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Myostatin HMP(Human Myostatin Propeptide(GDF-8)98% 2mg

Item 12626

Myostatin Identifiers Symbols MSTN; GDF8

External OMIM601788 MGI95691 HomoloGene3850

IDs GeneCards: MSTN Gene

 

Gene Ontology

Molecular function

receptor binding
receptor binding
cytokine activity
growth factor activity

Cellular component

extracellular region
extracellular space
cytoplasm

Biological process

transforming growth factor beta receptor signaling pathway
muscle organ development
response to heat
response to gravity
skeletal muscle atrophy
negative regulation of muscle hypertrophy
response to muscle activity
ovulation cycle process
response to testosterone stimulus
growth
skeletal muscle tissue regeneration
response to estrogen stimulus
response to ethanol
positive regulation of transcription, DNA-dependent
negative regulation of skeletal muscle tissue growth
response to glucocorticoid stimulus
response to electrical stimulus

 

Myostatin (also known as growth differentiation factor 8, abbreviated "GDF8") is a secreted TGF beta protein family member that inhibits muscle differentiation and growth. Myostatin is produced primarily in skeletal muscle cells, circulates in the blood and acts on muscle tissue, by binding a cell-bound receptor called the activin type II receptor.[1][2] In humans, myostatin is encoded by the MSTN gene.[3]

Animals lacking myostatin or animals treated with substances such as follistatin that block the binding of myostatin to its receptor have significantly larger muscles.[4]

Discovery and sequencing

The gene encoding myostatin was discovered in 1997 by geneticists Alexandra McPherron and Se-Jin Lee who also produced a strain of mutant mice that lack the gene. These myostatin "knockout" mice have approximately twice as much muscle as normal mice.[5] These mice were subsequently named "mighty mice".

Naturally occurring myostatin "nulls" have been identified in cattle, whippets, and humans; in each case the result is a dramatic increase in muscle mass. A mutation in the 3' UTR of the myostatin gene in Texel sheep creates target sites for the microRNAs miR-1 and miR-206. This is likely to cause the muscular phenotype of this breed of sheep.[6]

Effects of inactivated myostatin in cattle

After that discovery, several laboratories cloned and established the nucleotide sequence of a myostatin gene in two breeds of cattle Belgian Blue and Piedmontese, and found that these animals have mutations in that myostatin gene (various mutations in each breed) which in one way or another lead to absence of functional myostatin.[7] Unlike mice with a damaged myostatin gene, in these cattle breeds the muscle cells multiply rather than enlarge. People describe these cattle breeds as "double muscled", but the total increase in all muscles is no more than 40%.[8][9][10]

The double-muscle mutation in humans

Myostatin is active in muscles used for movement (skeletal muscles) both before and after birth. This protein normally restrains muscle growth, ensuring that muscles do not grow too large. Mutations that reduce the production of functional myostatin lead to an overgrowth of muscle tissue. Myostatin-related muscle hypertrophy has a pattern of inheritance known as incomplete autosomal dominance. People with a mutation in both copies of the MSTN gene in each cell (homozygotes) have significantly increased muscle mass and strength. People with a mutation in one copy of the MSTN gene in each cell (heterozygotes) also have increased muscle bulk, but to a lesser degree.

In 2004, a German boy was diagnosed with a mutation in both copies of the myostatin-producing gene, making him considerably stronger than his peers. His mother, a former sprinter, has a mutation in one copy of the gene.[11][12][13][14][15][16]

An American boy born in 2005 (Liam Hoekstra) was diagnosed with a clinically similar condition but with a somewhat different cause:[17] his body produces a normal level of functional myostatin, but because he is stronger and more muscular than most others his age, his doctor believes that a defect in his myostatin receptors prevents his muscle cells from responding normally to myostatin. Liam appeared on the television show World's Strongest Toddler.

A 2007 NIH study in PLOS Genetics[18] found a significant relationship in whippets between a myostatin mutation and racing performance. Whippets that were heterozygous for a 2 base pair deletion in the myostatin gene were significantly over-represented in the top racing classes. The mutation resulted in a truncated myostatin protein, likely resulting in an inactive form of myostatin.

Whippets with a homozygous deletion were apparently less able runners although their overall appearance was significantly more muscular. Whippets with the homozygous deletion also had an unusual body shape, with a broader head, pronounced overbite, shorter legs, and thicker tails. These whippets have also been called "bully whippets" by the breeding community due to their size. Despite the name "bully", these dogs tend have a friendly and positive demeanour towards people as usual for whippets.

This particular mutation was not found in other muscular dog breeds such as boxers and mastiffs, nor was it found in other sighthounds such as greyhounds, Italian greyhounds, or Afghan hounds. The authors of the study suggest that myostatin mutation may not be desirable in greyhounds, the whippets' nearest relative, because greyhound racing requires more significant endurance due to the longer races (900 meters for greyhounds vs. 300 meters for whippets).

Biochemistry

Myostatin is a member of the TGF beta superfamily of proteins.

Human myostatin consists of two identical subunits, each consisting of 109 amino acid residues. Its total molecular weight is 25.0 kDa. The protein is made in an inactive form. For it to be activated, a protease cleaves the NH2-terminal, or "pro-domain" portion of the molecule, resulting in the now-active COOH-terminal dimer.

Myostatin binds to the activin type II receptor, resulting in a recruitment of a coreceptor called Alk-3 or Alk-4. This coreceptor then initiates a cell signaling cascade in the muscle, which includes the activation of transcription factors in the SMAD family - SMAD2 and SMAD3. These factors then induce myostatin-specific gene regulation. When applied to myoblasts, myostatin inhibits their differentiation into mature muscle fibers.

Recently, myostatin has also been shown to inhibit Akt, a kinase that is sufficient to cause muscle hypertrophy, in part through the activation of protein synthesis.

Therefore, myostatin acts in two ways, by inhibiting muscle differentiation and by inhibiting Akt-induced protein synthesis.

Clinical significance

Further research into myostatin and the myostatin gene may lead to therapies for muscular dystrophy.[19] The idea is to introduce substances that block myostatin. In 2002, researchers at the University of Pennsylvania showed that monoclonal antibody specific to myostatin improves the condition of mice with muscular dystrophy, it is presumed, by blocking myostatin's action. Similar results in monkeys were published in 2009.[20]

In 2005, Lee showed that a two-week treatment of normal mice with soluble activin type IIB receptor, a molecule that is normally attached to cells and binds to myostatin, leads to a significantly increased muscle mass (up to 60%).[21] It is thought that binding of myostatin to the soluble activin receptor prevents it from interacting with the cell-bound receptors.

It remains unclear as to whether long-term treatment of muscular dystrophy with myostatin inhibitors is beneficial: The depletion of muscle stem cells could worsen the disease later on.

Myostatin levels are effectively decreased by creatine supplementation.[22]

A technique for detecting mutations in myostatin variants has been developed.[23]

In fiction

A fictional drug called myostatin is featured in the TV series The Incredible Hulk episode "A Death in the Family" (Season 1, Episode 2). This episode came out about 20 years before real myostatin was discovered.

In the Japanese manga Air Gear, the two characters Akira Udou and Mitsuru Bandou have this condition.

See also

References

1.       ^ Carnac G, Ricaud S, Vernus B, Bonnieu A (July 2006). "Myostatin: biology and clinical relevance". Mini Rev Med Chem 6 (7): 765–70. doi:10.2174/138955706777698642. PMID 16842126.

2.       ^ Joulia-Ekaza D, Cabello G (June 2007). "The myostatin gene: physiology and pharmacological relevance". Curr Opin Pharmacol 7 (3): 310–5. doi:10.1016/j.coph.2006.11.011. PMID 17374508.

3.       ^ Gonzalez-Cadavid NF, Taylor WE, Yarasheski K, Sinha-Hikim I, Ma K, Ezzat S, Shen R, Lalani R, Asa S, Mamita M, Nair G, Arver S, Bhasin S (December 1998). "Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting". Proc. Natl. Acad. Sci. U.S.A. 95 (25): 14938–43. Bibcode 1998PNAS...9514938G. doi:10.1073/pnas.95.25.14938. PMC 24554. PMID 9843994.

4.       ^ Kota J, Handy CR, Haidet AM, Montgomery CL, Eagle A, Rodino-Klapac LR, Tucker D, Shilling CJ, Therlfall WR, Walker CM, Weisbrode SE, Janssen PML, Clark KR, Sahenk Z, Mendell JR, Kaspar BK (2009). "Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates". Science Translational Medicine 1 (6): 6ra15–6ra15. doi:10.1126/scitranslmed.3000112. PMC 2852878. PMID 20368179.

5.       ^ McPherron AC, Lawler AM, Lee SJ (May 1997). "Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member". Nature 387 (6628): 83–90. Bibcode 1997Natur.387...83M. doi:10.1038/387083a0. PMID 9139826.

6.       ^ Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X, Bibé B, Bouix J, Caiment F, Elsen JM, Eychenne F, Larzul C, Laville E, Meish F, Milenkovic D, Tobin J, Charlier C, Georges M (2006). "A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep". Nat Genet 38 (7): 813–8. doi:10.1038/ng1810. PMID 16751773.

7.       ^ McPherron A., Lee S-J., 1997; Grobet L. et al., 1997; Kambadur R. et al., 1997

8.       ^ Photos of double muscled Myostatin, inhibited Belgian Blue Bulls

9.       ^ Kambadur R, Sharma M, Smith T, Bass J (1997). "Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle". Genome Res 7 (9): 910–6. doi:10.1101/gr.7.9.910. PMID 9314496.

10.   ^ McPherron A, Lee S (1997). "Double muscling in cattle due to mutations in the myostatin gene". Proc Natl Acad Sci USA 94 (23): 12457–61. Bibcode 1997PNAS...9412457M. doi:10.1073/pnas.94.23.12457. PMC 24998. PMID 9356471.

11.   ^ cevgenetica: Gene Mutation Makes German Boy Extra Strong Muscle Baby

12.   ^ Gina Kolota: A Very Muscular Baby Offers Hope Against Diseases, The New York Times, June 24, 2004. (Requires login)

13.   ^ Genetic mutation turns tot into superboy

14.   ^ Muscle Boy

15.   ^ One Strong Tyke: Gene mutation in muscular boy may hold disease clues

16.   ^ Schuelke M, Wagner K, Stolz L, Hübner C, Riebel T, Kömen W, Braun T, Tobin J, Lee S (2004). "Myostatin mutation associated with gross muscle hypertrophy in a child". N Engl J Med 350 (26): 2682–8. doi:10.1056/NEJMoa040933. PMID 15215484.

17.   ^ Associated Press (2007-05-30). "CTV.ca | Rare condition gives toddler super strength". CTVglobemedia. Retrieved 2009-01-21.

18.   ^ a b Mosher DS, Quignon P, Bustamante CD, Sutter NB, Mellersh CS, Parker HG, Ostrander EA (May 2007). "A Mutation in the Myostatin Gene Increases Muscle Mass and Enhances Racing Performance in Heterozygote Dogs". PLoS Genet. 3 (5): e79. doi:10.1371/journal.pgen.0030079. PMC 1877876. PMID 17530926.

19.   ^ Kate Ruder: Strong Boy Could Benefit Research on Muscular Dystrophy, Genome News Network, June 24, 2004.

20.   ^ http://www.npr.org/templates/story/story.php?storyId=120316010

21.   ^ Lee SJ, Reed LA, Davies MV, Girgenrath S, Goad ME, Tomkinson KN, Wright JF, Barker C, Ehrmantraut G, Holmstrom J, Trowell B, Gertz B, Jiang MS, Sebald SM, Matzuk M, Li E, Liang LF, Quattlebaum E, Stotish RL, Wolfman NM (December 2005). "Regulation of muscle growth by multiple ligands signaling through activin type II receptors". Proc. Natl. Acad. Sci. U.S.A. 102 (50): 18117–22. Bibcode 2005PNAS..10218117L. doi:10.1073/pnas.0505996102. PMC 1306793. PMID 16330774.

22.   ^ Saremi A, Gharakhanloo R, Sharghi S, Gharaati MR, Larijani B, Omidfar K (April 2010). "Effects of oral creatine and resistance training on serum myostatin and GASP-1". Mol. Cell. Endocrinol. 317 (1–2): 25–30. doi:10.1016/j.mce.2009.12.019. PMID 20026378.

23.   ^ US granted 6673534, Lee S-J, McPherron AC, "Methods for detection of mutations in myostatin variants", issued 2004-01-06, assigned to The Johns Hopkins University School of Medicine

External links

The Myostatin Gene

by Elzi Volk

Super Cows and Mighty Mice

In 1997, scientists McPherron and Lee revealed to the public the ‘secret’ of an anomaly that livestock breeders have capitalized since the late 1800’s: the gene responsible for big beefy cows (1). More than a century ago, livestock breeders in Europe observed that some of their cattle were more muscled than others. Being dabblers in genetics, they selectively bred these cattle to increase the progeny displaying this trait. Thus two breeds of cattle (Belgian Blue and Piedmontese) were developed that typically exhibit an increase in muscle mass relative to other conventional cattle breeds. Little did they know that many years later Mighty Mouse would be more than merely a cartoon.

A team of scientists led by McPherron and Lee at John Hopkins University was investigating a group of proteins that regulate cell growth and differentiation. During their investigations they discovered the gene that may be responsible for the phenomenon of increased muscle mass, also called ‘double-muscling’ (1, 2). Myostatin, the protein that the gene encodes, is a member of a superfamily of related molecules called transforming growth factors beta (TGF-b ). It is also referred to as growth and differentiation factor-8 (GDF-8). By knocking out the gene for myostatin in mice, they were able to show that the transgenic mice developed two to three times more muscle than mice that contained the same gene intact. Lee commented that the myostatin gene knockout mice "look like Schwarzenegger mice." (3).

Further exploration of genes present in skeletal muscle in the two breeds of double-muscled cattle revealed mutations in the gene that codes for myostatin. The double-muscling trait of the myostatin gene knockout mice and the double-muscled cattle demonstrates that myostatin performs the same biological function in these two species. Apparently, myostatin may inhibit the growth of skeletal muscle. Knocking out the gene in transgenic mice or mutations in the gene such as in the double-muscled cattle result in larger muscle mass. This discovery has paved the way for a plethora of futuristic implications from breeding super-muscled livestock to treatment of human muscle wasting diseases.

Researchers are developing methods to interfere with expression and function of myostatin and its gene to produce commercial livestock that have more muscle mass and less fat content. Myostatin inhibitors may be developed to treat muscle wasting in human disorders such as muscular dystrophy. However, several public media sources immediately raised the issue of abusing myostatin inhibitors by athletes. In addition, a hypothesis has been put forth that a genetic propensity for high levels of myostatin is responsible for the lack of muscle gain in weight trainees. Accordingly, this article presents a look at the science of myostatin and its implications for the athletic arena.

WHAT IS MYOSTATIN?

Growth Factors

Before we can understand the implications of tampering with myostatin and its gene, we must learn what myostatin is and what it does. Higher organisms are comprised of many different types of cells whose growth, development and function must be coordinated for the function of individual tissues and the entire organism. This is attainable by specific intercellular signals, which control tissue growth, development and function. These molecular signals elicit a cascade of events in the target cells, referred to as cell signaling, leading to an ultimate response in or by the cell.

Classical hormones are long-range signaling molecules (called endocrine). These substances are produced and secreted by cells or tissues and circulated through the blood supply and other bodily fluids to influence the activity of cells or tissues elsewhere in the body. However, growth factors are typically synthesized by cells and affect cellular function of the same cell (autocrine) or another cell nearby (paracrine). These molecules are the determinants of cell differentiation, growth, motility, gene expression, and how a group of cells function as a tissue or organ.

Growth factors (GF) are normally effective in very low concentrations and have high affinity for their corresponding receptors on target cells. For each type of GF there is a specific receptor in the cell membrane or nucleus. When bound to their ligand, the receptor-ligand complex initiates an intracellular signal inside of the cell (or nucleus) and modifies the cell’s function.

A GF may have different biological effects depending on the type of cell with which it interacts. The response of a target cell depends greatly on the receptors that cell expresses. Some GFs, such as insulin-like growth factor-I, have broad specificity and affect many classes of cells. Others act only on one cell type and elicit a specific response.

Many growth factors promote or inhibit cellular function and may be multifactoral. In other words, two or more substances may be required to induce a specific cellular response. Proliferation, growth and development of most cells require a specific combination of GFs rather than a single GF. Growth promoting substances may be counterbalanced by growth inhibiting substances (and vice versa) much like a feedback system. The point where many of these substances coincide to produce a specific response depends on other regulatory factors, such as environmental or otherwise.

Transforming Growth Factors

Some GFs stimulate cell proliferation and others inhibit it, while others may stimulate at one concentration and inhibit at another. Based on their biological function, GFs are a large set of proteins. They are usually grouped together on the basis of amino acid sequence and tertiary structure. A large group of GFs is the transforming growth factor beta (TGFb ) superfamily of which there are several subtypes. They exert multiple effects on cell function and are extensively expressed.

A common feature of TGFb s is that they are secreted by cells in an inactive complex form. Consequently, they have little or no biological activity until the latent complex is broken down. The exact mechanism(s) involved in activating these latent complexes is not completely understood, but it may involve specific enzymes. This further exemplifies how growth factors are involved in a complex system of interaction.

Another common feature of TGFb s is that their biological activity is often exhibited in the presence of other growth factors. Hence, we can see that the bioactivity of TGFb s is complex, as they are dependent upon the physiological state of the target cell and the presence of other growth factors.

Myostatin

There are several TGFb s subtypes which are based on their related structure. One such member is called growth and differentiation factors (GDF) and specifically regulates growth and differentiation. GDF-8, also called myostatin, is the skeletal muscle protein associated with the double muscling in mice and cattle.

McPherron et al detected myostatin expression in later stages of development of mouse embryos and in a number of developing skeletal muscles (1). Myostatin was also detected in adult animals. Although myostatin mRNA was almost exclusively detected in skeletal muscle, lower concentrations were also found in adipose tissue.

To determine the biological role of myostatin in skeletal muscle, McPherron and associates disrupted the gene that encodes myostatin protein in rats, leading to a loss its function. The resulting transgenic animals had a gene that was rendered non-functional for producing myostatin. The breeding of these transgenic mice resulted in offspring that were either homozygous for both mutated genes (i.e. carried both mutated genes), homozygous for both wild-type genes (i.e. carried both genes with normal function) or heterozygous and carrying one mutated and one normal gene. The main difference in resulting phenotypes manifested in muscle mass. Otherwise, they were apparently healthy. They all grew to adulthood and were fertile.

Homozygous mutant mice (often called gene knockout mice) were 30% larger than their heterozygous and wild-type (normal) littermates irregardless of sex and age. Adult mutant mice had abnormal body shapes with very large hips and shoulders and the fat content was similar to the wild-type counterparts. Individual muscles from mutant mice weighed 2-3 times more than those from wild-type mice. Histological analysis revealed that increased muscle mass in the mutant mice was resultant of both hyperplasia (increased number of muscle fibers) and hypertrophy (increased size of individual muscle fibers).

Since this discovery, McPherron and other researchers investigated the presence of myostatin and possible gene mutations in other animal species. Scientists have reported the sequences for myostatin in 9 other vertebrate animals, including pigs, chickens and humans (2, 4). Research teams separately discovered two independent mutations of the myostatin gene in two breeds of double-muscled cattle: the Belgian Blue and Piedmontese (2, 5). A deletion in the myostatin gene of the Belgian Blue eliminates the entire active region of the molecule and is non-functional; and this mutation causes hypertrophy and increased muscle mass. The Piedmontese coding sequence for myostatin contains a missense mutation. That is, a point in the sequence encodes for a different amino acid. This mutation likely leads to a complete or nearly completes loss of myostatin function.

McPherron et al analyzed DNA from other purebred cattle (16 breeds) normally not considered as double-muscled and found only one similar mutation in the myostatin gene (2). The mutation was detected in one allele a single animal which was non-double-muscled. Other mutations were detected but these did not affect protein function.

Earlier studies reported high levels of myostatin in developing cattle and rodent skeletal muscles (2, 7). Furthermore, mRNA expression varied in individual muscles. Consequently, it was thought that myostatin was relegated to skeletal muscle and that the gene’s role was restricted to the development of skeletal muscle. However, A New Zealand team of researchers recently reported the detection of myostatin mRNA and protein in cardiac muscle (8).

TGF-b superfamily members are found in a wide variety of cell types, including developing and adult heart muscle cells. Three known isoforms of TGF-b (TGF-b 1, -b 2, and -b 3) are expressed differentially at both the mRNA and protein levels during development of the heart (9). This suggests that these isoforms have different roles in regulating tissue development and growth. Therefore, Sharma and colleagues investigated distribution of the myostatin gene in other organ tissues using more sensitive detection techniques than that used by earlier researchers (8).

They found a DNA sequence in sheep and cow heart tissue that was identical to the respective skeletal muscle myostatin protein sequence, indicating the presence of myostatin gene in these tissues. In heart tissue from a Belgian Blue fetus, the myostatin gene deletion present in skeletal tissue was detected. They detected the unprocessed precursor and processed myostatin protein in normal sheep and cattle skeletal muscle, but not in that of the Belgian Blue. As well, only the unprocessed myostatin protein was found in adult heart tissue.

Animals with induced myocardial infarction (causing death of cells in heart tissue) displayed high levels of myostatin protein, even at 30 days postinfarct, in cells immediately surrounding the dead lesion. However, undamaged cells bordering the infarcted area contained very low levels of myostatin protein similar to control tissue. Considering the increase in other TGF-b levels in experimentally infarcted heart tissue (10), these growth factors may be involved in promotion of tissue healing.

Shaoquan and colleagues at Purdue University detected myostatin mRNA in the lactating mammary glands of pigs, possibly serving a regulatory role in the neonatal pig (12). They also detected similar mRNA is porcine skeletal tissue, but not in connective tissue. Most studies, in addition to this one, confirm that high levels of myostatin mRNA in prenatal animals and reduced levels postnatal at birth and postnatal reflect a regulatory role of myostatin in myoblast (muscle cell precursors) growth, differentiation and fusion.

A mutation in the myostatin gene in the two cattle breeds is not as advantageous as in mice. The cattle have only modest increases in muscle mass compared to the myostatin knockout mice (20-25% in the Belgian Blue and 200-300% in the null mice). Also, the cattle with myostatin mutations have reduced size of internal organs, reductions in female fertility, delay in sexual maturation, and lower viability of offspring (6). Although no heart abnormalities in myostatin-null mice were reported, the hearts in adult Belgian Blue cattle are smaller (11). Although the reduction in organ weight has been attributed to skeletal muscle mass increases, this has yet to be confirmed. Since there is evidence that the effects of myostatin mutation on heart tissue are variable in different species, there may be other possible tissue variabilities as well. Additionally, research detected myostatin mRNA in tissues other than skeletal muscle, demonstrating its expression is not relegated to skeletal muscle tissue as originally thought. Only further research will elucidate these possibilities.

Although several TGF-b superfamily members are found in skeletal and cardiac muscle tissue, their exact roles in development is not yet clear. Apparently, based on the early studies, the myostatin protein may have diverse roles in developmental and adult stage tissues. Sharma et al proposes that "myostatin has different functions at different stages of heart development" (8). As we shall see, the same can conceivably apply to skeletal muscle as well.

Myostatin and regulation of skeletal muscle

While many of the studies demonstrate that myostatin is involved with prenatal muscle growth, we know little of its association with muscle regeneration. Muscle regeneration of injured skeletal muscle tissue is a complex system and ability for regeneration changes during an animal’s lifetime. Exposure of tissues to various growth factors is altered during a lifetime. In embryos and young animals, hormones and growth factors favor muscle growth. However, many of these factors are downregulated in adults. Alteration in growth factors inside and outside of the muscle cells may diminish their capacity to maintain protein expression. Although protein mRNA may be detected within the cell, there are many sites of protein regulation beyond mRNA levels. As mentioned above, myostatin protein occurs in an unprocessed (inactive) and processed (active) form. Therefore, bioactivity of myostatin may be regulated at any point of its synthesis and secretion.

Keep in mind that nearly all regulatory systems in the body are under positive and negative control. This includes cardiac and skeletal muscle tissues. Myoblasts in developing animal embryos respond to different signals that control proliferation and cell migration. In contrast, differentiated muscle cells respond to another set of different signals. Distinct ratios of signals regulate the transition from undetermined cells to differentiated cells and ensure normal formation and differentiation in cellular tissues. However, many of the factors that regulate the various development pathways in muscle tissue are still poorly understood.

MyoD, IGF-I and myogenin (growth promoters in muscle cells) gene products are associated with muscle cell differentiation and activation of muscle-specific gene expression (14). Muscle-regulatory factor-4 (MRF-4) mRNA expression increases after birth and is the dominant factor in adult muscle. This growth factor is thought to play an important role in the maintenance of muscle cells. In addition to myostatin, there are other inhibitory gene products, such as Id (inhibitor of DNA binding). Although in vitro experiments are revealing the mechanisms of these specific proteins, we know less regarding their roles in vivo.

Although we know that lack of myostatin protein is associated with skeletal muscle hypertrophy in McPherron’s gene knockout mice and in double-muscled cattle, we know little about the physiological expression of myostatin in normal skeletal muscle. Recent studies in animal and human models indicate a paradox in myostatin’s role on growth of muscle tissue.

For example, evidence shows that myostatin may be fiber-type specific. Runt piglets, which have lower birth weights than their normal littermates, had lower proportions of Type I skeletal muscle fibers in specific muscles (12). Similar observations were made in rats where undetectable levels of myostatin mRNA in atrophied mice soleus (Type I fibers) (13). Transient upregulation of myostatin mRNA was detected in atrophied fast twitch muscles but not in slow twitch muscles. Thus, myostatin may modulate gene expression controlling muscle fiber type.

Studies also demonstrated lack of metabolic effects on myostatin expression in piglets and mice (12, 13). Food restriction in both piglets and mice did not affect myostatin mRNA levels in skeletal muscle. Neither dietary polyunsaturated fatty acids nor exogenous growth hormone administration in growing piglets altered myostatin expression (12). These and other studies strongly suggest that the physiological role of myostatin is mostly associated with prenatal muscle growth where myoblasts are proliferating, differentiating and fusing to form muscle fibers.

Although authors postulate that myostatin exerts its effect in an autocrine/paracrine fashion, serum myostatin has been detected demonstrating that it is also secreted into the circulation (8, 4). It is believed that the protein detected in human serum is of processed (active form) myostatin rather than the unprocessed form. High levels of this protein have been associated with muscle wasting in HIV-infected men compared to healthy normal men (4). However, this association does not necessarily verify that myostatin directly contributes to muscle wasting. We do not know if myostatin acts directly on muscle or on other regulatory systems that regulate muscle growth. Although several authors postulate that myostatin may present a larger role in muscle regeneration after injury, this has yet to be confirmed.

Myostatin and athletes

Further complicating the issue of myostatin’s role in regulation of muscle growth is the report by a team of scientists that mutations in the human myostatin gene had little impact on responses in muscle mass to strength training (15, unpublished data). Based on the report that muscle size is a heritable trait in humans (16), Ferrell and colleagues investigated the variations in the human myostatin gene sequence. They also examined the influence of myostatin variations in response of muscle mass to strength training.

Study subjects represented various ethnic groups and were classified by the degree of muscle mass increases they experienced after strength training. Included were competitive bodybuilders ranking in the top 10 world-wide and in lower ranks. Also included were football players, powerlifters and previously untrained subjects. Quadricep muscle volume of all subjects was measured by magnetic resonance imaging before and after nine weeks of heavy weight training of the knee extensors. Subjects were grouped and compared by degree of response and by ethnicity.

There were several genetic coding sequence variations detected in DNA samples from subjects. Two changes were detected in a single subject and another two were observed in two other individuals. They were heterozygous with the wild-type allele, meaning they had one allele with the mutation and the other allele was normal. The other variations were present in the general population of subjects and determined common. One of the variations was common in the group of mixed Caucasian and African-American subjects. However, the less frequent allele had a higher frequency in African-Americans. Although, as the authors comment, "these variable sites [in the gene sequence] have the potential to alter the function of the myostatin gene product and alter nutrient partitioning in individuals heterozygous for the variant allele", the data from this and other studies so far show that this may not occur. This study did not demonstrate any significant response between genotypes and response to weight training. Nor were there any significant differences between African-American responders to strength training and non-responders or between Caucasian responders and non-responders.

Further research will be necessary to determine whether myostatin has an active role in muscle growth after birth and in adult tissues. To ascertain benefit to human health, we also need to discover its role in muscle atrophy and regeneration after injury. Only extended research will reveal any such benefits.

The future of myostatin

Now that we have reviewed some of the biology of the myostatin protein, its gene, and the relevant scientific literature, what are the implications for its application?

Many authors of the myostatin studies have speculated that interfering with the activity of myostatin in humans may reverse muscle wasting disease associated with muscular dystrophy, AIDS and cancer. Some predict that manipulation of this gene could produce heavily muscled food animals. Indeed, current research is underway to investigate and develop these potentialities. Sure enough, a large pharmaceutical company has recently applied for a patent on an antibody vaccination for the myostatin protein.

A medical doctor and author of weight training articles asserts that overexpression of myostatin is to blame for weight lifters that have trouble gaining muscle mass. The spokesperson for a supplement and testing lab erroneously implied that the "rarest" form of mutation in the myostatin gene is responsible for a top competitive bodybuilder’s massive muscle gains, not taking into account the performance-enhancement substances the bodybuilder may be using. The public media has, of course, predicted that "steroid-popping" athletes will take advantage of myostatin inhibitors to gain competitive edge (3).

Many of these assertions are unfounded or they misrepresent the science. Granted, the possibility exists that manipulation of the myostatin gene in humans may be a key to reversing muscle-wasting conditions. However, too little is still yet unknown regarding myostatin’s role in muscle growth regulation. It is imperative that research demonstrates that the loss of myostatin activity in adults can cause muscle tissue growth. Likewise, research must also prove that overexpression or administration of myostatin causes loss of muscle mass. Also important is to know if manipulation of myostatin will interfere with other growth systems, especially in other tissues, and result in abnormal pathologies. Although McPherron’s gene knockout mice did not experience any other gross abnormalities, mice are not humans.

We do not fully understand the roles of myostatin in exercise-induced muscle hypertrophy or regeneration following muscle injury. Until we do, it may be premature to blame the lack of hypertrophy in weightlifters on overexpression of myostatin. Nor does the research support the claim that a top bodybuilder’s muscle mass gains are resultant of a detected mutation in the myostatin gene. The research simply does not advocate blaming genetic myostatin variations as a source of significant differences in human phenotypes.

Considering the history of the athlete’s propensity, in the public eye, to abuse performance-enhancement substances, the media’s prediction of myostatin-inhibitor may or may not be warranted. We all know that today’s athletic arena demands gaining the competitive edge to maintain top level competition. For many athletes, that is accomplished by supplementing hard training with substances that enhance growth or performance. Whether or not myostatin inhibitors will be added to the arsenal of substances is difficult to predict. Until science reveals the full nature of this growth factor and its role in the complex regulation of muscle tissue, and researchers determine its therapeutic implications, we can only surmise. Despite attempts to tightly control any pharmaceutical uses of myostatin protein manipulation, they will likely surface at some point in the black market world of bodybuilding supplements. Let us hope that science has determined the side effects and the benefits by that point.


References

McPherron, AC, AM Lawler, SJ Lee. Regulation of skeletal muscle mass in mice by a new TGF-b superfamily member. Nature 1997, 387:83.

McPherron, AC, SJ Lee. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 1997, 94:12457

John Hopkins Magazine, June 1997. URL: http://www.jhu.edu./~jhumag/0697/web/science.html 

Gonzalez-Cadavid, NF, WE Taylor, K Yarasheski, et al. Organization of the human myostatin gene and expression in healthy and HIV-infected men with muscle wasting. Proc Natl Acad Sci 1998, 95:14938.

Grobet, L, LJR Martin, D Poncelet, et al. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nature Genet 1997, 17:71.

Menissier, F. In: Muscle Hypertrophy of Genetic Origin and its Use to Improve Beef Production, eds. King, JWB and F Mennissier. Nijhoff, The Haugue, The Netherlands, pp. 23-53.

Kambadur R, M Sharma, TPL Smith, JJ Bass. Mutations in myostatin (GDF8) in double-muscled Belgian Bllue and Piedmontese cattle. Genome Res 1997, 7:910.

Sharma M, R Kambadur, KG Matthews, et al. Myostatin, a transforming growth factor-b superfamily member, is expressed in heart muscle and is upregulated in cardiomycetes after infarct. J Cell Physiol 1999, 180:1.

Millan FA, F Denhes, P Kondaiah, et al. Embryonic gene expression pattern of TGF-b 1, b 2, and b 3 suggest different developmental function in vivo. Development 1991, 111:131.

Sharma, HS, M Wunsch, T Brand, et al. Molecular biology of the coronary vascular and myocardial responses to ischemia. J Cardiovas Pharmacol 1992, 20:S23.

Bocard R. 1981. Facts and reflections on muscular hypertrophy in cattle: double muscling or culard. In: Developments in Meat Science, Vol. 2. Lawrie R, ed. Applied Science Publishers, London, pp. 1-28.

Shaoquan, J, RL Losinski, SG Cornelius, et al. Myostatin expression in porcine tissues: tissue specificity and developmental and postnatal regulation. Am J Physiol 1998, 275:R1265.

Carlson, JC, FW Booth, SE Gordon. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol 1999, 277:R601.

Marsh, DR, DS Criswell, JA Carson, FW Booth. Myogenic regulatory factors during regeneration of skeletal muscle in young, adult and old rats. J Appl Physiol 1997, 83:1270.

Ferrell, RE, V Conte, EC Lawrence, et al. Frequent sequence variation in the human myostatin (GDF8) gene as a marker for analysis of muscle related phenotypes. Genomics, in press.

Loos, R, M Thomis, HH Maes, et al. Gender-specific regional changes in genetic structure of muscularity in early adolescence. J Appl Physiol 1997, 82:1602.

Mutations in myostatin (GDF8) in Double-Muscled Belgian Blue and Piedmontese Cattle

  1. Ravi Kambadur1,2,
  2. Mridula Sharma1,
  3. Timothy P.L. Smith3, and
  4. John J. Bass
1.      AgResearch, Ruakura, Hamilton, New Zealand; 3 U.S. Department of Agriculture (USDA)/Agricultural Research Service (ARS) Meat Animal Research Center, Clay Center, Nebraska 68933-0166

 

Abstract

A visibly distinct muscular hypertrophy (mh), commonly known as double muscling, occurs with high frequency in the Belgian Blue and Piedmontese cattle breeds. The autosomal recessive mh locus causing double-muscling condition in these cattle maps to bovine chromosome 2 within the same interval as myostatin, a member of the TGF-β superfamily of genes. Because targeted disruption ofmyostatin in mice results in a muscular phenotype very similar to that seen in double-muscled cattle, we have evaluated this gene as a candidate gene for double-muscling condition by cloning the bovine myostatin cDNA and examining the expression pattern and sequence of the gene in normal and double-muscled cattle. The analysis demonstrates that the levels and timing of expression do not appear to differ between Belgian Blue and normal animals, as both classes show expression initiating during fetal development and being maintained in adult muscle. Moreover, sequence analysis reveals mutations in heavy-muscled cattle of both breeds. Belgian Blue cattle are homozygous for an 11-bp deletion in the coding region that is not detected in cDNA of any normal animals examined. This deletion results in a frame-shift mutation that removes the portion of the Myostatin protein that is most highly conserved among TGF-β family members and that is the portion targeted for disruption in the mouse study. Piedmontese animals tested have a G–A transition in the same region that changes a cysteine residue to a tyrosine. This mutation alters one of the residues that are hallmarks of the TGF-β family and are highly conserved during evolution and among members of the gene family. It therefore appears likely that the mh allele in these breeds involves mutation within the myostatin gene and that myostatin is a negative regulator of muscle growth in cattle as well as mice.

[The sequence data for bovine myostatin has been submitted to GenBank under accession no. AF019761.]

The muscular hypertrophy (mh), or double-muscle phenotype, is a heritable condition in cattle that primarily results from an increase in number of muscle fibers (hyperplasia) rather than the enlargement of individual muscle fibers (hypertrophy), relative to normal cattle (Hanset et al. 1982). The relative increase in fiber number is observed early in pregnancy (Swatland and Kieffer 1974) and results in a calf possessing nearly twice the number of muscle fibers at the time of birth. The occurrence of double muscling has been observed in several cattle breeds worldwide since it was first documented by Culley in 1807. The breed in which this muscular hypertrophy and its effects have been analyzed most extensively is the Belgian Blue breed, which has been systematically selected for double muscling to the point of fixation in many herds. Domestic animals other than cattle also show dramatic increases in muscle mass. Malignant hyperthermia of pigs with muscular hypertrophy (Brenig and Brem 1992) and muscle hypertrophy of cats associated with a dystrophin deficiency (Gaschen et al. 1992) have been analyzed at the molecular level.

Compared with normal cattle, Belgian Blue and Piedmontese animals have an increased proficiency to convert feed into lean muscle and produce a higher percentage of the most desirable cuts of meat (Casas et al. 1997). These animals have less bone, less fat, and 20% more muscle on average (Shahin and Berg 1985; Hanset 1986, 1991). However, problems associated with the trait, such as reduction in stress tolerance, fertility, and calf viability in Belgian Blue have hindered exploitation of the hypertrophy by classical genetic selection (Arthur 1995).

Segregation analysis has indicated a monogenic autosomal segregation pattern for the double-muscling trait (Hanset and Michaux 1985a,b; Charlier et al. 1995). The locus has been termed “partially recessive” because there is some effect of a single copy of the allele, but generally the truly double muscled phenotype requires that the animal be homozygous. A mapping study utilizing a panel of microsatellite markers to scan the bovine genome (Charlier et al. 1995) localized the mh locus in Belgian Blue cattle to the centromeric end of the bovine Chromosome 2 (BTA2) linkage group. The map position of the mh locus has been refined and extended to the Piedmontese breed using additional genetic and physical markers to a 3- to 5-cM interval near the centromere of BTA2 (Casas et al. 1997) close to the position of the α collagen type III (COL3AI) locus (Sonstegard et al. 1997).

A recent study demonstrated that mice lacking a normal copy of the myostatin gene (GDF8) display a phenotype with significant similarities to the double muscling seen in cattle. myostatinis a member of the transforming growth factor β (TGF-β) gene superfamily specifically expressed in skeletal muscle of adult mice, as well as during early development. In the mouse study, the third exon of the gene was replaced with a neo cassette, removing the portion of the protein that is highly conserved among the TGF-β superfamily of genes. Animals homozygous for the disruption display an increase in skeletal muscle mass similar to that observed in homozygousmh cattle.

The bovine myostatin gene recently has been mapped to the same interval as the mh locus by genetic linkage (Smith et al. 1997), which strongly suggests that it may be the gene causing double muscling in cattle. Here we report the sequence of bovine myostatin and evaluate it as a candidate gene by sequence and expression analysis. Although the expression of myostatin mRNA does not appear disrupted in Belgian Blue cattle, mutation analysis reveals an 11-bp deletion mutation in the coding region of the myostatin gene in Belgian Blue cattle that would be predicted to abolish the activity of the protein, as the truncated portion encodes the peptide sequence thought to mediate essential functions (McPherron et al. 1997). In addition, a transition mutation found in animals of the Piedmontese breed affects a conserved cysteine in exon 3 of the myostatin that is also likely to affect function of Myostatin.

RESULTS

Cloning and Sequencing of bovine myostatin cDNA

The first step in these experiments was to obtain a cDNA clone of bovine myostatin (bmyostatin) from a normal animal to use as a probe and to provide sequence information for comparison to the double-muscle allele. Primers were designed based on the murine sequence (GenBank accession no. U84005) of the 5′ and 3′-untranslated regions and used for RT–PCR to amplify the entire coding region of the bovine homolog (bmyostatin) from total RNA isolated from skeletal muscles of normal Friesian cattle. Comparison of the predicted amino acid sequence of murine and bmyostatin indicates that it is highly conserved, with 93% homology between the two proteins (Fig. 1A). All of the hallmarks of the TGF-β superfamily, including signal sequence for secretion, a proteolytic processing site, and a conserved pattern of cysteine residues in the carboxy-terminal region, are conserved between mouse and cattle forms (Fig. 1A).

Figure 1.Figure 1.Figure 1.Figure 1.

Figure 1.

Sequence analysis of normal- and double-muscled bovine myostatin. Sequencing was performed on three independent normal- and double-muscled alleles, and one representative sequence of both alleles is shown. (A) Amino acid sequence comparisons of mouse (MMYO.PRO) and bovine Myostatin (BMYO.PRO) proteins. Nonconserved amino acids are indicated by solid bars. The consensus amino acid sequence is shown at the top. The proteolytic processing site is underlined. (B) The deletion mutation is detected by fluorometric sequencing of myostatin cDNA from normal- and double-muscled cattle. The sequence of the double-muscled allele is shown above that of the normal allele (Control), and the position where 11 bp is deleted in the mutant allele is indicated by an arrow. The large bracket in the normal allele sequence denotes the region that is deleted in the double-muscled allele. (C) The amino acid sequence of Myostatin in normal cattle is shown above the predicted amino acid sequence of Myostatin in double-muscled cattle. The premature stop codon at amino acid position 288 in the double-muscle allele is indicated by an asterisk (*). (D) The predicted amino acid sequence of Myostatin in normal cattle is shown below that of the Piedmontese breed in the vicinity of the transition mutation. The altered residue in the Piedmontese allele is underlined. Asterisks (*) indicate two of the nine conserved cysteine residues in exon 3 of the normal bmyostatin allele.

Developmental Expression of the bmyostatin Gene in Normal and Belgian Blue Cattle

The phenotype of the myostatin knockout mice suggests that myostatin is a negative regulator of muscle growth, because mice lacking normal gene function displayed enlarged muscles. Therefore, any mutation that decreases the amount or activity of Myostatin at the critical developmental period could lead to an increase in muscle mass. The decrease could result either from changes in the mRNA expression pattern due to mutations that affect transcription/transcript stability or from changes in the translated portion that affect the function of the protein. First, we addressed the possibility that the mh allele affects transcription ofbmyostatin by comparing the pattern of expression of bmyostatin between normal and Belgian Blue animals.

RT–PCR analysis was performed on total RNA isolated from either whole embryo or M. semitendinosus muscle from various gestation stages of normal and Belgian Blue animals. Oligonucleotide primers were designed such that 513-bp coding region was amplified in a combined RT–PCR reaction (see Methods for primer sequences). As shown in Figure2A, the expression of bmyostatin gene was found at all developmental stages in normal cattle. Low levels of message were detected up to day 29 embryos in normal animals, and increased expression of bmyostatin was detected from day 31 onwards to late in gestation (260-day-old fetuses). bmyostatin cDNA was also detected in Belgian Blue 50-day-old fetuses onwards and late in gestation (day 260) (Fig 2B). bmyostatin mRNA expression in fetuses prior to day 50 was not analyzed in Belgian Blue cattle. No changes were noted in the level of expression of bmyostatin between normal and Belgian Blue animals at various gestation stages examined (Fig. 2A,B), and bmyostatin continued to be expressed in the skeletal muscle of adult animals.

Figure 2.Figure 2.Figure 2.

Figure 2.

(A,B) Agarose gel electrophoresis of PCR products (513 bp) obtained from RT–PCR using total RNA from embryos or fetuses of different normal (A)- or double (B)-muscled Belgian Blue bovine developmental stages. (M) Markers (1-kb ladder from GIBCO BRL). Different embryonic or fetal ages are indicated in corresponding lanes. The locations of the primers used to amplify 513-bp partial cDNA are from amino acid 202 to 208 (sense primer) and from amino acid 365 to 371 (antisense primer). (See Methods for primer sequence.) This 513-bp partial cDNA contains the 11-bp deletion observed in double-muscled Belgian Blue cattle. (C) Expression of myostatin in different adult bovine muscles. Fifteen micrograms of total RNA was electrophoresed on formaldehyde–agarose gel, blotted onto nylon membrane, and probed with bovine myostatin cDNA. (Lane 1) M. gastrocnemius; (lane 2) M. psoas major; (lane 3) M. longissimus dorsi; (lane 4) M. biceps femoris; (lane 5) M. diaphragm; (lane 6) M. semimembranosus; (lane 7) M. flexor digitorum longus; (lane 8) M. vastus medialis; (lane 9) M. vastus lateralis; (lane 10) heart; (lane 11) M. cutaneus trunci; (lane 12) M. semitendinosus; (lane 13) M. semitendinosus (normal, 260 day); (lane 14) M. semitendinosus (double-muscle, 260 day).

To compare the expression of bmyostatin in different skeletal muscles, we performed Northern analysis on total RNA isolated from adult tissues from normal animals. As shown in Figure 2C, the bmyostatin probe detected a single-message of 2.9-kilobase mRNA expressed in both axial and paraxial musculature. High levels of expression were observed in the hindlimb muscles M. semimembranosus and M. biceps femoris, whereas low levels of expression were detected in other hindlimb muscles (Fig. 2C). myostatin expression could not be detected in the heart or in the diaphragm muscle.

Mutations in bmyostatin in Belgian Blue Double-Muscled Cattle

Because no differences in expression of the bmyostatin gene could be detected by RT–PCR, we examined the sequence of the cDNA in normal- and double-muscled animals to evaluate possible changes in the protein and correlated any changes with the observed phenotype. Myostatin cDNA from animals in three unrelated double-muscled pedigrees were sequenced and screened for mutations in the coding region. This analysis revealed an 11-bp deletion in the open reading frame of the Belgian Bluebmyostatin allele, which results in the loss of three amino acids (275, 276, and 277) and a frameshift after amino acid 274 (Fig. 1B,C). The frameshift leads to a stop codon after amino acid 287 that is predicted to truncate the protein such that most of the same portion that was deleted in the heavy muscled knockout mice would not be translated (Fig. 1C). Another 17 pedigrees of Belgian Blue cattle were subsequently tested for this deletion by simple sizing of PCR products that include this portion of the gene, including 16 pedigrees in New Zealand and 4 in the United States. All double-muscled, purebred animals tested were homozygous for this deletion, wherease none of 11 different normal-muscled dairy and traditional beef breed cattle showed evidence of deletion in this region. On the basis of the results from the Belgian Blue analysis, myostatin mRNA from a Piedmontese breed animal was then assessed for deletions or nonsense mutations that would abnormally truncate the protein. The resulting Piedmontese cDNA sequence predicted that a full-length Myostatin protein was coded for in this breed. This result demonstrates that in contrast to the Belgian Blue breed, mh in Piedmontese animals is not the result of an abnormally truncated Myostatin protein. However, examination of the cDNA sequence revealed a G–A transiton mutation at position 941 of the coding region. This mutation predicts the replacement of cysteine at amino acid 314 with tyrosine (Fig. 1D). This cysteine is the fifth in a series of nine whose appearance and spacing is extremely conserved throughout the TGF-β and inhibin-β gene families; thus, it is likely that the observed mutation would interfere with normal function of the protein (McPherron et al. 1997).

To verify this result and assess its generality, three unrelated double-muscled pedigrees of the Piedmontese breed were then examined for the transition mutation. A primer was developed from the sequence of the second intron of the bovine gene and used in combination with a primer designed from the downstream untranslated region (see Methods). These primers amplify a 493-bp fragment containing the entire coding portion of the third exon of bmyostatin from amino acid residue 251 to the carboxyl terminus at 376. The amplified fragment encompasses all of the coding region downstream of the putative proteolytic processing site (McPherron et al. 1997), including the observed mutation. This allowed us to use archival DNA from Piedmontese sires to assess the generality of the mutation in herds in the United States. The predicted amino acid sequence for all three pedigrees contained the replacement of cysteine with tyrosine at residue 314. Multiple independent PCR reactions from each animal were used to generate sequence, and the mutation was consistently observed; thus it appears that Piedmontese animals are homozygous for this mutation.

DISCUSSION

We report the cloning of bovine myostatin and an evaluation of this TGF-β family member as a candidate gene formh. Recently the bovine myostatin gene has been mapped to the same interval as the mh locus that causes the double-muscle phenotype (Smith et al. 1997). Comparison of the bovine and murine proteins demonstrates that myostatin has been very highly conserved during mammalian evolution, suggesting an important role for this gene. This role has been demonstrated by the production of knockout mice, which develop greatly enlarged muscles (McPherron et al. 1997). The double-muscled trait in Belgian Blue and Piedmontese cattle has significant similarity to the phenotype of these mice, as both involve an increase in the muscle mass (Arthur 1995; McPherron et al. 1997). The increase in body weight found in mutant myostatin mice can be explained by an increase in muscle mass resulting from muscle fiber hyperplasia and hypertrophy. The increase in body weight of double-muscled cattle cannot be totally accounted for by an increase in muscle mass alone, as the weight of the skin, adipose and bone content, alimentary tract, and most other internal organs is reduced in double-muscled cattle. Also the increased musculature of double-muscled cattle results only from hyperplasia of muscle fibers and not muscle fiber hyperplasia and hypertrophy as found in the mutant mouse (Hanset et al. 1977). These differences in bovine and mouse phenotypes could be attributable to additional genes selected for during inbreeding of double-muscled cattle breeds.

How does myostatin negatively regulate myogenesis? Because myostatin expression is detected in the myotome early in myogenesis through to adult skeletal muscle, it may control fiber number and size during embryonic, fetal, and postnatal myogenesis. Based on the amino acid sequence, Myostatin is a secreted protein that is specifically synthesized by muscle tissue and therefore may be involved with autocrine or paracrine cell–cell communication that regulates proliferation and/or differentiation of myoblasts.

The phenotype of the knockout mice suggests that any mutation that affects myostatin production or activity could lead to muscle hyperplasia and hypertrophy. In both mice and cattle the heterozygotes display very mild abnormalities. The mild phenotypes displayed bymh/+ are probably caused by haploinsufficiency. Examination of the steady-state levels of myostatin mRNA through a range of developmental stages and in different adult muscles failed to reveal any significant difference in the levels of expression in double-muscled cattle, suggesting that differences inmyostatin transcription do not underlie the mhphenotype. However, sequence analysis of mh/mh animals revealed the presence of significant mutations in the third exon of the gene. Presumably the loss of a critical cysteine residue has a negative effect on the activity of Myostatin in Piedmontese cattle, although the extent to which the activity is diminished is uncertain. Certainly the truncation observed in Belgian Blue cattle is likely to severely impair Myostatin function, as it removes the majority of the part of the protein that was disrupted in the knockout mice. We conclude that myostatin is probably the mh locus and that it appears that a nonfunctional Myostatin protein is responsible for loss of control of muscle growth in double-muscled Belgian Blue and Piedmontese cattle as well as knockout mice.

METHODS

Sample Collections

Muscle biopsies (0.5 gram) were obtained from the Musculus (Mo) biceps femoris muscle from 18 adult double-muscled Belgian Blue cattle. The cattle biopsied were derived from 17 different sire lines with limited inbreeding over the last three generations. The pedigrees sampled represent germ-plasm from 17 (NZ) and 4 (USA) sire lines imported from Belgium, with each animal carrying at least one allele from a unique sire. Muscle samples from normal Friesian cattle were obtained after slaughter at the AgResearch abattoir. Normal bovine embryos from day 15 to 34 were collected in triplicate by flushing the uterine tracks of cows; fetuses from day 50 to day 260 were collected after slaughter. Double-muscled Belgian Blue fetuses (in triplicate) were obtained from the recipient cows, which were implanted with purebred Belgian Blue embryos. DNA was obtained from blood sample of Piedmontese bulls by salt extraction (Miller et al. 1988). Adult Piedmontese muscle was obtained from the M. biceps femoris immediately after slaughter in the MARC abbatoir.

RNA Extraction and RT–PCR

RNA from muscle or embryo tissue was extracted using Trizol (GIBCO BRL), according to the manufacturer’s protocol. First-strand cDNA was synthesized in a 20-μl reverse transcriptase (RT) reaction from 5 μg of total RNA using a Superscript preamplification kit (GIBCO-BRL), according to the manufacturers protocol.

Semiquantitative PCR was performed with 2 μl of the RT reaction at 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min for 35 cycles. To clone the bmyostatin entire coding sequence we used the following primers: 5′-ATGATGCAAAAACTGCAA-3′ and 5′-TCATGAGCACCCACA-3′ (1127 bp) and 5′-TCGGACGGACATGCACTAA-3′ and 5′-GTCTACTACCATGGCTGGAAT-3′ (1202 bp). The primers used to amplify the 513-bp cDNA shown in Figure 2, A and B, were 5′-GGTATTTGGCAGAGTATTGAT-3′ and 5′-GTCTACTACCATGGCTGGAAT-3′. To clone full-length cDNA from Piedmontese cattle we used alternate primers: 5′-TCACTTGGCATTACTCAAAAGC-3′ and 5′-TCGAAATTGAGGGGAAGACC-3′. The 493-bp product containing exon 3 of myostatin gene from Piedmontese cattle was amplified from genomic DNA using the reverse primer noted above and an intron-specific primer: 5′-TGAGGTAGGAGAGTGTTTTGGG-3′.

Sequencing and Sequence Analysis

PCR-amplified bovine myostatin cDNA fragments were run on a low melting point agarose gel. DNA fragments were excised from the gel, and DNA was purified using the Wizard kit (Promega) and directly sequenced on an ABI automated sequencer (model no. 377). Sequence alignments were performed by using DNA Laser Gene software (DNA STAR).

Northern Analysis

Northern analysis was performed according to Sambrook et al. (1989). Fifteen micrograms of total RNA from various muscles was run on a 1.0% formaldehyde–agarose gel and transferred to Hybond N+ membrane (Amersham). The membrane was prehybridized in 5× SSC, 50% formamide, 5× Denhardt’s solution, and 1% SDS, 0.25 mg/ml of Salmon sperm DNA for 2 hr, hybridized in the same solution withbmyostatin cDNA probe overnight, washed at 50°C for 15 min each with 2× SSC + 0.1% SDS, and then with 0.2× SSC + 0.1% SDS.

Recombinant myostatin (GDF-8) propeptide enhances the repair and regeneration of both muscle and bone in a model of deep penetrant musculoskeletal injury.

Hamrick MW, Arounleut P, Kellum E, Cain M, Immel D, Liang LF.

Source

Department of Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA. mhamrick@mail.mcg.edu

Abstract

BACKGROUND:

Myostatin (GDF-8) is known as a potent inhibitor of muscle growth and development, and myostatin is also expressed early in the fracture healing process. The purpose of this study was to test the hypothesis that a new myostatin inhibitor, a recombinant myostatin propeptide, can enhance the repair and regeneration of both muscle and bone in cases of deep penetrant injury.

METHODS:

We used a fibula osteotomy model with associated damage to lateral compartment muscles (fibularis longus and brevis) in mice to test the hypothesis that blocking active myostatin with systemic injections of a recombinant myostatin propeptide would improve muscle and bone repair. Mice were assigned to two treatment groups after undergoing a fibula osteotomy: those receiving either vehicle (saline) or recombinant myostatin propeptide (20 mg/kg). Mice received one injection on the day of surgery, another injection 5 days after surgery, and a third injection 10 days after surgery. Mice were killed 15 days after the osteotomy procedure. Bone repair was assessed using microcomputed tomography (micro-CT) and histologic evaluation of the fracture callus. Muscle healing was assessed using Masson trichrome staining of the injury site, and image analysis was used to quantify the degree of fibrosis and muscle regeneration.

RESULTS:

Three propeptide injections over a period of 15 days increased body mass by 7% and increased muscle mass by almost 20% (p < 0.001). Micro-CT analysis of the osteotomy site shows that by 15 days postosteotomy, bony callus tissue was observed bridging the osteotomy gap in 80% of the propeptide-treated mice but only 40% of the control (vehicle)-treated mice (p < 0.01). Micro-CT quantification shows that bone volume of the fracture callus was increased by 30% (p < 0.05) with propeptide treatment, and the increase in bone volume was accompanied by a significant increase in cartilage area (p = 0.01). Propeptide treatment significantly decreased the fraction of fibrous tissue in the wound site and increased the fraction of muscle relative to fibrous tissue by 20% (p < 0.01).

CONCLUSIONS:

Blocking myostatin signaling in the injured limb improves fracture healing and enhances muscle regeneration. These data suggest that myostatin inhibitors may be effective for improving wound repair in cases of orthopaedic trauma and extremity injury.

Myostatin: a modulator of skeletal-muscle stem cells.

Walsh FS, Celeste AJ.

Source

Wyeth Research, Collegeville, PA 19426, USA. walshfs@wyeth.com

Abstract

Myostatin, or GDF-8 (growth and differentiation factor-8), was first identified through sequence identity with members of the BMP (bone morphogenetic protein)/TGF-beta (transforming growth factor-beta) superfamily. The skeletal-muscle-specific expression pattern of myostatin suggested a role in muscle development. Mice with a targeted deletion of the myostatin gene exhibit a hypermuscular phenotype. In addition, inactivating mutations in the myostatin gene have been identified in 'double muscled' cattle breeds, such as the Belgian Blue and Piedmontese, as well as in a hypermuscular child. These findings define myostatin as a negative regulator of skeletal-muscle development. Myostatin binds with high affinity to the receptor serine threonine kinase ActRIIB (activin type IIB receptor), which initiates signalling through a smad2/3-dependent pathway. In an effort to validate myostatin as a therapeutic target in a post-embryonic setting, a neutralizing antibody was developed by screening for inhibition of myostatin binding to ActRIIB. Administration of this antimyostatin antibody to adult mice resulted in a significant increase in both muscle mass and functional strength. Importantly, similar results were obtained in a murine model of muscular dystrophy, the mdx mouse. Unlike the myostatin-deficient animals, which exhibit both muscle hypertrophy and hyperplasia, the antibody-treated mice demonstrate increased musculature through a hypertrophic mechanism. These results validate myostatin inhibition as a therapeutic approach to muscle wasting diseases such as muscular dystrophy, sarcopenic frailty of the elderly and amylotrophic lateral sclerosis.

Myostatin (GDF-8) as a key factor linking muscle mass and bone structure.

http://www.ncbi.nlm.nih.gov/gene/2660





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