A. Irintchev

Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts.

1. Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles of adult BALB/c mice. One to four months later isometric tension recordings were performed in vitro, and the male donor cells implanted into female hosts were traced on histological sections using a Y‐chromosome‐specific probe. The muscles were either mildly or severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles, 9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in regeneration of muscle tissue as judged from the reduced desmin‐positive cross‐sectional areas (34 and 66% of control, respectively). 2. Implantation of 10(6) myogenic cells into severely cryodamaged muscles more than doubled muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal). Absolute and relative amount of desmin‐positive muscle cross‐sectional areas were significantly increased indicating improved microarchitecture and less fibrosis. Newly formed muscle tissue was fully innervated since the tetanic forces resulting from direct and indirect (nerve‐evoked) stimulation were equal. Endplates were found on numerous Y‐positive muscle fibres. 3. As judged from their position under basal laminae of muscle fibres and the expression of M‐cadherin, donor‐derived cells contributed to the pool of satellite cells on small‐ and large‐diameter muscle fibres. 4. Myoblast implantation after mild cryodamage and in undamaged muscles had little or no functional or structural effects; in both preparations only a few Y‐positive muscle nuclei were detected. It is concluded that myoblasts from expanded primary cultures‐unlike permanent cell lines‐significantly contribute to muscle regeneration only when previous muscle damage is extensive and loss of host satellite cells is severe.

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 α (TNF‐α) 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.

Muscle damage and repair in voluntarily running mice: strain and muscle differences.

SummarySoleus, extensor digitorum longus and tibialis anterior muscles of mice voluntarily running in wheels for periods of 5 to 120 days were studied in spaced serial and serial cross-sections. Shortly after the onset of running and during the next 2 weeks, degeneration, necrosis, phagocytosis and regeneration of muscle fibers, satellite cell proliferation and cellular infiltration were found in soleus muscles of mice from all strains investigated (CBA/J, NMRI, C57b, NIH, SWS and Balb/c). Tibialis anterior but not extensor digitorum longus muscles were also damaged. Predominantly high-oxidative fibers were affected (both slow-oxidative and fast oxidative glycolytic in soleus, fast-oxidative glycolytic in tibialis anterior). Denervated soleus muscles that had been passively stretched during running were not damaged. Evidence was found that, during the early period of running, split fibers form by myogenesis within (regeneration) or outside (satellite cell proliferation) necrotic muscle fiber segments. Split fibers persisted in solei of long-term (2 to 3 months) exercised CBA/J but not NMRI mice. In 6 out of 20 solei of CBA/J runners exercised for 2 months or longer, fiber-type grouping was observed in the areas where extensive damage usually occurred in the early periods. The results show that different muscles are damaged and repaired to varying degrees and that marked interstrain and inter-individual differences are present. It appears that acute muscle injury occurring upon onset of voluntary running is a usual event in the adaptation of muscles to altered use.

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.

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 fibres 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.

Cellular and molecular reactions in mouse muscles after myoblast implantation.

SummaryImplantation of skeletal muscle precursor cells is a potential means of cell-mediated gene therapy. One unresolved question is the degree of immunogenicity of such myoblasts. We designed the extreme situation of implanting cells of a non-histocompatible myoblast cell line into cryodamaged, but regeneration-capable, muscles of adult mice. Without immuno-suppression donor cells are rejected within the first weeks. Immunosuppression with Cyclosporin A prevented invasion of T-lymphocytes and allowed differentiation of implanted myoblasts into myofibres as well as down-regulation of MHC expression. Still, withdrawal of Cyclosporin A after 4 weeks triggered lymphocyte invasion and cytotoxic cell reactions with rejection of donor tissue. Although the vast majority of muscle fibres was MHC-negative 1–4 days after Cyclosporin A withdrawal, single small desmin-positive profiles were weakly positive for donor MHC. Parallel with the increase in the number of lymphocytes, larger numbers of small and large muscle fibres expressed high levels of either donor, host or both, class I — but not class II — molecules. Surprisingly, immune reactions continued over several months, causing gradual loss of muscle tissue. Donor class I molecules persisted for more than 6 months after Cyclosporin A withdrawal, clearly indicating survival of donor muscle fibres despite ongoing rejection. Indirect evidence on the other hand suggests additional loss of host fibres, possibly caused by cytokine release from the immune cells (bystander damage). We conclude that transient treatment with Cyclosporin A induced a kind of tolerance related to the maturation and down-regulation of class I antigens in donor muscle fibres. It is suggested that the start of immune reaction following Cyclosporin A withdrawal is initiated by remaining small amounts of donor MHC molecules, possibly related to the continuous proliferation of the cell-lined-derived donor myoblasts.