Orna Halevy

Pattern of Pax7 Expression During Myogenesis in the Posthatch Chicken Establishes a Model for Satellite Cell Differentiation and Renewal

The paired‐box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7‐expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity. Developmental Dynamics 231:489–502, 2004.

Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rats age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.

HEPATOCYTE GROWTH FACTOR PLAYS A DUAL ROLE IN REGULATING SKELETAL MUSCLE SATELLITE CELL PROLIFERATION AND DIFFERENTIATION

The role of hepatocyte growth factor (HGF) and its receptor, c-met, in proliferation and differentiation of satellite cells was studied in primary cultures of chicken skeletal muscle satellite cells and a myogenic C2 cell line. HGF mRNA was expressed mainly in the myotubes of both cultures. The addition of conditioned medium derived from those cultures had a scattering effect on the canine kidney epithelial cell line, MDCK. In contrast, c-met mRNA levels decreased during cell differentiation of C2 and primary satellite cells. Application of exogenous HGF to chicken myoblasts resulted in their enhanced DNA synthesis. Among several growth factors, HGF was the first to induce DNA synthesis in quiescent satellite cells, thereby driving them into the cell cycle. Ectopic expression of chicken HGF in primary satellite cells suppressed the activation of muscle-regulatory gene reporter constructs MCK-CAT, MRF4-CAT, MEF2-CAT and 4Rtk-CAT, as well as the gene expression of MyoD and myogenin, and MHC protein expression. Ectopic MyoD reversed HGFs inhibitory effect on MCK transactivation. These data suggest that HGF inhibits cell differentiation by inhibiting the activity of basic helix-loop-helix (bHLH)/E protein heterodimers, thus inhibiting myogenic determination factor activity and subsequent muscle-specific protein expression. During muscle growth and regeneration, HGF plays a dual role in satellite-cell myogenesis, affecting both the proliferation and differentiation of these cells in a paracrine fashion.

Skeletal muscle cell activation by low-energy laser irradiation: A role for the MAPK/ERK pathway

Low‐energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum‐starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum‐starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI‐ and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N‐terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3‐sec irradiation induced ERK1/2 phosphorylation, a 12‐sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis α (TNF‐α) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

β-hydroxy-β-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.

Halofuginone: An inhibitor of collagen type I synthesis

The effect of halofuginone--a plant alkaloid used as a coccidiostat in birds--on collagen metabolism was studied in various avian and mammalian cell cultures. In avian skin fibroblasts halofuginone attenuated the incorporation of [3H]proline into collagenase-digestible proteins (CDP) at concentrations as low as 10(-11) M, without affecting production of [3H]collagenase-nondigestible proteins (NCDP), cell proliferation or collagen degradation. Halofuginone depressed specifically the expression of alpha 1 gene of collagen type I but not that of collagen type II. This was demonstrated in skin fibroblasts and growth-plate chondrocytes using probes containing inserts sequences corresponding to the alpha 1(I) and alpha 1(II) mRNAs. A slight inhibition of the expression of alpha 2(I) was observed in avian skin fibroblasts but not in growth-plate chondrocytes. The inhibition of gene expression of both polypeptides of collagen type I in skin fibroblasts resulted in a decrease in synthesis, as demonstrated by immunoprecipitation with specific type I collagen antiserum. In primary cultures of mouse skin fibroblasts, avian epiphyseal growth plate chondrocytes and a rat embryo cell line--all of which produce and secrete collagen type I--halofuginone inhibited the incorporation of [3H]proline into CDP, the Rat-1 line being the most sensitive to the drug. These results suggest that halofuginone affects specifically type I collagen synthesis by repressing gene-expression. The need for extremely low concentrations of halofuginone to inhibit collagen type I synthesis, regardless of the tissue or animal species, contributes to the potential usefulness of the substance in studying collagen metabolism.

Prevention of muscle fibrosis and improvement in muscle performance in the mdx mouse by halofuginone

Fibrosis is a known feature of dystrophic muscles, particularly the diaphragm, in the mdx mouse. In this study we evaluated the effect of halofuginone, a collagen synthesis inhibitor, on collagen synthesis in various muscles of young wild-type (C57/BL/6J) and mdx mice. Halofuginone prevented the age-dependent increase in collagen synthesis in the diaphragms of mdx with no effect on wild-type mice (n = 5 for each time point). This was associated with a decrease in the degenerated areas and number of central nuclei. Halofuginone also inhibited collagen synthesis in cardiac muscle. Moreover, enhanced motor coordination, balance and improved cardiac muscle function were observed implying reduced muscle injury. Halofuginone inhibited Smad3 phosphorylation downstream of TGFbeta in the diaphragm and cardiac muscles, in C2 cell line and in primary mouse myoblast cultures representing various muscular dystrophies. We suggest that via its effect on Smad3 phosphorylation, halofuginone inhibits muscle fibrosis and improves cardiac and skeletal muscle functions in mdx mice.

Thermal Manipulations During Broiler Embryogenesis: Effect on the Acquisition of Thermotolerance

Rapid growth rate has presented broiler chickens with serious difficulties when called on to thermoregulate efficiently in hot environmental conditions. Altering the incubation temperature may induce an improvement in the acquisition of thermotolerance (AT). This study aimed to elucidate the effect of thermal manipulations (TM) during the development of the thyroid and adrenal axis of broiler embryos on the potential of broilers to withstand acute thermal stress at marketing age. Cobb broiler embryos were subjected to TM at 39.5 degrees C and 65% RH from embryonic day 7 to 16 (inclusive), either continuously (24 h) or intermittently (12 h). After hatching chicks were raised under standard conditions to 35 d of age and then subjected to thermal challenge (35 degrees C for 5 h). Continuous TM caused a significant decline in hatchability, coupled with significantly lower BW and body temperature at hatching. The intermittent (12-h) chicks showed results similar to the controls but had significantly lower body temperature. Thermal challenge at marketing age demonstrated a significant improvement in AT in both the 12- and 24-h TM-treated broilers, which was characterized by a significantly lower level of stress (as evidenced by the level of plasma corticosterone) and rate of mortality. It was concluded that TM during the portion of embryogenesis when the thyroid and adrenal axis develop and mature had a long-lasting effect and improved the AT of broiler chickens. Whereas intermittent TM had no significant effect on hatchability and performance parameters, continuous TM negatively affected these parameters.

Low-energy laser irradiation enhances de novo protein synthesis via its effects on translation-regulatory proteins in skeletal muscle myoblasts.

Low-energy laser irradiation (LELI) drives quiescent skeletal muscle satellite cells into the cell cycle and enhances their proliferation, thereby promoting skeletal muscle regeneration. Ongoing protein synthesis is a prerequisite for these processes. Here, we studied the signaling pathways involved in the LELI regulation of protein synthesis. High levels of labeled [35S]methionine incorporation were detected in LELI cells as early as 20 min after irradiation, suggesting translation of pre-existing mRNAs. Induced levels of protein synthesis were detected up until 8 h after LELI implying a role for LELI in de novo protein synthesis. Elevated levels of cyclin D1, associated with augmented phosphorylation of the eukaryotic initiation factor 4E (eIF4E) and its inhibitory binding protein PHAS-I, suggested the involvement of LELI in the initiation steps of protein translation. In the presence of the MEK inhibitor, PD98059, eIF4E phosphorylation was abolished and levels of cyclin D1 were dramatically reduced. The LELI-induced PHAS-I phosphorylation was abolished after preincubation with the PI3K inhibitor, Wortmannin. Concomitantly, LELI enhanced Akt phosphorylation, which was attenuated in the presence of Wortmannin. Taken together, these results suggest that LELI induces protein translation via the PI3K/Akt and Ras/Raf/ERK pathways.

Inhibition of collagen type I synthesis by skin fibroblasts of graft versus host disease and scleroderma patients: effect of halofuginone.

The effect of halofuginone (a plant alkaloid) on collagen alpha 1(I) gene expression and collagen synthesis was evaluated in human skin fibroblasts from patients with chronic graft-versus-host disease (cGvHD) or scleroderma and from a normal individual. Halofuginone caused a dose-dependent inhibition in collagen alpha 1(I) gene expression and collagen synthesis in all cultures tested, the cGvHD fibroblasts being the least sensitive. In normal and scleroderma fibroblasts, concentrations of halofuginone as low as 10(-10) M and 10(-9) M were sufficient to cause a significant reduction in collagen alpha 1(I) gene expression and collagen synthesis, respectively. In addition, halofuginone also inhibited the transforming growth factor beta-induced collagen synthesis. Three days after halofuginone removal, collagen gene expression returned to control levels. The reduction of collagen mRNA transcript levels by halofuginone appeared to be dependent on new protein synthesis because simultaneous treatment of fibroblasts with protein synthesis inhibitors prevents the suppressive effect of halofuginone on collagen alpha 1(I) mRNA gene expression. The ability of extremely low concentrations of halofuginone to inhibit collagen alpha 1(I) synthesis specifically and transiently at the transcriptional level suggests that this material may be an important tool for studying collagen alpha 1(I) gene regulation and may be used as a novel and promising antifibrotic therapy.