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SteelWeaver
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This is long but interesting. W6 got me thinking when he asked MS if she'd tested for hers, and she mentioned Belgian Blue cattle a while back.
So, if you could get gene therapy to get bigger, would you? (Not saying myostatin inhibitors make this possible, just asking hypothetically).
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
So, if you could get gene therapy to get bigger, would you? (Not saying myostatin inhibitors make this possible, just asking hypothetically).
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
