Mridula Sharma

Myostatin, a transforming growth factor-β superfamily member, is expressed in heart muscle and is upregulated in cardiomyocytes after infarct

Myostatin is a secreted growth and differentiating factor (GDF‐8) that belongs to the transforming growth factor‐beta (TGF‐β) superfamily. Targeted disruption of the myostatin gene in mice and a mutation in the third exon of the myostatin gene in double‐muscled Belgian Blue cattle breed result in skeletal muscle hyperplasia. Hence, myostatin has been shown to be involved in the regulation of skeletal muscle mass in both mice and cattle. Previous published reports utilizing Northern hybridization had shown that myostatin expression was seen exclusively in skeletal muscle. A significantly lower level of myostatin mRNA was also reported in adipose tissue. Using a sensitive reverse transcription‐polymerase chain reaction (RT‐PCR) technique and Western blotting with anti‐myostatin antibodies, we show that myostatin mRNA and protein are not restricted to skeletal muscle. We also show that myostatin expression is detected in the muscle of both fetal and adult hearts. Sequence analysis reveals that the Belgian Blue heart myostatin cDNA sequence contains an 11 nucleotide deletion in the third exon that causes a frameshift that eliminates virtually all of the mature, active region of the protein. Anti‐myostatin immunostaining on heart sections also demonstrates that myostatin protein is localized in Purkinje fibers and cardiomyocytes in heart tissue. Furthermore, following myocardial infarction, myostatin expression is upregulated in the cardiomyocytes surrounding the infarct area. Given that myostatin is expressed in fetal and adult hearts and that myostatin expression is upregulated in cardiomyocytes after the infarction, myostatin could play an important role in cardiac development and physiology. J. Cell. Physiol. 180:1–9, 1999.

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-κB-independent, FoxO1-dependent mechanism†

Myostatin, a transforming growth factor‐beta (TGF‐β) super‐family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF‐κB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF‐κB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin‐mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin‐1, MuRF‐1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF‐1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy‐related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF‐κB‐independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT‐FoxO1 pathway. J. Cell. Physiol. 209: 501–514, 2006.

RETRACTED: The Ubiquitin Ligase Mul1 Induces Mitophagy in Skeletal Muscle in Response to Muscle-Wasting Stimuli

Recent research reveals that dysfunction and subsequent loss of mitochondria (mitophagy) is a potent inducer of skeletal muscle wasting. However, the molecular mechanisms that govern the deregulation of mitochondrial function during muscle wasting are unclear. In this report, we show that different muscle-wasting stimuli upregulated mitochondrial E3 ubiquitin protein ligase 1 (Mul1), through a mechanism involving FoxO1/3 transcription factors. Overexpression of Mul1 in skeletal muscles and myoblast cultures was sufficient for the induction of mitophagy. Consistently, Mul1 suppression not only protected against mitophagy but also partially rescued the muscle wasting observed in response to muscle-wasting stimuli. In addition, upregulation of Mul1, while increasing mitochondrial fission, resulted in ubiquitination and degradation of the mitochondrial fusion protein Mfn2. Collectively, these data explain the molecular basis for the loss of mitochondrial number during muscle wasting.

Myostatin regulation during skeletal muscle regeneration

Myostatin, a member of the TGF‐β superfamily, is a key negative regulator of skeletal muscle growth. The role of myostatin during skeletal muscle regeneration has not previously been reported. In the present studies, normal Sprague‐Dawley and growth hormone (GH)‐deficient (dw/dw) rats were administered the myotoxin, notexin, in the right M. biceps femoris on day 0. The dw/dw rats then received either saline or human‐N‐methionyl GH (200μg/100g body weight/day) during the ensuing regeneration. Normal and dw/dw M. biceps femoris were dissected on days 1, 2, 3, 5, 9 and 13, formalin‐fixed, then immunostained for myostatin protein. Immunostaining for myostatin revealed high levels of protein within necrotic fibres and connective tissue of normal and dw/dw damaged muscles. Regenerating myotubes contained no myostatin at the time of fusion (peak fusion on day 5), and only low levels of myostatin were observed during subsequent myotube enlargement. Fibres which survived assault by notexin (survivor fibres) contained moderate to high myostatin immunostaining initially. The levels in both normal and dw/dw rat survivor fibres decreased on days 2–3, then increased on days 9–13. In dw/dw rats, there was no observed effect of GH administration on the levels of myostatin protein in damaged muscle. The low level of myostatin observed in regenerating myotubes in these studies suggests a negative regulatory role for myostatin in muscle regeneration. J. Cell. Physiol. 184:356–363, 2000.

Prolonged absence of myostatin reduces sarcopenia

Sarcopenia is a progressive age‐related loss of skeletal muscle mass and strength. Parabiotic experiments show that circulating factors positively influence the proliferation and regenerative capacity of satellite cells in aged mice. In addition, we believe that negative regulators of muscle mass also serve to balance the signals that influence satellite cell activation and regeneration capacity with ageing. Myostatin, a negative regulator of pre‐ and postnatal myogenesis, inhibits satellite cell activation and muscle regeneration postnatally. To investigate the role of myostatin during age‐related sarcopenia, we examined muscle mass and regeneration in young and old myostatin‐null mice. Young myostatin‐null muscle fibers were characterized by massive hypertrophy and hyperplasia and an increase in type IIB fibers, resulting in a more glycolytic muscle. With ageing, wild‐type muscle became increasingly oxidative and fiber atrophy was prominent. In contrast no fiber type switching was observed and atrophy was minimal in aged myostatin‐null muscle. The effect of ageing on satellite cell numbers appeared minimal, however, satellite cell activation declined significantly in both wild‐type and myostatin‐null muscles. In young mice, lack of myostatin resulted in increased satellite cell number and activation compared to wild‐type, suggesting a greater propensity to undergo myogenesis, a difference maintained in the aged mice. In addition, muscle regeneration of myostatin‐null muscle following notexin injury was accelerated and fiber hypertrophy and type were recovered with regeneration, unlike in wild‐type muscle. In conclusion, a lack of myostatin appears to reduce age‐related sarcopenia and loss of muscle regenerative capacity. J. Cell. Physiol. 209: 866–873, 2006.

Titin-cap associates with, and regulates secretion of, Myostatin

Myostatin, a secreted growth factor, is a key negative regulator of skeletal muscle growth. To identify modifiers of Myostatin function, we screened for Myostatin interacting proteins. Using a yeast two‐hybrid screen, we identified Titin‐cap (T‐cap) protein as interacting with Myostatin. T‐cap is a sarcomeric protein that binds to the N‐terminal domain of Titin and is a substrate of the titin kinase. Mammalian two‐hybrid studies, in vitro binding assays and protein truncations in the yeast two‐hybrid system verified the specific interaction between processed mature Myostatin and full‐length T‐cap. Analysis of protein–protein interaction using surface plasmon resonance (Biacore, Uppsala, Sweden) kinetics revealed a high affinity between Myostatin and T‐cap with a KD of 40 nM. When T‐cap was stably overexpressed in C2C12 myoblasts, the rate of cell proliferation was significantly increased. Western analyses showed that production and processing of Myostatin were not altered in cells overexpressing T‐cap, but an increase in the retention of mature Myostatin indicated that T‐cap may block Myostatin secretion. Bioassay for Myostatin confirmed that conditioned media from myoblasts overexpressing T‐cap contained lower levels of Myostatin. Given that Myostatin negatively regulates myoblast proliferation, the increase in proliferation observed in myoblasts overexpressing T‐cap could thus be due to reduced Myostatin secretion. These results suggest that T‐cap, by interacting with Myostatin, controls Myostatin secretion in myogenic precursor cells without affecting the processing step of precursor Myostatin. J. Cell. Physiol. 193: 120–131, 2002.

Myostatin induces degradation of sarcomeric proteins through a Smad3 signaling mechanism during skeletal muscle wasting

Ubiquitination-mediated proteolysis is a hallmark of skeletal muscle wasting manifested in response to negative growth factors, including myostatin. Thus, the characterization of signaling mechanisms that induce the ubiquitination of intracellular and sarcomeric proteins during skeletal muscle wasting is of great importance. We have recently characterized myostatin as a potent negative regulator of myogenesis and further demonstrated that elevated levels of myostatin in circulation results in the up-regulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1). However, the exact signaling mechanisms by which myostatin regulates the expression of Atrogin-1 and MuRF1, as well as the proteins targeted for degradation in response to excess myostatin, remain to be elucidated. In this report, we have demonstrated that myostatin signals through Smad3 (mothers against decapentaplegic homolog 3) to activate forkhead box O1 and Atrogin-1 expression, which further promotes the ubiquitination and subsequent proteasome-mediated degradation of critical sarcomeric proteins. Smad3 signaling was dispensable for myostatin-dependent overexpression of MuRF1. Although down-regulation of Atrogin-1 expression rescued approximately 80% of sarcomeric protein loss induced by myostatin, only about 20% rescue was seen when MuRF1 was silenced, implicating that Atrogin-1 is the predominant E3 ligase through which myostatin manifests skeletal muscle wasting. Furthermore, we have highlighted that Atrogin-1 not only associates with myosin heavy and light chain, but it also ubiquitinates these sarcomeric proteins. Based on presented data we propose a model whereby myostatin induces skeletal muscle wasting through targeting sarcomeric proteins via Smad3-mediated up-regulation of Atrogin-1 and forkhead box O1.

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-κB

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro‐oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor‐β (TGF‐β) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor‐α (TNF‐α) signaling via NF‐κB and NADPH oxidase. Aged Mstn null (Mstn−/−) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF‐κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF‐α and hydrogen peroxide (H2O2) are potent inducers of Mstn and require NF‐κB signaling for Mstn induction. These results demonstrate that Mstn and TNF‐α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF‐α and NADPH oxidase, and the elevated TNF‐α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal‐mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.

Myostatin auto-regulates its expression by feedback loop through Smad7 dependent mechanism

Myostatin, a secreted growth factor, is a member of the TGF‐β superfamily and an inhibitor of myogenesis. Previously, we have shown that myostatin gene expression is regulated at the level of transcription and that myostatin is a downstream target gene of MyoD. Here we show that myostatin gene expression is auto‐regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of myostatin mRNA in the biceps femoris muscle of cattle that express a non‐ functional myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous myostatin decreases endogenous myostatin mRNA. Consistent with this result, wild type myostatin protein is able to repress myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non‐functional myostatin (Piedmontese) failed to repress the myostatin promoter suggesting that myostatin auto‐regulates its promoter by negative feedback inhibition. Auto‐regulation by myostatin appears 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 (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased myostatin mRNA indicating that Smad7 is a negative regulator of myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in myostatin expression is seen in myotubes that express non functional myostatin. In addition, interference with myostatin signaling prevents the induction of Smad7 promoter activity by myostatin. Based on these results, we propose that myostatin auto‐regulates its gene expression through a Smad7 dependent mechanism in myogenic cells.

Growth factors controlling muscle development

The enlarged muscles of certain breeds of cattle, such as the Belgian Blue, have been shown to result from a marked increase in the number of normal sized muscle fibers. Originally insulin-like growth factors (IGFs) were implicated in this myofiber hyperplasia, as IGFs have been shown to stimulate myoblast proliferation as well as maintain fiber differentiation. Recently it has been reported that mice lacking a myostatin gene, a member of the TGFbeta superfamily, have enhanced skeletal mass resulting from increased muscle fiber number and size. Mutations in this gene have been found in double-muscled cattle, indicating that myostatin is an inhibitor of muscle growth. Myostatin is expressed early in gestation and then maintained to adulthood in certain muscles. Myostatin expression in bovine muscle is highest during gestation when muscle fibers are forming and some of the myogenic regulatory factors have elevated expression over the same period as myostatin. Molecular expression of the IGF axis does not differ between Belgian Blue and normal muscled cattle, and IGF-II mRNA is increased throughout formation of secondary fibers in both breeds. However, myostatin and MyoD expression in muscle differ between normal and hypertrophied muscle cattle breeds. This evidence strongly suggests that lack of myostatin is associated with an increase in fiber number which then results in a marked increase in potential muscle mass in double-muscled cattle.