Nonmuscle stem cells fail to significantly contribute to regeneration of normal muscle.

Whole, normal extensor digitorum longus muscles (EDL) were orthtopically transplanted into transgenic mice, expressing nuclear localing beta-galactosidase (nlsbeta-gal) under control of a muscle-specific promoter, in order to determine the extent to which nonmuscle derived, multipotent stem cells (which under experimental conditions exhibit myogenic potential) are spontaneously recruited from distal, nonmuscle organs to participate in the graft's regeneration. The host's contribution to the graft's regeneration was determined by evaluating the number and distribution of beta-gal positive myonuclei in regenerated grafts. Fibers with beta-gal positive nuclei accounted for approximately 1% of the long-term (28- and 56-day) graft's myofibers. All were confined to the graft's periphery, adjacent to host's muscles. Failure to find myofibers with beta-gal positive nuclei across the revascularized graft's girth demonstrated that there was no meaningful recruitment of nonmuscle stem cells from distal host organs, which must arrive at the graft via the circulation. Rather, stem cells residing in the graft at the time of transplantation accounted for approximately 99.9% of the regenerated graft's myonuclei, with a minor contribution from the surrounding host muscles' myosatellite cells (that occurred when the epimysia of graft or host muscles were damaged during transplantation). The significance of these findings to gene therapy for Duchenne muscular dystrophy is discussed.

Effect of chronic denervation and denervation-reinnervation on cytoplasmic creatine kinase transcript accumulation.

The extensor digitorum longus (EDL) and soleus muscles of adult mice were chronically denervated or denervated and allowed to reinnervate. Muscles were evaluated 1, 5, 14, 21, and 52 days after sciaticectomy. In terms of weight loss, myofiber atrophy, degeneration, and fibrosis, the soleus muscle was more affected than the EDL by chronic denervation. Fifty-two days after chronic denervation, the number of molecules of MCK/ng total RNA in both muscles (determined with competitive PCR) decreased, with the soleus muscle being more affected. At that stage, BCK mRNA levels in the denervated soleus were unchanged, but they were increased (>50%) in the EDL. Reinnervation restored MCK transcript accumulation in the EDL, whereas, in the soleus MCK, transcripts exceeded control values by 57%, approaching levels in the reinnervated EDL. Despite restoration of MCK mRNA levels, the number of molecules of BCK mRNA/ng total RNA was four- to fivefold higher in reinnervated versus control muscles, suggesting that the genes encoding the CK mRNAs are not coordinately regulated in adult muscle. The role of denervation induced, fiber type changes in regulating CK mRNA accumulation has been evaluated. Electron microscopic analyses have established that fibrosis is not a factor that determines BCK mRNA levels in the chronically denervated or denervated-reinnervated muscles. CK isozyme analyses support the hypothesis that a greater proportion of BCK mRNA found in 52 day chronically denervated and denervated-reinnervated muscles is produced in myofibers vs. nonmuscle cells than in control muscles.

Enhancement of adult muscle regeneration by primary myoblast transplantation.

Extensor digitorum longus muscles (EDL) of SCID mice were induced to undergo degeneration-regeneration subsequent to orthotopic, whole-muscle transplantation. Two days after transplantation some of these muscles received injections of primary myoblasts derived from EDL muscles of transgenic mice, which express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter and enhancer. Nine weeks after transplantation, regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received no further treatment and to unoperated EDL muscles in order to determine the effect of myoblast transfer on muscle regeneration. Many myofibers containing donor derived myonuclei could be identified in the regenerated muscles that had received exogenous myoblasts. The mass of the muscles subjected to transplantation only was significantly less (31% less) than that of unoperated muscles. The addition of exogenous myoblasts to the regenerating EDL resulted in a muscle mass similar to that of unoperated muscles. The absolute twitch and tetanic tensions and specific twitch and tetanic tensions of transplant-only muscles were 28%, 36%, 32%, and 41%, respectively, of those of unoperated muscles. Myoblast transfer increased the absolute twitch and tetanic tensions of the regenerated muscles by 65% and 74%, respectively, and their specific twitch and tetanic tensions were increased by 41% and 48%, respectively. These data suggest a possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss.

Creatine kinase transcript accumulation: effect of nerve during muscle development.

To determine the role of the nerve in regulating the accumulation of cytoplasmic creatine kinase (CK) mRNAs in hindleg muscles of the developing mouse, the lumbosacral spinal cords of 14-day gestation mice (E14) were laser ablated, and the accumulation of muscle CK (MCK) and brain CK (BCK) mRNAs was evaluated just prior to birth with in situ hybridization. Numbers of molecules of each of these transcripts/ng total RNA in the soleus and extensor digitorum longus (EDL) muscles were determined with competitive PCR and compared to transcripts found in innervated crural muscles. Data suggest that: 1) the level of BCK mRNA accumulation in innervated hindlimb muscles peaks at E16.5 and remains at fetal levels until the second month postnatal, when it falls to the level found in the adult. Given that MCK transcripts meet or exceed adult levels by day 28 postnatal, the "down-regulation" of the BCK gene and the "up-regulation" of the MCK gene are not tightly coupled; 2) the developmental switch from BCK to MCK, as the dominant cytoplasmic CK mRNA, occurs in innervated and aneural leg muscles between E14 and E16.5, indicating this switch is not nerve dependent; 3) the absence of innervation has no effect on BCK mRNA accumulation. MCK transcripts/ng total RNA continue to increase in aneural muscle throughout the late fetal period, but from E16.5-E19.5 the MCK transcript levels in aneural muscles become progressively lower than in age-matched innervated muscles. Thus, the accumulation of the muscle specific cytoplasmic CK, but not BCK, transcripts is affected by the absence of innervation during the fetal period. Dev Dyn 1999;215:285-296.

DNA from both high-capacity and first-generation adenoviral vectors remains intact in skeletal muscle.

Previous studies of the use of adenoviral vectors in animal models of gene therapy have focused on the immune response against transduced cells as the major limiting factor to long-term transgene expression. In this study we eliminated the variable of immunity induced by expression of the transgene in order to investigate vector DNA stability of both first-generation and high-capacity adenoviral vectors after gene transfer to skeletal muscle. Transgene expression from a high-capacity adenoviral vector remained at a high level for at least 20 weeks and was accompanied by persistence of intact vector genomes. In contrast, transgene expression from a first-generation adenoviral vector markedly diminished by 6 weeks after gene transfer and was accompanied by mild and variable inflammatory cell infiltrates. Surprisingly, despite this loss of transgene expression, the first-generation adenoviral vector genomes persisted like the high-capacity adenoviral vector genomes. Therefore, in the absence of immunity to transgene proteins, loss of expression from the first-generation vector was due to inhibition of transgene expression rather than to the elimination of vector-containing cells. DNA stability and persistent expression of the high-capacity adenoviral vector supports the potential of this vector for clinical applications of muscle gene transfer.

Ex vivo gene transfer using adenovirus-mediated full-length dystrophin delivery to dystrophic muscles.

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle disease characterized by a lack of dystrophin expression. Myoblast transplantation and gene therapy have the potential of restoring dystrophin, thus decreasing the muscle weakness associated with this disease. In this study we present data on the myoblast mediated ex vivo gene transfer of full-length dystrophin to mdx (dystrophin deficient) mouse muscle as a model for autologous myoblast transfer. Both isogenic primary mdx myoblasts and an immortalized mdx cell line were transduced with an adenoviral vector that has all viral coding sequences deleted and encodes beta-galactosidase and full-length dystrophin. Subsequently, these transduced myoblasts were injected into dystrophic mdx muscle, where the injected cells restored dystrophin, as well as dystrophin-associated proteins. A greater amount of dystrophin replacement occurred in mdx muscle following transplantation of mdx myoblasts isolated from a transgenic mouse overexpressing dystrophin suggesting that engineering autologous myoblasts to express high amounts of dystrophin might be beneficial. The ex vivo approach possesses attributes that make it useful for gene transfer to skeletal muscle including: (1) creating a reservoir of myoblasts capable of regenerating and restoring dystrophin to dystrophic muscle; and (2) achieving a higher level of gene transfer to dystrophic muscle compared with adenovirus-mediated direct gene delivery. However, as observed in direct gene transfer studies, the ex vivo approach also triggers a cellular immune response which limits the duration of trans-gene expression.

Alteration in myosatellite cell commitment with muscle maturation.

Myosatellite cells are myoblasts found between the basal lamina and sarcolemma of myofibers of postnatal mice. The extent to which these cells are programmed, upon differentiation, to express isoforms of contractile protein genes specific to the type of fiber with which they are associated has been evaluated in vitro using myosatellite cells derived from the soleus and the extensor digitorum longus muscles (EDL) of 4-day-old and adult transgenic mice, which express nuclear localizing beta-galactosidase (nlsbeta-gal) under the control of the promoter and 3' enhancer of the gene encoding fast myosin light chain 3F (MLC3F) (Kelly et al. [1995] J. Cell Biol. 129:383-396). Cultures were allowed to differentiate either as myocytes (mononucleated cells), to prevent possible modification of the myosatellite phenotype by other myonuclei in mosaic myotubes, or as myotubes. Transgene expression was age related, with 90% and 70% of the myocytes derived from the neonatal EDL and soleus muscles (muscles that had not yet achieved their mature phenotype), respectively, having nuclei encoding beta-gal; 61% and 32% of the myocyte nuclei derived from myosatellite cells of the adult EDL (a fast muscle) and the adult soleus muscle (a mixed muscle containing many slow myofibers), respectively, expressed this transgene. Because myosatellite cells found in adult muscles are the progeny of those found in the neonate, an alteration of myosatellite cell commitment to express this transgene occurs with muscle maturation. Because expression of the transgene in neonatal and adult muscle in vivo reflects the expression of the endogenous MLC3F gene (Kelly et al. [1995] J. Cell Biol. 129:383-396), it is likely that expression of the transgene by differentiated myosatellite cells reflects the extent of commitment of these cells to produce MLC3F. A hypothesis is presented that MLC3F is widely expressed in developing muscles but eliminated in myofibers that undergo maturation toward a slower phenotype.

Role of the nerve in determining fetal skeletal muscle phenotype.

To determine the role of the nerve on the establishment of myofiber diversity in skeletal muscles, the lumbosacral spinal cord of 14-day gestation mice (E14) was laser ablated, and the accumulation of the myosin alkali light chains (MLC) mRNAs in crural (hindleg) muscles was evaluated just prior to birth with in situ hybridization. Numbers of molecules of each alkali MLC/ng total RNA in the extensor digitorum longus (EDL) and soleus muscles were determined with competitive polymerase chain reaction. Transcripts for all four alkali MLCs accumulate in aneural muscles. Data suggest that: (1) the absence of the nerve to either future fast or slow muscles results in less accumulation of MLC1V transcript. Moreover, the presence of the nerve is required for the enhanced accumulation of this transcript in future slow muscles; (2) the absence of innervation of future slow, but not fast, muscles decreases the accumulation of MLC1A transcript. Since increased accumulation of MLC1A and MLC1V transcripts are found in future slow muscles at birth, the nerve is necessary for the development of the slow phenotype during myogenesis; (3) MLC1F and MLC3F transcripts do not display any preferential accumulation in future fast muscles during the fetal period. Therefore, the establishment of the differential distribution of these mRNAs, based on fiber type, is a postnatal phenomenon. The nerve is required during the fetal period to allow accumulation of MLC3F messages above a basal level in future fast as well as slow muscles; whereas, the absence of the innervation to future fast, but not slow, muscles reduces the accumulation of MLC1F. Thus, the accumulation of the various alkali MLC mRNAs shows a differential, rather than coordinate, response to the absence of the nerve, and this response may vary depending on the future fiber type of the muscles.

Mass and functional capacity of regenerating muscle is enhanced by myoblast transfer.

Morphology and functional capacity of homotopically transplanted extensor digitorum longus muscles (EDL) of adult SCID mice that received 1 x 10(6) myoblasts [stably transfected to express nuclear localizing beta-galactosidase under the control of the myosin light-chain 3F promoter/enhancer] 2 days posttransplantation were evaluated 9 weeks after transplantation, to determine whether the injection of exogenous myoblasts had an effect on muscle regeneration. Regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received (a) no further treatment, or (b) sham injection of the vehicle (without myoblasts) and to unoperated EDL. Nine weeks after myoblast transfer, myofibers containing donor-derived nuclei could be identified after staining with X-gal solution. Judging from its size and poor functional performance compared to muscles subjected to transplantation only, sham injection provided a secondary trauma to the regenerating muscle from which it failed to fully recover. In comparison to the sham-injected muscle, the myoblast-injected muscles weighed 61% more and had 50% more myofibers and 82% more cross-sectional area occupied by myofibers at the muscles' widest girths. Their absolute twitch and tetanic tensions were threefold and twofold greater, respectively, and their specific twitch and tetanic tensions were 71% and 50% greater, respectively, than those of sham-injected muscles. In many parameters, the regenerating muscle subjected to myoblast transfer equaled or exceeded those of muscles that were transplanted only (received only one trauma). Absolute twitch and tetanic tensions were 73% and 65% greater, respectively, and specific twitch tensions of the muscles receiving myoblasts were 50% greater than forces generated by muscles subjected to whole-muscle transplantation only.

Limitations of nls beta-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer.

BACKGROUND: Nuclear localizing beta-galactosidase (nls beta-gal) is used as a marker for studying myoblast cell lineage and for evaluating myoblast survival after myoblast transfer, a procedure with potential use for gene complementation for muscular dystrophy. Usefulness of this construct depends on the establishment of the extent to which nls beta-gal or its mRNA may be translocated from the nucleus that encodes it to other non-coding myonuclei in hybrid myofibers and the ease with which the encoding and non-coding myonuclei can be distinguished. Previous in vitro studies (Ralston and Hall 1989. Science, 244:1066-1068) have suggested limited translocation of the fusion protein. We re-examined the extent to which nls beta-gal is translocated in hybrid myofibers, both in vitro and in vivo, and evaluated the extent to which one can rely on histochemistry to distinguish encoding from non-coding nuclei in these myofibers. METHODS: Myotubes formed in co-cultures of a myoblast line (MM14 cells), stably transfected with a construct consisting of a nls beta-gal under the control of the myosin light chain 3F promoter and 3' enhancer (3FlacZ10 cells), and [3H]-thymidine-labeled parental MM14 cells (plated at ratios of 1:6 or 1:20, respectively) were reacted with X-gal. After autoradiography, the distance over which nls beta-gal was translocated in hybrid myotubes was determined. In vivo translocation of nls beta-gal was evaluated by injecting [3H]-thymidine-labeled 3FlacZ10 myoblasts into the regenerating extensor digitorum longus muscle of immunosuppressed normal and mdx (dystrophin deficient) mice. Sections stained with X-gal and subjected to autoradiography permitted determination of the extent of nls beta-gal translocation in hybrid myofibers. RESULTS: In vitro: All nuclei in > 92% of hybrid myotubes showed evidence of nls beta-gal after exposure to X-gal, suggesting extensive translocation. Within hybrid myotubes, MM14-derived myonuclei approximately 350 microns from a 3FlacZ10-derived myonucleus showed evidence of nls beta-gal. In vivo: Similar translocation of nls beta-gal was observed in vivo. One week after myoblast transfer, donor-derived myonuclei were distinguishable from host-derived myonuclei containing nls beta-gal by the greater accumulation of reaction product in donor myonuclei after X-gal staining. However, 2 weeks after injection, host myonuclei often contained a significant amount of nls beta-gal, and accumulation of reaction product could not be used as the criterion for identification of donor myonuclei. CONCLUSIONS: Translocation of nls beta-gal (or its mRNA) is significantly greater than previously reported (Ralston and Hall 1989), resulting in large numbers of nls beta-gal positive non-coding myonuclei in hybrid myofibers. One week after myoblast transfer, distinguishing between nls beta-gal encoding and non-coding myonuclei in hybrid myofibers after X-gal staining of sectioned muscle is feasible; however, by 2 weeks, nls beta-gal increases in host myonuclei, making identification of donor-derived myonuclei problematic. Translocation of nls beta-gal to non-coding myonuclei in hybrid myofibers must be considered when nls beta-gal is used for studies of myogenic lineage or the efficacy of myoblast transfer therapy, particularly if long-term survival of hybrid myotubes is required.

Persistence in muscle of an adenoviral vector that lacks all viral genes.

Genetic correction of inherited muscle diseases, such as Duchenne muscular dystrophy, will require long term expression of the recombinant protein following gene transfer. We have shown previously that a new adenoviral vector that lacks all viral genes expressed both full-length dystrophin and beta-galactosidase in mdx (dystrophin-deficient) mouse muscle. We observed a significant histologic improvement of vector-transduced mdx muscle before the eventual loss of vector-encoded transgene expression. In this study, we investigated whether an immunological response against vector-encoded beta-galactosidase contributed to the loss of vector expression and affected vector persistence in muscle. Intramuscular vector injection in control normal mice resulted in an early and complete loss of beta-galactosidase expression accompanied by predominantly CD4+ and CD8+ lymphocytic infiltration and a significant loss of vector DNA. In contrast, intramuscular vector injection in lacZ transgenic mice resulted in persistent expression of beta-galactosidase for at least 84 days with no evidence of inflammation or significant loss of vector DNA. Our studies demonstrate that, in the absence of an immune response induced by beta-galactosidase expression, an adenoviral vector lacking all viral genes is stably maintained in muscle.

Muscle-specific gene expression during myogenesis in the mouse.

Over the past decade, significant advances in molecular biological techniques have substantially increased our understanding of in vivo myogenesis, supplementing the information that previously had been obtained from classical embryological and morphological studies of muscle development. In this review, we have attempted to correlate morphogenetic events in developing murine muscle with the expression of genes encoding the MyoD family of myogenic regulatory factors and the contractile proteins. Differences in the pattern of expression of these genes in murine myotomal and limb muscle are discussed in the context of muscle cell lineage and environmental factors. The differences in gene expression in these two types of muscle suggest that no single coordinated pattern of gene activation is required during the initial formation of the muscles of the mouse.

Gene complementation using myoblast transfer into fetal muscle.

Gene complementation by myoblast transfer into neonatal or adult muscle has been proposed as a therapy for primary myopathies as well as to augment non-muscle gene products that may be diminished in the adult circulation. This paper describes a technique whereby myoblasts have been injected into limb muscles of normal and dystrophin-deficient (mdx) fetal mice (during the period of active myotube formation and prior to the development of the host's immune competence) without significantly interfering with fetal viability or further maturation. More mosaic myofibers (myofibers containing both host- and donor-derived myonuclei) appear to result from these transfers than have been reported following myoblast transfer into neonatal muscle or adult muscle. The small size of the fetal hosts' muscles and the lack of well-defined connective tissue septa facilitate migration of donor myoblasts into muscle groups distal to the injection site. The use of donor myoblasts derived from a tetraploid variant of a mouse myogenic cell line (MM14) provides a convenient and permanent cytological marker for the recognition of donor myoblasts and donor-derived myonuclei. When MM14 myoblasts are injected into mdx fetuses, whose muscles lack dystrophin, mosaic myofibers contain sufficient dystrophin to be visualized with routine immunohistochemical techniques. The myoblast transfer system, using fetal hosts, described in this study will facilitate the evaluation of myoblasts as vectors to overcome genetic deficiencies that may be manifested during fetal development.

Modulation of contractile protein gene expression in fetal murine crural muscles: emergence of muscle diversity.

The modulation of contractile protein gene expression in mouse crural muscles (i.e., muscles located in the region between the knee and ankle) during the fetal period (defined as 15 days gestation to birth), resulting in diversity among and within these muscles, has been evaluated with in situ hybridization and correlated with morphogenetic events in the extensor digitorum longus and soleus muscles. During the fetal period extensive secondary myotube formation occurs in the crural muscles, and the myotubes become innervated (Ontell and Kozeka [1984a,b] Am. J. Anat. 171:133-148, 149-161; Ontell et al. [1988a,b] Am. J. Anat. 181:267-278, 181:278-288). At 15 days gestation, hybridization with 35S-labeled antisense cRNA probes demonstrates the accumulation of transcripts for alpha-cardiac and alpha-skeletal actin; MLC 1A, MLC 1F, and MLC 3F; and MHC emb, MHC pn, and MHC beta/slow. At 16 days gestation, accumulation of MHC emb transcripts is reduced (as compared with earlier developmental stages); intensity of signal following hybridization with the probe for alpha-skeletal actin is, for the first time, equal to that for the cardiac isoform; and MLC 1V mRNA accumulation is discernible. At this stage, variation in transcript accumulation for some mRNAs among and within crural muscles becomes evident. Two factors may play a role in the selective distribution of these transcripts: 1) the stage of muscle maturation; and 2) the future myofiber type. At 16 days gestation anterior crural muscles (which mature approximately 2 days before posterior crural muscles; Ontell and Kozeka [1984a,b], ibid., Ontell et al. [1988a,b], ibid.) exhibit a greater accumulation of transcripts for alpha-skeletal actin and for MLC 3F than is found in posterior crural muscles. In muscles that in the neonate are composed, in large part, of slow myofibers, MHC beta/slow and MLC 1V mRNAs accumulate in greater amounts, whereas MHC pn transcripts are less abundant in the soleus muscle than in other crural muscles. By 19 days gestation regionalization of transcript accumulation is more pronounced. The soleus muscle, a predominantly slow twitch muscle in the newborn mouse (Wirtz et al. [1983] J. Anat. 137:109-126) exhibits strong signal after hybridization with probes specific for MHC beta/slow and MLC 1V. While the level of transcript accumulation for the development isoforms, MHC emb, MLC 1A, and alpha-cardiac actin, is greatly reduced in most crural muscles at 19 days gestation, these transcripts persist in the soleus muscle at levels equal ot or exceeding their amount in limb muscles of 13 day gestation mouse embryos.(ABSTRACT TRUNCATED AT 400 WORDS)

Contractile protein gene expression in primary myotubes of embryonic mouse hindlimb muscles.

The time course of contractile protein [actin, myosin heavy chain (MHC) and myosin light chain (MLC)] gene expression in the hindlimb muscles of the embryonic mouse (< 15 days gestation) has been correlated with the expression of genes for the myogenic regulatory factors, myogenin and MyoD, and with morphogenetic events. At 14 days gestation, secondary myotubes are not yet present in crural muscles (M. Ontell and K. Kozeka (1984) Am. J. Anat. 171, 133-148; M. Ontell, D. Bourke and D. Hughes (1988) Am. J. Anat. 181, 267-278); therefore, all transcripts for contractile proteins found in these muscles must be produced in primary myotubes. In situ hybridization, with 35S-labeled antisense cRNAs, demonstrates the versatility of primary myotubes in that transcripts for (1) alpha-cardiac and alpha-skeletal actin, (2) MHCembryonic, MHCperinatal and MHC beta/slow, and (3) MLC1A, MLC1F and MLC3F are detectable at 14 days gestation. While the general patterns of early activation of the cardiac genes and early activation of the genes for the developmental isoforms are preserved in both myotomal and limb muscles (D. Sassoon, I. Garner and M. Buckingham (1988) Development 104, 155-164 and G. E. Lyons, M. Ontell, R. Cox, D. Sassoon and M. Buckingham (1990) J. Cell Biol. 111, 1465-1476 for myotomal muscle), there are a number of differences in contractile protein gene expression. For example, in the myotome, when myosin light chain genes are initially transcribed, hybridization signal with probe for MLC1A mRNA is greater than that with probe for MLC1F transcripts, whereas the relative intensity of signal with these same probes is reversed in the hindlimb. The order in which myosin heavy chain genes are activated is also different, with MHCembryonic and MHCperinatal preceding the appearance of MHC beta/slow transcripts in limb muscles, while MHCembryonic and MHC beta/slow appear simultaneously in the myotomes prior to MHCperinatal. In the myotome, an intense hybridization signal for alpha-cardiac and a weak signal for alpha-skeletal actin transcripts are detectable prior to myosin mRNAs, whereas in the limb alpha-cardiac actin transcripts accumulate with myosin transcripts before alpha-skeletal actin mRNA is detectable. These differences indicate that there is no single coordinate pattern of expression of contractile protein genes during initial formation of the muscles of the mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of growth hormone on diaphragmatic recovery from malnutrition.

A 25% weight loss was induced in adult Fisher 344 rats by nutritional deprivation. Subsequently, normal feeding was resumed. Refed animals were divided into three groups and received recombinant human growth hormone (rhGH) injections during 5 wk of refeeding, saline injections during 5 wk of refeeding, or 9 wk of refeeding without injections. The effects of nutritional deprivation and the various refeeding protocols on the cross-sectional areas (CSA) of each of the four types of myofibers [typed immunohistochemically with antibodies against four myosin heavy chain (MHC) isoforms known to be present in the rat diaphragm] were determined. Malnutrition decreased the CSA of myofibers containing MHC2X, MHC2B, and MHC2A (i.e., fast myofibers), with the greatest effect on muscle mass being due to the atrophy of fibers containing MHC2X. Fibers containing MHC beta/slow failed to undergo malnutrition-induced atrophy. Whereas refeeding for 5 wk in the absence of rhGH allowed the recovery of CSA of fibers containing MHC2A, fibers containing MHC2B and MHC2X remained smaller than fibers of similar type in control animals. In contrast, 5 wk of refeeding supplemented with rhGH returned all fiber CSAs to control values. Even when refeeding alone was extended to 9 wk to allow for weight stabilization, the CSA of the fibers containing MHC2B and MHC2X remained smaller than similar fibers in control muscle. Serum insulin-like growth factor, a marker of malnutrition (R. Reeves and J. Elders, J. Nutr. 109: 613-620, 1979), was significantly decreased after nutritional deprivation and returned to normal after 5 wk of refeeding and GH supplementation.

Modification of the dystrophic phenotype after transient neonatal denervation: role of MHC isoforms.

While it recently has been demonstrated that it is possible to modify the phenotypic expression of murine dystrophy (dy/dy) (i.e., prevent myofiber loss) by subjecting the extensor digitorum longus (EDL) muscle of 14-day-old dy/dy mice to transient neonatal denervation (Moschella and Ontell, 1987), the mechanism responsible for this phenomenon has not been determined. Since it has been suggested that the effects of dystrophy vary according to fiber type, the fiber type frequency in 100-day-old normal (+/+) and dy/dy EDL muscles subjected to transient neonatal denervation has been determined by immunohistochemical analysis of their myosin heavy chain (MHC) composition. This frequency has been compared with that found in the EDL muscles of 14- and 100-day-old unoperated +/+ and dy/dy mice, in order to determine whether the reinnervation of transiently denervated neonatal muscle results in a preponderance of fibers of the type that might be spared dystrophic deterioration. In unoperated dy/dy muscle there is a progressive decrease in the frequency and in the absolute number of fibers that express MHC2B, with 100-day-old dy/dy muscles having approximately 32% of the number of myofibers fibers containing MHC2B as is found in age-matched +/+ muscles. The number of fibers containing the other fast isoforms (MHC2A and MHC2X) is similar in +/+ and dy/dy muscles at this age, indicating that fibers with MHC2B are most affected by the dystrophic process. Reinnervation following transient neonatal denervation of both the +/+ and the dy/dy EDL muscles results in a similar decrease (approximately 62%) in the number of myofibers containing MHC2B and an increase in myofibers containing the other fast MHC isoforms (MHC2A and MHC2X). The selective effect of dy/dy on fibers containing MHC2B and the sparing of myofibers in transiently denervated dy/dy muscle (which contains a reduced frequency of fibers containing MHC2B) are consistent with, although not direct proof of, the hypothesis that alterations in the fiber type may play a role in the failure of myofibers in transiently denervated dy/dy muscles to undergo dystrophic deterioration. Evidence is presented suggesting that neurons that supply myofibers containing MHC2B may be at a selective disadvantage in their ability to reinnervate neonatally denervated muscles.

Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJdy/dy) muscle.

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration-regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJdy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) per milligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability.

Ablation of the fetal mouse spinal cord: the effect on soleus muscle cytoarchitecture.

A technique is described whereby it is possible to surgically ablate the lumbosacral spinal cord of a developing mouse fetus without interfering with fetal viability. The lumbosacral spinal cords of 14-day in utero, 129ReJ mice were ablated with a Cooper Nd-YAG laser, and the fetuses, enclosed in their membranes and attached to the uterus by their placentae, were allowed to develop in the abdominal cavity of the dam. The cytoarchitecture and the temporal pattern of organogenesis of aneural soleus muscles were studied in spaced, serial, transverse, ultrathin sections of muscles of 16- and 18-day gestation and newborn (20-day gestation) mice. At the time of surgery, the soleus muscle was a discrete mass consisting of primary myotubes and a pleomorphic population of mononucleated cells. Axon bundles and blood vessels were found at the muscle's periphery, but had not penetrated throughout the muscle mass. The organogenesis of the aneural muscle was remarkably similar to that of the innervated soleus muscle (Ontell et al., Am J Anat 181:267-278, 1988). In the aneural muscle, as in the innervated muscle, significant numbers of secondary myotubes formed all along the lengths of primary myotubes. Moreover, the time course of myotube formation, the dynamics of cluster formation and cluster dispersal, and the ultrastructural appearance of the myotubes mimicked that observed in innervated muscle. The frequency of necrotic myotubes was no greater in the aneural muscle than in the innervated soleus muscle. Myotube maturation was similar in aneural and innervated soleus muscles until 18 days gestation. However, at birth, aneural myotubes appeared to be slightly less mature than innervated myotubes. Thus, the major morphogenic phenomena that characterize the development of the soleus muscle appear to be independent of innervation.

Morphometric analysis of the developing, murine aneural soleus muscle.

The pattern of organogenesis of the aneural soleus muscle of the 129ReJ mouse [rendered aneural by laser ablation of the lumbosacral spinal cord at 14 days in utero (during the period of primary myotube formation, but prior to the formation of secondary myotubes)] was evaluated quantitatively with spaced, serial ultrathin sections and computer-assisted morphometric analysis. Aneural muscles from 16- and 18-day gestation and newborn mice were analyzed to determine age-related changes in a number of parameters including: muscles' maximal girths, numbers of myotubes, myotube diameter distributions, and cluster frequency. Data were compared with a similar study of the organogenesis of the normal soleus muscle (Ontell et al: Am J Anat 181:279-288, 1988). Basic patterns of morphogenesis of the soleus muscle were unchanged by spinal cord ablation, and differences in development between the aneural and innervated muscles were quantitative rather than qualitative. At birth, the aneural muscle contained approximately 76% of the myotubes found in the innervated muscle (approximately 840 myotubes in the innervated muscle and approximately 640 in the aneural muscle). Evidence is presented consistent with the hypothesis that primary myotube formation is reduced by approximately 32% in the aneural muscles and that while extensive secondary myotube formation occurs (approximately 78% of the myotube present at birth in these muscles are secondary myotubes), there is a significant reduction in the number of secondary myotubes in aneural muscles. It is suggested that the reduced numbers of secondary myotubes may be related to the reduction in the number of primary myotubes, which are known to act as scaffolds for secondary myotube formation. The time course of secondary myotube formation and of cluster formation and cluster dispersal and the number of cells per cluster are similar in age-matched, innervated and aneural muscles. The absence of innervation has little effect on myotube growth until birth, when comparison of the myotube diameter distributions reveals a slight alteration in myotube diameter distributions of aneural as compared with innervated muscles.