Martin P. Ontell

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.

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. Dev. Dyn. 1998;211:177–190.

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 β-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.

Limitations of nlsβ‐galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

Nuclear localizing β‐galactosidase (nlsβ‐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 nlsβ‐gal or its mRNA may be translocated from the nucleus that encodes it to other noncoding myonuclei in hybrid myofibers and the ease with which the encoding and noncoding 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 reexamined the extent to which nlsβ‐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 noncoding nuclei in these myofibers.

Neuronal Control of Myogenic Regulatory Factor Accumulation in Fetal Muscle

The lumbosacral spinal cords of 14.5‐day gestation mice (E14.5) were ablated. The number of molecules of each of the four myogenic regulatory factor (MRF) mRNAs per nanogram of total RNA were evaluated in innervated and aneural fetal crural muscles. Accumulation of all four MRF mRNAs was affected in aneural muscle, but was never more than threefold different than in innervated muscles, considerably less than after adult denervation. The effect of the nerve varied with the MRF, the fetal age, and with the muscle (extensor digitorum longus muscle [EDL] vs. soleus muscle), with the nerve having multiple effects including down‐regulation of certain MRF genes at specific periods (e.g., myoD and myogenin [E16.5–E18.5] and MRF4 in the EDL only [E18.5–E19.5]); limiting the up‐regulation of certain genes, which occurred in the absence of innervation (e.g., myf‐5 [E18.5–E19.5] and myogenin [E14.5–E16.5]); and even enhancing the accumulation of MRF4 mRNA (E14.5–E16.5). We hypothesize that factors other than nerve contribute to the down‐regulation of myf‐5 and myogenin mRNAs to adult levels. Innervation was required for the emergence of the slow, but not the fast, MRF mRNA profile at birth. MyoD, found in both the nuclear and cytoplasmic protein extracts of innervated fetal muscle, increased by ∼5‐fold in the nuclear extracts (∼2.5‐fold in the cytoplasmic) of E19.5 aneural muscles, significantly less than the 12‐fold increase found in the nuclear extract of 4‐day denervated adult muscle. This increase in aneural fetal muscle was due primarily to an increased concentration of myoD in muscle lineage nuclei, rather than to the presence of additional myoD+ muscle lineage nuclei. Developmental Dynamics 236:732–745, 2007.

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 β‐galactosidase (nlsβ‐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 β‐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. Dev. Dyn. 1998;211:141–152.

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.