Restoration of normal function in genetically defective myotubes by spontaneous fusion with fibroblasts

Muscular dysgenesis in mice is a genetic disease of skeletal muscle caused by the recessive mutation mdg. Muscle fibres in affected mice are paralysed because of the failure of excitation-contraction coupling. Unlike normal myotubes in primary culture, dysgenic myotubes do not contract, either spontaneously or in response to electrical stimulation. The deficiency results from mutation of the gene for the skeletal muscle dihydropyridine receptor, an essential sarcolemmal component both of excitation-contraction coupling and of the slow calcium-ion channel. It has recently been shown that the addition of fibroblasts from normal (but not dysgenic) mice to cultures of dysgenic myotubes can restore spontaneous contractions in a small fraction of these myotubes, but the mechanism for this 'rescue' was not determined. In principle, if fibroblast nuclei were able to incorporate into myotubes, such nuclei could then supply the missing muscle-specific gene product. We have now investigated this possibility using nuclear, cytoplasmic and plasmalemmal markers. We report that the rescue to contractile ability in genetically paralysed dysgenic muscle is mediated by the previously unrecognized ability of fibroblasts to fuse spontaneously with developing myotubes.     

An SCN9A channelopathy causes congenital inability to experience pain

The complete inability to sense pain in an otherwise healthy individual is a very rare phenotype. In three consanguineous families from northern Pakistan, we mapped the condition as an autosomal-recessive trait to chromosome 2q24.3. This region contains the gene SCN9A, encoding the alpha-subunit of the voltage-gated sodium channel, Na(v)1.7, which is strongly expressed in nociceptive neurons. Sequence analysis of SCN9A in affected individuals revealed three distinct homozygous nonsense mutations (S459X, I767X and W897X). We show that these mutations cause loss of function of Na(v)1.7 by co-expression of wild-type or mutant human Na(v)1.7 with sodium channel beta(1) and beta(2) subunits in HEK293 cells. In cells expressing mutant Na(v)1.7, the currents were no greater than background. Our data suggest that SCN9A is an essential and non-redundant requirement for nociception in humans. These findings should stimulate the search for novel analgesics that selectively target this sodium channel subunit.     

Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia

Malignant hyperthermia (MH) is a potentially lethal condition in which sustained muscle contracture, with attendant hypercatabolic reactions and elevation in body temperature, are triggered by commonly used inhalational anaesthetics and skeletal muscle relaxants. In humans, the trait is usually inherited in an autosomal dominant fashion, but in halothane-sensitive pigs with a similar phenotype, inheritance of the disease is autosomal recessive or co-dominant. A simple and accurate non-invasive test for the gene is not available and predisposition to the disease is currently determined through a halothane- and/or caffeine-induced contracture test on a skeletal muscle biopsy. Because Ca2+ is the chief regulator of muscle contraction and metabolism, the primary defect in MH is believed to lie in Ca2+ regulation. Indeed, several studies indicate a defect in the Ca2+ release channel of the sarcoplasmic reticulum, making it a prime candidate for the altered gene product in predisposed individuals. We have recently cloned complementary DNA and genomic DNA encoding the human ryanodine receptor (the Ca2(+)-release channel of the sarcoplasmic reticulum) and mapped the ryanodine receptor gene (RYR) to region q13.1 of human chromosome 19 (ref. 14), in close proximity to genetic markers that have been shown to map near the MH susceptibility locus in humans and the halothane-sensitive gene in pigs. As a more definitive test of whether the RYR gene is a candidate gene for the human MH phenotype, we have carried out a linkage study with MH families to determine whether the MH phenotype segregates with chromosome 19q markers, including markers in the RYR gene. Co-segregation of MH with RYR markers, resulting in a lod score of 4.20 at a linkage distance of zero centimorgans, indicates that MH is likely to be caused by mutations in the RYR gene.     

Inactivation of muscle chloride channel by transposon insertion in myotonic mice.

MYOTONIA (stiffness and impaired relaxation of skeletal muscle) is a symptom of several diseases caused by repetitive firing of action potentials in muscle membranes. Purely myotonic human diseases are dominant myotonia congenita (Thomsen) and recessive generalized myotonia (Becker), whereas myotonic dystrophy is a systemic disease. Muscle hyperexcitability was attributed to defects in sodium channels and/or to a decrease in chloride conductance (in Becker's myotonia and in genetic animal models). Experimental blockage of Cl- conductance (normally 70-85% of resting conductance in muscle) in fact elicits myotonia. ADR mice are a realistic animal model for recessive autosomal myotonia. In addition to Cl- conductance, many other parameters are changed in muscles of homozygous animals. We have now cloned the major mammalian skeletal muscle chloride channel (ClC-1). Here we report that in ADR mice a transposon of the ETn family has inserted into the corresponding gene, destroying its coding potential for several membrane-spanning domains. Together with the lack of recombination between the Clc-1 gene and the adr locus, this strongly suggests a lack of functional chloride channels as the primary cause of mouse myotonia.     

Existence of distinct sodium channel messenger RNAs in rat brain

The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel) and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000 (260K), whereas those purified from rat brain and skeletal muscle contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37-45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.     

The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle

Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are allelic X-linked muscle disorders in man. The gene responsible for the disease has been cloned from knowledge of its map location at band Xp21 on the short arm of the X chromosome. The product of the DMD gene, a protein of relative molecular mass 400,000 (Mr 400K) recently named dystrophin, has been reported to co-purify with triads of mouse and rabbit skeletal muscle when assayed using polyclonal antibodies raised against fusion proteins encoded by regions of mouse DMD complementary DNA. Here we show that antibodies directed against synthetic peptides and fusion proteins derived from the N-terminal region of human DMD cDNA strongly react with an antigen present in skeletal muscle sarcolemma on cryostat sections of normal human muscle biopsies. This immunoreactivity is reduced or absent in muscle fibres from DMD patients but appears normal in muscle fibres from patients with other myopathic diseases. The same antibodies specifically react with a 400K protein in sodium dodecyl sulphate (SDS) extracts of normal human muscle subjected to Western blot analysis. We conclude that the product of the DMD gene is associated with the sarcolemma rather than with the triads and speculate that it strengthens the sarcolemma by anchoring elements of the internal cytoskeleton to the surface membrane.     

Coexpression of two distinct muscle acetylcholine receptor alpha-subunits during development

The nicotinic acetylcholine receptor is a ligand-gated channel that mediates signalling at the vertebrate neuromuscular junction. It is a pentameric complex of four different subunits, assembled with a stoichiometry of alpha 2 beta gamma delta. Muscle-like alpha-subunits have been cloned from Torpedo, mouse, calf, rat, chicken, human and Xenopus, and only a single alpha-subunit complementary DNA from each species has been detected. We report here the cloning and characterization of a second muscle alpha-subunit cDNA from Xenopus, and show that this and a previously reported Xenopus alpha-subunit cDNA are encoded by distinct genes. The novel alpha-subunit reported here is expressed uniquely in oocytes; but both types of alpha-subunit are coexpressed throughout muscle development. This latter observation indicates that the expression of these two alpha-subunits is different from a previously reported developmental 'subunit-switch' mechanism used to generate channel diversity.     

Identification of an altered splice site in Ashkenazi Tay-Sachs disease

Tay-Sachs disease is an autosomal recessive genetic disorder resulting from mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A (ref. 1). A relatively high frequency of carriers (1/27) of a lethal, infantile form of the disease is found in the Ashkenazi Jewish population, but it is not yet evident whether this has resulted from a founder effect and random genetic drift or from a selective advantage of heterozygotes. We have identified a single-base mutation in a cloned fragment of the HEXA gene from an Ashkenazi Jewish patient. This change, the substitution of a C for G in the first nucleotide of intron 12 is expected to result in defective splicing of the messenger RNA. A test for the mutant allele based on amplification of DNA by the 'polymerase chain rection and cleavage of a DdeI restriction site generated by the mutation revealed that this case and two other cases of the Ashkenazi, infantile form of Tay-Sachs disease are heterozygous for two different mutations. The occurrence of multiple mutant alleles warrants further examination of the selective advantage hypothesis.     

Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide

Duchenne muscular dystrophy (DMD) is a debilitating X-linked muscle disease. We have used sequence information from complementary DNA clones, derived from the gene that is deleted in DMD patients, to generate an antiserum that stains the surface membrane of intact human and mouse skeletal muscle, but not that of DMD patients and mdx mice. Here we identify the protein reacting with this antiserum as a single component of relative molecular mass 210,000 (Mr = 210K) that fractionates with a low-ionic strength extract of intact human and mouse skeletal muscle. It is therefore distinct from the 400 K protein found in the heavy microsomal fraction of normal muscle and identified as a putative product of the DMD gene. We also analyse further the disease specificity of the antiserum. Positive staining is seen in normal controls, and in samples from patients with a wide range of muscular dystrophies other than DMD. Becker muscular dystrophy, which is allelically related to DMD, was the only other exception, and gave a sporadic staining pattern. The demonstration of a specific defect in the surface membrane of DMD muscle fibres substantiates the hypothesis that membrane lesions may initiate muscle degradation in DMD.     

Primary structure of the receptor for calcium channel blockers from skeletal muscle

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.     

Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs

The skeletal muscle dihydropyridine (DHP) receptor is essential in excitation-contraction (EC) coupling. The receptor is postulated to be the voltage sensor giving rise to the intramembrane current, termed charge movement. We have now tested this hypothesis using myotubes from mice with the muscular dysgenesis mutation, which alters the skeletal muscle DHP receptor gene and prevents its expression. Our results indicate that charge movement is deficient in dysgenic myotubes but is fully restored following injection of an expression plasmid carrying the rabbit skeletal muscle DHP receptor complementary DNA, strongly supporting the hypothesis that the DHP receptor is the voltage sensor for EC coupling in skeletal muscle. Additionally, our data obtained for normal and chimaeric DHP receptor constructs demonstrate that DHP receptors with widely differing abilities to function as calcium channels and to mediate EC coupling produce very similar charge movements.     

A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells

Contraction of a vertebrate skeletal muscle fibre is triggered by electrical depolarization of sarcolemmal infoldings termed transverse-tubules (t-tubules), which in turn causes the release of calcium from an internal store, the sarcoplasmic reticulum (SR). The mechanism that links t-tubular depolarization to SR calcium release remains poorly understood. In principle, this link might be provided by the prominent slow calcium current that has been described in skeletal muscle cells of adult frogs and rats. However, blocking this current does not abolish the depolarization-induced contractile responses of frog muscle, and the function of this slow calcium current is unknown. Here we describe measurements of calcium currents in developing skeletal muscle cells of normal rats and mice, and of mice with muscular dysgenesis, a mutation that causes excitation-contraction (E-C) coupling to fail. We find that a slow calcium current is present in skeletal muscle cells of normal animals but absent from skeletal muscle cells of mutant animals. The effect of the mutation is specific to the slow calcium current of skeletal muscle; a fast calcium current is present in developing skeletal muscle cells of both normal and mutant animals, and slow calcium currents are present in cardiac and sensory neurones of mutant animals. We believe this to be the first report of a mutation affecting calcium currents in a multicellular organism. The effects of the mutation raise important questions about the relationship between the slow calcium current and skeletal muscle E-C coupling.     

Restoration of dysgenic muscle contraction and calcium channel function by co-culture with normal spinal cord neurons

Muscular dysgenesis (mdg) is a spontaneous recessive lethal mutation in the mouse. The disease is characterized by a total lack of excitation-contraction coupling in embryonic skeletal muscle. This developmental abnormality is associated with a drastic deficiency in the expression of voltage-sensitive Ca2+ channels in skeletal muscle without alteration of the properties of voltage-sensitive Na+ channels or of voltage-sensitive Ca2+ channels in cardiac and neuronal cells. Membrane couplings between sarcoplasmic reticulum and the transverse tubules, known as triads, were also found to be drastically altered in embryonic muscle of the homozygous mutant (mdg/mdg). Triads in the mdg/mdg muscle were less numerous, disorganized and lacked spaced densities. This paper shows that co-culture of mdg/mdg myotubes with normal spinal cord neurons re-establishes Ca2+ channel activity, contraction and normal triad organization. The decrease thus cannot be due to a mutation of the Ca2+ channel as previously suggested. Normal nerve cells may supply an essential factor to mutant muscle cells.     

Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel.

Skeletal muscle is unusual in that 70-85% of resting membrane conductance is carried by chloride ions. This conductance is essential for membrane-potential stability, as its block by 9-anthracene-carboxylic acid and other drugs causes myotonia. Fish electric organs are developmentally derived from skeletal muscle, suggesting that mammalian muscle may express a homologue of the Torpedo mamorata electroplax chloride channel. We have now cloned the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl- channel from Torpedo. It encodes a 994-amino-acid protein which is about 54% identical to the Torpedo channel and is predominantly expressed in skeletal muscle. Messenger RNA amounts in that tissue increase steeply in the first 3-4 weeks after birth, in parallel with the increase in muscle Cl- conductance. Expression from cRNA in Xenopus oocytes leads to 9-anthracene-carboxylic acid-sensitive currents with time and voltage dependence typical for macroscopic muscle Cl- conductance. This and the functional destruction of this channel in mouse myotonia suggests that we have cloned the major skeletal muscle chloride channel.     

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.     

Early-onset Alzheimer's disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene

A mutation at codon 717 of the beta-amyloid precursor protein gene has been found to cosegregate with familial Alzheimer's disease in a single family. This mutation has been reported in a further five out of approximately 100 families multiply affected by Alzheimer's disease. We have identified another family, F19, in which we have detected linkage between the beta-amyloid precursor protein gene and Alzheimer's disease. Direct sequencing of exon 17 in affected individuals from this family has revealed a base change producing a Val----Gly substitution, also at codon 717. The occurrence of a second allelic variant at codon 717 linked to the Alzheimer's phenotype supports the hypothesis that they are pathogenic mutations.     

Calcium entry through stretch-inactivated ion channels in mdx myotubes.

Recent advances in understanding the molecular basis of human X-linked muscular dystrophies have come from the identification of dystrophin, a cytoskeletal protein associated with the surface membrane. Although there is little or virtually no dystrophin in affected individuals, it is not known how this causes muscle degeneration. One possibility is that the membrane of dystrophic muscle is weakened and becomes leaky to Ca2+. In muscle from mdx mice, an animal model of the human disease, intracellular Ca2+ is elevated and associated with a high rate of protein degradation. The possibility that a lack of dystrophin alters the resting permeability of skeletal muscle to Ca2+ prompted us to compare Ca2(+)-permeable ionic channels in muscle cells from normal and mdx mice. We now show that recordings of single-channel activity from mdx myotubes are dominated by the presence of Ca2(+)-permeable mechano-transducing ion channels. Like similar channels in normal skeletal muscle, they are rarely open at rest, but open when the membrane is stretched by applying suction to the electrode. Other channels in mdx myotubes, however, are often open for extended periods of time at rest and close when suction is applied to the electrode. The results show a novel type of mechano-transducing ion channel in mdx myotubes that could provide a pathway for Ca2+ to leak into the cell.     

Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.