Myostatin

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Myostatin (also known as growth differentiation factor 8, abbreviated GDF8) is a protein that in humans is encoded by the MSTN gene.<ref name="pmid9843994">Template:Cite journal</ref> Myostatin is a myokine that is produced and released by myocytes and acts on muscle cells to inhibit muscle growth.<ref>Template:Cite journal</ref> Myostatin is a secreted growth differentiation factor that is a member of the TGF beta protein family.<ref name="pmid16842126">Template:Cite journal</ref><ref name="pmid17374508">Template:Cite journal</ref>

Myostatin is assembled and produced in skeletal muscle before it is released into the blood stream.<ref name=":1" /> Most of the data regarding the effects of myostatin comes from studies performed on mice.<ref name=":2" />

Animals either lacking myostatin or treated with substances that block the activity of myostatin have significantly more muscle mass. Furthermore, individuals who have mutations in both copies of the myostatin gene (popularly called the "Hercules gene") have significantly more muscle mass and are stronger than normal. There is hope that studies into myostatin may have therapeutic application in treating muscle wasting diseases such as muscular dystrophy.<ref name="pmid18600566"/>

Discovery and sequencing

The gene encoding myostatin was discovered in 1997 by geneticists Se-Jin Lee and Alexandra McPherron who produced a knockout strain of mice that lack the gene, and have approximately twice as much muscle as normal mice.<ref name="McPherron_1997">Template:Cite journal</ref> These mice were subsequently named "mighty mice".

Naturally occurring deficiencies of myostatin of various sorts have been identified in some breeds of cattle,<ref name="Kambadur_1997">Template:Cite journal</ref> sheep,<ref name="pmid16751773">Template:Cite journal</ref> whippets,<ref name=Mosher/> and humans.<ref name= avery/> In each case the result is a dramatic increase in muscle mass.

Structure and mechanism of action

Template:One source Human myostatin consists of two identical subunits, each consisting of 109 (NCBI database claims human myostatin is 375 residues long) amino acid residues [note the full length gene encodes a 375AA prepro-protein which is proteolytically processed to its shorter active form].<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref><ref>Template:Cite journal</ref> Its total molecular weight is 25.0 kDa. The protein is inactive until a protease cleaves the NH2-terminal, or "pro-domain" portion of the molecule, resulting in the active COOH-terminal dimer. Myostatin binds to the activin type II receptor, resulting in a recruitment of either coreceptor Alk-3 or Alk-4. This coreceptor then initiates a cell signaling cascade in the muscle that 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 proliferation and either initiates differentiation or stimulates quiescence.

In mature muscle, myostatin inhibits Akt, a kinase that is sufficient to cause muscle hypertrophy, in part through the activation of protein synthesis while stimulating the production of ubiquitin ligases, proteins that regulate muscle protein breakdown. However, Akt is not responsible for all of the observed muscle hypertrophic effects, which are mediated by myostatin inhibition.<ref>Template:Cite journal</ref> Thus myostatin acts in two ways: by inhibiting Akt-induced protein synthesis and stimulating ubiquitin-regulated protein degradation.

Biological advantage

Many different mammalian and avian species produce myostatin, indicating that the ability to produce myostatin was positively selected for.<ref name=":0">Template:Citation</ref>

Effects in animals

Mutations

Mutations in myostatin do more than just affect the amount of muscle mass an organism can produce; they also have variable effects on other phenotypes for different species.<ref name=":0" />For example, a Belgian Blue bovine with a mutation that inhibits myostatin production will exhibit a dramatic increase in muscle mass but will also lead to dystocia.<ref name=":0" /> Other species with myostatin deficiency mutation such as humans or Whippet dogs do not get obstructed labor.<ref name=":2">Template:Citation</ref>

Double-muscled cattle

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Belgian Blue cattle

After the discovery of the gene encoding myostatin in 1997, several laboratories cloned and established the nucleotide sequence of a myostatin gene in two breeds of cattle, Belgian Blue and Piedmontese. They found mutations in the myostatin gene (various mutations in each breed) which in one way or another lead to absence of functional myostatin.<ref name="McPherron_1997"/><ref name="Kambadur_1997"/><ref name="Grobet_1997">Template:Cite journal</ref> Unlike mice with a damaged myostatin gene, in these cattle breeds, 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%.<ref name="Kambadur_1997"/><ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref><ref>Template:Cite journal</ref>

Animals lacking myostatin or animals treated with substances such as follistatin that block the binding of myostatin to its receptor have significantly larger muscles. Thus, reduction of myostatin could potentially benefit the livestock industry, with even a 20 percent reduction in myostatin levels potentially having a large effect on the development of muscles.<ref name="Kota_2009"/>

However, the animal breeds developed as homozygous for myostatin deficiency have reproduction issues due to their unusually heavy and bulky offspring, and require special care and a more expensive diet to achieve a superior yield. This negatively affects economics of myostatin-deficient breeds to the point where they do not usually offer an obvious advantage. While hypertrophic meat (e.g. from Piedmontese beef) has a place on the specialist market due to its high palatability and tenderness, at least for purebred myostatin-deficient strains the expenses and (especially in cattle) necessity of veterinary supervision place them at a disadvantage in the bulk market.<ref name="De_Smet_2004">Template:Cite encyclopedia</ref>

WhippetsTemplate:Anchor

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A "bully whippet" with a homozygous mutation in myostatin<ref name="Mosher">Template:Cite journal</ref>

Whippets can have a mutation of the myostatin which involves a two-base-pair deletion, and results in a truncated, and likely inactive, myostatin protein.

Animals with a homozygous deletion have an unusual body shape, with a broader head, pronounced overbite, shorter legs, and thicker tails, and are called "bully whippets" by the breeding community. Although significantly more muscular, they are less able runners than other whippets. However, whippets that were heterozygous for the mutation were significantly over-represented in the top racing classes.<ref name=Mosher/> In 2015 scientist used CRISPR/Cas9 to have the same homozygous deletion in Beagles that appears in "bully whippets".<ref>Template:Cite journal</ref><ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref>

Mice

Mice that produce large amounts of myostatin exhibit a significant loss of skeletal muscle and body fat compared to normal mice.<ref name=":1" /> Comparatively, mice that produced decreased levels of myostatin had more muscle mass, less adipose tissue, and were double the size of wild type mice.<ref name=":1" />

Rabbits and goats

In 2016, the CRISPR/Cas9 system was used to genetically engineer rabbits and goats with no functional copies of the myostatin gene.<ref>Template:Cite journal</ref> In both cases the resulting animals were significantly more muscular. However, rabbits without myostatin also exhibited an enlarged tongue, a higher rate of still births, and a reduced lifespan.

Pigs

A South Korean-Chinese team has engineered "double muscle" pigs, as with cattle, aiming for cheaper breeds for the meat market.<ref name="Cyranoski-2015" /> Similar health problems have resulted as with other mammals, such as birthing difficulties due to excessive size.<ref name="Cyranoski-2015" />

Fish

Myostatin-disabled red sea breams grow to 1.2 the natural average size with the same amount of food and are sold as food in Japan by a startup.<ref>Template:Cite journal</ref><ref>Template:Cite news</ref><ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref>

Clinical significance

Mutations

A technique for detecting mutations in myostatin variants has been developed.<ref>Template:Cite patent</ref> Mutations that reduce the production of functional myostatin lead to an overgrowth of muscle tissue. Myostatin-related muscle hypertrophy has an incomplete autosomal dominance pattern of inheritance. 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) have increased muscle bulk, but to a lesser degree.Template:Citation needed

In humans

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 has a mutation in one copy of the gene.<ref name=avery>Template:Cite news</ref><ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref><ref>Template:Cite journal</ref>

An American boy born in 2005 was diagnosed with a clinically similar condition, but with a somewhat different cause:<ref name="urlCTV.ca|Rare condition gives toddler super strength">Template:Cite news</ref> his body produces a normal level of functional myostatin, but because he is stronger and more muscular than most others his age, a defect in his myostatin receptors is thought to prevent his muscle cells from responding normally to myostatin. He appeared on the television show World's Strongest Toddler.<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref>

Therapeutic potential

Further research into myostatin and the myostatin gene may lead to therapies for muscular dystrophy.<ref name="pmid18600566">Template:Cite journal</ref><ref name="pmid15215484">Template:Cite journal

• Template:Cite magazine</ref> The idea is to introduce substances that block myostatin. A monoclonal antibody specific to myostatin increases muscle mass in mice<ref name="pmid12559968">Template:Cite journal</ref> and monkeys.<ref name="Kota_2009">Template:Cite journal
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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%).<ref name="pmid16330774">Template:Cite journal</ref> It is thought that binding of myostatin to the soluble activin receptor prevents it from interacting with the cell-bound receptors.Template:Citation needed In September 2020 scientists reported that suppressing activin type 2 receptors-signalling proteins myostatin and activin A via activin A/myostatin inhibitor ACVR2B – tested preliminarily in humans in the form of ACE-031 in the early 2010s<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref><ref>Template:Cite journal</ref> – can protect against both muscle and bone loss in mice. The mice were sent to the International Space Station and could largely maintain their muscle weights – about twice those of wild type due to genetic engineering for targeted deletion of the myostatin gene – under microgravity.<ref>Template:Cite news</ref><ref>Template:Cite journal</ref> Treating progeric mice with soluble activin receptor type IIB before the onset of premature ageing signs appear to protects against muscle loss and delay age related signs in other organs.<ref name="Alyodawi_2019">Template:Cite journal</ref>

It remains unclear as to whether long-term treatment of muscular dystrophy with myostatin inhibitors is beneficial, as the depletion of muscle stem cells could worsen the disease later on. Template:As of, no myostatin-inhibiting drugs for humans are on the market. An antibody genetically engineered to neutralize myostatin, stamulumab, which was under development by pharmaceutical company Wyeth,<ref>MYO-029 press release, mda.org, February 23, 2005.</ref> is no longer under development.<ref>Wyeth Won't Develop MYO-029 for MD Template:Webarchive, mda.org, March 11, 2008.</ref> Some athletes, eager to get their hands on such drugs, turn to the internet where fake "myostatin blockers" are being sold.<ref name="Kota_2009"/>

Resistance exercise and creatine supplementation lead to greater decreases in myostatin levels.<ref name="pmid20026378">Template:Cite journal</ref>

Myostatin levels can be temporarily reduced using a cholesterol-conjugated siRNA gene knockdown.<ref>Template:Cite journal</ref>

Athletic use

Inhibition of myostatin leads to muscle hypertrophy. Myostatin inhibitors can improve athletic performance and therefore there is a concern these inhibitors might be abused in the field of sports.<ref name="Haisma_2006">Template:Cite journal</ref> However, studies in mice suggest that myostatin inhibition does not directly increase the strength of individual muscle fibers.<ref name="pmid21565991">Template:Cite journal</ref> Myostatin inhibitors are specifically banned by the World Anti-Doping Agency (WADA).<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref> In an August 12, 2012, interview with NPR, Carlon Colker stated "when the myostatin inhibitors come along, they'll be abused. There's no question in my mind."<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref>

Lifestyle effects on myostatin

Myostatin expression is decreased in physically active individuals, while obesity is linked to having a higher level myostatin as well as plasma in circulation.<ref name=":1">Template:Citation</ref>

Effects

On bone formation

Due to myostatin's ability to inhibit muscle growth, it can indirectly inhibit bone formation by decreasing the load on the bone.<ref>Template:Cite journal</ref><ref name="Tarantino_2015">Template:Cite journal</ref> It has a direct signalling effect on bone formation<ref>Template:Cite journal</ref> as well as degradation.<ref name="Dankbar_2015">Template:Cite journal</ref><ref name="Tarantino_2015" /> Knockdown of myostatin has been shown to reduce formation of osteoclasts (multinucleated cells responsible for the breakdown of bone tissue) in mice modeling rheumatoid arthritis.<ref name="Dankbar_2015" /> Rheumatoid arthritis is an autoimmune disorder that, among other effects, leads to the degradation of the bone tissue in affected joints. Myostatin has not, however, been shown to be solely sufficient for the formation of mature osteoclasts from macrophages, only an enhancer.

Myostatin expression is increased around the site of a fracture. Suppression of myostatin at the fracture site leads to increased callus and overall bone size, further supporting the inhibitory effect of myostatin on bone formation. One study<ref name="Dankbar_2015" /> by Berno Dankbar et al., 2015 found that myostatin deficiency leads to a notable reduction in inflammation around a fracture site. Myostatin affects osteoclastogenesis by binding to receptors on osteoclastic macrophages and causing a signalling cascade. The downstream signalling cascade enhances the expression of RANKL-dependent integrin αvβ3, DC-STAMP, calcitonin receptors, and NFATc1 (which is part of the initial intracellular complex that starts the signaling cascade, along with R-Smad2 and ALK4 or ALK5).<ref name="Dankbar_2015" /><ref name="Tarantino_2015" />

An association between osteoporosis, another disease characterized by the degradation of bony tissue, and sarcopenia, the age-related degeneration of muscle mass and quality have also been found.<ref name="Tarantino_2015" /> Whether this link is a result of direct regulation or a secondary effect through muscle mass is not known.

A link in mice between the concentration of myostatin in the prenatal environment and the strength of offspring's bones, partially counteracting the effects of osteogenesis imperfecta (brittle bone disease) has been found.<ref name="Oestreich_2016">Template:Cite journal</ref> Osteogenesis imperfecta is due to a mutation that causes the production of abnormal Type I collagen. Mice with defective myostatin were created by replacing sequences coding for the C-terminal region of myostatin with a neomycin cassette, rendering the protein nonfunctional. By crossbreeding mice with the abnormal Type I collagen and those with the knockout myostatin, the offspring had "a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure" of their femurs as compared to the other mice with osteogenesis imperfecta, showing the positive effects of decreased myostatin on bone strength and formation.<ref>Template:Cite journal</ref>

On the heart

Myostatin is expressed at very low levels in cardiac myocytes.<ref name="Breitbart_2011">Template:Cite journal</ref><ref name="Heineke_2010">Template:Cite journal</ref> Although its presence has been noted in cardiomyocytes of both fetal and adult mice,<ref name="Sharma_1999">Template:Cite journal</ref> its physiological function remains uncertain.<ref name="Heineke_2010"/> However, it has been suggested that fetal cardiac myostatin may play a role in early heart development.<ref name="Sharma_1999"/>

Myostatin is produced as promyostatin, a precursor protein kept inactive by the latent TGF-β binding protein 3 (LTBP3).<ref name="Breitbart_2011"/> Pathological cardiac stress promotes N-terminal cleavage by furin convertase to create a biologically active C-terminal fragment. The mature myostatin is then segregated from the latent complex via proteolytic cleavage by BMP-1 and tolloid metalloproteinases.<ref name="Breitbart_2011"/> Free myostatin is able to bind its receptor, ActRIIB, and increase SMAD2/3 phosphorylation.<ref name="Breitbart_2011"/> The latter produces a heteromeric complex with SMAD4, inducing myostatin translocation into the cardiomyocyte nucleus to modulate transcription factor activity.<ref name="McKoy_2007">Template:Cite journal</ref> Manipulating the muscle creatinine kinase promoter can modulate myostatin expression, although it has only been observed in male mice thus far.<ref name="Breitbart_2011"/><ref name="Heineke_2010"/>

Myostatin may inhibit cardiomyocyte proliferation and differentiation by manipulating cell cycle progression.<ref name="Sharma_1999"/> This argument is supported by the fact that myostatin mRNA is poorly expressed in proliferating fetal cardiomyocytes.<ref name="Breitbart_2011"/><ref name="McKoy_2007"/> In vitro studies indicate that myostatin promotes SMAD2 phosphorylation to inhibit cardiomyocyte proliferation. Furthermore, myostatin has been shown to directly prevent cell cycle G1 to S phase transition by decreasing levels of cyclin-dependent kinase complex 2 (CDK2) and by increasing p21 levels.<ref name="McKoy_2007"/>

Growth of cardiomyocytes may also be hindered by myostatin-regulated inhibition of protein kinase p38 and the serine-threonine protein kinase Akt, which typically promote cardiomyocyte hypertrophy.<ref name="Morissette_2006">Template:Cite journal</ref> However, increased myostatin activity only occurs in response to specific stimuli,<ref name="Breitbart_2011"/><ref name="Morissette_2006"/> such as in pressure stress models, in which cardiac myostatin induces whole-body muscular atrophy.<ref name="Breitbart_2011"/><ref name="Sharma_1999"/>

Physiologically, minimal amounts of cardiac myostatin are secreted from the myocardium into serum, having a limited effect on muscle growth.<ref name="Heineke_2010"/> However, increases in cardiac myostatin can increase its serum concentration, which may cause skeletal muscle atrophy.<ref name="Breitbart_2011"/><ref name="Heineke_2010"/> Pathological states that increase cardiac stress and promote heart failure can induce a rise in both cardiac myostatin mRNA and protein levels within the heart.<ref name="Breitbart_2011"/><ref name="Heineke_2010"/> In ischemic or dilated cardiomyopathy, increased levels of myostatin mRNA have been detected within the left ventricle.<ref name="Breitbart_2011"/><ref name="Torrado_2010">Template:Cite journal</ref>

As a member of the TGF-β family, myostatin may play a role in post-infarct recovery.<ref name="Heineke_2010"/><ref name="Sharma_1999"/> It has been hypothesized that hypertrophy of the heart induces an increase in myostatin as a negative feedback mechanism in an attempt to limit further myocyte growth.<ref name="Wang_2008">Template:Cite journal</ref><ref name="Shyu_2005">Template:Cite journal</ref> This process includes mitogen-activated protein kinases and binding of the MEF2 transcription factor within the promoter region of the myostatin gene. Increases in myostatin levels during chronic heart failure have been shown to cause cardiac cachexia.<ref name="Breitbart_2011"/><ref name="Heineke_2010"/><ref name="Anker_2003">Template:Cite journal</ref> Systemic inhibition of cardiac myostatin with the JA-16 antibody maintains overall muscle weight in experimental models with pre-existing heart failure.<ref name="Heineke_2010"/>

Myostatin also alters excitation-contraction (EC) coupling within the heart.<ref name="Rodgers_2009">Template:Cite journal</ref> A reduction in cardiac myostatin induces eccentric hypertrophy of the heart, and increases its sensitivity to beta-adrenergic stimuli by enhancing Ca²⁺ release from the SR during EC coupling. Also, phospholamban phosphorylation is increased in myostatin-knockout mice, leading to an increase in Ca²⁺ release into the cytosol during systole.<ref name="Breitbart_2011"/> Therefore, minimizing cardiac myostatin may improve cardiac output.<ref name="Rodgers_2009"/>

See also

• Muscular dystrophy
• Muscle hypertrophy
• Myostatin-related muscle hypertrophy
• Superhuman strength

External links

• GeneReviews profile
• NPR.org: Myostatin Therapies Hold Hope for Muscle Diseases by Jon Hamilton
• Times Colonist Big Wendy the muscular whippet
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