Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle

Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P < 0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P < 0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P < 0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14 days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P < 0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P < 0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.     

Ammonia elicits a different myogenic response in avian and murine myotubes

Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10 mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10 mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48 h (P < 0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P < 0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2′, 7′- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24 h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P < 0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment.     

Polymorphisms of the Myostatin Gene and Its Relationship with Reproduction Traits in the Bian Chicken

Myostatin, or growth and differentiation factor 8, is a member of the transforming growth factor-β superfamily; it functions as a negative regulator of skeletal muscle development and growth in mammals. In this study, single nucleotide polymorphisms in the 5′ regulatory region and exon 1 of the myostatin gene were detected by PCR–SSCP in the Bian, Jinghai, Youxi, and Arbor Acre chickens, and the associations of the polymorphisms with reproduction traits were analyzed. Seven SNPs (A326G, C334G, C1346T, G1375A, A1473G, G1491A, and G2283A) were found in the myostatin gene. Association analysis showed that the G2283A were significantly associated with reproduction traits. Bian chickens of the GG genotype had a greater age at first egg than those of the GA and AA genotypes (P < 0.01). Correspondingly, Bian chickens of the GA and AA genotypes had larger egg number at 300 days than those of the GG genotype (P < 0.05 and P < 0.01, respectively). Bian chickens of the AA genotype had significantly higher body weight at 300 days than those of the GG genotype (P < 0.05). These results suggested that the myostatin gene may have certain effects on reproduction traits other than merely as a negative regulator of skeletal muscle development and growth in mammals previously reported.     

Effect of metabolic syndrome risk factors and MMP-2 genetic variations on circulating MMP-2 levels in childhood obesity

Matrix metalloproteinase-2 is involved in the development of the adipose tissue, and associated with cardiovascular diseases. Metabolic risk factors (MRFs) and functional polymorphisms in the MMP-2 gene may affect its expression and activity. We investigated whether traditional MRFs and two MMP-2 gene polymorphisms (C^?1306T; rs243865, and C^?735T; rs2285053) affect circulating MMP-2 levels in children and adolescents, and whether MMP-2 polymorphisms and/or haplotype are associated with susceptibility to childhood obesity. We studied 114 healthy controls, 43 obese, and 83 obese with ≥3 MRFs children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR. Plasma MMP-2 was measured using zymography. We found positive correlations between MMP-2 concentrations and mean blood pressure in all children and adolescents group (r = 0.132; P < 0.05) and in obese children and adolescents (r = 0.247; P < 0.01). We found that the CC genotype for the C^?1306T polymorphism was more common in subjects with higher MMP-2 concentrations in controls (P = 0.003) and in the obese group (P = 0.013). The CT genotype (OR = 0.40; P < 0.01) and the T allele (OR = 0.48; P < 0.01) for the C^?735T polymorphism were less common in obese children and adolescents than in controls. The haplotypes distribution did not show significant differences between control and obese (P > 0.05). Ours findings show that blood pressure is associated with circulating MMP-2 concentrations, and that the CC genotype for the C^?1306T polymorphism was more common subjects (controls and obese) with higher MMP-2 concentrations, whereas the CT genotype and the T allele for the C^?735T polymorphism are less common in obesity.     

Analysis on DNA sequence of KiSS-1 gene and its association with litter size in goats

Three pairs of primers were designed to clone the goat KiSS-1 and scan polymorphisms and four pairs to detect polymorphisms in sexual precocious and sexual late-maturing goat breeds. A 4118 bp DNA fragment was obtained, which contains an ORF of 408 bp and encodes 135 amino acids, having a high homology with other mammals. The protein was predicted containing a signal peptide of 17 amino acids. There are two mutations (G3433A [A86T] and C3688A) in exon 3, three mutations (G296C, G454T and T505A) in intron 1 and a 18 bp deletion (?)/insertion (+) (1960–1977) in intron 2 and no mutations in exon 2. The genotype distribution didn’t show obvious difference between sexual precocious and sexual late-maturing goat breeds and no consistency within the sexual late-maturing breeds. For the 296 locus, the Jining Grey goats with genotype CC had 0.80 (P < 0.01) or 0.77 (P < 0.01) kids more than those with genotype GG or GC, respectively. No significant difference (P > 0.05) was found in litter size between GG and GC. For the 1960–1977 locus, the Jining Grey goat does with genotype ?/? had 0.77 (P < 0.01) or 0.73 (P < 0.01) kids more than those with +/+ or +/?, respectively. No significant difference (P > 0.05) was found in litter size between +/+ and +/? genotypes. For the other four loci, no significant difference (P > 0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele C of the 296 locus and allele (?) of the 1960–1977 locus in KiSS-1 and high litter size in Jining Grey goats.     

Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference

Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P < 0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning.     

Muscle-specific transgenic expression of porcine myostatin propeptide enhances muscle growth in mice

Myostatin is a well-known negative regulator of skeletal muscle growth. Inhibition of myostatin activity results in increased muscle mass. Myostatin propeptide, as a myostatin antagonist, could be applied to promote meat production in livestock such as pigs. In this study, we generated a transgenic mouse model expressing porcine myostatin propeptide under the control of muscle-specific regulatory elements. The mean body weight of transgenic mice from a line expressing the highest level of porcine myostatin propeptide was increased by 5.4 % (P = 0.023) and 3.2 % (P = 0.031) in males and females, respectively, at 8 weeks of age. Weight of carcass, fore limb and hind limb was respectively increased by 6.0 % (P = 0.038), 9.0 % (P = 0.014), 8.7 % (P = 0.036) in transgenic male mice, compared to wild-type male controls at the age of 9 weeks. Similarly, carcass, fore limb and hind limb of transgenic female mice was 11.4 % (P = 0.002), 14.5 % (P = 0.006) and 14.5 % (P = 0.03) respectively heavier than that of wild-type female mice. The mean cross-section area of muscle fiber was increased by 17 % (P = 0.002) in transgenic mice, in comparison with wild-type controls. These results demonstrated that porcine myostatin propeptide is effective in enhancement of muscle growth. The present study provided useful information for future study on generation of transgenic pigs overexpressing porcine myostatin propeptide for improvement of muscle mass.     

An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study

Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90–95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3–4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p?<?0.01) of myogenic regulatory factors (i.e., myogenic determination factor 1, myogenic factor 5, and myogenin) and myostatin (MSTN) in the fetal as compared to the adult myoblasts. Consequently, higher proliferation and differentiation ability along with higher abundance of myogenic markers and MSTN make the fetal myoblasts a better in vitro model.     

Peroxisome Proliferator-Activated Receptor γ Ligands Retard Cultured Vascular Smooth Muscle Cells Calcification Induced by High Glucose

Peroxisome proliferator-activated receptor γ (PPARγ) and its ligands have profound effects on glucose homeostasis, cardiovascular diseases, and bone metabolism. To explore the pathophysiological roles of PPARγ in diabetes with concomitant vascular calcification, we investigated changes in PPARγ expression and the effect of the PPARγ ligands troglitazone and rosiglitazone on vascular smooth muscle cell (VSMC) calcification induced by high glucose (HG, 25 mmol/L). Compared with low glucose, HG-induced VSMC calcification, and PPARγ mRNA, protein level was decreased. Troglitazone and rosiglitazone treatment markedly attenuated the VSMC calcification, whereas PPARγ antagonist GW9662 abolished the effect of rosiglitazone on calcification. Pretreatment of VSMCs with rosiglitazone, but not troglitazone, restored the loss of lineage marker expression: the protein levels of α-actin and SM-22α were increased 52 % (P < 0.05) and 53.1 % (P < 0.01), respectively, as compared with HG alone. Troglitazone and rosiglitazone reversed the change in bone-related protein expression induced by HG: decreased the mRNA levels of osteocalcin, bone morphogenetic protein 2 (BMP2), and core binding factor α 1 (Cbfα-1) by 26.9 % (P > 0.05), 50.0 % (P < 0.01), and 24.4 % (P < 0.05), and 48.4 % (P < 0.05), 41.4 % (P < 0.01) and 56.2 % (P < 0.05), respectively, and increased that of matrix Gla protein (MGP) 84.2 % (P < 0.01) and 70.0 %, respectively (P < 0.05), as compared with HG alone. GW9662 abolished the effect of rosiglitazone on Cbfα-1 and MGP expression. PPARγ ligands can inhibit VSMCs calcification induced by high glucose.     

Plasma matrix metalloproteinase-9 levels, <Emphasis Type="Italic">MMP</Emphasis>-<Emphasis Type="Italic">9</Emphasis> gene haplotypes,and cardiovascular risk in obese subjects

Plasma matrix metalloproteinase (MMP)-9 is a predictor of cardiovascular mortality, and MMP-9 polymorphisms affect plasma MMP-9 levels. However, no study examined whether MMP-9 haplotypes affect MMP-9 levels in obese adults. We examined whether MMP-9 polymorphisms and haplotypes are associated with obesity, and whether they affect MMP-9 levels in obese subjects. We examined the plasma levels of MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in 105 subjects with normal weight (controls), 100 obese subjects, and 156 obese subjects with ≥3 metabolic risk factors (MRFs). We determined genotypes for three polymorphisms: C-1562T (rs3918242), Q279R (A>G, rs17576), and R668Q (G>A, rs17577). MMP-9 levels and activity (MMP-9/TIMP-1 ratio) were higher in obese subjects than in controls (P < 0.05). However, MMP-9 levels were higher in obese subjects with ≥3 MRFs than in obese subjects (P < 0.05). Obese subjects with ≥3 MRFs carrying the GA+AA genotypes for R668Q (G>A) polymorphism had higher MMP-9 levels than subjects carrying the AA genotype (P < 0.05). The “T, G, A” haplotype was more common in both groups of obese subjects than in controls (OR 3.95 and 4.39, respectively; P < 0.01). Notably, obese subjects with ≥3 MRFs carrying the “T, G, A” haplotype had higher MMP-9 levels than subjects carrying the “C, A, G” reference haplotype (P < 0.05). The “T, G, A” haplotype was associated with an increased risk of obesity and affected MMP-9 levels in obese subjects with ≥3 MRFs. Our findings suggest that plasma MMP-9 levels and MMP-9 haplotypes may help to discriminate obese subjects at an increased cardiovascular risk.     

The influence of maternal nutrition on expression of genes responsible for adipogenesis and myogenesis in the bovine fetus

The objective of this study was to determine whether altered maternal energy supply during mid-gestation results in differences in muscle histology or genes regulating fetal adipose and muscle development. In total, 22 Angus cross-bred heifers (BW=527.73±8.3 kg) were assigned randomly to the three dietary treatments providing 146% (HIGH; n=7), 87% (INT; n=7) or 72% (LOW; n=8) of the energy requirements for heifers from day 85 to day 180 of gestation. Fetuses were removed via cesarean section at day 180 of gestation and longissimus muscle (LM) and subcutaneous fat were collected and prepared for analysis of gene expression. Samples from the LM and semitendinosus (ST) were evaluated for muscle fiber diameter, area and number. The right hind limb was dissected and analyzed to determine compositional analysis. Fetal growth and muscle histology characteristics of the LM and ST were similar among treatments. Preadipocyte factor-1 expression was up-regulated in fetal LM (P<0.05) of HIGH fetuses as compared with INT, whereas LOW fetuses showed increased CCAAT/enhancer-binding protein-β (C/EBP-β) expression in LM as compared with INT (P<0.05). Peroxisome proliferator-activated receptor γand C/EBP-α did not differ as a result of dietary treatment in LM or subcutaneous fat samples. There was a tendency for increased expression of fatty acid synthase in LM of LOW fetuses as compared with INT (P<0.10). Myogenin was more highly expressed (P<0.05) in LM of the LOW fetuses, whereas μ-calpain expression was increased in the HIGH treatment compared with INT. A tendency for increased expression of IGF-II was observed for both LOW and HIGH fetuses compared with INT (P<0.10). Expression of stearoyl-CoA desaturase, myoblast determination protein 1, myogenic factor 5, myogenic regulatory factor-4, m-calpain, calpastatin, IGF-I and myostatin was similar between treatments. Collectively, these results suggest that fetal growth characteristics are not affected by the level of maternal nutritional manipulation imposed in this study during mid-gestation. However, differences in expression of fetal genes regulating adipose and muscle tissue growth and development could lead to differences in postnatal composition and warrants further investigation.     

Evaluation of Interferon Response Induced by Anti-Myostatin shRNA Constructs in Goat (Capra hircus) Fetal Fibroblasts by Quantitative Real TIME-Polymerase Chain Reaction

RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p < 0.05) of myostatin mRNA, respectively. Efficient suppression (42–86%) of MSTN gene (p < 0.05) was achieved even by reducing the concentration of shRNA constructs. The induction of classical interferon stimulated gene (Oligoandenylate Synthetase-1, OAS-1) was studied to analyze the immune response against shRNAs. Notably, a reduction in the potency of shRNAs to induce interferon response was observed at lower concentration for OAS1 gene. The results obtained in the study would be helpful in the abrogation of the bystander effects of RNAi for long term stable expression of anti-MSTN expression constructs in the muscle.     

Identification of novel SNPs in the Sarda breed goats POU1F1 gene and their association with milk productive performance

The aim of the study was to detect polymorphism in the POU1F1 gene in Sarda breed goat, as well as to establish if SNPs could be associated with milk productive traits. The research was conducted on 129 Sarda breed goats from 4 to 5 years old, multiparous, lactating and in their third to fifth lactation. We report nine exonic and seven non-coding regions SNPs within the Sarda goat POU1F1 gene, namely, Ex 1 61 G>C; Ex 1 108 G>A; Ex 3 C>T; Ex 3 92 C>T; Ex 4 110 A>G; Ex 5 34 G>A resulting in Arg213Lys change; IVS4 641 G>A, IVS4 643 A>C, IVS4 659 G>A, IVS4 677 A>C, IVS4 G699Del, IVS4 709 C>G, Ex 6 17 G>A resulting in Arg228Ser change, Ex 6 58 G>T, Ex 6 172 T>C, 3′UTR 110 T>C. A statistically significant association was found between genotype TT, in position 17 of the exon 6 (3.1 % of frequency), and increased milk yield (P < 0.01) while genotype GT (25.6 % of frequency) was associated with a higher fat content. Genotype TT in position 58 of the exon 6 (3.9 % of frequency) was found to be associated with a higher fat (P < 0.01) and protein content (P < 0.05). Twenty-eight haplotypes were detected, but no significant association between the haplotypes and the milk production traits have been found. Our data, as well as providing new SNPs extending the POU1F1 gene characterization, evidence a relationship between polymorphism and milk production traits in Sarda goat breed.     

ELISA examining urinary angiotensinogen as a potential indicator of intrarenal renin–angiotensin system (RAS) activity: a clinical study of 128 chronic kidney disease patients

In the current study, we measured urinary angiotensinogen (AGT) through enzyme-linked immunoadsordent assay (ELISA) and analyzed its correlation with intrarenal renin–angiotensin system (RAS) activity in 128 chronic kidney disease (CKD) patients. Urinary and plasma renin activity, AGT, angiotensin II (Ang II) and aldosterone levels were also measured by radioimmunoassay (RIA) or ELISA in these participants. Further, the expression level of intrarenal renin, AGT, Ang II and Ang II receptors were examined by immunohistochemistry staining (IHCS) in 72 CKD patients. Their correlations with urinary AGT were also analyzed. We found that the urinary AGT level was positively correlated with hypertension (ρ = 0.28, P < 0.01), urinary protein (r = 0.38, P < 0.01), urinary Ang II (r = 0.29, P < 0.05), urinary type IV collagen (Col IV) (r = 0.56, P < 0.01), and was negatively correlated with estimated glomerular filtration rate (eGFR) (r = ?0.28, P < 0.01), urinary sodium (r = ?0.22, P < 0.05) and serum AGT (r = ?0.27, P < 0.01). Multiple regression analysis indicated low serum AGT (P < 0.01), high urinary protein (P < 0.01), high urinary Ang II (P < 0.05) and high urinary Col IV (P < 0.01) were correlated significantly with high urinary AGT. Urinary AGT level was positively correlated with intrarenal expression level of AGT (ρ = 0.46, P < 0.01), Ang II (ρ = 0.56, P < 0.01) and Ang II type 1 receptor (ρ = 0.32, P < 0.01), as detected by IHCS. Together, these data suggest that urinary AGT might be a potential biomarker of intrarenal RAS and Ang II activities in CKD patients.     

Relationships Between Single Nucleotide Polymorphisms of the H-FABP Gene and Slaughter and Meat Quality Traits in Chicken

Using PCR-SSCP with five primer pairs, we detected six single nucleotide polymorphisms of the H-FABP gene: 332G → A, 534G → A, 783C → T, 835C → T, 1198T → C, and 2329C → T. Chi-square results showed significant differences (P < 0.05) in genotype frequency among breeds in Fragment 1 and extremely significant differences (P < 0.01) in Fragments 2–4. We found a significant association between Fragment 2 genotype and muscle fiber number, Arg and Thr (P < 0.05); between Fragment 3 genotype and living weight, carcass weight, breast muscle weight, abdominal fat weight, and abdominal fat percentage (P < 0.05); between Fragment 4 genotype and Thr, Phe, and inosinic acid (P < 0.05). It was concluded that H-FABP was the major gene influencing slaughter performance and meat quality or was linked with the major gene in these strains and that the C783T mutation could be used as a candidate molecular genetic marker for breeding selection. The combination M1C2–B2B2–D1D1 is an ideal model for breeding in these strains because it can improve slaughter and meat quality traits.