Myoblast Cultures (myoblast + culture)

Distribution by Scientific Domains

Selected Abstracts

Expression of phospholipase C beta family isoenzymes in C2C12 myoblasts during terminal differentiation,

Irene Faenza
In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLC,) family in muscle differentiation, given that nuclear PLC,1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLC,1, ,2, ,3, ,4 have been assessed. As further characterization, we investigated the localization of PLC, isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLC,4 was the most expressed isoform in the cytoplasm, whereas PLC,1 and ,3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLC,1 isoform was expressed at the highest extent. A marked decrease of PLC,4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLC,3 at both cytoplasmatic and nuclear level, whilst PLC,2 expression was almost undetactable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLC,1 and the decrease of cytoplasmatic PLC,4, during the establishment of myotube differentiation, is related to a spatial-temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLC,s. 2004 Wiley-Liss, Inc. [source]

Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity

M. Zimowska
Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N -acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity. 2005 Wiley-Liss, Inc. [source]

Myostatin down-regulates the IGF-2 expression via ALK-Smad signaling during myogenesis in cattle

ABSTRACT Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin-like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF-1 mRNA was similarly expressed in M. longissimus thoracis of double-muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF-2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF-2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM-myoblasts) as compared with differentiating NM derived myoblasts (NM-myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF-2 mRNA expression and decreased myotube formation, but did not effect IGF-1 mRNA expression. An activin-like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM-myoblasts, and restored the attenuated IGF-2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF-2 expression via ALK-Smad signaling. [source]

Differentiation rather than aging of muscle stem cells abolishes their telomerase activity

Matthew S. O'Connor
Abstract A general feature of stem cells is the ability to routinely proliferate to build, maintain, and repair organ systems. Accordingly, embryonic and germline, as well as some adult stem cells, produce the telomerase enzyme at various levels of expression. Our results show that, while muscle is a largely postmitotic tissue, the muscle stem cells (satellite cells) that maintain this biological system throughout adult life do indeed display robust telomerase activity. Conversely, primary myoblasts (the immediate progeny of satellite cells) quickly and dramatically downregulate telomerase activity. This work thus suggests that satellite cells, and early transient myoblasts, may be more promising therapeutic candidates for regenerative medicine than traditionally utilized myoblast cultures. Muscle atrophy accompanies human aging, and satellite cells endogenous to aged muscle can be triggered to regenerate old tissue by exogenous molecular cues. Therefore, we also examined whether these aged muscle stem cells would produce tissue that is "young" with respect to telomere maintenance. Interestingly, this work shows that the telomerase activity in muscle stem cells is largely retained into old age wintin inbred "long" telomere mice and in wild-derived short telomere mouse strains, and that age-specific telomere shortening is undetectable in the old differentiated muscle fibers of either strain. Summarily, this work establishes that young and old muscle stem cells, but not necessarily their progeny, myoblasts, are likely to produce tissue with normal telomere maintenance when used in molecular and regenerative medicine approaches for tissue repair. 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]