Why animals have different muscle fibre types

Animals have different muscle fibre types: slow fibres with a low maximum velocity of shortening (Vmax) and fast fibres with a high Vmax. An advantage conferred by the use of different fibre types during locomotion has been proposed solely on the basis of their in vitro properties. Isolated muscle experiments show that force generation, mechanical power production and efficiency are all functions of V/Vmax, where V is the velocity of muscle shortening. But it is not known whether animals actually use the different fibres at shortening velocities that are optimal for mechanical power production and efficiency. Here we compare the V of muscle fibres during locomotion with their Vmax. This comparison shows that during slow locomotion, the slow fibres shorten at a velocity that gives peak mechanical power and efficiency and the fast fibres shorten at their optimal velocity when powering maximal movements. Our results also show that maximal movements are impossible without fast fibres because the slow ones cannot shorten rapidly enough.     

Changes in spinal cord reflexes after cross-anastomosis of cutaneous and muscle nerves in the adult rat

Evidence exists that the specification of afferent nerves and their central connections in the embryo may depend in part on influences from the peripheral target innervated. We have now investigated whether such peripheral determination persists in the adult rat using the unmyelinated afferent system of C fibres, which differ chemically in the adult depending on their target. We have previously shown that if the cutaneous sural nerve and the muscle gastrocnemius nerve are cross-anastomosed so that they grow to each other's target, the A fibres establish functional endings and the C fibres change their chemistry to that which is appropriate for the new target. Here we report that in normal adult rats, a short train of stimuli to the cutaneous sural nerve produced a brief facilitation of the flexion reflex, lasting on average only 5 min, whereas similar stimulation of the gastrocnemius-muscle nerve enhanced this reflex for an average of 54 min. In cross-anastomosed animals, stimulation of the gastrocnemius nerve (innervating skin) induced a brief potentiation of the flexion reflex, lasting on average only 3 min. By contrast, stimulation of sural nerve (innervating muscle) produced a potentiation of this reflex lasting 57 min. Thus the ability of adult afferent nerves to potentiate the flexion reflex depends on the target with which they make contact. We propose that tissue-specific factors influence some of the central actions of primary afferent neurons in the adult.     

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.     

Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts

An important corollary to the recent advances in our understanding of the primary cause of Duchenne muscular dystrophy, is the validation of genuine genetic homologues as animal models of the disease in which potential therapies can be tested. The persistent skeletal muscle necrosis that characterizes human Duchenne muscular dystrophy is also seen in the mdx mouse and is, in both, a consequence of a deficiency of dystrophin, probably within the muscle fibres themselves. As injected muscle precursor cells of one genotype can fuse with host muscle fibres of a different genotype and express the donor genes, we decided to test grafts of normal muscle precursor cells to see if they could induce synthesis of dystrophin in innately dystrophin-deficient mdx muscle fibres. We show that injected normal muscle precursor cells can fuse with pre-existing or regenerating mdx muscle fibres to render many of these fibres dystrophin-positive and so to partially or wholly rescue them from their biochemical defect.     

Hsp72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.     

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.     

Migration of myoblasts across basal lamina during skeletal muscle development

Basal lamina is a sheet of extracellular matrix that separates cells into topologically distinct groups during morphogenesis and is thought to form a barrier to cell migration. We have examined whether, during normal muscle development, myoblasts--mononucleate muscle precursor cells--can cross the basal lamina that surrounds each multinucleate muscle fibre. We marked myoblasts in vivo by injecting replication-defective retroviral vectors encoding LacZ into muscle tissue and analysed the fate of their progeny by the expression of beta-galactosidase. A dual labelling method with broad application to retroviral lineage-marking studies was developed to ensure that most clusters of labelled cells were clones derived from a single precursor cell. Most of the myoblasts that were infected at a late stage of rat hindlimb development, when each fibre with its satellite myoblasts is individually encased in a basal lamina sheath, gave rise to clones that contributed to several labelled fibres. Our results show that myoblasts from healthy fibres migrate across basal lamina during normal development and could contribute to the repair of fibres damaged by injury or disease.     

Membrane currents that govern smooth muscle contraction in a ctenophore

Ctenophores are transparent marine organisms that swim by means of beating cilia; they are the simplest animals with individual muscle fibres. Predatory species, such as Beroe ovata, have particularly well-developed muscles and are capable of an elaborate feeding response. When Beroe contacts its prey, the mouth opens, the body shortens, the pharynx expands, the prey is engulfed and the lips then close tightly. How this sequence, which lasts 1 s, is accomplished is unclear. The muscles concerned are structurally uniform and are innervated at each end by a neuronal nerve net with no centre for coordination. Isolated muscle cells studied under voltage-clamp provide a solution to this puzzle. We find that different groups of muscle cells have different time-dependent membrane currents. Because muscle contraction depends upon calcium entry during each action potential, these different currents produce different patterns of contraction. We conclude that in a simple animal such as a ctenophore, a sophisticated set of membrane conductances can compensate for the absence of an elaborate system of effectors.     

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.     

Nerve sheaths and motoneurone collateral sprouting

When disease or injury causes partial loss of innervation from a muscle, the remaining axons sprout and form new connections to the denervated muscle fibres. Sprouting can occur in two ways: from axon terminals (terminal sprouting) or from the intramuscular axons themselves, probably from the nodes of Ranvier (collateral sprouting). Terminal sprouting has been induced experimentally using various methods, including partial denervation, nerve conduction block and nerve transmission block. A common factor in the induction of terminal sprouting seems to be changes in the surface membrane of muscle fibres; these changes and terminal sprouting are prevented by direct stimulation of the muscle. Collateral sprouting has been induced only by partial denervation and is not prevented by direct stimulation. This has been taken as evidence for an earlier suggestion that products of nerve or axon degeneration may be a direct stimulus for collateral sprouting. We report here that axon degeneration products alone are probably not the stimulus for collateral sprouting.     

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.     

Expression of a mammalian Na-H exchanger in muscle fibres of the giant barnacle

It is now well established that the internal pH (pHi) of mammalian cells is regulated by means of a plasma membrane transport system that exchanges extracellular Na+ for intracellular H+ (ref. 1). Furthermore, modulation of the activity of the Na-H exchanger seems to have a crucial role in the action of various mitogens and growth factors. The possibility that such a mammalian Na-H exchanger might be efficiently expressed in a giant invertebrate cell was suggested to us by recent results of Barnard and Miledi and colleagues, who demonstrated in frog oocytes the expression of various plasma membrane channels that presumably were encoded by the mammalian messenger RNA wih which the oocytes had been injected. We used muscle fibres of the giant barnacle, which normally have no demonstrable Na-H exchanger activity, and report here that, when injected with poly(A)+ RNA isolated from rabbit liver, the muscle fibres express a Na-H exchanger. No such expression is observed, however, when the injected material is pretreated with ribonuclease A. As hepatocytes are known to possess a Na-H exchanger, the most straightforward interpretation of our data is that a mammalian Na-H exchanger has been expressed in the muscle fibre of an invertebrate.     

Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse

Human X-linked Duchenne and Becker muscular dystrophies are due to defects in dystrophin, the product of an exceptionally large gene. Although dystrophin has been characterized as a spectrin-like submembranous cytoskeletal protein, there is no experimental evidence for its function in the structural maintenance of muscle. Current hypotheses attribute necrosis of dystrophin-less fibres in situ to mechanical weakening of the outer membrane, to an excessive influx of Ca2+ ions, or to a combination of these two mechanism, possibly mediated by stretch-sensitive ion channels. Using hypo-osmotic shock to determine stress resistance and a mouse model (mdx) for the human disease, we show that functional dystrophin contributes to the stability of both cultured myotubes and isolated mature muscle fibres.     

Functional improvement of dystrophic muscle by myostatin blockade

Mice and cattle with mutations in the myostatin (GDF8) gene show a marked increase in body weight and muscle mass, indicating that this new member of the TGF-beta superfamily is a negative regulator of skeletal muscle growth. Inhibition of the myostatin gene product is predicted to increase muscle mass and improve the disease phenotype in a variety of primary and secondary myopathies. We tested the ability of inhibition of myostatin in vivo to ameliorate the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). Blockade of endogenous myostatin by using intraperitoneal injections of blocking antibodies for three months resulted in an increase in body weight, muscle mass, muscle size and absolute muscle strength in mdx mouse muscle along with a significant decrease in muscle degeneration and concentrations of serum creatine kinase. The functional improvement of dystrophic muscle by myostatin blockade provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD, and circumvents the major problems associated with conventional gene therapy in these disorders.     

Fibre type composition of single motor units during synapse elimination in neonatal rat soleus muscle

Skeletal motor neurones innervate the specialized 'types' of fibres comprising most mammalian muscles in a characteristic fashion: each motor neurone forms a 'motor unit' by innervating a set of fibres all of the same type. Because the type expression of adult muscle fibres is plastic and apparently controlled by their innervation, each motor neurone is thought to impose a common type differentiation on all the fibres in its motor unit. However, the situation in developing muscles cannot be this simple. Muscle fibres in neonates receive synaptic input from several motor neurones and achieve the adult, single innervation only after a period of 'synapse elimination. Despite this polyneuronal innervation, differentiated fibre types are present in neonatal muscles. This means either that the motor neurones polyneuronally innervate fibres in a random fashion and type expression is not determined by innervation or that the polyneuronal innervation is ordered in such a way that each fibre could receive unambiguous instructions for type differentiation. We have investigated these possibilities here by determining the fibre type composition of motor units in neonatal rat soleus muscle. We find that even during the time of polyneuronal innervation each motor neurone confines its innervation to largely one of two fibre types present in the muscle. Therefore, some mechanism during early development segregates the synapses of two groups of soleus motor neurones onto two separate populations of soleus muscle fibres.     

Slow inward calcium currents have no obvious role in muscle excitation-contraction coupling

It has been proposed that an influx of calcium ions into twitch muscle fibres during an action potential might initiate contraction. However, when external Ca2+ is lowered to 10(-8) M with EGTA, the fibres can produce normal twitches for many minutes. Nevertheless, a clear Ca2+ influx during contraction has been demonstrated, and it has been found that phasic skeletal muscle has an inward calcium current (ICa) which can give rise to calcium spikes. In certain conditions, a reduction in external Ca2+ with 80-90 mM EGTA results in reversible blockade of excitation-contraction (e-c) coupling, leading some authors to suggest that extracellular Ca2+ moved into the myoplasm due to ICa may be involved in the e-c coupling mechanism that triggers contraction. This proposition was further supported by the localization of ICa in the T-system, which circumvented the problem of the delay due to calcium diffusion from the surface membrane. We have now investigated whether ICa has a clear role in initiating or sustaining contractions in twitch muscle fibres. Our approach was to decrease or eliminate ICa with the calcium-blocking agent diltiazem (Herbesser) and to see how the twitch, tetanic and potassium-contracture tensions were affected. We found that ICa could be decreased or cancelled with the calcium-blocking agent, but that the same concentration of the drug potentiated the twitch, tetanus and contractures. We conclude, therefore, that ICa has no role in e-c coupling. A preliminary report of these results has been presented elsewhere.     

A Cl- conductance activated by hyperpolarization in Aplysia neurones

Although many voltage-gated cation channels have been described and extensively studied in biological membranes, there are very few examples of voltage-gated anion channels. Chloride conductances activated by depolarization have been observed in skate electroplaque and in frog and chick skeletal muscle. A Cl- conductance activated by hyperpolarization has been suggested both for frog muscle treated with acid (pH 5) solutions, and for crayfish muscle where it could account for the fact that the pronounced inward-going rectification of the I-V curve disappears if the fibres have been soaked in a Cl(-)-free solution. More recently, voltage-dependent anion channels extracted from biological membranes have been incorporated into artificial membranes. I now report that in Aplysia neurones, and in particular those in which the internal Cl- concentration has been increased, a Cl- conductance can be observed which is slowly activated by hyperpolarization and shows a vary steep voltage dependence. This time- and voltage-dependent Cl- conductance probably exists also in many other cells. Its presence might explain why it is difficult when using KCl-filled microelectrodes to maintain prolonged hyperpolarizations. This Cl- conductance constitutes a new type of inward-going rectification distinct both from the classical "anomalous rectification' which involves selective K+ channels and from the current termed if in heart muscle that is presently attributed to a cationic conductance.     

Alteration in crossbridge kinetics caused by mutations in actin

The generation of force during muscle contraction results from the interaction of myosin and actin. The kinetics of this force generation vary between different muscle types and within the same muscle type in different species. Most attention has focused on the role of myosin isoforms in determining these differences. The role of actin isoforms has received little attention, largely because of the lack of a suitable cell type in which the myosin isoform remains constant yet the actin isoforms vary. An alternative approach would be to examine the effect of actin mutations, however, most of these cause such gross disruption of muscle structure that mechanical measurements are impossible. We have now identified two actin mutations which, despite involving conserved amino acids, can assemble into virtually normal myofibrils. These amino-acid changes in actin significantly affect the kinetics of force generation by muscle fibres. One of the mutations is not in the putative myosin-binding site, demonstrating the importance of long-range effects of amino acids on actin function.