The ontogeny of soleus muscles in mdx and wild type mice.

The satellite cell, the organotypic muscle stem cell, is the key element in ontogenetic and load induced muscle fibre growth and repair. It is therefore possible that the satellite pool becomes exhausted with age, especially in mdx mice where dystrophin deficiency results in skeletal muscle degeneration. We compared structural criteria and satellite cell frequencies in soleus muscles of 26 mdx and 23 wild type mice aged between 26 and 720 days. The total number of muscle fibres was similar in both groups and remained stable throughout life, except for an early increase in wild type mice. However, in mdx muscles there was always a proportion of small-diameter fibres which resulted in a reduction in the effective myogenic area on cross-section, whereas total cross-sectional area and muscle weights were increased relative to controls throughout life. In adult animals, the frequency and numbers of satellite cells remained stable with age and were similar in both animal groups. Satellite cell numbers showed some considerable variation between individual animals, although with a markedly smaller variability between results of the same animal, pointing to the satellite cell pool being an individual variant.

[Regeneration capacity of skeletal muscle]. / Die Regenerationsfähigkeit der Skelettmuskulatur.

The organotypic stem cell of skeletal muscle has previously been known as satellite cell. They allow muscle fiber growth during ontogenesis, enable fiber hypertrophy and are responsible for the very efficient repair of muscle fibers. This efficient apparatus is to some degree counterbalanced by an enormous use of the satellite cell pool: fiber atrophy probably is accompanied by loss of myonuclei such that every reversal of atrophy is bound to use new myonuclei i.e. satellite cells. How often in life does this occur? Hard to say. Moreover, the potent repair capacity is challenged by an unexpected vulnerability of skeletal muscle fibers: Passive stretching of contracted muscles may cause multiple "microdamage," disruption of contractile elements or tiny areas of true necrosis (focal necrosis). How often does this happen? Well, for many of us at least once per year when we go up and down mountains during vacation time, followed by sour muscles. Others may decide to change his/her (locomotor) behaviour by severe onset of jogging; it may happen that they suffer kidney failure on Monday due to muscle microdamage and the transfer of myoproteins into the serum over weekend. Also 20 minutes of stepping up and down something like a chair will do: There is a remarkable increase in kreatin kinase and other muscle derived proteins which lasts for days and is bound to reflect some muscle damage. How about sportsmen and worker who repeatedly use their muscles in such a way? We don't have answers yet to most of these questions, but considerable amount of information has been collected over the last years both in animal and--less--in human. What is common in all cases of growth and repair is the proliferation of the satellite cells and their consequent incorporation and fusion with the parent fiber. This way focal damage is repaired often without visible reminders. We would run out of satellite cells were they not stem cells: After division one daughter remains a satellite cell while the other is free to divide. Divide how often? Important for the human cells since the cell ages and proliferates slower and slower till it stops to divide at all, at least in culture. The same is true for the new satellite cell. This we know from recent experiments in which human biopsies derived myogenic cells were grown in vitro and in vivo (by implanting them into skeletal muscles of immunoincompetent mice): Growth correlates negatively with age of the donor. Between age 2 and some 70 years, about two divisions are performed by each satellite cell in human vastus lateralis and biceps brachii muscle in 10 years in the average. Most important for the older among us: at age 76 there are still some 13 divisions left before complete exhaustion. However, there are diseases like Duchenne Muscular Dystrophy (DMD) in which muscle fibers lack a structural protein with the effect of enhanced vulnerability to mechanical stress. There the enhanced use of the satellite cell pool makes the remaining growth capacity in an 8-years-old child as low as otherwise found at age 80. Some time ago, implantation of genetically intact myoblasts obtained from healthy relatives has been proposed as a treatment of DMD. Every logic would have predicted that some local implantation of whatever numbers of cells was bound to fail rescue the complete masculature or at least the muscles for breathing. The human as guinea pig? Now, even years later, we still collect the basic information on growth of human myoblasts and start thinking of ways for systemic application and quantitatively relevant incorporation of the myogenic stem cell or other--possibly pluripotent--stem cells derived from bone marrow.

Muscle satellite (stem) cell activation during local tissue injury and repair.

In post-mitotic tissues, damaged cells are not replaced by new cells and hence effective local tissue repair mechanisms are required. In skeletal muscle, which is a syncytium, additional nuclei are obtained from muscle satellite (stem) cells that multiply and then fuse with the damaged fibres. Although insulin-like growth factor-I (IGF-l) had been previously implicated, it is now clear that muscle expresses at least two splice variants of the IGF-I gene: a mechanosensitive, autocrine, growth factor (MGF) and one that is similar to the liver type (IGF-IEa). To investigate this activation mechanism, local damage was induced by stretch combined with electrical stimulation or injection of bupivacaine in the rat anterior tibialis muscle and the time course of regeneration followed morphologically. Satellite cell activation was studied by the distribution and levels of expression of M-cadherin (M-cad) and related to the expression of the two forms of IGF-I. It was found that the following local damage MGF expression preceded that of M-cad whereas IGF-IEa peaked later than M-cad. The evidence suggests therefore that an initial pulse of MGF expression following damage is what activates the satellite cells and that this is followed by the later expression of IGF-IEa to maintain protein synthesis to complete the repair.

Recruitment of spinal motor pools during voluntary movements versus stepping after human spinal cord injury.

We investigated the activation of lower limb motor pools by supraspinal and spinal networks after human spinal cord injury (SCI). We compared electromyographic (EMG) activity from six muscles during voluntarily attempted non-weight-bearing single-joint movements, multijoint movements approximating stepping in a supine position, and weight-bearing stepping on a treadmill with body weight support (BWST) in seven clinically incomplete and three clinically complete SCI subjects. Seven SCI subjects had previously completed Laufband therapy (a specific step training using variable levels of body weight support and manual assistance). Significant coactivation of agonists and antagonists and multijoint flexion or extension movements of the entire limb occurred during attempts at isolated knee or ankle single-joint movements in clinically incomplete SCI subjects. Further, some muscles that were not recruited during voluntary attempts at single-joint movements were activated during voluntary step-like multijoint movements (5/16 comparisons). This suggests that the residual voluntary motor control in incomplete SCI subjects evokes more generalized motor patterns (limb flexion or extension) rather than selective activation of individual muscles. Clinically incomplete and clinically complete SCI subjects could achieve greater activation of motor pools and more reciprocal patterns of activity between agonists and antagonists during weight bearing stepping than during non-weight-bearing voluntary movements. The EMG mean amplitudes were higher during stepping than during voluntary movements in 50/60 muscles studied (p < 0.05). These results suggest that stepping with knee and hip extension and flexion and alternating lower limb loading and unloading provides proprioceptive inputs to the spinal cord that increases motor recruitment and improves reciprocity between agonists and antagonists compared to voluntary efforts.

A model system for studying postnatal myogenesis with tetracycline-responsive, genetically engineered clonal myoblasts in vitro and in vivo.

The aim of this work was to introduce a tetracycline-responsive (Tet-off) gene expression system into myoblasts in order to regulate a reporter gene not only in vitro but also particularly in muscles implanted with these engineered myoblasts. Mouse myoblasts from a long-term culture (i28 cells) were transfected initially to generate and characterize two stable master clones expressing tetracycline-responsive transactivator protein tTA. Like parental i28 myoblasts, these clones differentiated well in vitro. The second step introduced the firefly (Photinus pyralis) luciferase gene into one of the stable tTA clones producing double transfectants expressing luciferase in the absence of tetracycline. Addition of tetracycline (1 microg ml(-1)) resulted in at least 100-fold decreases in luciferase activity within 8 h in both growing and differentiating myoblast cultures. Enzyme activity was rapidly restored after tetracycline was removed (8 h). After successful implantation of these myoblasts into damaged mouse muscles, luciferase expression in the matured progeny cells could be regulated by oral application of doxycycline for at least 1 month. The tetracycline-responsive master clones are potentially powerful tools for studying the function of various genes in postnatal myogenesis.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Tenascin-R is expressed by Schwann cells in the peripheral nervous system.

The extracellular matrix glycoprotein tenascin-R (TN-R) has been implicated in a variety of cell-matrix interactions involved in the molecular control of axon guidance and neural cell migration during development and regeneration of the central nervous system (CNS). Whereas TN-R is amply expressed in the early postnatal and adult mammalian CNS, the protein has so far not been detected in different compartments of the peripheral nervous system (PNS). Here we provide first evidence that TN-R (predominantly TN-R 160 isoform) is transiently expressed in the sciatic nerve of late embryonic (E14-18) and neonatal mice, while at later developmental stages, both protein and mRNA are downregulated. In vitro, TN-R protein was found to be expressed by both undifferentiated and neuronally differentiated PC12 cells and by L1-positive Schwann cells (SC), but not by other neural and non-neural cell types in cell cultures derived from embryonic (E17/18) hindlimbs and neonatal sciatic nerves. In the developing PNS, TN-R expression correlated with axon growth and SC migration during the period of skeletal muscle innervation. Based on different in vitro approaches, we found that the substrate-bound glycoprotein selectively inhibits the fibronectin-dependent: (1) neurite outgrowth from dorsal root ganglion neurons (strongly expressing alpha5beta1 integrin and the disialoganglioside GD3) by a ganglioside-sensitive signaling mechanism; and (2) migration of primary myoblasts and other non-neuronal cells in a ganglioside-independent manner. Our findings suggest the functional role of TN-R in PNS pattern formation during distinct stages of axon pathfinding and skeletal muscle innervation.

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway.

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

Growth factor supplemented matrigel improves ectopic skeletal muscle formation--a cell therapy approach.

Following damage to skeletal muscle, satellite cells become activated, migrate towards the injured area, proliferate, and fuse with each other to form myotubes which finally mature into myofibers. We tested a new approach to muscle regeneration by incorporating myoblasts, with or without the exogenous growth factors bFGF or HGF, into three-dimensional gels of reconstituted basement membrane (matrigel). In vitro, bFGF and HGF induced C2C12 myoblast proliferation and migration and were synergistic when used together. In vivo, C2C12 or primary i28 myoblasts were injected subcutaneously together with matrigel and growth factors in the flanks of nude mice. The inclusion of either bFGF or HGF increased the vascularization of the gels. Gels supplemented with bFGF showed myogenesis accompanied by massive mesenchymal cell recruitment and poor organization of the fascicles. Samples containing HGF showed delayed differentiation with respect to controls or bFGF, with increased myoblast proliferation and a significantly higher numbers of cells in myotubes at later time points. HGF samples showed limited mesenchymal cell infiltration and relatively good organization of fascicles. The use of both bFGF and HGF together showed increased numbers of nuclei in myotubes, but with bFGF-mediated fibroblast recruitment dominating. These studies suggest that an appropriate combination of basement membrane components and growth factors could represent a possible approach to enhance survival dispersion, proliferation, and differentiation of myogenic cells during muscle regeneration and/or myoblast transplantation. This model will help develop cell therapy of muscle diseases and open the future to gene therapy approaches.

Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.

Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majority.

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

Regenerative capacity and the number of satellite cells in soleus muscles of normal and mdx mice.

Satellite cells are potential myogenic cells that participate in repair and growth of muscle fibres. In this investigation, the change in the number of satellite cells following severe muscle damage was monitored in soleus muscle of age-matched mdx and C57Bl/10 mice. Satellite cells were identified immunohistochemically in the light microscope by their association with a recently described marker protein, M-cadherin, and their location between the muscle fibre's sarcolemma and the surrounding basal lamina. In cross-sections of untreated soleus muscle of C57Bl/10 mice at 11-14. 5 months of age, nuclei of M-cadherin positive satellite cells on average amounted to 3.4% of the total number of myonuclei. Surprisingly, significantly higher numbers of satellite cell nuclei, both in absolute numbers (mean 24+/-11 versus 40+/-11 satellite cells per section) and relative to the total number of myonuclei (5. 3%), were found in similarly aged animals in which severe muscle damage had been inflicted 3-6 months before. Cross-sectional area, muscle tissue area and myonuclei counts had recovered to control values. In untreated muscles of age-matched mdx mice satellite cell counts were not different (2.7% of myonuclei) from C57Bl/10 mice. However, regeneration showed marked deficits, as there was a loss of about 36% total cross-sectional area, about 48% total muscle fibre area and about 43% myonuclei per section compared to the untreated mdx muscles. Furthermore, the absolute number of satellite cells decreased from 20+/-11 to 12+/-8 per section. The relative number of satellite cell nuclei remained comparable to, but did not exceed, the undamaged muscles. The poor recovery of muscle and the missing post-regeneration rise in satellite cell numbers may indicate the reproductive limits of the satellite pool.

Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles.

1. Pretreatment of muscles with ionising radiation enhances tissue formation by transplanted myoblasts but little is known about the effects on muscle function. We implanted myoblasts from an expanded, male-donor-derived, culture (i28) into X-ray irradiated (16 Gy) or irradiated and damaged soleus muscles of female syngeneic mice (Balb/c). Three to 6 months later the isometric contractile properties of the muscles were studied in vitro, and donor nuclei were visualised in muscle sections with a Y chromosome-specific DNA probe. 2. Irradiated sham-injected muscles had smaller masses than untreated solei and produced less twitch and tetanic force (all by about 18 %). Injection of 106 myoblasts abolished these deficiencies and innervation appeared normal. 3. Cryodamage of irradiated solei produced muscle remnants with few (1-50) or no fibres. Additional myoblast implantation led to formation of large muscles (25 % above normal) containing numerous small-diameter fibres. Upon direct electrical stimulation, these muscles produced considerable twitch (53 % of normal) and tetanic forces (35 % of normal) but innervation was insufficient as indicated by weak nerve-evoked contractions and elevated ACh sensitivity. 4. In control experiments on irradiated muscles, reinnervation was found to be less complete after botulinum toxin paralysis than after nerve crush indicating that proliferative arrest of irradiated Schwann cells may account for the observed innervation deficits. 5. Irradiation appears to be an effective pretreatment for improving myoblast transplantation. The injected cells can even produce organised contractile tissue replacing whole muscle. However, impaired nerve regeneration limits the functional performance of the new muscle.

Laufband (treadmill) therapy in incomplete paraplegia and tetraplegia.

Recent reports indicate that intensive training of upright walking on a treadmill (German: Laufband, LB), significantly improves walking capability in spinal cord-damaged persons. The aids provided initially are body weight support by a harness and passive setting of one or both limbs by therapists. To facilitate stepping and evoke motor automatisms, "rules of spinal locomotion" need to be applied during training. The effects of this novel locomotion therapy on patients with chronic and acute incomplete paralysis are summarized and discussed here. Many patients with chronic paralysis, still wheelchair-bound and not capable of walking without help from others, became independent and learned to walk for some distance without help. Assessment of voluntary muscle activity in resting position before and after the period of therapy often showed only small increases, rendering the involvement of complex motor reflexes (motor programs) and better utilization of remaining muscle function during walking as main sources for the improvements in locomotion. This idea is supported by electromyographic recordings. Follow-up assessments performed 0.5 to 6.5 years after discharge from the hospital show that the significant improvements achieved by LB-therapy in patients with initially chronic paralysis can be maintained under domestic surrounding. Patients with initially acute paralysis improved their walking capabilities even further. It is suggested that LB therapy may be generally applied in the motor rehabilitation of persons with acute and chronic incomplete paraplegia and tetraplegia. Its use in other diseases is discussed.

Cross-talk mechanisms in the development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake.

Insulin resistance in skeletal muscle is one of the earliest symptoms associated with non-insulin-dependent diabetes mellitus (NIDDM). Tumour necrosis factor (TNF) and nonesterified fatty acids have been proposed to be crucial factors in the development of the insulin-resistant state. We here show that, although TNF downregulated insulin-induced insulin receptor (IR) and IR substrate (IRS)-1 phosphorylation as well as phosphoinositide 3-kinase (PI3-kinase) activity in pmi28 myotubes, this was, unlike in adipocytes, not sufficient to affect insulin-induced glucose transport. Rather, TNF increased membrane expression of GLUT1 and glucose transport in these muscle cells. In contrast, the nonesterified fatty acid palmitate inhibited insulin-induced signalling cascades not only at the level of IR and IRS-1 phosphorylation, but also at the level protein kinase B (PKB/Akt), which is thought to be directly involved in the insulin-induced translocation of GLUT4, and inhibited insulin-induced glucose uptake. Palmitate also abrogated TNF-dependent enhancement of basal glucose uptake, suggesting that palmitate has the capacity to render muscle cells resistant not only to insulin but also to TNF with respect to glucose transport by GLUT4 and GLUT1, respectively. Our data illustrate the complexity of the mechanisms governing insulin resistance of skeletal muscle, questioning the role of TNF as a direct inhibitor of glucose homoeostasis in this tissue and shedding new light on an as yet unrecognized multifunctional role for the predominant nonesterified fatty acid palmitate in this process.