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Physiological Factors Controlling Myostatin Gene Expression – Part I

Nothing makes a bodybuilder cringe more than hearing the word “MYOSTATIN.” It’s downright frightening to hear about a muscle-specific gene that inhibits muscle growth. There is an inverse correlation between serum levels of myostatin and lean muscle mass (29), meaning that the higher levels of myostatin a person has floating around in his serum, the lower their muscle mass. Myostatin is a secreted protein that is mainly site-specific to muscle, although it is produced in other organs to a smaller extent (i.e. adipose tissue and heart). Myostatin, previously known as Growth Differentiation Factor-8, and a suggested member of the transforming growth factor (TGF- ?) super family, is predominantly expressed throughout life in skeletal muscle, from the early stages of life until late adulthood.

Myostatin seems to mainly inhibit myoblasts (cells recognizable as immediate precursors of skeletal muscle fibers) and prevent proliferation (the reproduction or multiplication of similar muscle precursors). So unless you’re a Belgian Blue Cow and don’t express myostatin, how the hell do we keep this evil little gene in check – or more importantly – what have researchers documented so far about its effects? In the January issue of the American Journal of Physiology-Endocrinology and Metabolism, a group of researchers reported that an acute bout of heavy resistance exercise (i.e. 3 sets of 8-12 repetitions to volitional fatigue for squats, leg press, and knee extension) downregulated myostatin mRNA expression in young and older males and young females. Older women, however, demonstrated no decrease in myostatin expression like their younger counterparts (1). It is somewhat surprising that younger males and females had similar changes in myostatin expression; one’s initial thought might be that males would have greater decreases in myostatin since males generally have larger muscle mass than females. But suppressing myostatin is just one of many genes associated with muscle hypertrophy. When comparing muscle gene expression through the use of a micro-array, which has the capacity to measures thousands of genes, significant gender differences are observed. On average, males expressed 200 identified muscle-specific genes that were ~75% higher than females. Scientists have yet to discover the exact mechanism in which myostatin suppresses muscle growth but it seems that satellite cells are a main mechanism. Muscle mass is a balance between anabolic factors (i.e. IGF-1, growth hormone, testosterone, act.) and catabolic factors (i.e. glucocorticoids, cytokines, [mediators of systemic inflammatory responses], and, of course, myostatin). Several research studies have documented that without satellite cell activation there is no muscle hypertrophy even in the presence of overload. (In other words we’d all be wearing Gap Kids clothing.) Well, take a guess at one of the primary mechanisms in which myostatin appears to inhibit muscle growth? Try inhibiting satellite cell activation (14). Myostatin also seems to be heavily influenced through glucocorticoid receptor mediated mechanisms (24). Additionally, it has been established that myostatin inhibits muscle proliferation (i.e. an increase in the number of cells as a result of cell growth and cell division) and inhibits DNA and protein synthesis (4). A possible mechanism of myostatin could include the impairment of the ability of muscle cells to regenerate. Improved healing, reduced scarring, and enhanced muscle cell regeneration has been observed in myostatin deficient mice, suggesting that the muscle inflammatory response is significantly accelerated when myostatin is absent (23). It would be easy to hypothesize that the reason that all pro-bodybuilders are so massive is that they were given good genetics and have some form of myostatin deficiency. Yet present day research does not validate myostatin gene deficiency as a main cause for the muscle mass demonstrated among top ranked bodybuilders (34). Before the mechanisms of myostatin are discussed, a brief background on the history of myostatin will be discussed in the next segment of this article.

Physiology of “double-muscled” animals

Mutations, defined as any change in the base sequence of DNA, can either occur naturally or be induced (i.e. genetic manipulation). For nearly 200 years, Belgian Blue and Piedmontese cattle has amazed live stock producers and scientist alike by their heavy degree of muscle mass and low body fat. What bodybuilder would not due to have twice the muscle mass and low body fat to match without ever hitting the gym? The partially recessive myostatin gene causes an average increase in muscle mass by ~25%; however there is a decrease in muscle mass in most organs (5). The extreme muscularity of the Belgian Blur is enhanced by the lack of visible bodyfat anywhere on the Belgian Blue bulls giving them a bodybuilder like appearance. Myostatin is expressed in skeletal muscle, cardiac muscle, and to a lesser extent adipose tissue (2). The lack of functional levels of the myostatin gene causes almost complete absence of subcutaneous fat. The adipocytes of Belgian Blue seem to be smaller than that of normal cattle. Other unusual physiological traits that make Belgian Blue unique are they posses lower bone mass, reduced fertility, increased stress susceptibility, and less connective tissue, despite their extremely hypertrophied muscle. During forced exercise, myostatin deficient cattle demonstrate faster levels of fatigue than normal cattle due to their higher proportion of type IIb anaerobic fibers and low amounts of type I aerobic fibers (36).

In 1997, two researchers at John Hopkins (i.e. Se Jin Lee and Alexandra Mc Pherron) discovered a new gene looking for super family’s of growth factors within the TGF- ? family. When they discovered this new novel gene that regulated muscle mass, they termed this gene “Myostatin.” Members of the TGF- ? super family regulate cytokines. Cytokines act by binding to specific membrane receptors, which then signal the cell via second messengers, often tyrosine kinases, to alter gene expression. Responses to cytokines include increasing or decreasing expression of membrane proteins (including cytokine receptors), proliferation, and secretion of effector molecules. Mutations in the TGF- ? signaling pathway leads to cancer, primary pulmonary hypertension, and other diseases which negatively impact health. Myostatin knockout mice, increase in size much more dramatically than Belgian Blue whom have a natural genetic variation of myostatin. Knockout mice are ~200-300% larger than their normal littermates (3). It’s interesting that myostatin deficient mice have increases in both muscle hypertrophy and muscle hyperplasia, whereas the “double muscled” cattle have increases only in muscle hyperplasia (6). Not only are the myostatin deficient mice more muscular that there counterpart’s but they are stronger as well, suggesting improved muscle regeration (7). The Chosen One!! A Child Born With a Myostatin Deficiency In 2004, in the New England Journal of Medicine researchers documented a baby born in Germany with a myostatin deficiency; his weight was in the 75th percentile and demonstrated extreme muscularity and low body-fat (8). Several family members of the child have been reported to “unusually” strong. At the age of 5 years the child grew in muscle size and strength extraordinarily fast, with him being able to hold a 7 pound dumbbell in the horizontal position with his arms extended!! At the age of 10, the cross sectional area of his quadriceps was 7.2 times greater than boys his age!!! It seems that this kid is a mini-Belgian Blue; the child apparently seems to be healthy however the risks to this child in the future are questionable as myostatin is also expressed in the heart, which means the child is at risk for cardiomyopathy (i.e. enlarged heart). It seems that there is a high correlation between muscle strength and myostatin gene expression. In a study of linking myostatin pathways to muscle strength and muscle mass, 329 siblings were tested for muscle mass and muscle strength by knee extension on a Cybex isokinetic dynamometer. To the researchers’ surprise, genetic linkage was found mainly for muscle strength, but only marginal evidence for its effect on estimated muscle cross sectional area (20).

Myostatin as a Regulator of Muscle Hypertrophy

The functional role of myostatin in control of muscle mass has been well documented however the mechanism in which myostatin controls muscle fiber number is poorly understood. So what causes this gene to stop muscle growth in its tracks? Myostatin regulates muscle mass by acting directly on muscle cells. Myostatin is expressed in developing and adult muscle, but myostatin seems to stop new muscle cells from their normal regeration cycle (9). If you add myostatin to a culture of satellite cells, it will cause growth inhibition (10). Myostatin is expressed in satellite cells and regulates their activation and self-renewal, so basically myostatin is the brakes on satellite cells, removes the brakes (a.k.a. myostatin) and the gas is pushed (a.k.a. satellite cells) you have more cells being formed for muscle (14). In addition, in rats that are genetically manipulated to be myostatin deficient, there is complete absence of myostatin expression in regenerating regions where satellite cells are most abundant (14). Satellite cells are stimulated by damage to muscle; they are basically muscle precursor cells that repair damaged muscle tissue by regulating muscle growth factors. Interestingly, myostatin seems to have higher gene expression in fast twitch fibers, which has a lower amount of satellite cells (10). Remember, fast twitch fiber types (IIb fibers) are dominant in explosive lifts such as power cleans, performing plyometrics, ect; whereas slow twitch fiber types (type I) are utilized in aerobic activities.

What Causes Myostatin Gene Expression to Increase?

Inactivity, catabolic hormones, and certain disease states seem to be the potent stimulators of myostatin production. Contrary to the latest acute resistance exercise study discussed earlier in which acute resistance exercise caused a decrease in myostatin gene expression, chronic adaptations of myostatin are different. Willboughby (33) reported that when 22 young untrained men performed a 12 week resistance training study (i.e. 3 days a week performing 3 sets of 6 to 8 repetitions at 85-90% of a 1-RM), at the end of 12 weeks there was an increase in myostatin mRNA gene expression along with glucocorticoid receptor expression despite an increase in lean muscle mass, strength, and thigh volume and mass. Interestingly, although myostatin mRNA expression increased so did follistatin-like related gene expression which actively binds myostatin negating its activity. It seems that adaptations to resistance exercise many induce muscle regulatory growth factors that both positively and negatively regulate muscle hypertrophy.

Researchers have used a model called “hind limb unloading” to study the effect of what happens when you don’t use your muscles. Basically, the tail of the mice and its back legs are suspended in the air while the front arms are free to move around. The mice can get water, eat, and do just about everything except use its back legs. This model can basically mimic people that are hospitalized after injury or bedridden. Hind limb suspension causes rapid atrophy in muscles, so it’s reasonable to assume that if muscle atrophy is occurring during hind limb suspension that it would have to be regulated by increases in myostatin gene expression. Results indicate that myostatin expression is not strongly associated with muscle atrophy in hind-limb unloading. In part, myostatin does not regulate muscle fiber size directly, but may influence other muscle pathways that influence muscle fiber size. For example, in mice that were subjected to muscle hind-limb unloading, myostatin expression was unregulated in fast twitch fibers by ~67% at day 1, but myostatin expression did not change thereafter. On day 7 there was still no change in myostatin expression in fast twitch fibers, yet significant atrophy was demonstrated. To further complicate matters, there was no change in myostatin expression in the soleus (i.e. type I muscles) muscle yet it demonstrated the greatest degree of muscle atrophy (21). Additionally, when muscles are denervated (i.e. basically means they remove the nerve from the muscle causing complete muscle paralysis) there was a increase in mRNA myostatin expression by ~31% but by day 14 when the muscle had decreased in size by 50% there was a 34% decrease in mRNA myostatin expression (27). The lack of association between myostatin and muscle atrophy is not new. McMahon et al. (32) observed that after 7 days of hindlimb suspension, muscle atrophy occurrence was not associated with increased protein expression of myostatin. To further complicate matters, myostatin deficient animals that are exposed to hindlimb suspension lose more muscle mass than normal mice. The researchers hypothesized that since myostatin deficient mice have increased type IIb fibers compared to their normal mice, that the greater losses in type IIb that occurred in the myostatin deficient mice could have been due to an overabundance of these fibers compared to normal mice. Spaceflight is another model of atrophy that uses anti-gravity to induce muscle mass loss. The pathophysiology of muscle mass loss during spaceflight is multi-factorial. So what happens to myostatin when you dress rats in Star-Trek outfits and blast them into space for 17 days? Myostatin muscle gene expression increased significantly in response to weightless ness, but when they returned to earth had a decrease in myostatin values that returned to normal by 13 days post-flight (22) Again, the data suggest that myostatin is associated with muscle mass loss, but does not prove it directly causes muscle loss. The muscle atrophy resulting from catabolic factors, such as glucocorticoids, starvation, and illness, could be the result of inhibition of protein synthesis or stimulation of protein breakdown in skeletal muscle. Glucocorticoids are steroid based and possess anti-inflammatory and immunosuppressive properties. Glucocorticoids are normally produced normally by the adrenal cortex and provide for the response to stress. Dexamethasone is a glucocorticoid commonly used for the treatment of a vast array of diseases such as chronic inflammatory disease, lupus, and rheumatoid arthritis. Dexamethasone is highly catabolic and long term use causes muscle atrophy in humans and animals alike. There is a dose dependent increase in myostatin mRNA which coincides with decreases in myosin heavy chain protein (i.e. a protein found in muscle fibers that is involved in muscle contraction) expression when Dexamethasone is administered (24). To be precise, in single dose of Dexamethasone caused a 60% (4 hours after) and a 270% (24 hours later) increase in myostatin mRNA expression in muscle (25). It is interesting that administration of RU-486 (i.e. a drug that inhibits glucocorticoids receptor binding) along with Dexamethasone significantly reduced myostatin mRNA gene expression but not completely abolish its activity (24). Rats that are pre-treated with RU-486 and then exposed to thermal injury (i.e. thermal injury is a nice way of saying we burned the crap out of some rats) almost completely prevented an increase in myostatin expression compared to control rats who had a ~ fourfold increase (25). This suggests that the muscle wasting effects of Dexamethasone are mediated by an upregulation of the myostatin gene. Continued next week…