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The Myostatin Gene
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'.
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.
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.
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.
WHAT IS MYOSTATIN?
Growth Factors
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 signalling, leading to an ultimate response in or
by the cell.
Classical hormones are long-range signalling 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. Myostatin was also detected in adult animals.
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. Research teams separately
discovered two independent mutations of the myostatin gene in two breeds of double-muscled cattle: the Belgian Blue and Piedmontese. 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. 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. 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.
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. 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. 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 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, 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. 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 and200-300% 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. Although no heart
abnormalities in myostatin-null mice were reported, the hearts in adult
Belgian Blue cattle are smaller. 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". 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. 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.
Studies also demonstrated lack of metabolic effects on myostatin expression
in piglets and mice. 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. 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. 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. Based on the report that muscle size is a heritable trait in
humans, 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.
Granted, the possibility exists that manipulation of the myostatin gene in
humans may be a key to reversing muscle-wasting conditions. However, too
little is still yet unknown regarding myostatin's role in muscle growth
regulation. It is imperative that research demonstrates that the loss of
myostatin activity in adults can cause muscle tissue growth. Likewise,
research must also prove that overexpression or administration of myostatin
causes loss of muscle mass. Also important is to know if manipulation of
myostatin will interfere with other growth systems, especially in other
tissues, and result in abnormal pathologies. Although McPherron's gene
knockout mice did not experience any other gross abnormalities, mice are not
humans.
We do not fully understand the roles of myostatin in exercise-induced muscle
hypertrophy or regeneration following muscle injury. The research does not
support the claim that a top bodybuilder's muscle mass gains are resultant
of a detected mutation in the myostatin gene. The research simply does not
advocate blaming genetic myostatin variations as a source of significant
differences in human phenotypes.
Considering the history of the athlete's propensity, in the public eye, to
abuse performance-enhancement substances, the media's prediction of
myostatin-inhibitor may or may not be warranted. We all know that today's
athletic arena demands gaining the competitive edge to maintain top level
competition. For many athletes, that is accomplished by supplementing hard
training with substances that enhance growth or performance. Whether or not myostatin inhibitors will be added to the arsenal of substances is difficult
to predict. Until science reveals the full nature of this growth factor and
its role in the complex regulation of muscle tissue, and researchers
determine its therapeutic implications, we can only surmise. Despite
attempts to tightly control any pharmaceutical uses of myostatin protein
manipulation, they will likely surface at some point in the black market
world of bodybuilding supplements. Let us hope that science has determined
the side effects and the benefits by that point.
...Thermogenics investigator...
...Jesus Christ forgave the bastards. But I can't. I hate them....
misOO
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