Search

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice. inflammation; limb-girdle muscular dystrophy type 2B; Miyoshi myopathy; muscle injury doi: 10.1152/ajpcell.00122.2009

We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and So18). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2[alpha] increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPAR[alpha]) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-[gamma] coactivator-1[alpha] (PGC1[alpha]), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1[alpha]-related coactivator and PGC1[beta] was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter (~2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity. citrate synthase; muscle; transcription factor; coactivator; metabolism

The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulares the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context.

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway--activated by both epidermal and fibroblast growth factor receptors--functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. We have investigated whether these opposing signals directly interact with each other, and we have examined how they are integrated by the responding cells to specify their unique fates. Our findings reveal that Ras and Notch do not function independently; rather, we have uncovered several modes of cross-talk between these pathways. Ras induces Notch, its ligand Delta, and the epidermal growth factor receptor antagonist, Argos. We show that Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells. Key Words: signal integration; myogenesis; cardiogenesis; EGF receptor; FGF receptor; Notch; Ras; Drosophila development; cell fate specification; equivalence group.

Research has been conducted on the mutations of the gene required for the nonstriated muscle fate specification in Caenorhabditis elegans. The genetic screen for the mutations altering specification of uterine and vulval muscle fates and gene identification required for patterning in the M lineage have been investigated and described.

Sp homozygous mice were used as experimental models to study the programming of skeletal myogenesis. The results of the loss-of-function approach demonstrated that the bHLH myogenic regulatory factor Myf-5 and the paired box transcription factor Pax-3 are required by progenitor cells to generate muscles. Another bHLH myogenic regulatory factor, MyoD, on the other hand, was found to act genetically downstream of both Pax-3 and Myf-5 for the establishment of skeletal muscles in the body.

The molecular mechanism that regulates muscle gene expression, particularly the differentiation of skeletal muscles, has been the subject of recent research which led to the discovery of transcription factors such as MyoD, myogenin, Myf-5 and MRF4. It has also been recognized that Pax-3 is a major regulator of skeletal muscle development and that it is both necessary and sufficient for MyoD gene expression.

nautilus (nau), one member of the myogenic regulatory family of bHLH-encoding genes, is expressed in a subset of muscle precursors and differentiated fibers in the Drosophila embryo. To elucidate the role of nautilus in myogenesis, we have misexpressed it using the GAL4-targeted system. We find that ectopic expression results in lethality throughout Drosophila development. We analyzed the effects of embryonic expression in mesodermal tissues that include the cardioblasts of the dorsal vessel as well most, if not all, of the presumptive somatic muscle precursors. Immunohistochemical staining for muscle MHC revealed abnormalities that include an absence of cardial cells, coincident with the appearance of novel muscle fibers adjacent to the dorsal vessel. Moreover, many cardioblasts express increased levels of muscle-specific genes such as myosin, actin 57B, and Mlp60A, a protein that is restricted to the somatic, visceral, and pharyngeal muscles. These data suggest that the missing cardial cells have been transformed into cells with properties similar to those of the somatic muscles. In addition, ubiquitous expression of nautilus in somatic muscle cells of these embryos resulted in muscle pattern defects. Specifically, muscles that do not normally express nautilus were frequently absent, and novel fibers were observed in positions reminiscent of nau-expressing muscles. These data imply that nautilus can alter the developmental program of muscle precursors. In summary, we suggest that nautilus induces myogenic differentiation in vivo when ectopically expressed in developing cardioblasts and may affect the myogenic differentiation program of specific muscle fibers.

Activin-A and fibroblast growth factor-2 (FGF-2) are involved in the development of the heart in chick embryos. These compounds are both secreted by the anterior endoderm. Samples of developing heart tissue were taken from stage 6 chick embryos and cultured. The addition of activin-A and FGF-2 caused the tissue to begin contracting rhythmically and to produce cardiac alpha-actin mRNA.

Examination of the dependence of somite myogenesis on neural tube/notochord, using a newly developed in vitro assay, reveals that myogenesis in single somite explants is stimulated by neural tube and this stimulation requires close somite neural tube proximity. Myogenesis induced by neural tube does not require somite segmentation. Myogenic cell numbers in somites are increased by continuous exposure to neural tube. A different mechanism explains the promotion of in vitro myogenesis in single somite explants by notochord.

Immunohistochemistry of cultured adult rat fibers reveals high levels of temporal expression of MyoD in the developing satellite cells during myogenesis. The myogenesis resembles the sequential expression of sarcomeric proteins such as alpha-smooth muscle actin and developmental sarcomeric myosin heavy chains. Basic fibroblast growth factor regulates the number of developing satellite cells.

Short range interactions influence the formation and segregation of myogenic and chondrogenic precursors in transfilter micromass cultures. The segregation depends on autocrine activity in the cell to extracellular matrix or on cell to cell contact in body living cells. The autocrine signal is transferred across the filter pores in the cultures to initial prechondrogenic condensations.

Emergence of committed cells during gastrulation of myogenic cell lineages in avian embryos requires morphogenetic movement or DNA replication. DNA replication is explained by 'intrinsic' and 'extrinsic' models containing asymmetrical cell divisions and cellular interaction as the main features. Endothelial cells do not require DNA replication during commitment of myogenic cells.

Changed states of intrinsic activation of arterioles enhances myogenic responsiveness, associated with hypertensive rate. The increased venous pressure in these animals results in greater arteriolar construction and increases myogenic reactivity or sensitivity to shear rate which is the causative factor. In vitro data reveal that the pressure-diameter curve has a significantly higher slope value in hypertensive arterioles than in normotensive arterioles. The increased myogenic responses of the skeletal muscle arterioles contrast with the diminished responses of cerebral arterioles during hypertension.

The MyoD family of proteins are DNA-binding proteins which have been localized in skeletal muscles and embryonic muscle progenitor cells. The effects of retinoic acid (RA) on the activity of these MyoD proteins in the differentiation-competent rhabdomyosarcoma cell line BA-Han-1C were investigated. The results showed that MyoD1 is constitutively expressed in this cell line. RA treatment resulted in myogenin gene expression which was dependent on protein and DNA synthesis. The results suggest that RA induces myogenic differentiation by activating myogenin gene expression. In addition, a mechanism that inhibits MyoD may also be involved.

A mitochondria-targeted rosamine library in C2C12 myogenesis was applied to identify the compound that regulates the muscle differentiation during the remarkable changes undergone by mitochondria. The findings from the study provide insight into the potential application of fluorescent small molecules in the discovery of a bioactive probe that induces a specific cellular response.

Myogenin is part of the helix-loop-helix family of skeletal muscle-specific transcription factors. The pig myogenin in chromosomal location 9q2.1.-q2.6 conforms with a segment of the human chromosome 1q31-qter which indicates its localization. It is determined through genetic mapping which shows the correspondence between the pig and human chromosomes. Such an analysis presents that myogenin physically maps to porcine chromosome 9q2.1-q2.6.