Myostatin Group Paper

8/13/2019 Myostatin Group Paper

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By: Caroline Carswell , Nicole Hartman, M ichelina Heise, M ike McNeil l, Katheri ne Vary, and

Ryan Wil genkamp.

Introduction:

Myostatin was discovered in 1997 by McPherron and Lee when they produced mice that

had twice as much muscle as normal mice. These mice were knockouts meaning that they did not

have the gene that encoded myostatin. They are said to have doubling muscle disease. It is a

negative regulator of muscle growth and is produced in skeletal muscle cells predominantly. A

functional myostatin gene and protein work to limit muscle growth during all stages of life. Over

expression of the myostatin gene can decrease muscle mass whereas under expression can lead to

increased muscle mass. Agriculturally, there are many studies to see if it is commercially

beneficial to have double muscled animals. These animals include cattle, poultry, trout, and

whippets. Of course a mutation in the myostatin gene can affect humans as well. These humans

have a 10 to 40% increased muscle mass. Before the physical effects that are produced it is

important to understand the gene organization.

Structure of Myostatin:

Myostatin is a Growth Differentiation Factor- (GDF-). Myostatin is a member of the

Transforming Growth Factor- (TGF-) superfamily. TGF- superfamily is a large family of

structurally related cell regulatory proteins that control proliferation, differentiation, and other

functions in many types of cells of several different species. The TGF- superfamily includes

ligands for bone morphogenetic proteins, growth and differentiation factors, Anti-mullerian

hormone, TGF-s, and activin. Activins are involved in embryogenesis and osteogenesis and

regulation of hormones. TGF-s are also involved in embryogenesis, apoptosis, and cell

differentiation. Myostatin exhibits all of the structural characteristics that are common to the


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TGF- family: nine invariant cysteine residues, and RXXR furin-type proteolytic processing

site, and a bioactive C-terminal domain.

Myostatin is found on chromosome 2: base pairs 190,920,425 to 190,927,454. The

genomic organization includes three similarly sized exons separated by two prevailing introns

whose sizes appear to be conserved (~2kb) in birds and mammals. Intron sizes are smaller

among salmonid MSTN-2 genes, which is consistent with an increased susceptibility to relaxed

selection because the probable development of null mutations within coding regions is greater

among genes with smaller noncoding regions. Both rainbow trout and Atlantic salmon MSTN-2b

paralogs contain in-frame stop codons in their first exons and lack a 51-bp cassette from the

second exon that is common to all other fish MSTN genes. It is unclear whether similar indels

and polymorphisms exist in myostatin genes from other salmonid species. This data also

suggests that only three of the four myostatin genes are functionally active in modern salmonids

because rtMSTN-2b appears to be a pseudogene.

Fig. 1: Chromosomal Location of the Myostatin Gene.

Comparison of myostatin sequences revealed that myostatin was extremely well

conserved throughout evolution. Remarkably, the murine, rat, human, porcine, chicken and

turkey myostatin sequences are all identical in the active C-terminal region of the protein,

suggesting that the function of this gene might be conserved in all vertebrates. However, this has

not been tested.


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Fig. 2: Myostatin genes of various species.

Alignments of myostatin amino acid sequences from multiple vertebrate species show

that the proteins are well conserved overall, and within related taxa. It is unknown whether the

intermolecular interactions that occur between mammalian myostatin and LAP peptides also

occur in other vertebrates. Small, but notable, differences within the bioactive domains of fish

myostatin (MSTN)-1 and -2 orthologs exist and include methionine for lysine and histidine for

arginine substitutions in the carboxy-terminal domains of fish MSTN-2 proteins. The former

substitution occurs in a region hypothesized to have contributed to enhance musculature in

domesticated and wild bovid.

Many species show similar sequence conservation of myostatin. The species that differ

slightly would be baboon, bovine and ovine. The myostatin gene, in these species, contains only

one to three amino acid differences in the mature protein. Also the zebra fish myostatin would be

the most diverse. Compared to the other species, it is only 88% identical.


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Myostatin is a unique negative regulator of muscle growth. Myostatin has many salient

features in skeletal muscles including activating activin receptor type II B/SMAD and nuclear

factor B signaling, and reducing insulin like growth factor 1/phosphoinositide 3 kinase/Akt

pathway.

Scientists who conducted the Women's Health and Aging Study II, revealed six

different variants in the myostatin gene that are polymorphic; this was proved in their study

population of 286 women, age 70 to 79. Of these, 81.1% were Caucasian, 18.8% were African

American, and 0.2% were Asian or Hispanic (Seibert). The overall mean strength of these

participants that those women with a K genotype had higher/more muscle strength than those

women with an R genotype. Another study focused on the myostatin variations in average men

(with no previous athletic training). They found that there were several polymorphic variations

in this gene. The Lys(K)153Arg(R) polymorphism in exon 2 (rs1805086, 2379 A>G

replacement) of the myostatin (MSTN) gene is a candidate to influence skeletal muscle

phenotypes. This is stating that the polymorphism in the genes

are very likely responsible for the variation in the muscle

phenotype of the men that were studied. In the myostatin gene,

there are two domains identified. One is the pro-terminal

knownas the NH2-terminal. This is a site on myostatin where it

is activated by a protease, which leaves it as an active site. In

Figure three, the myostatin propeptide domain is shown. This is

from a company called abcam, who put the domain on the

market for sale for thepurposes of research .

Fig 3: Propeptide domain

of Myostatin Gene.


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Gene Expression and Regulation:

Myostatin circulates in the blood in its inactive form before it is cleaved by a propeptide.

Once cleaved, the active myostatin acts on muscle tissue by binding a cell-bound receptor called

the activin type II receptor. After binding to the activin type II receptor, a coreceptor called

Alk-3 or Alk-4 is recruited. The coreceptor then initiates a cell signaling cascade in the muscle

including activation of transcription factors SMAD2 and

SMAD3. These two factors are bound to SMAD4 in order

to be carried to the nucleus where they can influence the

gene regulation in the myoblast cells and inhibit their

differentiation into mature muscle fibers. SMADs are

intracellular proteins in charge of activating downstream

TGF- gene transcription in the nucleus. In the nucleus, these proteins interact with different

cellular partners, bind to DNA, and regulate transcription of various downstream response genes

related to skeletal muscle growth.

Myostatin is active in muscles both before and after birth, restraining the muscle growth

so that they do not become too large. During embryogenesis, myostatin is expressed by cells in

the myotome and developing skeletal muscle to regulate the final

number of muscle fibers that are produced. During adult life,

myostatin is predominantly produced in the skeletal muscle and

circulates in the blood.

If the myostatin gene is damaged, it causes the absence of

functional myostatin. In cows this leads to multiplication of muscle

cells and in mice it leads to growth of muscle cells. Animals

Fig. 4: SMAD proteins bind to

DNA to influence gene expression.

Fig. 5: Mice treated with

follistatin exhibit

increasedmuscle mass.


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treated with substances like follistatin have a larger mass of muscle because it blocks the binding

ability of myostatin to bind to the receptor.

An October 2002 study

suggested that the myostatin gene is

a downstream target gene of MyoD

(a protein that plays a key role in

muscle differentiation). Comparing

synchronized myoblasts and

reserve cells, results of the study revealed that the myostatin promoter activity is higher during

the G1 phase of the cell cycle. MyoD expression was lacking in the reserve cells of the cycle and

had a significant reduction in myostatin promoter activity. MyoD could be triggering myoblast

withdraw from the cell cycle by regulating the myostatin gene expression.

A study published in theJournal of Physiologic Genomics in 2009 looked at the effect of

loss of myostatin signaling on gene expression in muscle. They analyzed RNA from mice with

postdevelopmental myostatin knockout in oligonucleotide arrays. Myostatin was undetectable in

muscle within two weeks of Cre recombinase activation in four-month-old male mice. Three

months after knocking out the myostatin gene, muscle mass had increased by 26%. The

expression of several hundred genes differed in knockout mice compared to control mice. They

found that mice with postdevelopmental knockout of the myostatin gene did not downregulate

expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins

involved in energy metabolism. In addition, several collagen genes were expressed at 20-50%

lower rates in myostatin-deficient mice, which suggests that myostatin regulates collagen

production in muscle.

Fig. 6: Role of MyoD and Myostatin during muscle growth and

differentiation.


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A study published inExperimental Biology and Medicine in 2003 examined changes in

myostatin gene expression in response to strength training. Seven men and eight women

completed a 9-week strength training program. Muscle biopsies were obtained from each

participant before and after the training. Myostatin mRNA levels, muscle volume, and muscle

strength were measured. They found a 37% decrease in myostatin expression in all subjects

combined, and the decline was similar regardless of age or gender. There were no significant

correlations between myostatin expression and muscle strength or volume in this data. While

further work is necessary, due to the small sample size, the data did demonstrate a decrease in

myostatin mRNA levels in response to heavy-resistance strength training in humans.

An August 2012 study shows that the myostatin gene is transcriptionally regulated by

multiple genes including MEF2. The study investigated transcriptional regulation of MEF2 on

porcine Mstn promotor activity. The study found that MEF2C restrains myogenesis by Mstn

activation and Mstn-dependent gene processing in porcine.

Satellite cells are skeletal muscle stem cells that contribute to postnatal growth and

muscle regeneration after injury or disease. Targeting Mstn in satellite cells has potential for

application to livestock performance. This study investigated the effect of the bioactive

component sulforaphane (SFN) on Mstn in satellite cells. SFN supplementation in vitro

inhibited histone deacetylace (HDAC) and DNA methyltransferase (DNMT) in porcine satellite

cells. SFN treatment represses Mstn expression and increases expression of negative feedback

inhibitors of the Mstn signaling pathway. SFN regulation of Mstn is epigenetic and weakens the

Mstn signaling pathway.

MicroRNAs are noncoding RNAs that play rose in skeletal muscle development and in

regulation of muscle cell proliferation and differentiation. Expression of miR-27a in increased


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during proliferation of C2C12 myoblasts. Overexpression of miR27-a promotes myoblast

proliferation by reducing expression of myostatin. MiR-27a targets myostatin 3'UTR.

In a 2013 publication, it was found that the

myostatin gene could be regulated by shRNAs in

chicken embryonic myoblast cells. Seven

different shRNAs constructs were produced by

using the techniques of Reverse Transcription

quantitative real time PCR. The silencing

efficiency of the myostatin constructs were first

tested in human embryonic kidney cell lines and

revealed a 30-75.6 percent reduction in the

myostatin gene. Next, the shRNAs were used in

the chicken embryo myoblast cells and revealed a 55 percent reduction in myostatin expression.

To prove that myostatin controls muscle formation, Janaiah Kota et. al. injected

follistatin, an antagonist of myostatin, into nonhuman primates. When injected into the

quadriceps of monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1

vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength.

Long-term expression of the transgene did not produce any abnormal changes in the morphology

or function of key organs, indicating the safety of gene delivery by intramuscular injection of an

AAV1 vector. The study concluded that, along with the findings in mice and the monkeys, it is

possible to use follistatin to improve muscle mass and functional therapy in patients with certain

degenerative muscle disorders. This study serves as evidence that myostatin is in charge of

controlling the differentiation and formation of muscle mass and function.

Fig. 7: Follistatin injection into primates.


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In another study by L.A. Whittemore, JA16, a

neutralizing monoclonal antibody to myostatin, was

administered to mice. The mass of muscles for the JA16

treated mice were greater than the muscle mass for the

control antibody treated mice. For example, the quadriceps

were 30% larger and the peak force, in a grip test, was 10%

greater in the JA16 treated mice. This shows that

pharmacological inhibition of myostatin increases muscle

mass.

Myostatin Function:

The consequences of the gain of function and

loss of function mutations in the myostatin gene are

opposite reactions. The gain of function mutation

occurs in the myostatin gene when the gene becomes

over expressed in an organism. The overexpression

spikes the level of myostatin binding to activin type II

receptors and inhibits more myoblasts than its normal concentration, resulting in a decrease in

total muscle mass of an organism. On the other side of the spectrum, the loss of function

mutation occurs in the myostatin gene when the gene is mutated to the point that the protein can

no longer bind to the activin type II receptors. When this occurs, the signaling cascade is not

activated and the SMADs are not able to change the gene expression of myoblasts. This

Fig. 8: Mice treated with a

monoclonal antibody exhibit

increased muscle mass.

Fig. 9: As myostatin increases,

muscle mass decreases.


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inhibition results in continuous rapid growth and division of myoblasts, ultimately creating a

dramatic increase in muscle tissue density.

Biotechnological Applications:

Commercially many companies market nutritional supplements that presumably block or

neutralize myostatin. These companies include Most of these products are composed of sulfated

polysaccharides isolate from a brown marine plant that bind to myostatin. A study showed that

after 12 weeks of taking this compound with heavy resistance training the body composition did

not change as much as a placebo group. Although most of these companies make the myostatin

blocker for humans it is the largest commercial market.

Expression of Myostatin Mutation in Animals:

Agriculturally, double muscling cattle frequently require cesarean delivery due to large

calf size. Therefore there is a higher cost of veterinary care. This has prevented the widespread

acceptance of these double muscled breeds in cattle. Not only is there a problem in calving, there

is also effects in the taste of the meat. For example, in South Devon Cattle, no myostatin reduced

fat levels specifically saturated and monounsaturated fats. This decreased flavor and decreased

overall liking and increased abnormal taints. It is unknown whether potential loss of fat would

offset commercial gains. There are greater commercial gains in fish and fowl because they are

egg-laying vertebrates. A more thorough assessment of myostatin nonmuscle actions in fish is

needed to determine whether blocking its actions is feasible and commercially beneficial.


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This mutation is very favorable when it comes to a certain breed of dogs. A racing dog

that has just a single copy of the gene can be a very valuable dog. Whippets, which are

commonly used as racing dogs become

much leaner and are referred to as bully

whippets. These dogs are very fast and

because of this, the gene is now present

in many racing dogs. One of the first

documented cases was for a whippet

named Wendy.

This mutation can also be favorable in fish. Trout were genetically modified by a team

lead by Terry Bradley of the University of Rhode Island. His experiment took ten years and the

end was result was fish that appear to have a six-pack abdominal

muscles. This trout incorporated the same genes that produce the

myostatin inhibiting protein found in the Belgian Blue

cattle. This genetically modified trout is the first proof that

myostatin inhibition has similar effects on both fish and

mammals. Right now the trout are not approved for

consumption, however, if they do get approved, it would mean

very cheap trout because fish would get larger without having to

feed them more. There are trout that are approved with altered

genes, but trout with DNA from other species have not been approved for commercial use.

A slight advantage to the meat industry with regards to the myostatin mutation is that

researchers found heterozygotes produced slightly more favorable trans- 18:1 and CLA fatty acid

Fig. 10: Wendy exhibits one copy of the myostatin gene.

Fig. 11: Trout (left) that is

genetically modified for

myostatin inhibition.


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bred at optimum times. The delay in puberty is due to the overall delay in growth typically seen

by double muscled animals due to their abnormal body composure. Double muscled animals also

have reduced fertility due to the common mortality of double muscled embryos. Due to the

mutation in the gene, the embryo does not always begin to form right with the added muscles.

This ultimately leads to death in some embryos.

In addition to fertility issues, double muscled animals experience many calving issues as

well. Most of the research involving double muscling and birth has been done in the bovine

industry, so cows will be used to further demonstrate the double muscling birthing issues. A cow

is predisposed to birth a certain size calf. When calves become too big, dystocia or stress can

result and an assisted birth is necessary. The assisted birth ties back into the monetary issues of

raising double muscled animals. Needing a veterinarian to intercede in a birth will cost a lot

more than an unassisted birth of a normal calf. Double muscled calves typically have higher birth

weights due to their abnormal muscles. A recent study showed that for each kilogram increase in

birth weight, there will be a 0.7% increase in birthing difficulty. In addition to the size of the calf

being a problem, double muscled mothers have a different internal composition that the normal

cow. High levels of muscling in the pelvis can prevent the distension and springing needed to

birth a calf.

Myostatin in Human Health:

If one copy of the gene is inactive you get a moderate increase in strength and muscle. If

both copies of the gene are inactive then no myostatin will be produced and there will be a large

increase in muscle mass. The use of so many muscle cells to build up this heavy muscle mass

early in life could lead to depletion of muscle stem cells. Doctors worry that heart problems


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could occur down the road. Double muscling requires that you

consume a larger amount of food in order to sustain the muscle

mass. Lack of food could have resulted in the mortality rates of

double muscled children in developing countries. The double

muscling mutation doesn't seem to have too many side effects on

humans besides increases strength and muscle tone.

Human Examples of Double Muscling:

Double muscling was first documented in a human in

2004 with the discovery of a boy in Germany born with

protruding muscles in his legs and forearms. He could supposedly lift 7 pound weights by age 5.

As an infant he would jerk his limbs seemingly involuntary, making his parents wonder if

something was wrong with him. The boy's doctor had heard of research done by Dr. Se-Jin Lee

on mice with myostatin mutations. When

discussed with the boy's mother, she

recalled how her father had always been

very strong and agreed to test her son

and her for the mutations. He was shown

to have a mutation in both copies of his

myostatin genes. His mother had one

copy of the mutation. Another boy

named Liam Hoekstra was found in 2005 to have similar characteristics. He was said to have

started doing pull-ups and sit-ups at only a few months old. Liam had a slightly different cause to

Fig. 12: Child that only

expresses one copy of

myostatin, with moderateincrease in muscle mass.

Fig. 13: German child documented as double muscled.


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his condition, having normal levels of myostatin in his blood. His doctors have suggested he may

have problems with the myostatin receptors in his body.

Myostatin Related Diseases:

There are numerous muscle diseases that can affect both children and adults alike.

Researchers have found that proinflammatory cytokines play an important role in muscle

wasting. Muscle wasting diseases are mainly characterized by the progressive weakening in

muscles, which is often more fatal in young children. Since the myostatin gene is a vital

component in the formation of muscle, scientists believe that it can be pivotal in the treatment of

muscle wasting diseases such as muscular dystrophy, inflammatory muscle diseases, cachexia

and myasthenia gravis. Some of the diseases directly related to myostatin in some form are

further discussed in more detail below.

Myostatin-related Muscle Hypertrophy is a condition that involves decreased body fat

and increased muscle size. Usually people with this disease inherited it via incomplete

autosomal dominance and display twice the muscle mass as normal people. Research has shown

that no other medical problems occur in conjunction with this diseasecardiac function and

intellectual were normal in people possessing the mutation. Besides an increase in muscle size,

individuals with the disease are known to display an increase in strength as well. Ultrasound

examination, DEXA, or MRI are all tools that can be used to diagnose the disease by measuring

skeletal muscle size in suspected individuals.

Muscular Dystrophyis a genetic disorder characterized by progressive muscle and

wasting. There are numerous types of the disease that can occur in childhood or adulthood,

including Becker muscular dystrophy, Duchenne muscular dystrophy, Myotonia congenital, and


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Myotonic dystrophy. Symptoms vary with each type of the disease and can affect all muscles or

just groups of muscles. Mental retardation, slow muscle weakening, delayed development of

muscle motor skills, ptosis (eyelid drooping), drooling, loss of strength, and problems walking

are only some of the symptoms experienced by individuals with the devastating disease.

Scoliosis, joint contractures and hypotonia (low muscle tone) are all signs used by doctors to

diagnose the disease. Unfortunately, there are no known cures for the disease, but current

research into the Myostatin mutation offers hope.

Cachexiais another condition that mysostatin research shows potential in treating.

Occurring at terminal stages of diseases (i.e. cancer, AIDS, chronic heart failure, chronic kidney

failure, etc.), cachexia usually causes a loss of body weight resulting from pathological changes

in certain metabolic pathways. Muscle hypotrophya decrease in muscle mass and fatis a

characteristic of the disorder that leads to increased morbidity and mortality in those affected.

Currently, there are no treatments for cachexia.

Use of Myostatin as a Treatment:

Clinical research has been done introducing substances that will potentially block

myostatin production and treat those with muscular dystrophy. Studies done by Whittemore et

al, in mice and by Kota et al, in monkeys used monoclonal antibodies aimed specifically towards

myostatin. These antibodies attacked myostatin and increased the muscle mass of both these

species. Another possible substance that has been studied in mice by Lee et al is the activin type

IIB receptor. It binds to myostatin and in the study, muscle mass increased up to 60%. Even

though all this research is being done with these two substances, researches are still not sure

whether or not treatments will be beneficial in the long run. There is no treatment for double


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muscling at this time. Studies have shown that epigenetic silencing blocks myostatin

production and transcriptional gene silencing may possibly be able to treat muscle wasting

disorders.

One such type IIB activin receptor is follistatin, which binds to myostatin in skeletal

muscle and inhibits its expression. Another related follistatin gene binds to myostatin, not in

skeletal muscle, but in serum where myostatin is circulating in. Lee et al, showed that if mice

overexpress these follistatin genes then their muscle mass increases remarkably.

Other inhibitors of myostatin other than type IIB activin receptors, and myostatin

antibodies include myostatin propeptides and soluble decoy myostatin receptors. The myostatin

antibodies, myostatin propeptides, and the soluble decoy myostatin receptors have all been

experimented with using animal models and found to actually prevent bone fractures. The

myostatin soluble receptor and antibody have actually been tested in humans, too and both

proved to increase the size of muscle. One such antibody tested in mice is the PF-354 antibody

that decreased muscle fatigue and improved the treadmill running time of these mice.

Researchers are thinking about using these findings to help children who have Duchenne

muscular dystrophy because these children have a higher risk of bone fractures and the elderly,

so they are not so prone to falling and fracturing bones.

There were experiments that looked at the overexpression of the myostatin gene to better

understand what its involvement was in muscle and adipogenesis. Zimmers et al, studied the

effects of treating animals with recombinant myostatin and revealed that it caused muscle

wasting. Reisz-Porszasz et al, studied the effects of myostatin transgenic overexpression and

found that this overexpression decreased muscle mass. There has also been research done

through in vitro that shows a deficiency in myostatin has positive effects on osteogenesis and in


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vivoresearch has shown bone formation and bone density are positively affected by myostatin

deficiency as well.

Through all of the research done on myostatin inhibitors, the FDA has not approved

anything for use. More research must be done, but there are many findings that show how

myostatin inhibitors can effectively help in many ways. There are no myostatin treatments for

double muscling because there is no proof that this disease needs a treatment. The research is

mainly done on inhibiting myostatin because the animals and even the two cases of little boys

having double muscling show so much improvement on both the muscle and the reduction of

fat. Researchers want to predominantly focus on improving muscle wasting diseases and other

diseases associated with the muscle to help reverse or delay the effects of these diseases.

Myostatin Group Paper

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