Bovine Myoblasts Research Articles

Myostatin alters glucose transporter-4 (GLUT4) expression in bovine skeletal muscles and myoblasts isolated from double-muscled (DM) and normal-muscled (NM) Japanese shorthorn cattle

The purpose of this study was to determine whether myostatin alters glucose transporter-4 (GLUT4) expression in bovine skeletal muscles and myoblasts isolated from double-muscled (DM) and normal-muscled (NM) Japanese Shorthorn cattle. Plasma concentrations of glucose were lower in DM cattle than in NM cattle (P < 0.01). The expression of GLUT4 messenger RNA (mRNA) in the skeletal muscle ex vivo and in myoblasts at 72 h after differentiation in vitro was higher in DM cattle than in NM cattle (P < 0.01). In contrast, the NM and DM cattle did not differ with respect to skeletal muscle expression of GLUT1 and myocyte enhancer factor-2c (MEF2c), a transcription factor of GLUT4. In differentiated myoblasts, the expression of GLUT1, GLUT4, and MEF2c mRNAs was greater in DM cattle than in NM cattle (P < 0.01). In the presence and absence of insulin, glucose uptake in myoblasts was increased in DM cattle relative to that of NM cattle (P < 0.01). The addition of myostatin decreased the expression of GLUT4 and MEF2c mRNAs in DM myoblasts (P < 0.05). Results of the present study suggest that myostatin inhibits the expression of GLUT4 mRNA possibly via MEF2c and that the greater ability of the DM cattle to produce muscle relative to the NM cattle may be due to their greater sensitivity to insulin and greater use of glucose.

Expression analysis of Gli1 and Gli2 in different tissues and muscle-derived cells of Qinchuan cattle.

The Hedgehog (Hh) signaling pathway regulates the differentiation of many kinds of cells and plays a critical role in many embryonic and postnatal developmental processes. Gli1 and Gli2 are two transcription factors of the Hh signaling pathway. In this study, we used quantitative real-time polymerase chain reaction to detect the relative expression of Gli1 and Gli2 in 13 tissues from three two-year-old purebred Qinchuan cattle, as well as in different cell populations derived from muscle and different stages of myogenic differentiation of myoblasts. The expression levels of Gli1 and Gli2 in muscle were the lowest of the 13 tissues (P < 0.05), and they declined predominantly from preplate (pp)1 to pp6 cells. However, the expression of Gli2 was elevated during myogenic differentiation until the 6th day. We speculated that Hh signaling was negatively activated in myocytes and quiescent myoblasts. The increased expression of Gli1 and Gli2 in the early days of myogenic differentiation suggested that Hh signaling would be activated when the quiescent bovine myoblast was stimulated to initiate myogenic differentiation.

Role of caspase‐9 in the effector caspases and genome expressions, and growth of bovine skeletal myoblasts

Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48h. Results indicated that all three siRNAs could silence the caspase-9 mRNA expression significantly. Particularly, the mRNA expression level of caspase-9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase-9 led to the significant increases in the mRNA and protein expressions of effector caspase-3, whereas the reduction in mRNA and protein expressions of caspase-7. The microarray results showed that the suppression of caspase-9 resulted in significant upregulations of cell proliferation-, adhesion-, growth-, development- and division-regulating genes, whereas the reduction in the expressions of cell death program- and stress response-regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase-9 could play an important role in the control of cell growth, and knockdown of caspase-9 may have genuine potential in the treatment of skeletal muscle atrophy.

한우(Bos taurus coreanae) 유래 myoblast에서 전사인자 과발현에 의한 지방세포로의 교차 분화 유도

본 연구는 한우유래 myoblast에서 지방세포분화 유도 전사인자들을 과발현시켜 지방세포로의 교차분화를 유도하기 위하여 실시하였다. 한우 유래 satellite cell을 배양한 후 adipogenic transcription factor인 <TEX>$PPAR{\gamma}$</TEX>, C/<TEX>$EBP{\alpha}$</TEX>, SREBP1c, KLF5등을 단독 또는 co-transfection을 실시하여 세포에 과발현을 유도하였다. 이들 세포들은 adipogenic differentiation medium에서 2일간 배양한 후growth medium에서 8일간 추가로 배양하였다. 지방세포로의 교차분화 유무는 Oil-red O염색과 지방세포 마커 유전자들의 발현으로 확인하였다. <TEX>$PPAR{\gamma}$</TEX>과 C/<TEX>$EBP{\alpha}$</TEX>를 각각 단독으로 과발현을 유도한 경우myoblast에서 지방세포로의 교차분화를 유도하기에는 충분하지 못하였다. 그러나 <TEX>$PPAR{\gamma}$</TEX>와 C/<TEX>$EBP{\alpha}$</TEX>을 co-transfection을 실시한 경우 지방세포로의 교차분화가 유도되었고, 세포내지방구형성, 지방세포 마커유전자의 발현, 근세포 마커유전자의 발현 감소 등이 확인되었다. KLF5 와 <TEX>$PPAR{\gamma}$</TEX>를 동시에 과발현할 경우 지방세포로의 교차분화를 볼 수 있었지만 KLF단독의 경우는 교차분화를 유도하지 못하였다. 할성형SREBP1c (tSREBP1c)의 경우, 단독으로 myoblast에 과발현을 처리한 경우만으로 지방세포로의 교차분화를 유도할 수 있었다. 이들 결과는 한우유래 satellite cell을 이용하여 지방세포분화 전사인자를 단독 혹은 조합하여 이들 세포에 과발현 시킬 경우 지방세포로의 교차분화를 유도할 수 있음을 보여 주었다. The present study was conducted to investigate whether myoblasts can be transdifferentiated into adipocytes by ectopic expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-<TEX>${\gamma}$</TEX> (<TEX>$PPAR{\gamma}$</TEX>), CCAAT/enhancer-binding protein-<TEX>${\alpha}$</TEX> (C/<TEX>$EBP{\alpha}$</TEX>), sterol regulatory element binding protein-1c (SREBP1c), and Krueppel-like factor 5 (KLF5), in primary bovine satellite cells. Transcription factors were transiently transfected into primary bovine myoblasts, and the cells were cultured with adipogenic differentiation medium for 2 days and then cultured on growth medium for an additional 8 days. Ectopic expression of <TEX>$PPAR{\gamma}$</TEX> or C/<TEX>$EBP{\alpha}$</TEX> alone was insufficient to induce adipogenesis in myoblasts. However, overexpression of both <TEX>$PPAR{\gamma}$</TEX> and C/<TEX>$EBP{\alpha}$</TEX> in myoblasts was able to induce adipogenic transdifferentiation as indicated by the appearance of mature adipocytes, the induction of adipogenic gene expressions, and the suppression of myogenic gene expressions. In addition, KLF5 and <TEX>$PPAR{\gamma}$</TEX> co-transfected bovine myoblasts were converted to adipocytes but not in cells transfected with only KLF5 expression vector. Overexpression of SREBP1c alone was sufficient to induce transdifferentiation from myoblasts into adipocytes. These results demonstrate that primary bovine satellite cells can be transdifferentiated into adipocytes either by single ectopic expression or combined expression of adipogenic transcription factors in a culture system.

Co-culture of bovine muscle satellite cells with preadipocytes increases PPARγ and C/EBPβ gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes

We hypothesized that preadipocyte differentiation would be depressed by differentiating myoblasts, whereas preadipocytes would promote adipogenic gene expression in myoblasts in a co-culture system. We also determined the effects of arginine, a biological precursor of nitric oxide, and/or trans-10, cis-12 conjugated linoleic acid (CLA) on adipogenic gene expression during differentiation of bovine preadipocytes and myoblasts. Bovine semimembranosus satellite cells (BSC) and subcutaneous preadipocytes were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's modified Eagle medium (DMEM) and 1% antibiotics during the 3-day proliferation period. After proliferation, BSC and preadipocytes were treated for 3 days with 3% horse serum/DMEM and 5% FBS/DMEM with antibiotics, respectively. Media also contained 100 μM oleic acid, 10 μg/ml insulin, 1 μg/ml pioglitazone and 1 μg/ml dexamethasone. Subsequently, the differentiating myoblasts and adipocytes were cultured in their respective media with 5 mM arginine and/or 40 μM trans-10, cis-12 CLA for 4 days. Finally, myoblasts and adipocytes were single- or co-cultured for 2 h singly or in combination. Arginine stimulated SCD gene expression, whereas CLA depressed SCD gene expression in adipocytes and myoblasts (P=.002). Co-culture of adipocytes and myoblasts elicited an increase in C/EBPβ and PPARγ gene expression in differentiated myoblasts (P≤.01) and an increase in GPR43 gene expression in adipocytes (P=.01). Expression of AMPKα and CPT1ß was unaffected by co-culture, although SCD gene expression tended (P=.12) to be depressed by co-culture. These experiments demonstrated that co-culture of adipocytes with myoblasts increased adipogenic gene expression in the myoblastic cells.

Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression1

Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.

Ghrelin stimulates myogenic differentiation in a mouse muscle satellite cell line and in primary cultures of bovine myoblasts

Ghrelin is an acylated hormone that influences food intake, energy metabolism and reproduction, among others. Ghrelin may also stimulate proliferating myoblast cell differentiation and multinucleated myotube fusion. The aim of this work was to assess the effect of human ghrelin (hGHRL) and human ghrelin fragment 1-18 (hGHRL1-18) on myoblast differentiation by means of mRNA expression and protein level. Two types of cells were tested, the cell line i28 obtained from mouse skeletal muscle and primary cultures of bovine myoblasts. Both ghrelin and its N-terminal fragment hGHRL1-18 were used at concentrations of 0, 0.01, 0.1, 1, 10 and 100 nm. Treatments were applied to pre-confluent cultures and were maintained for 4 days. We determined that between 0.1 and 100 nm, hGHRL and hGRHL1-18 had similar effects on myogenic differentiation of i28 cells (p < 0.01). On the other hand, only the higher concentrations (10 and 100 nm) of hGHRL stimulated bovine myoblast differentiation. These results could be attributed to the presence, in both i28 cells and in bovine myoblasts, of the mRNA for GHS-R1a and CD36 receptors. The use of ghrelin in livestock production is still questionable because of the limited effects shown in this study, and additional research is needed in this field.

Enhanced proliferation of human skeletal muscle precursor cells derived from elderly donors cultured in estimated physiological (5%) oxygen

Human skeletal muscle precursor cells (myoblasts) have significant therapeutic potential and are a valuable research tool to study muscle cell biology. Oxygen is a critical factor in the successful culture of myoblasts with low (1-6%) oxygen culture conditions enhancing the proliferation, differentiation, and/or viability of mouse, rat, and bovine myoblasts. The specific effects of low oxygen depend on the myoblast source and oxygen concentration; however, variable oxygen conditions have not been tested in the culture of human myoblasts. In this study, muscle precursor cells were isolated from vastus lateralis muscle biopsies and myoblast cultures were established in 5% oxygen, before being divided into physiological (5%) or standard (20%) oxygen conditions for experimental analysis. Five percent oxygen increased proliferating myoblast numbers, and since low oxygen had no significant effect on myoblast viability, this increase in cell number was attributed to enhanced proliferation. The proportion of cells in the S (DNA synthesis) phase of the cell cycle was increased by 50%, and p21(Cip1) gene and protein expression was decreased in 5 versus 20% oxygen. Unlike in rodent and bovine myoblasts, the increase in myoD, myogenin, creatine kinase, and myosin heavy chain IIa gene expression during differentiation was similar in 5 and 20% oxygen; as was myotube hypertrophy. These data indicate for the first time that low oxygen culture conditions stimulate proliferation, whilst maintaining (but not enhancing) the viability and the differentiation potential of human primary myoblasts and should be considered as optimum conditions for ex-vivo expansion of these cells.

Melengestrol acetate alters muscle cell proliferation in heifers and steers1

In vitro experiments were performed to investigate the effects of melengestrol acetate (MGA) or progesterone (P4) on bovine muscle satellite cells and C2C12 myoblasts. Addition of MGA at physiological and supraphysiological concentrations resulted in a dose-dependent decrease (P < 0.05) in DNA synthesis as measured by [3H]-thymidine incorporation (TI). Similarly, P4 addition (0.01 nM) reduced (P < 0.05) TI. Addition of MGA (10 nM) increased (P < 0.05) IGF-I mRNA abundance but did not affect myogenin mRNA. Progesterone addition (10 nM) increased myogenin mRNA abundance (P < 0.05). In C2C12 cultures, P4 addition resulted in a dose-dependent decrease in TI. The antiprogestin RU486, in combination with MGA or P4, also resulted in reduced (P < 0.05) TI. Treatment with RU486 alone had a negative effect (P < 0.05) on TI that was similar to the progestins. Treatment of C2C12 myoblasts with MGA (100 nM) resulted in an increase (P < 0.05) in myogenin mRNA. These studies suggest that progestins may reduce satellite cell proliferation, ultimately affecting carcass composition.

Effect of phase limited inhibition of MyoD expression on the terminal differentiation of bovine myoblasts: No alteration of Myf5 or myogenin expression

To investigate the roles played by MyoD in the terminal differentiation of satellite cell-derived myoblasts, the effect of antisense inhibition of MyoD expression was examined in bovine adult myoblast culture, in which inhibition treatment was limited to the terminal differentiation phase. MyoD antisense oligonucleotide DNA (AS-mD) suppressed the formation of multinucleated myotubes in the cell culture. Myotube formation was suppressed even when AS-mD treatment was limited to the period preceding the onset of myotube formation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that treatment with AS-mD suppressed the expression of myosin heavy chain embryonic isoform and troponin T isoforms at 4 days after the induction of differentiation. AS-mD also suppressed the expression of MRF4, but did not alter the expression of either Myf5 or myogenin, in contrast to previous results using mouse cells possessing MyoD(-/-) genetic background. These findings suggest that MyoD controls myogenesis but not Myf5 or myogenin mRNA expression during the terminal differentiation phase. Furthermore, among the alpha4, alpha5, alpha6, and alpha7 integrins, alpha4, alpha5, and alpha7 integrin expression was suppressed by AS-mD treatment, in parallel with the suppression of myotube formation, which suggests that MyoD controls myotube formation by regulating the expression of alpha4, alpha5, and alpha7 integrins.

Comparison of in vitro proliferation and differentiation of bovine myoblasts from different genetic types

Comparison of in vitro proliferation and differentiation of bovine myoblasts from different genetic types

Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-α and -β1 action on culture of L6 and fetal bovine myoblasts

1. 1. α-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-α or -β 1 in L6 and fetal bovine myoblasts. 2. 2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and Sdenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-α and EGF. 3. 3. TGF-β 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-β 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-α, and -β 1 in L6 and fetal bovine myoblasts.

Comparison of metabolic effects of EGF, TGF-α, and TGF-β1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts

1. 1. Comparative studies of EGF, TGF-α, and TGF-βl action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells. 2. 2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells. 3. 3. The dynamics of stimulation of protein synthesis by TGF-α was greater than by EGF in both examined types of cell cultures. 4. 4. The maximal response of fetal bovine myoblasts to TGF-α in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control. 5. 5. In contrast to EGF, TGF-α significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-α may indicate changes toward cell differentiation. 6. 6. TGF-β 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-β 1 was more distinct in fetal bovine myoblasts than in the L6 cell line. 7. 7. Increasing concentrations of TGF-β l diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.

Effect of fibroblast growth factor on the division and fusion of bovine myoblasts.

The effect of fibroblast growth factor (FGF) on the rate of proliferation and fusion of bovine myoblast has been examined. Addition to the cultures of 0.1 mug-1 mug/ml of FGF stimulates the rate of proliferation and delays the fusion of primary cultures of bovine myoblasts cultured in 10% serum. Final cell densities reached in the presence of 0.1 mug/ml of FGF were fivefold higher than in controls; with 1 mug/ml, they were 10-fold higher. Increases in cell density were paralleled by increases in acetylcholine receptor sites as measured by the binding of 125I-alpha-bungarotoxin. Both fusion and the appearance of acetylcholine receptor sites were delayed in the presence of FGF. Growth hormone, insulin and testosterone, which have been reported to be mitogenic for rat and chick embryo myoblasts, did not have significant effects on DNA synthesis in bovine myoblasts when compared to the FGF. Conversely, FGF did not stimulate the proliferation of chick embryo myoblasts, indicating that it is not active in all vertebrate species.