulates their shape and mobility in vitro. This responsiveness of secondary myoblasts to laminin corresponds to the time when laminin is a major component of the extracellular matrix, when there is an expansion of the population of secondary myoblasts, and when the alpha 7 integrin is expressed on secondary myoblasts in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

H36-alpha 7 is a novel integrin alpha chain that is developmentally regulated during skeletal myogenesis.

H36 is a 120,000-D membrane glycoprotein that is expressed during the differentiation of skeletal muscle. H36 cDNA clones were isolated from a lambda UniZapXR rat myotube cDNA library and sequenced. The deduced amino acid sequence demonstrates that H36 is a novel integrin alpha chain that shares extensive homology with other alpha integrins that includes: (a) the GFFKR sequence found in all alpha integrins; (b) a single membrane spanning region; (c) conservation of 18 of 22 cysteines; and (d) a protease cleavage site found in the non-I region integrin alpha chains. The cytoplasmic domain of H36 is unique and additional regions of nonhomology further indicate H36 is distinct from all other alpha chains. In keeping with current nomenclature we designate this alpha chain alpha 7. Northern blots demonstrate that expression of H36-alpha 7 mRNA is regulated both early in the development of the myogenic lineage and later, during terminal differentiation. Detection of H36-alpha 7 mRNA coincides with conversion of H36- myogenic precursor cells to H36+ cells. H36-alpha 7 mRNA is present in replicating myoblasts: expression increases upon terminal differentiation and is markedly reduced in developmentally defective myoblasts. In addition, H36-alpha 7 mRNA is not detected in C3H10T1/2 cells. It is in myotubes derived from myoblasts obtained by treatment of 10T1/2 cells with azacytidine or transfection with MRF4. Immunoblots and immunofluorescence demonstrate that the H36-alpha 7 chain is associated with integrin beta 1. Affinity chromatography demonstrates that H36-alpha 7 beta 1 selectively binds to laminin. The expression of H36-alpha 7 on secondary myoblasts during the development of the limb in vivo corresponds with the appearance of laminin in the limb, with the responsiveness of secondary myoblast proliferation to laminin, and with the onset of increased muscle mass, suggesting that H36-alpha 7 modulates this stage in limb development. We conclude that H36-alpha 7 is a novel alpha integrin laminin binding protein whose expression is developmentally regulated during skeletal myogenesis.

In vitro development of precursor cells in the myogenic lineage.

Expression of the muscle-specific integral membrane protein H36 and the intermediate filament protein desmin, detected by immunofluorescence, was used to identify cells at distinct stages in the skeletal myogenic lineage. These proteins were coordinately expressed in cultures of rat hindlimb myoblasts from 17- and 19-day fetuses and newborn pups, and in satellite cells from juveniles. Both H36+ and desmin+ cells were present in cultures from 13.5- and 15-day embryonic hindlimbs, but desmin expression was more prevalent: H36-/desmin+ myoblasts predominate during this early stage of development. H36 was not detected in Day 12 embryo hindlimb bud cells in vivo nor in cultures soon after plating. Initially, only 1% of the Day 12 limb bud cells expressed desmin. When these cells were serially passaged every 3-4 days, cells with all three possible myogenic phenotypes developed: that is, H36+/desmin-, H36+/desmin+, and H36-/desmin+ cells. There was a progressive increase in the frequency of H36+ cells, with 75% of cells positive by passage 6 (Day 27 in vitro). The maximum frequency of cells that expressed desmin occurred in passage 5 (Day 23 in vitro). These results demonstrate that precursors to the cells that express H36 and desmin are present in the 12-day embryo hindlimb bud and that the transition from H36-/desmin- precursors to cells with a myogenic phenotype can occur in vitro. MyoD1 and myogenin were not detected in these cells, suggesting that the initial expression of H36 and desmin in the myogenic lineage may precede and/or is independent of these regulatory proteins. The conversion of precursor cells in the 12-day limb bud to a more advanced stage of development serves to define additional cells in the myogenic lineage. The ability to monitor in vitro these stages of development affords the opportunity to study how they are regulated.

Localization of anti-clathrin antibody in the sarcomere and sensitivity of myofibril structure to chloroquine suggest a role for clathrin in myofibril assembly.

Immunofluorescence microscopy has been used to demonstrate that X22, a monoclonal antibody specific for clathrin heavy chain, localizes in repetitive bands that appear soon after the fusion of skeletal myoblasts into multinucleate fibers. This organization has been found in cultures containing myotubes that develop in vitro from explants of newborn rat hindlimb cells and in myotubes derived from the L8E63 myogenic line. Bands were also prominent in skinned fibers prepared from adult rat soleus muscle and in cardiac myocytes grown in vitro from 4-day heart ventricles. Immunofluorescence banding was localized in the sarcomere as a doublet, with one element on either side of the Z line. Evidence that supports the conclusion that the reaction with X22 antibody is specific and indicative of the localization of clathrin in the sarcomere includes: (1) Identical titration of X22 antibody reactivity with the determinant in coated vesicles and in the sarcomere. (2) Conditions (eg., pH and Tris) that disrupt clathrin baskets or prevent its assembly likewise disrupt the localization of X22 in bands. (3) Chloroquine inhibits both the normal trafficking of clathrin in the cell and X22 banding in the sarcomere. (4) Immunoblot analysis of myotube lysates reveals a single band with an electrophoretic mobility identical to the 180,000-Da clathrin heavy chain. (5) The assembly of clathrin into sarcomeric bands occurs early in the development of the myofibrillar apparatus. Quantitation of the appearance of X22 banding in primary cultures of myotubes indicates that it precedes that of other myofibrillar proteins and that assembly takes place in the following order: X22, titin, myosin heavy chain, actin, and desmin. The assembly of myosin, titin, and actin into sarcomeric bands, as well as X22, is inhibited by chloroquine. Upon prolonged exposure to chloroquine previously assembled proteins are drastically reduced or no longer evident in the sarcomere. On the basis of these results and considering the role of clathrin in intracellular transport and its capacity to interact with actin and alpha-actinin, we suggest that clathrin may have diverse roles in the assembly, integrity, and functioning of the sarcomere and its integration with the sarcolemma. The early organization of X22 into bands further suggests that clathrin may also function early in the assembly of the contractile system.

Membrane-cytoskeleton associations during myogenesis deviate from traditional definitions.

Plasma membrane-cytoskeleton associations involving four membrane proteins (A5, H58, H36, and I20) were studied in developing L8E63 rat skeletal muscle cells using immunofluorescence microscopy and photometry on the basis of three criteria: Triton-insolubility, colocalization with cytoskeletal components, and sensitivity to cytoskeleton-directed drugs. The results presented demonstrate that there are developmental stage-specific associations between membrane proteins and the cytoskeleton during skeletal myogenesis. Several inconsistencies were found with traditional expectations of membrane-cytoskeleton associations. For example, although A5 is Triton-insoluble and sensitive to cytochalasin, its distribution generally does not correspond with any known cytoskeletal structure. Furthermore, the topography of A5 is dependent on the integrity of the plasma membrane. H36 and I20 are completely soluble in Triton and therefore by accepted definitions would not be expected to be associated with any cytoskeletal component. Yet H36 and actin codisrupt in the presence of cytochalasin, while I20, whose distribution does not correspond with microtubules, is uniquely sensitive to their disruption. These results demonstrate that (i) neither Triton-solubility nor colocalization alone predicts all membrane-cytoskeleton associations; some associations between the membrane and cytoskeleton are unstable in nonionic detergent; (ii) the native distribution of proteins in the membrane may not reflect their cytoskeletal associations; and (iii) the topography of some membrane proteins with no apparent association with the cytoskeleton may be greatly influenced by the cell cytoskeleton.