Biomedical subjects

M P Ontell

Publications and source records attributed to M P Ontell.

13 recordsLinked to original sources

PubMed2004

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed1999

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.

Age Factors↗

PubMed1998

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1995

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.

Animals↗

PubMed1993

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)

Animals↗

PubMed1993

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)

Actins↗

PubMed1992

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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1991

Emergence of the mature myosin phenotype in the rat diaphragm muscle.

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.

Age Factors↗

PubMed1980

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.

DNA Replication↗

PubMedDate not supplied

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.

Animals↗