Differential expression patterns of myogenic regulatory factors in the postnatal longissimus dorsi muscle of Jeju Native Pig and Berkshire breeds along with their co-expression with Pax7

BACKGROUND: Myogenic regulatory factors (MRFs) such as MyoD, Myf6 and Myf5 play a vital role in the growth and development of muscles. Jeju Native Pig (JNP) is the top ranker in Korea amongst the indigenous livestock reared for meat purpose. Few studies covering transcript abundance of the MRFs and related to their co-expression with Pax7 in JNP have been conducted. Despite having better quality pork, JNP does not have a comparative growth rate with respect to western breeds. Therefore, the present study was designed with the objective to study the relative transcript levels of MRFs in the postnatal myogenesis of longissimus dorsi muscles in JNP and Berkshire breeds. RESULTS: Relative transcript levels were analyzed by qRT-PCR and blot expression analysis through Western blotting. Immunocytochemistry was performed to analyze their expressions at cellular levels. ToppCluster aided in the analysis of gene ontology of biological processes. The quantitative transcript levels of MyoD and Pax7 were significantly (P < 0.05) higher in Berkshire than in JNP. Myotube formation was observed under the co-expression of MyoD and Pax7. ToppCluster helped in the understanding of the linking of biological processes of the MRFs with the different signaling pathways. MyBPH had significantly (P < 0.05) high transcript levels during the chosen age groups in JNP than Berkshire. CONCLUSIONS: The current study can be helpful in understanding the genetic basis for myogenesis in postnatal stage. Moreover, it can act as stepping stone for the identification of marker genes related to body growth and meat quality in JNP.

Regulation of myostatin promoter activity by myocyte enhancer factor 2 / 生物工程学报

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

Myogenic Differentiation of Human Adipose-Derived Stem Cells

OBJECTIVE: Cell therapy has been extensively studied as a gene complementation approach in muscular dystrophy including Duchenne muscular dystrophy (DMD), and adipose tissue has recently been identified as a uniquely abundant and adequately accessible source of pluripotent cells. In the present work, we investigated myogenic potentials of adipose-derived stem cells (ADSCs) depending on culture media and isolation with using surface markers. METHOD: Human ADSCs were obtained by liposuction and cultured in two different media; control and myogenic media. In addition we attempted to isolate ADSCs by utilizing surface markers: CD45 and CD133. The following observations were made to evaluate myogenic differentiation as the expression of myogenic regulatory factors (MyoD, Myf-5 and Myf-6) and desmin by RT-PCR and immunoflurescence study. RESULTS: Conversion of ADSCs to myogenic phenotype was observed by indirect immunoflurescence study of MyoD and Myf-5 in regardless of media type and isolation method. In addition mRNA of MyoD and Myf-5 were positive in both culture media, and there were no differences of MyoD and Myf-5 responses between CD45- and CD45-CD133-ADSCs. However, secondary myogenic regulatory factor (Myf-6) was not expressed constantly, and desmin were negative in all cultural condition. CONCLUSION: Our findings suggest that human ADSCs might have myogenic potentials. However, further studies are needed to express the secondary myogenic regulatory factors and proteins in myoblasts.

Adaptações morfofuncionais e respostas moleculares do músculo esquelético de ratos submetidos ao treinamento resistido / Morphofunctional adaptations an molecular responses of skeletal muscle of rats submitted to resistance training

Embora fortes evidências demonstrem que os fatores de regulação miogênica (MRFs) e o fator de crescimento semelhante à insulina (IGF-I) apresentem um importante papel na resposta hipertrófica após treinamento resistido (TR) agudo, permanece desconhecido se a resposta dos MRFs e IGF-I também ocorre durante a adaptação ao TR a longo-prazo. Portanto, o objetivo deste estudo foi testar a hipótese de que a resposta hipertrófica e modulação das fibras do músculo esquelético após TR a longo-prazo poderia estar associada ao aumento na expressão gênica dos MRFs e IGF-I. Ratos Wistar (80 dias de idade, 250-300 g) foram divididos em quatro grupos: Controle 8 semana (C8, n = 8), Treinado 8 semanas (T8, n = 8), Controle 12 semanas (C12, n = 8) e Treinado 12 semanas (T12, n = 8). Os grupos T8 e T12 foram submetidos a um programa de TR progressivo (3 dias/semana) durante 8 e 12 semanas, respectivamente. O protocolo de treinamento consistiu de quatro séries de 10-12 repetições, com um período de descanso de 40 segundos entre cada série, realizado a 65-75% de uma repetição máxima (1RM). Ao término do experimento, os animais foram sacrificados e o músculo plantar coletado para as análises morfológica e molecular. O TR durante 8 e 12 semanas não promoveu qualquer alteração (p > 0,05) significante no ganho de peso corporal e consumo alimentar dos grupos T8 e T12 em relação aos grupos C8 e C12, respectivamente...

Although strong evidence show that the myogenic regulatory factors (MRFs) and insulin-like growth factor (IGF-I) have important roles in the hypertrophy response after acute resistance training, it is still unclear if response of MRFs and IGF-I also occurs during the adaptation to prolonged periods of resistance training (RT). Therefore, the purpose of this study was to test the hypothesis that fiber-types transition and hypertrophy during long-term RT could be associated with increased MRFs and IGF-I mRNA expression in the skeletal muscle. Male Wistar rats (80 days old, 250-300 g) were divided into four groups: 8 weeks control (C8, n = 8), 8-weeks trained (T8, n = 8), 12-weeks control (C12, n = 8), 12-weeks trained (T12, n = 8). T8 and T12 groups were submitted to a progressive RT program (3 day/week) for 8 and 12 weeks, respectively. The training protocol consisted of four sets of 10–12 repetitions, with a 40 s rest period between each set, performed at 65–75% of one repetition maximum (1RM). At the end of the experiment, animals were sacrificed and the plantaris muscle collected for morphological and molecular analysis. The RT did not change (p > 0.05) in body weight gain and food intake in the T8 and T12 compared to the C8 and C12 groups, respectively...

Expression of MEF2D on nasopharyngeal carcinoma tissues and its influence of prognostic / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the expression of MEF2D in NPC tissues, study the relationship between the expression and prognostic.@*METHOD@#Specimens from 101 NPC patients who were follow-up visited 1 to 7 years were analyzed for MEF2D by using immunohistochemistry.@*RESULT@#(1) The expression of MEF2D was higher in the higher clinical stage. (2) Density and Grey of MEF2D was negative correlated (|r| = 0.865, P < 0.01). (3) NPC patients' survival rate after therapies was 52.5%, the survival curve of 1th clinical stage was higher than 4th. (4) The survival curves of MEF2D stages were no statistical significance.@*CONCLUSION@#There's statistical significance of the MEF2D expression in clinical stages, but not in survival curve, which indicated that MEF2D concerned with invasion and metastatic of NPC.

Chronic ethanol feeding impairs AMPK and MEF2 expression and is associated with GLUT4 decrease in rat myocardium

Chronic and heavy alcohol consumption is one of the causes of heart diseases. However, the effects of ethanol on insulin sensitivity in myocardium has been unclear. To investigate the effects of ethanol on the expression of AMP-activated protein kinase (AMPK), myocyte enhancer factor 2 (MEF2) and glucose transporter 4 (GLUT4), all of which are involved in the regulation of insulin sensitivity, in the myocardium, we performed three parts of experiments in vivo and in vitro. I: Rats were injected with 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR, 0.8 mg.kg(-1)) for 2 h. II: Rats received different dose (0.5, 2.5 or 5 g.kg(-1).d(-1)) of ethanol for 22-week. III: Primary neonatal rat cardiomyocytes were isolated and treated with or without 100 mM ethanol or 1 mM AICAR for 4 h. The cardiac protein and mRNA expression of AMPKalpha subunits, MEF2 and GLUT4 were observed by western-blotting and RT-PCR, respectively. Serum TNFalpha levels were assessed by ELISA. The results showed chronic ethanol exposure induced insulin resistance. Ethanol decreased the mRNA levels of AMPKalpha1 and alpha2, the protein levels of total- and phospho-AMPKalpha in cardiomyocytes. Similarly, ethanol showed inhibitory effects on both the mRNA and protein levels of MEF2A and 2D, and GLUT4 in a dose-response-like fashion. Correlation analysis implied an association between phospho-AMPKalpha and MEF2A or MEF2D, and between the levels of MEF2 protein and GLUT4 transcription. In addition, ethanol elevated serum TNFalpha level. Taken together, chronic ethanol exposure decreases the expression of AMPKalpha and MEF2, and is associated with GLUT4 decline in rat myocardium.

Myocyte-specific enhancer binding factor 2C (MEF2C) expression in the dentate gyrus during development and after pilocarpine-induced status epilepticus: a preliminary report / Realce miócito específico ligado a expressão do fator 2C (MEF2C) no giro denteado durante o desenvolvimento e após uso de pilocarpina induzindo status epilepticus: estudo preliminar

OBJECTIVE: As axon outgrowth and dentate granule cell neurogenesis are hallmarks of hippocampal development and are also the two morphologic changes in the structure of the dentate gyrus after status epilepticus (SE), we hypothesized that molecules involved in normal development may also play a role during epileptogenesis. METHOD: Using in situ hybridization, we have characterized mRNA expression of myocyte-specific enhancer binding factor 2C (MEF2C) in the dentate gyrus during development (P0, P3, P7, P14 and P28) and at multiple time points following pilocarpine-induced SE (3, 7, 14, 28 days after SE). RESULTS: It was demonstrated that MEF2C is up-regulated during development (P0, P3, P7, P14 and P28) and in the adult rat dentate gyrus following SE (3, 7, 14, 28 days after SE). CONCLUSIONS: The molecules controlling cell-fate decisions in the developing dentate gyrus are also operative during epileptogenesis.

OBJETIVO: Como o crescimento axonal e a neurogênese do giro denteado são características intrínsecas do hipocampo durante o processo de desenvolvimento, e também são duas alterações morfológicas na estrutura do giro denteado após o status epilepticus (SE), nós hipotetizamos que as moléculas envolvidas no processo normal do desenvolvimento hipocampal também podem participar do processo de epileptogênese. MÉTODO: Utilizando hibridização in situ, caracterizamos a expressão do RNAm do fator de transcrição myocyte-specific enhancer binding factor 2C (MEF2C) no giro denteado durante o desenvolvimento (P0, P3, P7, P14 e P28) e em diferentes períodos após o SE (3, 7, 14, 28 dias após SE). RESULTADOS: Foi demonstrado um aumento da expressão de MEF2C no giro denteado durante o desenvolvimento e no giro denteado de animais adultos após o SE. CONCLUSÃO: As moléculas que controlam o destino celular durante o processo de desenvolvimento também estão operativas durante o processo de epileptogênese.

Study of the 482G/A variation in PGC-1alpha gene domain MEF2C as possible mechanism of type 2 diabetes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association of the 482G/A polymorphism of the PGC-1alpha gene with type 2 diabetes by family-based study in the Han population in South China, and to analyze the quantitative and qualitative binding force changes between the PGC-1alpha domain mutant and MEF2C, as well as to evaluate the possibility of PGC-1alpha -MEF2C-GLUT4 pathway in the pathogenesis of type 2 diabetes.</p><p><b>METHODS</b>Blood samples were collected from 350 patients with type 2 diabetes and their first-degree relatives. Genomic DNA was extracted and polymorphic PGC-1alpha genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and direct DNA sequencing. The results were analyzed by family-based transmission disequilibrium test (TDT) and haplotype relative risk (HRR). The protein-protein interaction between PGC-1alpha and MEF2C was detected by means of the site-directed mutagenesis kit and bacteriomatch two-hybrid system kit.</p><p><b>RESULTS</b>In the family-based study, HRR analyses demonstrated that the 482A allele was more often transmitted to patients than predicted by chance (chi (2)= 7.2170, P= 0.0072, HRR= 1.4496). TDT-extended test(ETDT) analyses also revealed that PGC-1alpha 482A allele was significantly deviated from 0.5 from heterozygous parents to patients than expected (219 trios, P= 0.0310; 350 trios, P= 0.0292). BacterioMatch Two-Hybrid System showed that 482A variation could lead to decreased binding force between PGC-1alpha and MEF2C (62.1+/- 8.97, P< 0.05).</p><p><b>CONCLUSION</b>The 482A polymorphism increases the risk of developing type 2 diabetic mellitus in the South China Han population, which might be mediated by the PGC-1alpha -MEF2C-GLUT4 pathway.</p>

The cSNPs analysis in whole extron-wide of PGC-1alpha gene in Chinese population and the domain MEF2C bioinformatics study / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze distribution characteristics of PGC-1alpha gene coding single nucleotide polymorphisms (cSNPs), and to investigate the association between cSNPs and type 2 diabetes mellitus, and to study biological information about PGC-1alpha domain muscle enhancer factor 2C (MEF2C) in Chinese Han population.</p><p><b>METHODS</b>These cSNPs were identified by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA direct sequencing technology in a total of 263 type 2 diabetic patients and 282 normal glucose tolerant controls. The possible association was analyzed between type 2 diabetes mellitus and the specific cSNPs and their haplotypes by case-control method. The tertiary structure of PGC-1alpha domain MEF2C was predicated and analyzed for possible biological information by a series of bioinformatics soft wares.</p><p><b>RESULTS</b>Four variants were found in whole extron-wide of PGC-1alpha gene in Chinese Han diabetic population. They were 394G/A, 482G/A, 528A/G and 612C/T. The 482G/A polymorphism was remarkably associated with type 2 diabetes (chi2 = 14.2025, P= 0.0002). Haplotypes analysis shown that distribution frequency of haplotypes had a statistical difference between type 2 diabetes mellitus and normal glucose tolerance control groups (chi2 = 59.9, P< 0.01) and haplotype 394A-482A-528A had a linkage disequilibrium with type 2 diabetes (t= 2.361, P< 0.05). The tertiary simulant structure of PGC-1alpha domain MEF2C was established successfully by computer. The 482G/A variant accompanied with hydrogen bonds breaking might decrease hydrophobicity and lead to an incompact space configuration which was very critical to function.</p><p><b>CONCLUSION</b>The 482G/A variant could decrease binding force between PGC-1alpha and MEF2C and increase the risk of type 2 diabetes in Chinese Han population by PGC-1alpha -MEF2C-GLUT-4 pathway.</p>

Association between peroxisome proliferator-activated receptor-gamma coactivator-1alpha gene polymorphisms and type 2 diabetes in southern Chinese population: role of altered interaction with myocyte enhancer factor 2C / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Some single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha gene have been reported to be associated with type 2 diabetes in different populations, and studies on Chinese patients yielded controversial results. The objective of this case-control study was to explore the relationship between SNPs of PGC-1alpha and type 2 diabetes in the southern Chinese population and to determine whether the common variants: Gly482Ser and Thr394Thr, in the PGC-1alpha gene have any impacts on interaction with myocyte enhancer factor (MEF) 2C.</p><p><b>METHODS</b>The SNPs in all exons of the PGC-1alpha gene was investigated in 50 type 2 diabetic patients using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Thereafter, 263 type 2 diabetic patients and 282 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A bacterial two-hybrid system and site-directed mutagenesis were used to investigate whether Gly482Ser and Thr394Thr variants in the PGC-1alpha gene alter the interaction with MEF2C.</p><p><b>RESULTS</b>Three frequent SNPs (Thr394Thr, Gly482Ser and Thr528Thr) were found in exons of the PGC-1alpha gene. Only the Gly482Ser variant had a different distribution between diabetic patients and healthy subjects, with the 482Ser allele more frequent in patients than in controls (40.1% vs 29.3%, P < 0.01). Even in controls, the 482Ser (A) carriers were more likely to have higher levels of total cholesterol and low-density lipoprotein cholesterol than the 482Gly (G) carriers. The 394A-482G-528A haplotype was associated with protection from diabetes, while the 394A-482A-528A was associated with the susceptibility to diabetes. The bacterial two-hybrid system and site-directed mutagenesis revealed that the 482Ser variant was less efficient than the 482Gly variant to interact with MEF2C, whereas the 394Thr (A) had a synergic effect on the interaction between 482Ser variant and MEF2C.</p><p><b>CONCLUSIONS</b>The results suggested that the 482Ser variant of PGC-1alpha conferred the susceptibility to type 2 diabetes in the southern Chinese population. The underlying mechanism may be attributable, at least in part, to the altered interaction between the different variants (Gly482Ser, Thr394Thr) in the PGC-1alpha gene and MEF2C.</p>

Sequence characterization of coding regions of the myostatin gene ( GDF8 ) from Brazilian Murrah buffaloes ( Bubalus bubalis ) and comparison with the Bos taurus sequence

Within about 30 years the Brazilian buffalo (Bubalus bubalis) herd will reach approximately 50 million head as a result of the great adaptive capacity of these animals to tropical climates, together with the good productive and reproductive potential which make these animals an important animal protein source for poor and developing countries. The myostatin gene (GDF8) is important in the physiology of stock animals because its product produces a direct effect on muscle development and consequently also on meat production. The myostatin sequence is known in several mammalian species and shows a high degree of amino acid sequence conservation, although the presence of non-silent and silent changes in the coding sequences and several alterations in the introns and untranslated regions have been identified. The objective of our work was to characterize the myostatin coding regions of B. bubalis (Murrah breed) and to compare them with the Bos taurus regions looking for variations in nucleotide and protein sequences. In this way, we were able to identify 12 variations at DNA level and five alterations on the presumed myostatin protein sequence as compared to non double-muscled bovine sequences.

MEF2A gene and susceptibility to coronary artery disease in the Chinese people / 中南大学学报(医学版)

OBJECTIVE@#To explore MEF2A gene and susceptibility to coronary artery disease in the Chinese.@*METHODS@#One hundred seventy-five coronary artery disease (CAD) patients and 228 normal subjects were recruited and their blood samples were amplified to detect sequences of all 11 exons of MEF2A gene by PCR. Single-strand conformational polymorphism (SSCP) analysis was used to detect the mutation. The amplified products were purified and sequenced.@*RESULTS@#The tri-nucleotide (CAG) length polymorphism in the last coding exon of MEF2A in the Chinese was revealed and 4 of the 175 (2.3%) CAD samples containing 4 prolines were due to one proline deletion in MEF2A gene. But all the 228 normal subjects contained 5 prolines. The mutation in both 175 CAD samples and 228 normal subjects was not found in other exons.@*CONCLUSION@#The deletion mutation in exon 11 in MEF2A gene may be related to CAD susceptibility in the Chinese population.

Study on novel mutations of MEF2A gene in Chinese patients with coronary artery disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the mutations of MEF2A gene in Chinese patients with coronary artery disease(CAD).</p><p><b>METHODS</b>With polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA direct sequencing, the mutation analysis of exon 11 of MEF2A gene was performed to 156 patients with CAD and 93 normal controls.</p><p><b>RESULTS</b>By DNA sequence analyzing the samples of abnormal mobility shift of SSCP, the MEF2A gene mutations were found in three patients with CAD. One of mutations was 147130(C>A)(P431Q), and the second one was 21 bases deletion(147108-147128) which was leading to the absence of 7 amino acids (424QQQQQQQ430), and the third was 147191(G>T). Three mutations were all found in one patient, but meanwhile 21 bases deletion was found in the other two patients.</p><p><b>CONCLUSION</b>Mutations in exon 11 of MEF2A gene exist in the patients with CAD, and the mutations may be pathological.</p>

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping á-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19 percent was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Acetyl-CoA carboxylase beta expression mediated by MyoD and muscle regulatory factor 4 is differentially affected by retinoic acid receptor and retinoid X receptor

Mammals have two major isoforms of acetyl-CoA carboxyase (ACC). The 275 kDa beta-form (ACC beta) is predominantly in heart and skeletal muscle while the 265 kDa alpha-form (ACC alpha) is the major isoform in lipogenic tissues such as liver and adipose tissue. ACC alpha is thought to control fatty acid oxidation by means of the ability of malonyl-CoA to inhibit carnitine palmitoyl-CoA transferase-1 (CPT-1), which is a rate-limiting enzyme of fatty acid oxidation in mitochondria. Previously, it was reported that MyoD and other muscle regulating factors (MRFs) up-regulate the expression of ACC beta by interactions between these factors and several cis-elements of ACC beta promoter. We described here that ACC beta expression mediated by MRFs is regulated by retinoic acids. Endogenous expression of ACCb in differentiated H9C2 myotube was significantly increased by retinoic acid treatment. However, on transient transfection assay in H9C2 myoblast, ACC beta promoter activity was suppressed by RXRa and more severely by RAR alpha. These effects on ACCb expression in myoblasts and myotubes by RXR alpha and RAR alpha seem to be mediated by their interactions with MRFs because no consensus sequence for RXR alpha and RAR alpha has been found in ACC beta promoter and retinoic acid receptors did not affect this promoter activities by itself. In transient transfection in NIH3T3 fibroblast, the activation of ACC beta promoter by MyoD, main MRF in myoblast, was significantly suppressed by RAR alpha and to a less extent by RXR alpha while the RXR alpha drastically augmented the activation by MRF4, major MRF in myotube. These results explained that retinoic acids differentially affected the action of MRFs according to their types and RXR alpha specially elevates the expression of muscle specific genes by stimulating the action of MRF4.