How Does Myostatin Inhibit Muscle Development & Hypertrophy?
by: Karl Hoffman
Myostatin is a member of the family of growth factors known as the transforming growth factor-beta (TGF-beta) family. It is an important negative regulator of embryonic andpostnatal muscle growth. By now we have all seen photographs of the famous myostatin null mice. These mice lackmyostatin, which normally puts the brakes on muscular development. Myostatin null mice generated by gene targetingshow a dramatic and widespread increase in skeletal muscle mass. Individual muscles in myostatin null mice weigh 2- to3-fold more than those of wild-type mice, primarily due to an increased number of muscle fibers without a correspondingincrease in the amount of fat. As well as an increased number of muscle fibers (hyperplasia), the individual muscle fibersin these mice are larger than normal (hypertrophy).
Mutations in the myostatin gene leading to “overmuscled” humans havebeen reported (1). See figure 1 below
Figure 1. Photographs of the Child at the Ages of SixDays and Seven Months (Panel A), Ultrasonograms (Panel B) and Morphometric Analysis (Panel C) of the Muscles of thePatient and a Control Infant, and the Patient’s Pedigree (Panel D).
The arrowheads in Panel A indicate the protruding muscles of the patient’s thigh andcalf. In Panel B, an ultrasonographic transverse section (linear transducer, 10 MHz) through the middle portion of the thighreveals differences between the patient and a control infant of the same age, sex, and weight. VL denotes vastus lateralis,VI vastus intermedius, VM vastus medialis, RF rectusfemoris, and F femur. In Panel C, retracings of the muscle outlinesand results of the morphometric analysis of the muscle cross-sectional planes of the two infants
also reveal markeddifferences. Panel D shows the patient’s pedigree. Solid symbols denote family members who are exceptionally strong,according to information in their clinical history. Square symbols denote male family members, and circles female family members. (Adapted from Schuelke et.al)
There are several reported ways by which myostatin may regulate muscular development. During growth, muscle precursor cells (known as myoblasts) enter the cell cycle and proliferate until a cascade of signals (initiated by the myogenic regulatory factor MyoD) causes myoblasts to withdraw from the cell cycle, differentiating and fusing into multinucleated myotubes. These mature into fully developed muscle fibers. Myostatin appears to downregulate the activity and expression levels of MyoD, thus preventing the differentiation of myoblasts into
myotubes (2). See figure 2 below.
Fig. 2. A model for the role of myostatin during muscle growth and differentiation. A model from Thomas et al. (24) for the role of myostatin during muscle growth, adapted to include the role of myostatin during myogenic differentiation. During myogenic embryogenesis, Myf5 and MyoD specify cells to adopt the myogenic fate. Myoblast proliferation is regulated by myostatin via the up-regulation of p21 and inactivation of Cdk (cyclin-dependent kinase) activity resulting in retinoblastoma (Rb)hypophosphorylation and myoblast cell cycle arrest. In response to a differentiation cue, MyoD becomes fully functional, activating downstream myogenic gene expression, including myogenin and p21, resulting in committed myoblasts that fuse into multinucleated myotubes. Myostatin regulates this process by inhibiting MyoD expression via Smad 3 (another protein in the TGF-beta family) after the differentiation cue resulting in the loss of downstream myogenic gene expression and myogenicdifferentiation. (Adapted from Langley et al)
Satellite cells are myogenic precursor (stem) cells that are quiescent in mature muscle.Injury to the surrounding muscle activates the nearby satellite cells which then reenter the cell cycle, and express myogenic regulatory factors, proliferate, develop into myoblasts, and fuse to the injured muscle, affecting its repair. Satellite cells are responsible for the postnatal growth of muscle. When we damage muscle by performing resistance exercise, we activate satellite cells which then contribute to the observed hypertrophy. Myostatin has been shown to inhibit reentry of satellite cells into the cell cycle, thus rendering them inactive (3).
McCrosky et.al. (3) performed an interesting experiment designed to demonstrate the effects of myostatin on satellite cells. To show the direct inhibitory effects of myostatin on myoblast proliferation, tissue-dissociated satellite cells were isolated from both wild-type and myostatin-null mice. The same number of wild-type and myostatin-null myoblasts were cultured in media, and myostatin was added in increasing concentrations to only myostatin-null cultures. After 48 h of proliferation, the cells were fixed with 10% formal saline and stained with the methylene blue. When recombinant myostatin was added to the media in increasing amounts, the enhanced proliferation rate seen in the myostatin-null myoblasts was reduced to that of the wild-type myoblasts.
Figure 3. A model for the role of Myostatin in postnatal muscle growth. Quiescent satellite cells on muscle fibers are activated in response to muscle injury to give rise to myoblasts. Proliferating myoblasts can either fuse with the existing fiber or differentiate into a nascent myotube. A portion of proliferating myoblasts, however, can revert to become quiescent satellite cells, thus resulting in self-renewal. As myostatin is a negative regulator of cell cycle progression, high levels of myostatin in satellite cells block the activation to maintain quiescence.(From McCroskey et al.)
Myostatin acts in yet another way to inhibit muscle growth. When androgens such as testosterone bind to the androgen receptor (AR), the AR undergoes a series of conformational (shape) changes that allow it to interact with androgen response elements (binding sites) in androgen target genes. ARA70 (androgen receptor associated protein 70) is a so-called AR coregulator that stabilizes the ligand (androgen)-bound AR enhancing the ability of the AR to induce transcription of target genes. Siriett et. al. were recently able to demonstrate that ARA70 is expressed in myoblasts during myogenesis and that myostatin is a potent ownregulator of ARA70 gene expression (4). Siriett et. al. thus propose that the hypertrophy seen in animals that lack myostatin could not only be due to the mechanisms outlined above but could also be due to increased protein synthesis due to enhanced AR activity resulting from increased expression of ARA70. Although the authors of (4) performed their experiments with mice, they showed that murine ARA70 has about an 80% sequence homology to human ARA70 and propose that the same mechanism at work in mice alsosuppresses ARA70 expression in humans.
In addition to ARA70, Siriett et.al. discovered numerous other key genes controlling muscle development and various metabolic pathways that are upregulated in myostatin-null mice. These include the
genes for actin and tropomyosin, both myofibril proteins; muscle creatine kinase which is involved in energy metabolism; phosphorylase kinase, an enzyme involved in glycogen metabolism; ATP synthase, an enzyme involved in mitochondrial ATP synthesis, to name but a few, as well as a number of novel genes with unknown functions.
These findings led them to conclude that an increase in myostatin gene expression by glucocorticoids might contribute to the pathogenesis of glucocorticoid-induced skeletal muscle atrophy. Specifically, the intramuscular myostatin mRNA expression in rats treated with dexamethasone for 5 days was significantly (4.50-fold, P < 0.01) higher than that in their pair-fed controls. The myostatin protein expression in these rats was also significantlyhigher (2.6-fold, P < 0.01) than that in their pair-fed controls. Further confirming dexamethasone’s effect on myostatin, administration of RU-486, a potent glucocorticoid antagonist, was given to the dexamethasone treated animals. The action of dexamethasone treatment on myostatin mRNA expression was effectively nullified by RU-486 administration.
Interestingly, the majority of muscle loss in the dexamethasone treated animals occurred in the first 5 days of treatment, whereas animals treated for 10 days did not experience significantly greater muscle loss than those treated for 5 days. This coincides with the observation that, although the marked upregulation of myostatin mRNA and protein expression induced by dexamethasone could be seen for 5 days after treatment, this overexpression
was not sustained by extending the treatment to 10 days. The counterregulatory mechanism is unknown.
We discussed above how myostatin inhibits the normally hypertrophic activity of satellite cells. Growth hormone, on the other hand, may exert its stimulatory effects on muscle protein synthesis, in part, by activating satellite cells (6). Marcell et al (7) observed a significant inverse relation between myostatin levels and GH receptor levels in healthy but aging (>65). It is also well known that GH levels decline with age. Thus it’s possible that the combination of decreasing GH/GHR and increasing myostatin that accompanies aging may contribute to age related muscle loss.
Collectively the data suggest that GH excess in youth may override myostatin’s inhibition of satellite cell activation rather than GH directly inhibiting myostatin promoter activity. For example Brill et al (8) examined the effects of GH and/or testosterone administration on body composition, performance, mood, sexual function, bone turnover, and muscle-gene expression in healthy older men. Of relavence to our current discussion, myostatin gene expression was unaffected by either treatment.
In addition to GH/IGF-1 keeping myostatin in check, a negative feedback autoregulatory system for myostatin gene expression appears to exist (9). As previously mentioned, myostatin is a member of the TGF-beta family of growth factors. Human SMAD7 is a 426 amino acid protein. Smad7 acts as an antagonist of TGF-beta signaling. It functions in several different ways. It interacts with the TGF-beta receptor, interfering with the activation of the downstream signaling factors Smad2/3. In addition, it recruits E3-ubiquitin ligases, Smurf1/2, to the activated TGF-beta receptor, resulting in receptor ubiquitination and degradation. Smad7 expression is induced by TGF-beta itself, resulting in an autoregulatory feedback loop in TGF-beta signaling.
Since myostatin belongs to the TGF-beta family, Forbes et al (9) tested the logical proposition that myostatin expression might be autoregulated via Smad7. Indeed, the researchers discovered that auto-regulation by myostatin does appear to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity
In summary, we have seen several proposed mechanisms through which myostatin regulates muscle growth. It is not known which if any is the predominant mode through which myostatin works, and it is likely that all contribute to myostatin’s action.
1) 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
2) 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
3) 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.
4) Siriett V, Nicholas G, Berry C, Watson T, Hennebry A, Thomas M, Ling N, Sharma M, Kambadur R. Myostatin negatively regulates the expression of the steroid receptor co-factor ARA70. J Cell Physiol. 2005 Aug 18; [Epub ahead of print
5) 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
6) Le Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis: 2001. Endocr Rev. 2001Feb;22(1):53-74.
by: Karl Hoffman
Myostatin is a member of the family of growth factors known as the transforming growth factor-beta (TGF-beta) family. It is an important negative regulator of embryonic andpostnatal muscle growth. By now we have all seen photographs of the famous myostatin null mice. These mice lackmyostatin, which normally puts the brakes on muscular development. Myostatin null mice generated by gene targetingshow a dramatic and widespread increase in skeletal muscle mass. Individual muscles in myostatin null mice weigh 2- to3-fold more than those of wild-type mice, primarily due to an increased number of muscle fibers without a correspondingincrease in the amount of fat. As well as an increased number of muscle fibers (hyperplasia), the individual muscle fibersin these mice are larger than normal (hypertrophy).
Mutations in the myostatin gene leading to “overmuscled” humans havebeen reported (1). See figure 1 below
Figure 1. Photographs of the Child at the Ages of SixDays and Seven Months (Panel A), Ultrasonograms (Panel B) and Morphometric Analysis (Panel C) of the Muscles of thePatient and a Control Infant, and the Patient’s Pedigree (Panel D).
The arrowheads in Panel A indicate the protruding muscles of the patient’s thigh andcalf. In Panel B, an ultrasonographic transverse section (linear transducer, 10 MHz) through the middle portion of the thighreveals differences between the patient and a control infant of the same age, sex, and weight. VL denotes vastus lateralis,VI vastus intermedius, VM vastus medialis, RF rectusfemoris, and F femur. In Panel C, retracings of the muscle outlinesand results of the morphometric analysis of the muscle cross-sectional planes of the two infants
also reveal markeddifferences. Panel D shows the patient’s pedigree. Solid symbols denote family members who are exceptionally strong,according to information in their clinical history. Square symbols denote male family members, and circles female family members. (Adapted from Schuelke et.al)
There are several reported ways by which myostatin may regulate muscular development. During growth, muscle precursor cells (known as myoblasts) enter the cell cycle and proliferate until a cascade of signals (initiated by the myogenic regulatory factor MyoD) causes myoblasts to withdraw from the cell cycle, differentiating and fusing into multinucleated myotubes. These mature into fully developed muscle fibers. Myostatin appears to downregulate the activity and expression levels of MyoD, thus preventing the differentiation of myoblasts into
myotubes (2). See figure 2 below.
Fig. 2. A model for the role of myostatin during muscle growth and differentiation. A model from Thomas et al. (24) for the role of myostatin during muscle growth, adapted to include the role of myostatin during myogenic differentiation. During myogenic embryogenesis, Myf5 and MyoD specify cells to adopt the myogenic fate. Myoblast proliferation is regulated by myostatin via the up-regulation of p21 and inactivation of Cdk (cyclin-dependent kinase) activity resulting in retinoblastoma (Rb)hypophosphorylation and myoblast cell cycle arrest. In response to a differentiation cue, MyoD becomes fully functional, activating downstream myogenic gene expression, including myogenin and p21, resulting in committed myoblasts that fuse into multinucleated myotubes. Myostatin regulates this process by inhibiting MyoD expression via Smad 3 (another protein in the TGF-beta family) after the differentiation cue resulting in the loss of downstream myogenic gene expression and myogenicdifferentiation. (Adapted from Langley et al)
Satellite cells are myogenic precursor (stem) cells that are quiescent in mature muscle.Injury to the surrounding muscle activates the nearby satellite cells which then reenter the cell cycle, and express myogenic regulatory factors, proliferate, develop into myoblasts, and fuse to the injured muscle, affecting its repair. Satellite cells are responsible for the postnatal growth of muscle. When we damage muscle by performing resistance exercise, we activate satellite cells which then contribute to the observed hypertrophy. Myostatin has been shown to inhibit reentry of satellite cells into the cell cycle, thus rendering them inactive (3).
McCrosky et.al. (3) performed an interesting experiment designed to demonstrate the effects of myostatin on satellite cells. To show the direct inhibitory effects of myostatin on myoblast proliferation, tissue-dissociated satellite cells were isolated from both wild-type and myostatin-null mice. The same number of wild-type and myostatin-null myoblasts were cultured in media, and myostatin was added in increasing concentrations to only myostatin-null cultures. After 48 h of proliferation, the cells were fixed with 10% formal saline and stained with the methylene blue. When recombinant myostatin was added to the media in increasing amounts, the enhanced proliferation rate seen in the myostatin-null myoblasts was reduced to that of the wild-type myoblasts.
Figure 3. A model for the role of Myostatin in postnatal muscle growth. Quiescent satellite cells on muscle fibers are activated in response to muscle injury to give rise to myoblasts. Proliferating myoblasts can either fuse with the existing fiber or differentiate into a nascent myotube. A portion of proliferating myoblasts, however, can revert to become quiescent satellite cells, thus resulting in self-renewal. As myostatin is a negative regulator of cell cycle progression, high levels of myostatin in satellite cells block the activation to maintain quiescence.(From McCroskey et al.)
MYOSTATIN AND ARA70
Myostatin acts in yet another way to inhibit muscle growth. When androgens such as testosterone bind to the androgen receptor (AR), the AR undergoes a series of conformational (shape) changes that allow it to interact with androgen response elements (binding sites) in androgen target genes. ARA70 (androgen receptor associated protein 70) is a so-called AR coregulator that stabilizes the ligand (androgen)-bound AR enhancing the ability of the AR to induce transcription of target genes. Siriett et. al. were recently able to demonstrate that ARA70 is expressed in myoblasts during myogenesis and that myostatin is a potent ownregulator of ARA70 gene expression (4). Siriett et. al. thus propose that the hypertrophy seen in animals that lack myostatin could not only be due to the mechanisms outlined above but could also be due to increased protein synthesis due to enhanced AR activity resulting from increased expression of ARA70. Although the authors of (4) performed their experiments with mice, they showed that murine ARA70 has about an 80% sequence homology to human ARA70 and propose that the same mechanism at work in mice alsosuppresses ARA70 expression in humans.
In addition to ARA70, Siriett et.al. discovered numerous other key genes controlling muscle development and various metabolic pathways that are upregulated in myostatin-null mice. These include the
genes for actin and tropomyosin, both myofibril proteins; muscle creatine kinase which is involved in energy metabolism; phosphorylase kinase, an enzyme involved in glycogen metabolism; ATP synthase, an enzyme involved in mitochondrial ATP synthesis, to name but a few, as well as a number of novel genes with unknown functions.
GLUCOCORTICOIDS UPREGULATE MYOSTATIN
Glucocorticoids are well known to induce muscle atrophy in animals and humans, although the exact mechanism(s) are not well understood. Ma and colleagues (5) recently cloned and characterized the 5′-upstream regulatory region of the human myostatin gene and found that the promoter contains a number of response elements important for muscle growth, including seven putative glucocorticoid response elements (GREs). GREs are sites where the glucocorticoid/receptor complex bind and control gene transcription. They also demonstrated thatdexamethasone dose-dependently increases endogenous myostatin transcription in myocytes through a glucocorticoid receptor-mediated mechanism.These findings led them to conclude that an increase in myostatin gene expression by glucocorticoids might contribute to the pathogenesis of glucocorticoid-induced skeletal muscle atrophy. Specifically, the intramuscular myostatin mRNA expression in rats treated with dexamethasone for 5 days was significantly (4.50-fold, P < 0.01) higher than that in their pair-fed controls. The myostatin protein expression in these rats was also significantlyhigher (2.6-fold, P < 0.01) than that in their pair-fed controls. Further confirming dexamethasone’s effect on myostatin, administration of RU-486, a potent glucocorticoid antagonist, was given to the dexamethasone treated animals. The action of dexamethasone treatment on myostatin mRNA expression was effectively nullified by RU-486 administration.
Interestingly, the majority of muscle loss in the dexamethasone treated animals occurred in the first 5 days of treatment, whereas animals treated for 10 days did not experience significantly greater muscle loss than those treated for 5 days. This coincides with the observation that, although the marked upregulation of myostatin mRNA and protein expression induced by dexamethasone could be seen for 5 days after treatment, this overexpression
was not sustained by extending the treatment to 10 days. The counterregulatory mechanism is unknown.
MYOSTATIN AND GROWTHHORMONE/IGF-1
We discussed above how myostatin inhibits the normally hypertrophic activity of satellite cells. Growth hormone, on the other hand, may exert its stimulatory effects on muscle protein synthesis, in part, by activating satellite cells (6). Marcell et al (7) observed a significant inverse relation between myostatin levels and GH receptor levels in healthy but aging (>65). It is also well known that GH levels decline with age. Thus it’s possible that the combination of decreasing GH/GHR and increasing myostatin that accompanies aging may contribute to age related muscle loss.
Collectively the data suggest that GH excess in youth may override myostatin’s inhibition of satellite cell activation rather than GH directly inhibiting myostatin promoter activity. For example Brill et al (8) examined the effects of GH and/or testosterone administration on body composition, performance, mood, sexual function, bone turnover, and muscle-gene expression in healthy older men. Of relavence to our current discussion, myostatin gene expression was unaffected by either treatment.
MYOSTATIN AUTOREGULATION
In addition to GH/IGF-1 keeping myostatin in check, a negative feedback autoregulatory system for myostatin gene expression appears to exist (9). As previously mentioned, myostatin is a member of the TGF-beta family of growth factors. Human SMAD7 is a 426 amino acid protein. Smad7 acts as an antagonist of TGF-beta signaling. It functions in several different ways. It interacts with the TGF-beta receptor, interfering with the activation of the downstream signaling factors Smad2/3. In addition, it recruits E3-ubiquitin ligases, Smurf1/2, to the activated TGF-beta receptor, resulting in receptor ubiquitination and degradation. Smad7 expression is induced by TGF-beta itself, resulting in an autoregulatory feedback loop in TGF-beta signaling.
Since myostatin belongs to the TGF-beta family, Forbes et al (9) tested the logical proposition that myostatin expression might be autoregulated via Smad7. Indeed, the researchers discovered that auto-regulation by myostatin does appear to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity
In summary, we have seen several proposed mechanisms through which myostatin regulates muscle growth. It is not known which if any is the predominant mode through which myostatin works, and it is likely that all contribute to myostatin’s action.
References
1) 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
2) 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
3) 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.
4) Siriett V, Nicholas G, Berry C, Watson T, Hennebry A, Thomas M, Ling N, Sharma M, Kambadur R. Myostatin negatively regulates the expression of the steroid receptor co-factor ARA70. J Cell Physiol. 2005 Aug 18; [Epub ahead of print
5) 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
6) Le Roith D, Bondy C, Yakar S, Liu JL, Butler A. The somatomedin hypothesis: 2001. Endocr Rev. 2001Feb;22(1):53-74.
