어류 유래 마이오스타틴 프로도메인 단백질에 의한 시마연어(Oncorhychus masou) 성장효과

Jeong Hwan Kim(김정환),Sang Beum Lee(이상범),Mi Jin Cho(조미진),Ji Young Ahn(안지영),Suk Keun Lee(이석근),Sung-Youl Hong(홍성열),Ki Baik Seong(성기백),Hyung-Joo Jin(진형주) 한국생명과학회 2011 생명과학회지 Vol.21 No.8

성장과 분화를 조절하는 인자인 myostatin은 포유류에서 주로 골격근에 분포하며 근육성장을 억제하는 것으로 알려져 있다. Myostatin은 포유류에서뿐만 아니라 어류에 있어서도 그 기능이 유사하며 본 연구에서는 넙치와 조피볼락 유래 재조합 myostatin 단백질을 생산하여 시마연어에 침지방법을 통해 처리하였다. 처리 결과 시마연어의 무게와 생화학 분석에서는 유의성이 나타날 정도의 증가는 없었지만 근(muscle) 조직학적 분석에서 넙치와 조피볼락 유래 재조합 myostatin prodomain에 의해 12주째에는 세포의 수가 증가하는 hyperplasia가 일어났으며 22주째에는 조피볼락 유래의 재조합 myostatin prodomain을 처리한 군에서만 hypertrophy가 일어났다. 결론적으로 어류 유래 재조합 myostatin prodomain이 시마연어 근육성장 시 hyperplasia와 hypertrophy가 순차적으로 유도되는 것으로 확인되었다. Myostatin (MSTN) belongs to the transforming growth factor-β superfamily or growth and differentiation factor 8 (GDF-8), and functions as a negative regulator of skeletal muscle development and growth. Previous studies in mammals have suggested that myostatin knock-out increased muscle mass and decreased fat content compared to those of the wide type. Recently, several studies on myostatin have beenconducted on the block myostatin signal pathway with myostatin antagonists and the MSTN regulation with RNAi to control myostatin function. This study was performed to analyze growth and muscle alteration of Oncorhychus masou by treatment with recombinant myostatin prodomains derived from fish. We designed myostatin prodomains derived from P. olivaceus (pMALc2x-poMSTNpro) and S. schlegeli (pMALc2x-sMSTNpro) in a pMALc2x expression vector, and then purified the recombinant proteins using affinity chromatography. The purified recombinant proteins were treated in O. masou through an immersion method. Recombinant protein treated groups did not show a significant difference in weight, protein, or lipid composition compared to the control. However, there was a difference in the average number and area for histological analyses in the muscle fiber. At twelve and twenty-two weeks from the initial treatment, there were differences in average fiber number and area between the 0.05 ㎎/ℓ treated-group and the control, but the numbers were similar to those of the control during the same time period. At twelve weeks, however, 0.2 ㎎/ℓ treated-group had an increase in average fiber number and decrease in average fiber area compared to the control. At twenty-two weeks, the pMALc2x-sMSTNpro 0.2 ㎎/ℓ treated-group was induced and showed a decrease in average fiber number and increase in average fiber area. The results between twelve and twenty-two weeks showed that the fiber numbers had decreased, whereas average fiber area had increased due to sMSTNpro. It is understood that the sMSTNpro induced only hyperplasia at twelve weeks, after which it induced hypertrophy. Recombinant myostatin prodomains derived from fish may induce hyperplasia and hypertrophy in O. masou depending upon the time that has elapsed.

자연과학편 : 근저항 운동이 신체조성 및 IGF-1과 Myostatin에 미치는 영향

이윤서(YunSeoLee),강성훈(SungHwunKang),이성수(SungSooLee) 한국체육학회 2006 한국체육학회지 Vol.45 No.5

본 연구는 24주간 근저항 운동을 통해 신체조성 및 IGF-1과 myostatin의 변화를 알아보는데 목적이 있다. 실험대상자는 20대 남자들로서 근저항 운동그룹(n=7, year=25.30±0.89)과 대조군(n=7, year=25.40±0.79)으로 나누어 연구하였다. 신체조성검사는 Inbody 4.0을 이용하였으며, IGF-1의 경우 125I RIA(INCSTRA Co., USA)를 이용하였고, myostatin은 Human myostatin ELISA(효소면역흡착검사)를 통하여 분석하였다. 근저항 운동프로그램은 24주간 주3회 1일 80분간 실시하였으며, 운동강도는 1RM의 80%로 실시하였다. 그 결과 신체조성의 모든 변인에서 그룹과 시간에 따라 교호작용이 나타나 두 그룹간 변화 패턴이 다르며 그룹간의 차이가 나타났다. IGF-1과 myostatin역시 그룹과 시간에 따른 교호작용이 나타나 두 그룹간의 변화 패턴이 다르며 그룹간의 차이가 나타났으며, 사후검증 결과 운동전 보다 운동 24주후 IGF-1과 myostatin 모두 유의한(p<0.01) 차이가 나타났다. 이상의 결과를 종합해 보면 24주간 근저항 운동은 myostatin의 감소를 가져오며, 반대로 IGF-1의 증가를 가져와 근비대의 원인이 되며, 짧은 기간보다도 장기간의 근저항운동이 더욱 근육의 성장과 발달에 긍정적인 영향을 주는 것으로 판단된다. 따라서 6개월 이상의 지속적인 근저항운동이 근비대를 유발하는 것으로 사료된다. The purpose of this study was to investigate the effects of muscle resistance exercise on IGF-1 and myostatin in men. Subjects were recruited and divided into 2 groups. One group of subjects(n=7, year=25.30±0.89) was trained only muscle resistance exercise during 24 weeks and other group was(n=7, year=25.40±0.79) treated with control. Body composition used through Venus 5.5 and blood samples were obtained from the antecubital vein while the subjects fasted. IGF-1 measured to 125I RIA and myostatin measured to Human myostatin ELISA. Muscle resistance exercise practiced for 24 weeks, per week 3, per day 80min and exercise intensity was 80%RM. The results showed that body composition, IGF-1 and myostatin was groups and times interaction. Then, IGF-1(p<0.01) and myostatin(p<0.01) in post-hoc was changed significantly. Therefore, muscle resistance exercise during 24 weeks was decreased of myostatin, which caused of muscle hypertrophy through increased IGF-1. That effect of long than short muscle resistance on muscle growth and development.

Myostatin-2 isolation and spatiotemporal expression comparison between myostatin-1 and -2 in Larimichthys crocea

Zhifei Liu,Liangyi Xue,Sheng Sun,Zhen Xu,Hong Yu 한국유전학회 2014 Genes & Genomics Vol.36 No.5

The 3,110-bp myostatin-2 was obtained from amarine teleost, Larimichthys crocea (L. crocea), by PCRamplication coupled with 50- and 30-rapid amplification ofcDNA ends. Myostatin-2 contains a 71-bp 50-untranslatedregion, a 328-bp exon I, an 895-bp intron I, a 371-bp exonII, a 382-bp intron II, a 381-bp exon III, and a 682 bp 30-untranslated region, and it encodes a protein of 359 aminoacid residues. The deduced amino acid sequence has typicalcharacteristics of TGF-b family members with nineconserved cysteine residues and a RXXR proteolytic processingsite within the conserved C-terminal portion, andmore than 90 % similarity with other fish myostatin-2proteins. Quantitative assessment revealed that levels ofmyostatin-1 transcripts fluctuated markedly, whereas myostatin-2 expression remained very low during most stagesof embryonic development. In addition, both myostatin-1and -2 transcripts were detected in all 10 examined tissuesof juvenile and adult individuals, but they exhibited differentexpression patterns and responses to fasting. Expression of myostatin-1 was highest in skeletal muscle,which was higher than myostatin-2 by 2–3 orders ofmagnitude. Myostatin-2 transcripts were greatest in brain. Myostatin-1 expression increased significantly after 3 daysof fasting, whereas myostatin-2 expression was unaffected. Our results imply that myostatin-1 and -2 may have differentroles in skeletal muscle growth and development ofL. crocea.

도담탕(導痰湯)이 $C_{2}C_{12}$세포주로부터 myostatin발현에 의한 심근에 미치는 영향

이유승,신유정,박종혁,김승모,백경민,박치상,Lee, You-Seung,Shin, Yoo-Jeong,Park, Jong-Hyuk,Kim, Seung-Mo,Paek, Kyung-Min,Park, Chi-Sang 대한한방내과학회 2008 大韓韓方內科學會誌 Vol.29 No.1

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. To determine MyoD expression by Dodamtang treatment, we compared the expression pattern of $C_{2}C_{12}$ mouse myoblasts that constitutively express myostatin with control cells. In vitro, increasing concentrations of Dodamtang reversibly prevented the myogenic blockage of myoblasts by myostatin expression. ELISA assay, Western and confocal analysis indicated that treatment of Dodamtang to the low serum culture media increased the levels of MyoD leading to the inhibition of myogenic differentiation by myostatin. The stable transfection of $C_{2}C_{12}$ myoblasts with myostatin expressing constructs did rescue MyoD-induced myogenic differentiation. Consistent with this, the treatment of Dodamtang rescued the expression of a MyoD in $C_{2}C_{12}$ myoblasts treated with myostatin. Taken together, these results suggest that induction of MyoD by Dodamtang inhibits myostatin activity and expression via SMAD3 resulting in the rescue of the myoblasts to differentiate into myotubes. Thus we propose that myostatin action by Dodamtang plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that blockage of functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

Myostatin Inhibitors: Panacea or Predicament for Musculoskeletal Disorders?

서준호,Yun-Sil Lee 대한골대사학회 2020 대한골대사학회지 Vol.27 No.3

Myostatin, also known as growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member that functions to limit skeletal muscle growth. Accordingly, loss-of-function mutations in myostatin result in a dramatic increase in muscle mass in humans and various animals, while its overexpression leads to severe muscle atrophy. Myostatin also exerts a significant effect on bone metabolism, as demonstrated by enhanced bone mineral density and bone regeneration in myostatin null mice. The identification of myostatin as a negative regulator of muscle and bone mass has sparked an enormous interest in developing myostatin inhibitors as therapeutic agents for treating a variety of clinical conditions associated with musculoskeletal disorders. As a result, various myostatin-targeting strategies involving antibodies, myostatin propeptides, soluble receptors, and endogenous antagonists have been generated, and many of them have progressed to clinical trials. Importantly, most myostatin inhibitors also repress the activities of other closely related TGF-β family members including GDF11, activins, and bone morphogenetic proteins (BMPs), increasing the potential for unwanted side effects, such as vascular side effects through inhibition of BMP 9/10 and bone weakness induced by follistatin through antagonizing several TGF-β family members. Therefore, a careful distinction between targets that may enhance the efficacy of an agent and those that may cause adverse effects is required with the improvement of the target specificity. In this review, we discuss the current understanding of the endogenous function of myostatin, and provide an overview of clinical trial outcomes from different myostatin inhibitors.

Chimeric Myostatin - Tetanic Toxin Epitopes and Heterologous Prime-boost Immunization Improve Immune Response Stimulating Muscle Growth in Mice

Vianey Ramírez Andoney,Amanda Gayosso Vázquez,Juan Pablo Pintor Ríos,Jorge Enrique Vázquez Buchelli,Rogelio A. Alonso Morales 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.5

Myostatin is a transforming growth factor-β family member who acts as a negative regulator of skeletal muscle growth. The interference of its biological activity could increase skeletal muscle growth with clinical and animal production applications. A strategy to block the myostatin action is by the induction of an immune response against it. In this work, we evaluated as an immunogen a recombinant myostatin fused to the tetanic toxin T- helper epitopes P2 and P30. Genetic constructs of the chimeric myostatin were cloned in an expression vector and used as a DNA vaccine. Besides, a chimeric genetic construct, P2-miostatin–P30 was expressed in Escherichia coli, obtaining a recombinant chimeric antigen. To find out the functionality of these genetic constructs as a vaccine in inducing muscle growth responses, experimental groups of BALB/c mice were DNA immunized with the myostatin fused to P2, P30 or both. Furthermore, to improve the immune response, a heterologous prime–boost immunization scheme was evaluated where the DNA inoculation was followed by immunization with the recombinant antigen P2-myostatin-P30. The different body segments weight was recorded in control and vaccinated mice groups, finding increased muscle masses in the vaccinated groups. These experiments showed the effectiveness of the P2 and P30 Thelper epitopes in inducing an immune response to the fused myostatin, leading to muscle growth. The heterologous prime-boost immunization protocol is a promising vaccination strategy reducing the time and amount of antigen used to induce a immune response to myostatin.

Molecular characterization of myostatin-like genes expressed highly in the muscle tissue from Morotoge shrimp, <i>Pandalopsis japonica</i>

Kim, Kyoung Sun,Kim, Young-Ji,Jeon, Jeong Min,Kang, Yang Soon,Kang, Young Shil,Oh, Chul Woong,Kim, Hyun-Woo Blackwell Publishing Ltd 2010 Aquaculture research Vol.41 No.11

<P>Abstract</P><P>Myostatin is one of the transforming growth factor (TGF)-β family members and plays inhibitory roles in the development and growth of muscle in mammals. Mammalian myostatins have been studied intensively, considering its medical and industrial potential use. Still, limited information is available about myostatin homologues in crustaceans. In the present study, we isolated for the first time cDNA that encodes for myostatin-like protein (Pj-MSTN) from Morotoge shrimp, <I>Pandalopsis japonica</I>. The putative mature peptide of Pj-MSTN was composed of 109 amino acids, which contains an additional amino acid residue compared with mammalian myostatins. Pj-MSTN exhibited 32% amino acid sequence identity and 52% similarity to human myostatin. Multiple sequence alignment analysis indicated that Pj-MSTN shared the conserved proteolytic cleavage site (RXXR) for its maturation and nine cysteine residues for disulphide bridges. These results suggest that Pj-MSTN has conserved the three-dimensional structure of TGF-β family members in vertebrates. Phylogenetic analysis suggests that Pj-MSTN is a primitive form of vertebrate myostatin and GDF11. The expression of Pj-MSTN was not just identified in muscular tissues, suggesting that Pj-MSTN functions differently from mammalian myostatin. Ablation of the X-organ/sinus gland complex significantly reduced the expression of Pj-MSTN in most tissues, suggesting its potential association with moulting.</P>

A Myostain-like Gene Expressed Highly in the Muscle Tissue of Chinese mitten crab, Eriocheir sinensis

Kim, Kyoung-Sun,Jeon, Jeong-Min,Kim, Hyun-Woo The Korean Society of Fisheries and Aquatic Scienc 2009 Fisheries and Aquatic Sciences Vol.12 No.3

A complete cDNA, which encodes for a myostatin-like protein (Es-MSTN), was isolated from the Chinese mitten crab, Eriocheir sinensis. Es-MSTN was composed of 2,397 nucleotides and the open reading frame (ORF) specified a protein containing 468 amino acids. Es-MSTN exhibited 32% amino acid sequence identity and 52% similarity to human myostatin. Multiple sequence alignment analysis indicated that Es-MSTN possessed the conserved proteolytic cleavage site (RXXR) for maturation of the protein and nine cysteine residues for disulfide bridges. Besides the conserved structural features, Es-MSTN also exhibits its unique characters; a longer N-terminal domain which is involved in protein folding and latent form of myostatin and absence of the cleavage site for BMP-1/tolloid family of metalloproteinase to activate mature myostatin. Phylogenetic analysis suggests that Es-MSTN showed the closely related to both vertebrate myostatin and GDF11. Es-MSTN is expressed highly in the claw muscle, leg muscle, thoracic muscle and heart, and moderately in the hindgut suggesting that Es-MSTN may play important roles in the muscle tissues. As homolog of mammalian myostatin and GDF11, Es-MSTN may be involved in development of muscular tissue and further study will help to produce high-quality seafood.

A Myostain-like Gene Expressed Highly in the Muscle Tissue of Chinese mitten crab, Eriocheir sinensis

김경선,전정민,김현우 한국수산과학회 2009 Fisheries and Aquatic Sciences Vol.12 No.3

A complete cDNA, which encodes for a myostatin-like protein (Es-MSTN), was isolated from the Chinese mitten crab, Eriocheir sinensis. Es-MSTN was composed of 2,397 nucleotides and the open reading frame (ORF) specified a protein containing 468 amino acids. Es-MSTN exhibited 32% amino acid sequence identity and 52% similarity to human myostatin. Multiple sequence alignment analysis indicated that Es-MSTN possessed the conserved proteolytic cleavage site (RXXR) for maturation of the protein and nine cysteine residues for disulfide bridges. Besides the conserved structural features, Es-MSTN also exhibits its unique characters; a longer N-terminal domain which is involved in protein folding and latent form of myostatin and absence of the cleavage site for BMP-1/tolloid family of metalloproteinase to activate mature myostatin. Phylogenetic analysis suggests that Es-MSTN showed the closely related to both vertebrate myostatin and GDF11. Es-MSTN is expressed highly in the claw muscle, leg muscle, thoracic muscle and heart, and moderately in the hindgut suggesting that Es-MSTN may play important roles in the muscle tissues. As homolog of mammalian myostatin and GDF11, Es-MSTN may be involved in development of muscular tissue and further study will help to produce high-quality seafood.

저강도 운동과 Silymarin의 투여가 MyoD, Myostatin과 PEPCK유전자에 미치는 영향

최은주(Choi, Eun-Ju),이창진(Lee, Chang-Jin) 한국체육과학회 2012 한국체육과학회지 Vol.21 No.1

The purpose of this study was to investigate how low-intensity exercise and silymarin administration effect into a gene on the MyoD, Myostatin and PEPCK in skeletal muscle. Subjects of investigation were classified into two groups in Sprage-Dawley rats. One group of subjects was performed with only exercise which was divided exercise group(EX), and the other group was performed with both silymarin administration and exercise(SIL+EX). Treadmill was used for training by rats which were given silymarin administration (50㎎/g/day) 30 minutes before exercising, and they were trained a low-intensity exercise (8m/min, 60min/day, 5 times a week). After analyzing with Real time-PCR, this experiment showed us that the revelation of mRNA of PEPCK at soleus muscle (SOL) was significantly increased more in SIL+EX than in EX. But, there was not significant difference between the two groups in Gastrocnemius muscle(GA). A mRNA revelation of Myostatin was not significant difference between EX and SIL+EX in SOL and GA. In MyoD, the revelation of mRNA at SOL was increased more in SIL+EX than in EX. But, there was not significant difference between the two groups in Gastrocnemius muscle(GA). As a result, SIL+EX in SOL are considered to be an improvement for exercising capabilities as they SIL+EX effect into a gene of MyoD, Myostatin and PEPCK which involves exercising capabilities.