Myostatin: The Muscle Buster
Physiological Factors Controlling Myostatin Gene Expression
by Robbie Durand
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’s an inverse correlation between serum levels of myostatin and fat-free mass,29 meaning the higher levels of myostatin a person has floating around in his serum, the lower the muscle mass.
This Evil Little Gene
Myostatin is a secreted protein that’s mainly site specific to muscle although it’s 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 in skeletal muscle throughout life, from the early stages to 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., three sets of eight to 12 repetitions to volitional fatigue for squats, leg press and knee extension) downregulated myostatin mRNA expression in young and older males and young females. However, older women demonstrated no decrease in myostatin expression like their younger counterparts.1 It’s somewhat surprising younger males and females had similar changes in myostatin expression; one’s initial thought would be that males have greater decreases in myostatin since males generally have larger muscle mass than females, however suppressing myostatin is just one of many genes associated with muscle hypertrophy. When comparing muscle gene expression thru the use of a micro-array, which has the capacity to measures thousands of genes, significant gender differences are observed.
One study reported that after nine weeks of heavy strength training the biggest difference in muscle gene expression attributed to gender was independent of age or strength training.31 On average, the males expressed 200 identified muscle specific genes that were approximately 75 percent higher than females. Scientists have yet to discover the exact mechanism by which myostatin suppresses muscle growth, but it seems that satellite cells are a main mechanism. (See Fig. 1 for a simplified version of how myostatin regulates muscle growth).
Satellite Cell Activation
In the last two installments of this column, the importance of satellite cell activation and how IGF-I is a potent simulator of satellite cell activation was discussed. Muscle mass is a balance between anabolic factors (i.e., IGF-1, growth hormone, testosterone, etc.) 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’s no muscle hypertrophy even in the presence of overload. 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 It also 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 So a possible mechanism could be that myostatin impairs 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 all pro bodybuilders are so massive is because of good genetics and having some form of myostatin deficiency, yet present day research doesn’t 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, I will give a brief background.
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 have amazed livestock producers and scientists alike by their heavy degree of muscle mass and low body fat. What bodybuilder wouldn’t want 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 approximately 25 percent; however there’s a decrease in muscle mass in most organs.5 The extreme muscularity of the Belgian Blue is enhanced by the lack of visible body fat anywhere, 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 normal cattle. Other unusual traits making the Belgian Blue unique include 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 lower amounts of type-I aerobic fibers.36
In 1997, researchers Se Jin Lee and Alexandra McPherron at Johns Hopkins, discovered a new gene within the TGF-?? family. 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) and proliferation and secretion of effector molecules. Mutations in the TGF-?? signaling pathway lead to cancer, primary pulmonary hypertension and other diseases. Myostatin knockout mice, increase in size more dramatically than Belgian Blue, who have a natural genetic variation of myostatin. Knockout mice are approximately 200 to 300 percent larger than their normal littermates.3
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 than their counterparts, but they’re stronger as well, suggesting improved muscle regeration.7
The Chosen One!! A Child Born With a Myostatin Deficiency
Big Ronnie Coleman should be sweating when he reads about this mutant child. In 2004, in the New England Journal of Medicine, researchers reported on 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 as “unusually” strong. At the age of five the child grew in muscle size and strength extraordinarily fast and he was able to hold a seven-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 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).
Myostatin as a Regulator of Muscle Hypertrophy
The functional role of myostatin in controlling muscle mass has been well documented, however the mechanism by which myostatin controls the muscle fiber number is poorly understood. What causes this gene to stop muscle growth? Myostatin regulates muscle mass by acting directly on muscle cells. Myostatin is expressed in developing and adult muscle, but seems to stop new muscle cells from their normal regeneration 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 activation and self-renewal, so basically myostatin is the brakes; remove the brakes (aka myostatin), the gas is pushed (aka satellite cells) and you have more cells being formed for muscle.14 In addition, in rats that are genetically manipulated to be myostatin deficient, there’s 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, whereas slow-twitch fiber types (type-I) are utilized in aerobics.
Why Does Myostatin Increase?
Inactivity, catabolic hormones and certain diseases seem to be potent stimulators of myostatin production. Contrary to the latest acute resistance exercise study discussed earlier, chronic adaptations of myostatin are different. Willboughby33 reported that when 22 young, untrained men performed a 12-week resistance training study (i.e., three days a week performing three sets of six to eight repetitions at 85 to 90 percent 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 binds myostatin, negating its activity. It seems that adaptations to resistance exercise may induce muscle regulatory growth factors that positively and negatively regulate muscle hypertrophy.
Researchers have used a model called “hind limb unloading” to study 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. The mice can get water, eat and do just about everything except use its back legs. This model mimics the bedridden or those hospitalized after injury. Hind limb suspension causes rapid atrophy in muscles, so it’s reasonable to assume that if muscle atrophy is occurring during hind limb suspension, it would have to be regulated by increases in myostatin gene expression (You know what they say about the word assume).
Results indicate that myostatin expression isn’t strongly associated with muscle atrophy in hind limb unloading. In part, myostatin doesn’t regulate muscle fiber size directly, but may influence other muscle pathways that influence muscle fiber size. For example, in mice subjected to muscle hind limb unloading, myostatin expression was unregulated in fast-twitch fibers by approximately 67 percent at day one, but didn’t change thereafter. On day seven 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., the nerve is removed from the muscle causing muscle paralysis) there was a increase in mRNA myostatin expression by approximately 31 percent, but by day 14, when the muscle had decreased in size by 50 percent, there was a 34 percent 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 seven days of hind limb suspension, muscle atrophy occurrence wasn’t associated with increased protein expression of myostatin. To further complicate matters, myostatin-deficient animals exposed to hind limb suspension lose more muscle mass than normal mice. The researchers hypothesized that since myostatin-deficient mice have increased type-IIb fibers compared to normal mice, the greater losses in type-IIb that occurred 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 weightlessness, but when they returned to earth, the mice had a decrease in myostatin values, which returned to normal by 13 days post-flight.22 Again, the data suggest that myostatin is associated with muscle mass loss, but doesn’t 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 produced 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’s 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’s involved in muscle contraction) expression when Dexamethasone is administered.24 To be precise, a single dose of Dexamethasone caused a 60 percent (four hours after) and a 270 percent (24 hours later) increase in myostatin mRNA expression in muscle.25 It’s 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 didn’t abolish its activity.24 Rats that are pre-treated with RU-486 and then exposed to thermal injury (we burned the crap out of some rats) almost completely prevented an increase in myostatin expression compared to control rats that had an approximate fourfold increase.25 This suggests that the muscle-wasting effects of Dexamethasone are mediated by an upregulation of the myostatin gene.
How the Hell Do I Turn It Off?
Besides taking a black market pharmaceutical-grade myostatin inhibitor, GH might be a potential inhibitor of myostatin production. Here’s another one of those catch-22 studies. In GH-deficient males, administration of GH (five µg/kg per day) resulted in a significant inhibition (approximately 31 percent) of myostatin muscle mRNA compared to the control group.11 Surprisingly, systemic administration of neither GH nor testosterone to healthy elderly males resulted in no change in myostatin expression; however a combination of both GH and testosterone almost reached a significant effect.12
In a 2001 study, 27 healthy men (less than 65 years of age) had muscle biopsies and blood samples taken to determine if there was any correlation between GH, testosterone, IGF-1 with mRNA myostatin expression. Results of the study showed there were no significant relationships between age, lean body mass, or percent body fat and transcript levels of growth hormone receptors, IGF-I and rogen receptor, or myostatin. Moreover, there were no significant correlations of serum GH, IGF-I, or T with their corresponding target mRNA levels (GHR, intramuscular IGF-I, or AR) in skeletal muscle. There was a negative relationship between the amount of mRNA muscle myostatin expressed in muscle and GH receptor gene expression.27 There’s a documented rise in serum myostatin levels with aging. The author hypothesized since there’s a significant down-regulation of the GH axis with aging, possibly the decrease in GH may lead to increased myostatin gene expression. GH may be able to down-regulate myostatin expression, but more research needs to be conducted.
There’s controversy as to whether heavy resistance exercise decreases or increase myostatin gene expression. As mentioned previously, Willboughby33 reported that when 22 young, untrained men performed a 12-week resistance-training program, at the end of 12 weeks there was an increase in myostatin mRNA gene expression. Contrary to these results, another study found that when untrained young and old men and women were subjected to a nine-week heavy resistance training routine, an approximate 37 percent decrease in myostatin gene expression was observed in all subjects.16 Interestingly, there were no gender differences between males and females. One would suspect that testosterone would have some kind of impact on myostatin, but as mentioned in an earlier study, administration of testosterone has no effect on myostatin production.12
There’s something really interesting about resistance exercise effects on myostatin. As mentioned earlier, hind limb suspension in mice will cause an increase in myostatin mRNA expression. After 10 days of muscle unloading in mice, there was a 110 percent increase in myostatin mRNA in muscle however, hind limb unloading combined with intermittent bouts of muscle loading (i.e., 30 minutes on a treadmill with a 20 percent grade) causes a significant rise in myostatin (approximately 55 percent increase), but no significant muscle mass loss.18 Results of the study indicate that myostatin expression can be increased during modified muscle load, yet not cause significant decreases in muscle mass.
I know what you’re thinking: I can take a myostatin inhibitor supplement. Well sure, maybe if you stack it with some Boron you’ll become extra huge!!! A research group in the exercise and biochemical nutrition laboratory at Baylor University did all us bodybuilders a favor and buried that notion. The study examined 12 weeks of resistance training and Cystoseira canariensis supplementation (a supplement that’s supposed to bind to myostatin) on serum levels of myostatin and muscle strength and body composition. After 12 weeks of heavy resistance training and 1,200 milligrams per day of Cystoseira canariensis there was no difference between the control group and the resistance training group in terms of muscle strength, fat loss, or changes in lean muscle mass. Sorry…don’t shoot the messenger!!
Myostatin and Adipose Tissue
Only a small quantity of myostatin is expressed in adipose tissue compared to muscle, but it seems to have some rather potent effects of regulating adipose tissue size. When myostatin-deficient mice were compared to there normal mice counterparts some interesting features were discovered. For up to two months of age there were no differences in the amount of fat between the two groups, but by six months of age there were. After six months, the normal mice had fat pads (i.e., lumps of fat) that were 2.4 to four times greater than that of the myostatin-deficient mice. After nine months, the normal mice had fat pads nine-fold greater than that of myostatin-deficient mice. Here’s what’s really interesting, so while myostatin-deficient mice are aging and staying lean, they have normal food intake and normal body temperatures, but have a slightly lower metabolic rate (myostatin-deficient mice had lower rates of total oxygen and resting oxygen when expressed as a percentage of bodyweight).15 So what about humans? In obese subjects who underwent an operation called a billiopancreatic diversion (a form of gastric bypass surgery in which portions of the stomach are removed) there was a significant decrease in myostatin expression mRNA after weight loss. Also of interest is the possibility of using myostatin to help treat diabetes. It’s hypothesized myostatin might reduce the incidence of diabetes by reducing the amount of fatty acids that accumulate in the beta cells of the pancreas, which cause insulin release. So far, treatment with myostatin looks promising as mice experimentation with myostatin inhibitors reduced obesity as well as type II diabetes symptoms.
Myostatin, Aging and Wasting Disease
Aging is associated with a reduction in lean muscle mass with a concomitant increase in fat mass. This partly may be related to the decline in circulating levels of anabolic hormones that might be affecting gene transcription. It’s widely believed that thousands of genes and their products (i.e., RNA and proteins) function in a complicated and orchestrated way. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means there’s a very limited view of the "whole picture" of gene function.
In the past several years, a new technology called DNA microarray has attracted tremendous interest among biologists. This technology promises to monitor the whole genome on a single chip so researchers can have a better picture of the interactions among thousands of genes simultaneously. When comparing gene expression from biopsies of younger and older men, older muscle had reduced expression of genes involved in energy metabolism (i.e., electron chain transport, ATP synthesis) and increased expression of genes involved in oxidative stress and inflammation. Also reported, the gene with the greatest expression in older men was follistatin, which binds myostatin and inhibits its activity. The author hypothesized that increased follistatin expression may be unregulated to compensate for increased serum levels of myostatin, which occurs with aging.28 Elevated serum levels of myostatin not only occur with aging, but in many catabolic disease states.
The serum concentrations of myostatin have been found to be higher in HIV-infected men than in healthy men; furthermore, myostatin levels are even higher in men who meet the definition of AIDS wasting syndrome.29 When healthy mice are systemically administered myostatin, they develop muscle wasting and fat loss just like many human cachexia syndromes (i.e., wasting diseases).
In conclusion, myostatin is a muscle-specific protein that negatively regulates muscle mass. Based on the research, increases in myostatin accompany muscle atrophy though significant increases in myostatin don’t necessarily produce muscle atrophy. There’s growing evidence that adaptation to exercise isn’t mediated by one signal transduction pathway or mechanism, but by several transcription factors binding to enhancers or silencers. Myostatin antagonists seem to be a therapeutic means to alleviating sarcopenia, muscle atrophy and other wasting diseases.
Muscular dystrophy is documented by extensive muscle inflammation, muscle injury and replacement of muscle tissue with connective tissue and fat. Researchers have long sought to find agents that block the molecular pathway to muscular dystrophy by using “booster genes” to support muscle growth and cope with the otherwise damaging steps leading to muscular dystrophy. There are some potential benefits to using myostatin inhibitors as one study showed when a strain of mice who are myostatin deficient, but also contain the muscular dystrophy gene, have increased muscle mass and increased muscle strength, yet there’s still no reduction in inflammation or tissue injury.21
The safety issues of myostatin are still being investigated. It’s been suggested by researchers that since myostatin inhibitors cause dramatic increases in muscle mass (approximately 20 to 40 percent), it’s questionable whether human bone can withstand such a dramatic increase in muscle mass in such a short period of time. This probably wouldn’t be a problem for a healthy person, but what about a frail elderly person? Finally, myostatin is expressed in the heart as well. The suppression of myostatin could possibly lead to cardiac enlargement. Furthermore, following a heart attack, myostatin expression is upregulated in the heart. It should be mentioned that myostatin-deficient mice seem to be healthy although more long-term research needs to be conducted.
References:
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2. Arnold HA, Della-Ferra MA, Baile C (2001). Review of myostatin history, physiology and applications. Life XY, 2001(1): 1014.
3. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature, 1997 May 1;387(6628):83-90.
4. Taylor WE, Bhasin S, Artaza J, Byhower F, Azam M, Willard DH Jr, Kull FC Jr, Gonzalez-Cadavid N. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. Am J Physiol Endocrinol Metab, 2001 Feb;280(2):E221-8.
5. McPherron AC, Lee SJ.. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA. 1997 Nov 11;94(23):12457-61.
6. Kambadur R, Sharma M, Smith TP, Bass JJ.. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res, 1997 Sep;7(9):910-6.
7. Wagner KR, McPherron AC, Winik N, Lee SJ. Loss of myostatin attenuates severity of muscular dystrophy in mdx mice. Ann Neurol, 2002 Dec;52(6):832-6.
8. Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee SJ. Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med. 2004 Jun 24;350(26):2682-8.
9. Thomas M, Langley B, Berry C, Sharma M, Kirk S, Bass J, Kambadur R. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem, 2000 Dec 22;275(51):40235-43.
10. Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem, 2002 Dec 20;277(51):49831-40.
11. Liu W, Thomas SG, Asa SL, Gonzalez-Cadavid N, Bhasin S, Ezzat S. Myostatin is a skeletal muscle target of growth hormone anabolic action. J Clin Endocrinol Metab, 2003 Nov;88(11):5490-6.
12. Brill KT, Weltman AL, Gentili A, Patrie JT, Fryburg DA, Hanks JB, Urban RJ, Veldhuis JD. Single and combined effects of growth hormone and testosterone administration on measures of body composition, physical performance, mood, sexual function, bone turnover and muscle gene expression in healthy older men. J Clin Endocrinol Metab, 2002 Dec;87(12):5649-57.
13. Wehling M, Cai B, Tidball JG. Modulation of myostatin expression during modified muscle use. FASEB J, 2000 Jan;14(1):103-10.
14. McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R. Myostatin negatively regulates satellite cell activation and self-renewal. J Cell Biol, 2003 Sep 15;162(6):1135-47. 2003 Sep 8.
15. McPherron AC, Lee SJ. Suppression of body fat accumulation in myostatin-deficient mice. J Clin Invest, 2002 Mar;109(5):595-601.
16. Roth SM, Martel GF, Ferrell RE, Metter EJ, Hurley BF, Rogers MA. Myostatin gene expression is reduced in humans with heavy-resistance strength training: a brief communication. Exp Biol Med (Maywood), 2003 Jun; 228(6):706-9.
17. Willoughby DS. Effects of an alleged myostatin-binding supplement and heavy resistance training on serum myostatin, muscle strength and mass and body composition. Int J Sport Nutr Exerc Metab, 2004 Aug;14(4):461-72.
18. Wehling M, Cai B, Tidball JG. Modulation of myostatin expression during modified muscle use. FASEB J, 2000 Jan;14(1):103-10.
19. Huygens W, Thomis MA, Peeters MW, Aerssens J, Janssen R, Vlietinck RF, Beunen G. Linkage of myostatin pathway genes with knee strength in humans. Physiol Genomics, 2004 May 19;17(3):264-70.
20. Carlson CJ, Booth FW, Gordon SE. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol, 1999 Aug;277(2 Pt 2):R601-6.
21. Engvall E, Wewer UM. The new frontier in muscular dystrophy research: booster genes. FASEB J, 2003 Sep;17(12):1579-84. Review.
22. Lalani R, Bhasin S, Byhower F, Tarnuzzer R, Grant M, Shen R, Asa S, Ezzat S, Gonzalez-Cadavid NF. Myostatin and insulin-like growth factor-I and -II expression in the muscle of rats exposed to the microgravity environment of the NeuroLab space shuttle flight. J Endocrinol, 2000 Dec;167(3):417-28.
23. McCroskery S, Thomas M, Platt L, Hennebry A, Nishimura T, McLeay L, Sharma M, Kambadur R. Improved muscle healing through enhanced regeneration and reduced fibrosis in myostatin-null mice. J Cell Sci, 2005 Aug 1;118(Pt 15):3531-41.
24. Ma K, Mallidis C, Bhasin S, Mahabadi V, Artaza J, Gonzalez-Cadavid N, Arias J, Salehian B. Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression. Am J Physiol Endocrinol Metab, 2003 Aug;285(2):E363-71.
25. Lang CH, Silvis C, Nystrom G, Frost RA. Regulation of myostatin by glucocorticoids after thermal injury. FASEB J, 2001 Aug;15(10):1807-9.
26. Baumann AP, Ibebunjo C, Grasser WA, Paralkar VM. Myostatin expression in age and denervation-induced skeletal muscle atrophy. J Musculoskelet Neuronal Interact, 2003 Mar;3(1):8-16.
27. Marcell TJ, Harman SM, Urban RJ, Metz DD, Rodgers BD, Blackman MR. Comparison of GH, IGF-I and testosterone with mRNA of receptors and myostatin in skeletal muscle in older men. Am J Physiol Endocrinol Metab, 2001 Dec;281(6):E1159-64.
28. Welle S, Brooks AI, Delehanty JM, Needler N, Thornton CA. Gene expression profile of aging in human muscle. Physiol Genomics, 2003 Jul 7;14(2):149-59.
29. Gonzalez-Cadavid NF, Taylor WE, Yarasheski K, Sinha-Hikim I, Ma K, Ezzat S, Shen R, Lalani R, Asa S, Mamita M, Nair G, Arver S, Bhasin S. Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting. Proc Natl Acad Sci USA, 1998 Dec 8;95(25):14938-43.
30. Zimmers TA, Davies MV, Koniaris LG, Haynes P, Esquela AF, Tomkinson KN, McPherron AC, Wolfman NM, Lee SJ. Induction of cachexia in mice by systemically administered myostatin. Science, 2002 May 24; 296(5572):1486-8.
31. Roth SM, Ferrell RE, Peters DG, Metter EJ, Hurley BF, Rogers MA. Influence of age, sex and strength training on human muscle gene expression determined by microarray. Physiol Genomics, 2002 Sep 3;10(3):181-90.
32. McMahon CD, Popovic L, Oldham JM, Jeanplong F, Smith HK, Kambadur R, Sharma M, Maxwell L, Bass JJ. Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension. Am J Physiol Endocrinol Metab, 2003 Jul;285(1):E82-7.
33. Willoughby DS. Effects of heavy resistance training on myostatin mRNA and protein expression. Med Sci Sports Exerc, 2004 Apr;36(4):574-82.
34. Ferrell RE, Conte V, Lawrence EC, Roth SM, Hagberg JM, Hurley BF. Frequent sequence variation in the human myostatin (GDF8) gene as a marker for analysis of muscle-related phenotypes. Genomics, 1999 Dec 1;62(2):203-7.
35. Ma K, Mallidis C, Artaza J, Taylor W, Gonzalez-Cadavid N, Bhasin S. Characterization of 5'-regulatory region of human myostatin gene: regulation by dexamethasone in vitro. Am J Physiol Endocrinol Metab, 2001 Dec;281(6):E1128-36.
36. R. H. S. Bellinge, D. A. Liberles, S. P. A. Iaschi, P. A. O'Brien and G. K. Tay. Myostatin and its implications on animal breeding: a review. Animal Genetics, Volume 36 Issue 1 Page 1, February 2005
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Physiological Factors Controlling Myostatin Gene Expression
by Robbie Durand
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’s an inverse correlation between serum levels of myostatin and fat-free mass,29 meaning the higher levels of myostatin a person has floating around in his serum, the lower the muscle mass.
This Evil Little Gene
Myostatin is a secreted protein that’s mainly site specific to muscle although it’s 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 in skeletal muscle throughout life, from the early stages to 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., three sets of eight to 12 repetitions to volitional fatigue for squats, leg press and knee extension) downregulated myostatin mRNA expression in young and older males and young females. However, older women demonstrated no decrease in myostatin expression like their younger counterparts.1 It’s somewhat surprising younger males and females had similar changes in myostatin expression; one’s initial thought would be that males have greater decreases in myostatin since males generally have larger muscle mass than females, however suppressing myostatin is just one of many genes associated with muscle hypertrophy. When comparing muscle gene expression thru the use of a micro-array, which has the capacity to measures thousands of genes, significant gender differences are observed.
One study reported that after nine weeks of heavy strength training the biggest difference in muscle gene expression attributed to gender was independent of age or strength training.31 On average, the males expressed 200 identified muscle specific genes that were approximately 75 percent higher than females. Scientists have yet to discover the exact mechanism by which myostatin suppresses muscle growth, but it seems that satellite cells are a main mechanism. (See Fig. 1 for a simplified version of how myostatin regulates muscle growth).
Satellite Cell Activation
In the last two installments of this column, the importance of satellite cell activation and how IGF-I is a potent simulator of satellite cell activation was discussed. Muscle mass is a balance between anabolic factors (i.e., IGF-1, growth hormone, testosterone, etc.) 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’s no muscle hypertrophy even in the presence of overload. 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 It also 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 So a possible mechanism could be that myostatin impairs 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 all pro bodybuilders are so massive is because of good genetics and having some form of myostatin deficiency, yet present day research doesn’t 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, I will give a brief background.
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 have amazed livestock producers and scientists alike by their heavy degree of muscle mass and low body fat. What bodybuilder wouldn’t want 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 approximately 25 percent; however there’s a decrease in muscle mass in most organs.5 The extreme muscularity of the Belgian Blue is enhanced by the lack of visible body fat anywhere, 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 normal cattle. Other unusual traits making the Belgian Blue unique include 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 lower amounts of type-I aerobic fibers.36
In 1997, researchers Se Jin Lee and Alexandra McPherron at Johns Hopkins, discovered a new gene within the TGF-?? family. 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) and proliferation and secretion of effector molecules. Mutations in the TGF-?? signaling pathway lead to cancer, primary pulmonary hypertension and other diseases. Myostatin knockout mice, increase in size more dramatically than Belgian Blue, who have a natural genetic variation of myostatin. Knockout mice are approximately 200 to 300 percent larger than their normal littermates.3
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 than their counterparts, but they’re stronger as well, suggesting improved muscle regeration.7
The Chosen One!! A Child Born With a Myostatin Deficiency
Big Ronnie Coleman should be sweating when he reads about this mutant child. In 2004, in the New England Journal of Medicine, researchers reported on 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 as “unusually” strong. At the age of five the child grew in muscle size and strength extraordinarily fast and he was able to hold a seven-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 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).
Myostatin as a Regulator of Muscle Hypertrophy
The functional role of myostatin in controlling muscle mass has been well documented, however the mechanism by which myostatin controls the muscle fiber number is poorly understood. What causes this gene to stop muscle growth? Myostatin regulates muscle mass by acting directly on muscle cells. Myostatin is expressed in developing and adult muscle, but seems to stop new muscle cells from their normal regeneration 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 activation and self-renewal, so basically myostatin is the brakes; remove the brakes (aka myostatin), the gas is pushed (aka satellite cells) and you have more cells being formed for muscle.14 In addition, in rats that are genetically manipulated to be myostatin deficient, there’s 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, whereas slow-twitch fiber types (type-I) are utilized in aerobics.
Why Does Myostatin Increase?
Inactivity, catabolic hormones and certain diseases seem to be potent stimulators of myostatin production. Contrary to the latest acute resistance exercise study discussed earlier, chronic adaptations of myostatin are different. Willboughby33 reported that when 22 young, untrained men performed a 12-week resistance training study (i.e., three days a week performing three sets of six to eight repetitions at 85 to 90 percent 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 binds myostatin, negating its activity. It seems that adaptations to resistance exercise may induce muscle regulatory growth factors that positively and negatively regulate muscle hypertrophy.
Researchers have used a model called “hind limb unloading” to study 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. The mice can get water, eat and do just about everything except use its back legs. This model mimics the bedridden or those hospitalized after injury. Hind limb suspension causes rapid atrophy in muscles, so it’s reasonable to assume that if muscle atrophy is occurring during hind limb suspension, it would have to be regulated by increases in myostatin gene expression (You know what they say about the word assume).
Results indicate that myostatin expression isn’t strongly associated with muscle atrophy in hind limb unloading. In part, myostatin doesn’t regulate muscle fiber size directly, but may influence other muscle pathways that influence muscle fiber size. For example, in mice subjected to muscle hind limb unloading, myostatin expression was unregulated in fast-twitch fibers by approximately 67 percent at day one, but didn’t change thereafter. On day seven 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., the nerve is removed from the muscle causing muscle paralysis) there was a increase in mRNA myostatin expression by approximately 31 percent, but by day 14, when the muscle had decreased in size by 50 percent, there was a 34 percent 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 seven days of hind limb suspension, muscle atrophy occurrence wasn’t associated with increased protein expression of myostatin. To further complicate matters, myostatin-deficient animals exposed to hind limb suspension lose more muscle mass than normal mice. The researchers hypothesized that since myostatin-deficient mice have increased type-IIb fibers compared to normal mice, the greater losses in type-IIb that occurred 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 weightlessness, but when they returned to earth, the mice had a decrease in myostatin values, which returned to normal by 13 days post-flight.22 Again, the data suggest that myostatin is associated with muscle mass loss, but doesn’t 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 produced 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’s 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’s involved in muscle contraction) expression when Dexamethasone is administered.24 To be precise, a single dose of Dexamethasone caused a 60 percent (four hours after) and a 270 percent (24 hours later) increase in myostatin mRNA expression in muscle.25 It’s 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 didn’t abolish its activity.24 Rats that are pre-treated with RU-486 and then exposed to thermal injury (we burned the crap out of some rats) almost completely prevented an increase in myostatin expression compared to control rats that had an approximate fourfold increase.25 This suggests that the muscle-wasting effects of Dexamethasone are mediated by an upregulation of the myostatin gene.
How the Hell Do I Turn It Off?
Besides taking a black market pharmaceutical-grade myostatin inhibitor, GH might be a potential inhibitor of myostatin production. Here’s another one of those catch-22 studies. In GH-deficient males, administration of GH (five µg/kg per day) resulted in a significant inhibition (approximately 31 percent) of myostatin muscle mRNA compared to the control group.11 Surprisingly, systemic administration of neither GH nor testosterone to healthy elderly males resulted in no change in myostatin expression; however a combination of both GH and testosterone almost reached a significant effect.12
In a 2001 study, 27 healthy men (less than 65 years of age) had muscle biopsies and blood samples taken to determine if there was any correlation between GH, testosterone, IGF-1 with mRNA myostatin expression. Results of the study showed there were no significant relationships between age, lean body mass, or percent body fat and transcript levels of growth hormone receptors, IGF-I and rogen receptor, or myostatin. Moreover, there were no significant correlations of serum GH, IGF-I, or T with their corresponding target mRNA levels (GHR, intramuscular IGF-I, or AR) in skeletal muscle. There was a negative relationship between the amount of mRNA muscle myostatin expressed in muscle and GH receptor gene expression.27 There’s a documented rise in serum myostatin levels with aging. The author hypothesized since there’s a significant down-regulation of the GH axis with aging, possibly the decrease in GH may lead to increased myostatin gene expression. GH may be able to down-regulate myostatin expression, but more research needs to be conducted.
There’s controversy as to whether heavy resistance exercise decreases or increase myostatin gene expression. As mentioned previously, Willboughby33 reported that when 22 young, untrained men performed a 12-week resistance-training program, at the end of 12 weeks there was an increase in myostatin mRNA gene expression. Contrary to these results, another study found that when untrained young and old men and women were subjected to a nine-week heavy resistance training routine, an approximate 37 percent decrease in myostatin gene expression was observed in all subjects.16 Interestingly, there were no gender differences between males and females. One would suspect that testosterone would have some kind of impact on myostatin, but as mentioned in an earlier study, administration of testosterone has no effect on myostatin production.12
There’s something really interesting about resistance exercise effects on myostatin. As mentioned earlier, hind limb suspension in mice will cause an increase in myostatin mRNA expression. After 10 days of muscle unloading in mice, there was a 110 percent increase in myostatin mRNA in muscle however, hind limb unloading combined with intermittent bouts of muscle loading (i.e., 30 minutes on a treadmill with a 20 percent grade) causes a significant rise in myostatin (approximately 55 percent increase), but no significant muscle mass loss.18 Results of the study indicate that myostatin expression can be increased during modified muscle load, yet not cause significant decreases in muscle mass.
I know what you’re thinking: I can take a myostatin inhibitor supplement. Well sure, maybe if you stack it with some Boron you’ll become extra huge!!! A research group in the exercise and biochemical nutrition laboratory at Baylor University did all us bodybuilders a favor and buried that notion. The study examined 12 weeks of resistance training and Cystoseira canariensis supplementation (a supplement that’s supposed to bind to myostatin) on serum levels of myostatin and muscle strength and body composition. After 12 weeks of heavy resistance training and 1,200 milligrams per day of Cystoseira canariensis there was no difference between the control group and the resistance training group in terms of muscle strength, fat loss, or changes in lean muscle mass. Sorry…don’t shoot the messenger!!
Myostatin and Adipose Tissue
Only a small quantity of myostatin is expressed in adipose tissue compared to muscle, but it seems to have some rather potent effects of regulating adipose tissue size. When myostatin-deficient mice were compared to there normal mice counterparts some interesting features were discovered. For up to two months of age there were no differences in the amount of fat between the two groups, but by six months of age there were. After six months, the normal mice had fat pads (i.e., lumps of fat) that were 2.4 to four times greater than that of the myostatin-deficient mice. After nine months, the normal mice had fat pads nine-fold greater than that of myostatin-deficient mice. Here’s what’s really interesting, so while myostatin-deficient mice are aging and staying lean, they have normal food intake and normal body temperatures, but have a slightly lower metabolic rate (myostatin-deficient mice had lower rates of total oxygen and resting oxygen when expressed as a percentage of bodyweight).15 So what about humans? In obese subjects who underwent an operation called a billiopancreatic diversion (a form of gastric bypass surgery in which portions of the stomach are removed) there was a significant decrease in myostatin expression mRNA after weight loss. Also of interest is the possibility of using myostatin to help treat diabetes. It’s hypothesized myostatin might reduce the incidence of diabetes by reducing the amount of fatty acids that accumulate in the beta cells of the pancreas, which cause insulin release. So far, treatment with myostatin looks promising as mice experimentation with myostatin inhibitors reduced obesity as well as type II diabetes symptoms.
Myostatin, Aging and Wasting Disease
Aging is associated with a reduction in lean muscle mass with a concomitant increase in fat mass. This partly may be related to the decline in circulating levels of anabolic hormones that might be affecting gene transcription. It’s widely believed that thousands of genes and their products (i.e., RNA and proteins) function in a complicated and orchestrated way. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means there’s a very limited view of the "whole picture" of gene function.
In the past several years, a new technology called DNA microarray has attracted tremendous interest among biologists. This technology promises to monitor the whole genome on a single chip so researchers can have a better picture of the interactions among thousands of genes simultaneously. When comparing gene expression from biopsies of younger and older men, older muscle had reduced expression of genes involved in energy metabolism (i.e., electron chain transport, ATP synthesis) and increased expression of genes involved in oxidative stress and inflammation. Also reported, the gene with the greatest expression in older men was follistatin, which binds myostatin and inhibits its activity. The author hypothesized that increased follistatin expression may be unregulated to compensate for increased serum levels of myostatin, which occurs with aging.28 Elevated serum levels of myostatin not only occur with aging, but in many catabolic disease states.
The serum concentrations of myostatin have been found to be higher in HIV-infected men than in healthy men; furthermore, myostatin levels are even higher in men who meet the definition of AIDS wasting syndrome.29 When healthy mice are systemically administered myostatin, they develop muscle wasting and fat loss just like many human cachexia syndromes (i.e., wasting diseases).
In conclusion, myostatin is a muscle-specific protein that negatively regulates muscle mass. Based on the research, increases in myostatin accompany muscle atrophy though significant increases in myostatin don’t necessarily produce muscle atrophy. There’s growing evidence that adaptation to exercise isn’t mediated by one signal transduction pathway or mechanism, but by several transcription factors binding to enhancers or silencers. Myostatin antagonists seem to be a therapeutic means to alleviating sarcopenia, muscle atrophy and other wasting diseases.
Muscular dystrophy is documented by extensive muscle inflammation, muscle injury and replacement of muscle tissue with connective tissue and fat. Researchers have long sought to find agents that block the molecular pathway to muscular dystrophy by using “booster genes” to support muscle growth and cope with the otherwise damaging steps leading to muscular dystrophy. There are some potential benefits to using myostatin inhibitors as one study showed when a strain of mice who are myostatin deficient, but also contain the muscular dystrophy gene, have increased muscle mass and increased muscle strength, yet there’s still no reduction in inflammation or tissue injury.21
The safety issues of myostatin are still being investigated. It’s been suggested by researchers that since myostatin inhibitors cause dramatic increases in muscle mass (approximately 20 to 40 percent), it’s questionable whether human bone can withstand such a dramatic increase in muscle mass in such a short period of time. This probably wouldn’t be a problem for a healthy person, but what about a frail elderly person? Finally, myostatin is expressed in the heart as well. The suppression of myostatin could possibly lead to cardiac enlargement. Furthermore, following a heart attack, myostatin expression is upregulated in the heart. It should be mentioned that myostatin-deficient mice seem to be healthy although more long-term research needs to be conducted.
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