Expression, purification of recombinant flounder MRF4 protein in Escherichia coli and analysis of its polyclonal antibodies

MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (130). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.

牙鲆成肌因子myogenin和MRF4的研究

本文从牙鲆中克隆到其肌肉调节因子myogenin和MRF4的基因，并对它们的表达、启动子活性及功能进行分析。 研究结果表明牙鲆myogenin和MRF4都由三个外显子和两个内含子组成，其cDNA编码的氨基酸序列分别与几种鱼类的同源基因亲缘关系较近。 原位杂交显示最早在胚胎5-6个体节时体节中部的细胞检测到myogenin的表达，随着发育的进行，它的表达向体节两侧和体后延伸，最后在整个体节表达。当发育到30个体节的时候，在躯干的表达迅速下降，只在胚胎的尾部能检测到信号。 RT-PCR的结果显示，MyoD和Myf5的表达要早于myogenin，而MRF4的表达晚于myogenin，并且myogenin、MRF4只在成鱼的肌肉中表达，这进一步证明其在肌肉发育过程中的作用。 myogenin和MRF4的启动子中含有保守的肌肉特异调控序列，瞬时表达分析的结果证实两段启动子序列足以驱动GFP在斑马鱼胚胎肌肉中的特异表达。首次对鱼类MRF4启动子进行了调控序列的分析，发现在启动子的-181到-506的序列中含有调节MRF4表达的必需位点。 首次应用原核表达系统对MRF4蛋白进行重组表达，并利用纯化的重组蛋白制备多克隆抗体，通过western杂交分析显示所得到的MRF4多克隆抗体能够识别牙鲆内源性的MRF4蛋白。

Heart failure alters MyoD and MRF4 expressions in rat skeletal muscle

Heart failure (HF) is characterized by a skeletal muscle myopathy with increased expression of fast myosin heavy chains (MHCs). The skeletal muscle-specific molecular regulatory mechanisms controlling MHC expression during HF have not been described. Myogenic regulatory factors (MRFs), a family of transcriptional factors that control the expression of several skeletal muscle-specific genes, may be related to these alterations. This investigation was undertaken in order to examine potential relationships between MRF mRNA expression and MHC protein isoforms in Wistar rat skeletal muscle with monocrotaline-induced HF. We studied soleus (Sol) and extensor digitorum longus (EDL) muscles from both HF and control Wistar rats. MyoD, myogenin and MRF4 contents were determined using reverse transcription-polymerase chain reaction while MHC isoforms were separated using polyacrylamide gel electrophoresis. Despite no change in MHC composition of Wistar rat skeletal muscles with HF, the mRNA relative expression of MyoD in Sol and EDL muscles and that of MRF4 in Sol muscle were significantly reduced, whereas myogenin was not changed in both muscles. This down-regulation in the mRNA relative expression of MRF4 in Sol was associated with atrophy in response to HF while these alterations were not present in EDL muscle. Taken together, our results show a potential role for MRFs in skeletal muscle myopathy during HF. © 2006 Blackwell Science Ltd.

Análise da expressão gênica de MyoD, MRF4, miogenina e miostatina nos músculos Bíceps femoris e Gastrocnemius lateralis em duas linhagens de Gallus gallus (corte e postura)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Functional analysis of MRF4 during mouse embryogenesis

MRF4 is one of four skeletal muscle specific regulatory genes, (the other three genes being MyoD, myf5, and myogenin), each of which has the unique ability to orchestrate an entire program of muscle-specific transcription when introduced into diverse cell types. These findings have led to the notion that these factors function as master regulators of muscle cell fate. Analysis of mice lacking MyoD, myf5, and myogenin have further defined their roles in the commitment and differentiation of myotomal progenitor cells. Current data strongly supports the model that MyoD and myf5 share functional redundancy in determining the muscle cell lineage, while myogenin acts downstream of MyoD and myf5, to initiate myoblast differentiation. Unlike other myogenic bHLH genes, MRF4 is expressed predominantly in the adult, suggesting that it may function to regulate adult muscle maturation and maintenance. To test this hypothesis and to eventually incorporate MRF4 into a general model for muscle specification, differentiation, maturation and maintenance, I deleted the MRF4 gene. MRF4-null mice are viable and fertile, however, they show mild rib anomalies. In addition, the expression of myogenin is dramatically upregulated only in the adult, suggesting that myogenin may compensate for the loss of MRF4 in the adult, and MRF4 may normally suppress the expression of myogenin after birth. MRF4 is also required during muscle regeneration after injury.^ To determine the degree of genetic redundancy between MRF4-myogenin; and MRF4-MyoD, I crossed the MRF4-null mice with MyoD- and myogenin-null mice respectively. There are no additional muscle phenotypes in double-null progeny from a MRF4 and myogenin cross, suggesting that the existence of residual fibers in myogenin-null mice is not due to the presence of MRF4. MRF4 expression also cannot account for the ability of myogenin-null myoblasts to differentiate in vitro. However, the combination of the MRF4-null mutation with the myogenin-null mutation results in a novel rib phenotype. This result suggests that MRF4 modifies the myogenin-null rib phenotype, and MRF4 and myogenin play redundant roles in rib development.^ MRF4 also shares dosage effects with MyoD during mouse development. (MyoD+/$-$;MRF4$-$/$-$)mice are fertile and viable, while (MyoD$-$/$-$;MRF4+/$-$) mice die between birth and two weeks after birth, and have a small skeletal structure. The double homozygous mice for MRF4 and MyoD mutations are embryonic lethal and die at around E10.5. These results suggest that MRF4 and MyoD share overlapping functions during mouse embryogenesis. ^

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.

Chronic heart failure-induced skeletal muscle atrophy, necrosis, and changes in myogenic regulatiory factors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Down-regulation of MyoD gene expression in rat diaphragm muscle with heart failure

Diaphragm myopathy has been described in patients with heart failure (HF), with alterations in myosin heavy chains (MHC) expression. The pathways that regulate MHC expression during HF have not been described, and myogenic regulatory factors (MRFs) may be involved. The purpose of this investigation was to determine MRF mRNA expression levels in the diaphragm. Diaphragm muscle from both HF and control Wistar rats was studied when overt HF had developed, 22 days after monocrotaline administration. MyoD, myogenin and MRF4 gene expression were determined by RT-PCR and MHC isoforms by polyacrylamide gel electrophoresis. Heart failure animals presented decreased MHC IIa/IIx protein isoform and MyoD gene expression, without altering MHC I, IIb, myogenin and MRF4. Our results show that in HF, MyoD is selectively down-regulated, which might be associated with alterations in MHC IIa/IIx content. These changes are likely to contribute to the diaphragm myopathy caused by HF.

Mecanismos celulares e moleculares que controlam o desenvolvimento e o crescimento muscular

O músculo estriado esquelético é formado pela associação de fibras musculares com a matriz extracelular. Esse tecido possui alta plasticidade e o conhecimento das características morfológicas, da miogênese, e da dinâmica do crescimento é importante para o entendimento da morfofisiologia bem como para a seleção de animais visando a melhoria na produção de carne. A maioria dos músculos estriados originam-se de células precursoras do mesoderma a partir dos somitos do embrião e o controle da diferenciação ocorre pela ação de fatores indutores ou inibidores. Um grupo de fatores transcricionais, pertencentes à família MyoD tem um papel central na diferenciação muscular. Coletivamente chamados de Fatores de Regulação Miogênica (MRFs), são conhecidos quatro tipos: MyoD, myf-5, miogenina e MRF4. Esses fatores ligam-se à seqüências de DNA conhecidas como Ebox (CANNTG) na região promotora de vários genes músculo-específicos, levando à expressão dos mesmos. As células embrionárias com potencial para diferenciação em células musculares (células precursoras miogênicas) expressam MyoD e Myf-5 e são denominadas de mioblastos. Essas células proliferam, saem do ciclo celular, expressam miogenina e MRF4, que regulam a fusão e a diferenciação da fibra muscular. Uma população de mioblastos que se diferencia mais tardiamente, as células miossatélites, são responsáveis pelo crescimento muscular no período pós natal, que pode ocorrer por hiperplasia e hipertrofia das fibras. As células satélites quiescentes não expressam os MRFs, porém, sob a ação de estímulos como fatores de crescimento ou citocinas, ocorre a ativação desse tipo celular que prolifera e expressa os MRFs de maneira similar ao que ocorre com as células precursoras miogênicas durante a miogênese. Os mecanismos de crescimento muscular são regulados pela expressão temporal dos (MRFs), que controlam a expressão dos genes relacionados com o crescimento muscular.

Heart failure-induced skeletal myopathy in spontaneously hypertensive rats

Background: Although skeletal muscle atrophy and changes in myosin heavy chain (MyHC) isoforms have often been observed during heart failure, their pathophysiological mechanisms are not completely defined. In this study we tested the hypothesis that skeletal muscle phenotype changes are related to myogenic regulatory factors and myostatin/follistatin expression in spontaneously hypertensive rats (SHR) with heart failure. Methods: After developing tachypnea, SHR were subjected to transthoracic echocardiogram. Pathological evidence of heart failure was assessed during euthanasia. Age-matched Wistar-Kyoto (WKY) rats were used as controls. Soleus muscle morphometry was analyzed in histological sections, and MyHC isoforms evaluated by electrophoresis. Protein levels were assessed by Western blotting. Statistical analysis: Student's t test and Pearson correlation. Results: All SHR presented right ventricular hypertrophy and seven had pleuropericardial effusion. Echocardiographic evaluation showed dilation in the left chambers and left ventricular hypertrophy with systolic and diastolic dysfunction in SHR. Soleus weight and fiber cross sectional areas were lower (WKY 3615±412; SHR 2035±224 μm2; P < 0.001), and collagen fractional volume was higher in SHR. The relative amount of type I MyHC isoform was increased in SHR. Myogenin, myostatin, and follistatin expression was lower and MRF4 levels higher in SHR. Myogenin and follistatin expression positively correlated with fiber cross sectional areas and MRF4 levels positively correlated with I MyHC isoform. Conclusion: Reduced myogenin and follistatin expression seems to participate in muscle atrophy while increased MRF4 protein levels can modulate myosin heavy chain isoform shift in skeletal muscle of spontaneously hypertensive rats with heart failure. © 2012 Elsevier B.V.

Expressão dos fatores de regulação miogênica, composição das miosinas e trofismos no músculo esquelético periférico de ratos com insuficiência cardíaca crônica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo do músculo diafragma em ratos com insuficiência cardíaca crônica: composição das miosinas e expressão dos fatores de regulação miogênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Differential Expression of Myogenic Regulatory Factor Genes in the Skeletal Muscles of Tambaqui Colossoma macropomum (Cuvier 1818) from Amazonian Black and Clear Water

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Heart Failure-Induced Diaphragm Myopathy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)