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A study on the transdetermination can serve as a model for mechanisms of stem-cell commitment was conducted. The data provided suggests that pluripotency of cells within regenerating tissues is constrained by homeotic products.

In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer [N.sup.G]-nitro-D-arginine methyl ester HC1 (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which call activate satellite cells. muscle regeneration

Current approaches to tissue regeneration are limited by the death of most transplanted cells and/or resultant poor integration of transplanted cells with host tissue. We hypothesized that transplanting progenitor cells within synthetic microenvironments that maintain viability, prevent terminal differentiation, and promote outward migration would significantly enhance their repopulation and regeneration of damaged host tissue. This hypothesis was addressed in the context of muscle regeneration by transplanting satellite cells to muscle laceration sites on a delivery vehicle releasing factors that induce cell activation and migration (hepatocyte growth factor and fibroblast growth factor 2) or transplantation on materials lacking factor release. Controls included direct cell injection into muscle, the implantation of blank scaffolds, and scaffolds releasing factors without cells. Injected cells demonstrated a limited repopulation of damaged muscle and led to a slight improvement in muscle regeneration, as expected. Delivery of cells on scaffolds that did not promote migration resulted in no improvement in muscle regeneration. Strikingly, delivery of cells on scaffolds that promoted myoblast activation and migration led to extensive repopulation of host muscle tissue and increased the regeneration of muscle fibers at the wound and the mass of the injured muscle. This previously undescribed strategy for cell transplantation significantly enhances muscle regeneration from transplanted cells and may be broadly applicable to the various tissues and organ systems in which provision and instruction of a cell population competent to participate in regeneration may be clinically useful. fibroblast growth factor 2 | hepatocyte growth factor | myoblast | satellite cell | tissue engineering

Muscle precursor cells (MPCs) are required for the regrowth, regeneration, and/or hypertrophy of skeletal muscle, which are deficient in sarcopenia. In the present investigation, we have addressed the issue of age-associated changes in MPC differentiation. MPCs, including satellite cells, were isolated from both young and old rat skeletal muscle with a high degree of myogenic purity (>90% MyoD and desmin positive). MPCs isolated from skeletal muscle of 32-mo-old rats exhibited decreased differentiation into myotubes and demonstrated decreased myosin heavy chain (MHC) and muscle creatine kinase (CK-M) expression compared with MPCs isolated from 3-mo-old rats. [p27.sup.Kip1] is a cyclin-dependent kinase inhibitor that has been shown to enhance muscle differentiation in culture. Herein we describe our finding that [p27.sup.Kip1] protein was lower in differentiating MPCs from skeletal muscle of 32-mo-old rats than in 3-mo-old rat skeletal muscle. Although MHC and CK-M expression were ~50% lower in differentiating MPCs isolated from 32-mo-old rats, MyoD protein content was not different and myogenin protein concentration was twofold higher. These data suggest that there are inherent differences in cell signaling during the transition from cell cycle arrest to the formation of myotubes in MPCs isolated from sarcopenic muscle. Furthermore, there is an age-associated decrease in muscle-specific protein expression in differentiating MPCs despite normal MyoD and elevated myogenin levels. satellite cells; skeletal muscle; [p27.sup.Kip1]; myogenic regulatory factors

The successful use of myogenic progenitor cells for therapeutic applications requires an understanding of the intrinsic and extrinsic cues involved in their regulation. Herein we demonstrate the expression pattern and transcriptional regulation of Rad, a prototypical member of a family of novel Ras-related GTPases, during mammalian development and skeletal muscle regeneration. Rad was identified using microarray analysis, which revealed robust upregulation of its expression during skeletal muscle regeneration. Our current findings demonstrate negligible Rad expression with resting adult skeletal muscle; however, after muscle injury, Rad is expressed within the myogenic progenitor cell population. Rad expression is significantly increased and localized to the myogenic progenitor cell population during the early phases of regeneration and within the newly regenerated myofibers during the later phases of regeneration. Immunohistochemical analysis demonstrated that Rad and MyoD are coexpressed within the myogenic progenitor cell population of regenerating skeletal muscle. This expression profile of Rad during skeletal muscle regeneration is consistent with the proposed roles for Rad in the inhibition of L-type C[a.sup.2+] channel activity and the inhibition of Rho/RhoA kinase activity. We also have demonstrated that known myogenic transcription factors (MEF2, MyoD, and Myf-5) can increase the transcriptional activity of the Rad promoter and that this ability is significantly enhanced by the presence of the C[a.sup.2+]-dependent phosphatase calcineurin. Furthermore, this enhanced transcriptional activity appears to be dependent on the presence of a conserved NFAT binding motif within the Rad promoter. Taken together, these data define Rad as a novel factor within the myogenic progenitor cells of skeletal muscle and identify key regulators of its transcriptional activity. satellite cell; myoblast; stem cell; transcriptional regulation; RGK protein

We assessed viable Pax[7.sup.-/-] mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However, a small number of regenerated myofibers were detected, suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax[3.sup.+]/Myo[D.sup.+] myoblasts were recovered from Pax[7.sup.-/-] muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally, we identified Pax[3.sup.+] cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore, we conclude that interstitial Pax[3.sup.+] cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells.

Planaria possess remarkable powers of regeneration. After bisection, one blastema regenerates a head, while the other forms a tail. The ability of previously-adjacent cells to adopt radically different fates could be due to long-range signaling allowing determination of position relative to, and the identity of, remaining tissue. However, this process is not understood at the molecular level. Following the hypothesis that gap-junctional communication (GJC) may underlie this signaling, we cloned and characterized the expression of the Innexin gene family during planarian regeneration. Planarian innexins fall into 3 groups according to both sequence and expression. The concordance between expression-based and phylogenetic grouping suggests diversification of 3 ancestral innexin genes into the large family of planarian innexins. Innexin expression was detected throughout the animal, as well as specifically in regeneration blastemas, consistent with a role in long-range signaling relevant to specification of blastema positional identity. Exposure to a GJC-blocking reagent which does not distinguish among gap junctions composed of different Innexin proteins (is not subject to compensation or redundancy) often resulted in bipolar (2-headed) animals. Taken together, the expression data and the respecification of the posterior blastema to an anteriorized fate by GJC loss-of-function suggest that innexin-based GJC mediates instructive signaling during regeneration. Keywords: Planaria; Regeneration; Innexins; Gap-junctional communication; Patterning

Muscle satellite cells contribute to muscle regeneration. We have used a [Pax3.sup.GFP/+] mouse line to directly isolate (Pax3)(green fluorescent protein)--expressing muscle satellite cells, by flow cytometry from adult skeletal muscles, as a homogeneous population of small, nongranular, Pax7+, CD34+, CD45-, Sca1-cells. The flow cytometry parameters thus established enabled us to isolate satellite cells from wild-type muscles. Such cells, grafted into muscles of mdx nu/nu mice, contributed both to fiber repair and to the muscle satellite cell compartment. Expansion of these cells in culture before engraftment reduced their regenerative capacity., Satellite cells are skeletal muscle progenitor cells responsible for postnatal growth and repair (1). The difficulty of isolating pure populations of satellite cells in sufficient number has precluded their use [...]

The historical basis and evidence for the identification of satellite cells as adult muscle stem cells is described and contributions of other cells to adult myogenesis is evaluated. Data regarding the origins, genetic markers, and molecular regulation of satellite cells is normal, diseased, and aged muscles are summarized.

Myostatin is an endogenous inhibitor of muscle conserved across diverse species. In the absence of myostatin, there is massive muscle growth in mice, cattle, and humans. Previous studies in the mdx mouse model of muscular dystrophy demonstrate that inhibiting myostatin attenuates several features of dystrophic muscle. These findings have encouraged the development of human therapies to block myostatin. However, little is known of the long-term effects on muscle of myostatin blockade. To evaluate potential sequelae from the prolonged absence of myostatin, senescent myostatin null ([mstn.sup.-/-]) mice were studied. Senescent [mstn.sup.-/-] mice continue to have normal muscle with increased mass and strength relative to controls. Muscles of senescent [mstn.sup.-/-] mice regenerate robustly from both chronic and acute injury. Early markers of regeneration are enhanced in the absence of myostatin, suggesting a mechanism for the attenuation of dystrophic features found in mdx mice lacking myostatin. aging | muscular dystrophy

Under normal circumstances, mammalian adult skeletal muscle is a stable tissue with very little turnover of nuclei. However, upon injury, skeletal muscle has the remarkable ability to initiate a rapid [...]

To investigate the cause of skeletal muscle weakening during aging we examined the sequence of cellular changes in murine muscles. Satellite cells isolated from single muscle fibers terminally differentiate progressively less well with increasing age of donor. This change is detected before decline in satellite cell numbers and all histological changes examined here. In MSVski transgenic mice, which show type IIb fiber hypertrophy, initial muscle weakness is followed by muscle degeneration in the first year of life. This degeneration is accompanied by a spectrum of changes typical of normal muscle aging and a more marked decline in satellite cell differentiation efficiency. On a myoD-null genetic background, in which satellite cell differentiation is defective, the MSVski muscle phenotype is aggravated. This suggests that, on a wild-type genetic background, satellite cells are capable of repairing MSVski fibers and preserving muscle integrity in early life. We propose that decline in myogenic cell differentiation efficiency is an early event in aging-related loss of muscle function, both in normal aging and in some late-onset muscle degenerative conditions. muscle regeneration; MyoD; myonuclear domain size

This article discusses the relationship between muscle mass, protein synthesis, and genetics. An analysis of phosphorylation, insulin receptors, and amino acid metabolism is presented.

This article discusses the relationship between muscle protein synthesis, physical activity, and amino acid metabolism. An analysis of food habits, muscle mass, and leucine metabolism is presented.

This article discusses the relationship between gene expression and resistance exercise in aged muscles. An analysis of DNA repair, cell proliferation, vascular endothelial growth factor, and heat shock proteins is presented.

Two important indices of myogenic differentiation are the formation of syncytial myotubes and the postmitotic arrest from the cell cycle, both of which occur after fusion of mononucleate cells. We show here that these indices are reversed in the environment of the urodele limb regeneration blastema. In order to introduce an integrated (genetic) marker into newt myotubes, we infected mononucleate cells in culture with a pseudotyped retrovirus expressing human placental alkaline phosphatase (AP). After fusion the myotubes expressed AP and could be purified by sieving and micromanipulation so as to remove all mononucleate cells. When such purified retrovirus-labelled myotubes were implanted into a limb blastema they gave rise to mononucleate progeny with high efficiency. Purified myotubes labelled with fluorescent lipophilic cell tracker dye also gave rise to mononucleate cells; myotubes which were double labelled with the tracker dye and a nuclear stain gave rise to double-labelled mononucleate progeny. Nuclei within retrovirus-labelled myotubes entered S phase as evidenced by widespread labelling after injection of implanted newts with BrdU. The relation between the two aspects of plasticity is a critical further question.

Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair, and maintenance of skeletal muscle. Recent studies in mice, however, have revealed the potential for highly purified hematopoietic stem cells from bone marrow to participate in muscle regeneration. Perhaps more significantly, a population of putative stem cells isolated directly from skeletal muscle efficiently reconstitutes the hematopoietic compartment and participates in muscle regeneration following intravenous injection in mice. The plasticity of muscle stem cells has raised important questions regarding the relationship between the muscle-derived stem cells and the skeletal muscle satellite cells. Furthermore, the ability of hematopoietic cells to undergo myogenesis has prompted new investigations into the embryonic origin of satellite cells. Recent developmental studies suggest that a population of satellite cells is derived from progenitors in the embryonic vasculature. Taken together, these studies provide the first evidence that pluripotential stem cells are present within adult skeletal muscle. Tissue-specific stem cells, including satellite cells, may share a common embryonic origin and possess the capacity to activate diverse genetic programs in response to environmental stimuli. Manipulation of such tissue-specific stem cells may eventually revolutionize therapies for degenerative diseases, including muscular dystrophy.

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.

We tested the hypothesis that after skeletal muscle regeneration in old compared with young rats damage to the motor nerve rather than damage to muscle fibers determines the magnitude of the deficits in muscle mass and maximum force ([P.sub.o]). The mass and [P.sub.o] of extensor digitorum longus (EDL) muscles of young (4 months) and old (24 months) male rats were compared two months following (i) Marcaine treatment plus simultaneous motor nerve transection, (ii) motor nerve transection alone, and (iii) Marcaine treatment alone (from data compiled previously). In both the nerve transection-only and Marcaine with nerve transection groups the recovery of mass and [P.sub.o] was significantly greater in young than in old rats. This is in contrast to our previous data showing that in the absence of nerve damage Marcaine-treated muscle in old rats regenerates as well as that in young rats. Our hypothesis was supported, and we conclude that impaired axonal regeneration, re-establishment of nerve-muscle contact, or both, is the critical component in the impaired regeneration of muscle grafts in old as compared with young rats.

Researchers have investigated the temporal expression pattern of messenger RNAs for insulin-like growth factor (IGF)-I, IGF-I receptor and IGF-II in the regenerating tibialis anterior muscle following injection of the myotoxic anesthetic bupivacaine in young, adult and old rats. Five days after injection, IGF-II mRNA was boosted 46-fold in young rats, but only fourfold in adult rats. The results indicate that the impairment of muscle regeneration is a maturation effect, rather than an aging effect.

The specific role of weight bearing on the expression of myosin heavy chain (MHC) isoforms in regenerative fibers after muscle degeneration was investigated using rat soleus muscles. Immunocytochemical analysis showed that almost all fibers reacted positively with the MHC antibody, which has been reported to be associated with slow-twitch muscle fibers. However, removal of weight bearing resulted in the dramatic increase in MHC expression. These results suggested that the regulation of expression of MHC isoforms is more complete and homogenous in regenerating than degenerate muscles.

Hypertension in spontaneously hypertensive rats (SHR) has no effect on the revascularization of a nerve-intact graft of the extensor digitorum longus muscle, but muscle regeneration and maturation are severely impaired. Reinnervation and neuromuscular transmission of the graft in the SHR are similar to those of a graft in Wistar-Kyoto rats. The slower regeneration in the SHR may be due to insulin resistance. Muscle insulin receptors bind the insulin-like growth factor I, which helps in development.

Heart muscle cells die en masse after injury, yet the adult mammalian heart retains little capacity to regenerate them. Regulatory microRNA sequences may stimulate self-renewal of these muscle cells. See [...]

Gamma irradiation-induced inhibition of muscle regeneration leads to loss of contractile strength in dytrophic muscles from mdx mice, which is stopped by treatment with clenbuterol, an adrenergic beta-receptor. Myosin concentrations and muscle mass are more in the clenbuterol-treated irradiated muscles than in non-irradiated muscles, indicating that beta2-agonists inhibit degenerative processes in dystrophin-depleted muscle fibers.

Study of mice with a targeted mutation in the skeletal muscle ryanodine receptor gene reveals that the ryanodine receptor is required for muscular maturation and excitation-contraction (E-C) coupling. Other subtypes of the receptor cannot replace the function of the ryanodine receptor in E-C coupling. The ryanodine receptors facilitate calcium ion release from the sarcoplasmic reticulum in mutant muscle.

An analysis of the regulatory mechanism involved in voltage-induced sodium channels (NaChs) at synaptic sites of regenerating muscles revealed the redundant role of nerve terminal during the accumulation of NaCh at neuromuscular synapses. This indicates that the signal responsible for inducing NaCh aggregation initiates from Schwann cells or basal lamina. Transitory change in expression occurring during development of immature NaCh isoform is partly localized on regenerating muscles when innervation or nerve stimulus is absent.

The closer muslce in the paired claws of the lobster Homarus americanus become determined into their asymmetric form of a cutter and crusher type claw during the 4th and 5th juvenile stages and differentiate their fiber composition accordingly in subsequent juvenile stages. Our aim was to study the effects of claw loss during this critical juvenile period on muscle regeneration. Hence the fiber composition of the paired closer muscles in newly regenerated claws was examined histochemically following removal of both claws either in the 4th and 5th stages or in the 4th through 7th stages. The newly regenerated muscle was retarded compared to its original counterpart in both cases. In the former case, however, the retardation was temporary as the muscle composition in later stages resembled the original. Recovery in the latter was not apparent in later stages, suggesting that retardation is more permanent. Also in both protocols the newly regenerated closer muscle occasionally displayed a mosaic distribution, with slow fibers interspersed among fast fibers in a central band that is normally homogenously fast. Therefore, loss of the paired claws during a developmentally sensitive period affects the phenotype of the regenerated muscle with the change persisting for shorter or longer periods depending on how often the claws are lost.

Growth and repair of skeletal muscle are normally mediated by the satellite cells that surround muscle fibers. In regenerating muscle, however, the number of myogenic precursors exceeds that of resident satellite cells, implying migration or recruitment of undifferentiated progenitors from over sources. Transplantation of genetically marked bone marrow into immunodeficient mice revealed that marrow-derived cells migrate into areas of induced muscle degeneration, undergo myogenic differentiation, and participate in the regeneration of the damaged fibers. Genetically modified, marrow-derived myogenic progenitors could potentially be used to target therapeutic genes to muscle tissue, providing an alternative strategy for treatment of muscular dystrophies., In postnatal life, growth and repair of skeletal muscle fibers are mediated by a resident population of mononuclear myogenic precursors, the satellite cells. These cells, which are located between the [...]

Byline: K. Agrawal, S. Agrawal This paper reviews the various aspects of tissue regeneration during the process of tissue expansion. 'Creep' and mechanical and biological 'stretch' are responsible for expansion. [...]

Local treatment of fibroblast growth factor-2 (FGF-2) on amputated stage 25 chick limb bud stimulates the regeneration of digit-like structures. Location of the heparan sulfate conjugated beads, loaded with FGF-2, on the limb stump is related to the regenerative response. Without the use of FGF-2, regeneration is not possible in the chick limb after amputation.