The Regulation of Myostatin and Its Relationship with Muscle Atrophy
Date Issued
2011
Date
2011
Author(s)
Han, Der-Sheng
Abstract
Introduction
Myostatin (growth and differentiation factor 8, GDF8), a member of transforming growth factor beta family, is a potent negative regulator of muscle growth and differentiation. Myostatin knock-out mice have 3 times larger muscle mass and reduced adiposity than wild type. On the other hand, increased secretion of myostatin protein systemically causes cachexia-like phenotype. Human myostatin gene is located on 2q32.2, and its mutation causes increased muscularity. Myostatin mutation also causes double-muscle phenotype in cattle, sheep, dog, and zebrafish. Myostatin peptide sequence is conserved among vertebrates. The amino acid sequence of the active C-terminal peptide is identical in rodents, chicken, turkey, pig, and human. The translated product, prepropeptide, is composed of 375 amino acids. It becomes full-length myostatin after removing the 21-amino-acid signal peptide. The full-length myostatin is cleaved by peptidase into N-terminal propeptide and C-terminal 110-amino-acid active peptide. The C-terminal active peptide binds non-covalently with propeptide, follistatin, and product of follistatin-related gene to become latent. The myostatin acts through autocrine and endocrine fashion. It binds with activin type II receptor on the membrane, and phosphorylates intracellular transcription factors-- Smad2 and Smad3.
Since myostatin can inhibit muscle growth, it may have therapeutic role in the muscle related diseases and metabolic syndrome. The muscle related diseases include neuromuscular diseases (eg. muscular dystrophy, myopathy), sarcopenia, cachexia caused by metabolic diseases or malignancy, muscle atrophy caused by denervation or disuse/immobilization, and rhabdomyosarcoma. These diseases have different etiology, but the common final presentation is muscle atrophy. In 2002, two groups of researchers applied different methods to inhibit myostatin in mdx mice, a rodent model of Duchenne and Becker muscular dystrophy, and found improved symptoms and function in these animals. Minetti injected trichostatin A (TSA) intraperitoneally into mdx mice, and also found improved symptoms and decreased myostatin expression. Inhibition of myostatin in mouse model of amyotrophic lateral sclerosis improved pathological characteristics in the diaphragm and increased muscle power and mass temporally and significantly, but did not lengthen survival. There are still debates on the expression of myostatin during ageing. Cachexia is the clinical condition of losing muscle and fat tissue, caused by severe trauma, malignancy, infection, or other medical diseases. The decrease of muscle mass can reach 75% in patients with malignancy, which will cause weakness, fatigue, disuse, and death. The typical pathological manifestation is smaller muscle fiber diameter. Cachexia is also a poor prognostic factor, which increases medical expenses and demand for rehabilitation. The secondary muscle atrophy usually incurred major disability and direct or indirect socioeconomic loss, even if the primary disease is cured. The mainstream of treatment for cachexia is rehabilitation, electric stimulation, or palliative therapy. Myostatin modulation might shed light for the treatment of cachexia. The relationship between cachexia and myostatin has been explored in chronic renal failure, heart failure, AIDS, COPD, Addison’s disease, Cushing’s syndrome, liver cirrhosis, cancer, calorie restriction, and burn. Generally speaking, the myostatin mRNA expression increased during the process of muscle wasting; however, its expression in chronic stage was still unclear. On the first day after sciatic neurectomy, the myostatin expression in gastrocnemius of rat increased 31%. It decreased 34% 14 days after operation, when the muscle size decreased 50%. The change of myostatin protein level in gastrocnemius was similar to that of mRNA. The results implied that the mechanism governing denervation-induced muscle atrophy was complex. The effect of myostatin might be variable in the different time frame. After hind limb suspension in rat for 10 days, the myostatin mRNA and protein increased 110% and 37%, and the muscle mass decreased to 84%. Those parameters normalized after reload for 4 days. Reload by walking for 30 minutes every day could prevent muscle atrophy caused by high-limb suspension, but could not prevent myostatin elevation. This suggested that increase of myostatin would not cause muscle atrophy in healthy adult rat. Myostatin mRNA expression increased and the area of type IIA and IIX fiber decreased in the patients having hip joint osteoarthritis. Rhabdomyosarcoma is the overgrowth of the undifferentiated muscle cells. The effect of myostatin on rhabdomyosarcoma is still in debate. Myostatin not only inhibited muscle growth, but also affected adipose tissue. Myostatin knock-out mice had decreased fat deposit. The cause might be the increased fat burner—muscle. On the other hand, systemic administration of myostatin caused decrease of white adipose tissue. The possible reason is that myostatin competes with ActRIIB for BMP7. Decreased BMP7 expression then inhibits lipogenesis.
Serum myostatin level related to the intramuscular expression of this gene. Some reports using radioimmunoassay (RIA) as a measurement for serum myostatin showed that the level of serum myostatin was inversely related with the total skeletal muscle mass in patients having acquired immunodeficiency syndrome. The serum myostatin level also increased in those bed-ridden patients. Due to environmental and occupational issues on radioactive substance, the application of RIA is limited in many ways. On the other hand, enzyme-linked immunosorbent assay (ELISA) is widely used because of its high accessibility and economy, and no radioactive waste. Some studies employed ELISA to quantify myostatin, but the discrepancy between them is obvious. Using antibody-antigen pair with high specificity is crucial to ELISA.
Histone deacetylase inhibitor (HDACI) facilitates the acetylation of histone, unwinds packed chromatin, and activates related genes. It also increases myoblast fusion, and myofiber cross-section area. Our lab found the HDACI, including trichostatin A (TSA) and valproic acid, could activate the expression of myostatin over 40 times in differentiated mouse myoblast than the untreated cells. However, the mechanism is still unclear.
Our specific aims are to elucidate the mechanism and involving signal pathways governing myostatin expression in order to found suitable modulators to treat muscle related diseases, and to establish ELISA system for serum myostatin to investigate whether it could be a new biomarker for systemic muscle atrophy and regeneration in chronic renal failure and Pompe disease.
There are two hypotheses. First, TSA activates myostatin through MAPK and Akt/mTOR pathway, and we can decrease myostatin expression by inhibiting those pathways. Besides, serum myostatin level is related to muscle strength/mass. Patients using high-flux dialyzer will have lower myostatin level and thus higher muscle strength/mass than those using low-flux dialyzer. Before enzyme replacement therapy (ERT), Pompe disease patients will have lower myostatin and IGF-1 level due to muscle atrophy. After ERT, the level of those serum markers will increase.
Material and Method
We used differentiated mouse C2C12 myoblast as a model to measure the expression of myostatin in different treatment condition with quantitative real-time PCR. The Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum was the maintaining media, and the DMEM containing 2% horse serum was the differentiation media. After 4-day differentiation, the C2C12 myotubes were pretreated with p38MAPK inhibitor (SB203580), ERK inhibitor (PD98059), JNK inhibitor (SP600125), PI3K inhibitor (PIK-75), Akt inhibitor (LY294002 and AKT INH VIII), or mTOR inhibitor (rapamycin) for 1 hour, and subsequently treated with 50nM TSA for 24 hours. The RNA polymerase inhibitor (Actinomycin D) was added as pretreatment to verify transcriptional level control. Besides, p38 MAPK and JNK activator (anisomycin) will be added to induce myostatin expression without TSA. The phosphorylated form of the components in the two signal pathways were quantified with western blot. In order to inhibit respective pathway more specifically, we employed C2C12 stably transfected with short hairpin RNA plasmids aimed at p38MAPK, Akt1, and Akt2 to see if the induction of myostatin would be inhibited.
In the study of chronic renal failure, we recruited 41 healthy controls and 60 patients receiving maintenance hemodialysis (MHD) for over half year. Twenty-nine of them used high-flux dialyzer, and 31 used low-flux dialyzer. The healthy controls received blood sampling, and body composition determination with multi-channel bioimpedance analyzer. The hemodialysis patients accepted blood sampling before and after single episode of hemodialysis session, and body composition determination. Dominant hand grip strength was measured in all subjects. The serum myostatin level was determined by home-made ELISA kit. IGF-1 was measured with commercial ELISA kit. Pearson correlation analysis was used to find the relationship between myostatin and other parameters. The linear regression analysis was applied to find the major determinants of myostatin and grip strength. Binary logistic regression was employed to find the odds ratio of low grip strength.
In the study of Pompe disease, we recruited 16 patients with Pompe disease and 16 sex-, age-matched neonate screening subjects as control. We performed quadriceps muscle biopsy and blood sampling to measure the myostatin, follistatin, creatine kinase, and IGF-1 level to monitor the change of serum markers before and one year after ERT. The median duration of follow-up was 11.7 months (range: 6-23 months). There were 10 patients having complete muscle and blood samples. Six were infant-onset Pompe disease, and 4 were late-onset Pompe disease. The gross and fine motor development was evaluated with Peabody Developmental Motor Scale (PDMS). The muscle sample was biopsied, processed and stained, and was evaluated by an experienced pathologist. The percentage of intramuscular vacuoles was calculated. The test of mean between two variables was performed by Wilcoxon rank-sum test nonparametrically. The pre- and post-ERT comparison of serum markers was performed with Wilcoxon signed-rank test.
Results
TSA increased myostatin mRNA expression up to 40-fold after treatment for 24 hours. Pretreatment with actinomycin D reduced the TSA-induced myostatin mRNA by 93%, suggesting TSA induced myostatin expression mainly at the transcriptional level. Pretreatment with p38 MAPK and JNK inhibitors, but not ERK inhibitor, blocked TSA-induced myostatin expression respectively by 72% and 43%. Knockdown of p38MAPK by RNAi inhibited the TSA-induced myostatin expression by 77% in C2C12 myoblasts. The protein levels of phosphorylated p38 MAPK, JNK, but not ERK, increased with TSA treatment in differentiated C2C12 cells. Direct activation of p38 MAPK and JNK by anisomycin in the absence of TSA increased myostatin mRNA by 4-fold. The phosphorylated form of the kinase MKK3/4/6 and ASK1, upstream cascades of p38 MAPK and JNK, also increased with TSA treatment. We concluded that the induction of myostatin by TSA treatment in differentiated C2C12 cells is in part through ASK1-MKK3/6-p38 MAPK and ASK1-MKK4-JNK signaling pathways. Activation of p38 MAPK and JNK axis is necessary, but not sufficient for TSA-induced myostatin expression.
We also explored the role of the PI3K-Akt-mTOR axis in myostatin induction. We confirmed that phosphorylated Akt was induced after TSA treatment. Pretreatment with respective PI3K, Akt, and mTOR inhibitors partially blocked the TSA-induced myostatin expression 66%, 82%, and 90%, respectively. The shRNA aimed to knock-down Akt1 and Akt2 also inhibited TSA-induced myostatin expression 56% and 82%, suggesting that TSA-induced transcription activation of myostatin is in part through Akt pathway.
The MHD patients had lower body mass index, IGF-1 level, and grip strength than the normal controls. The patients using the high-flux dialyzer had better grip strength than those using the low-flux (25.5 vs. 19.2 kg). The pre-dialysis myostatin level was higher in low-flux dialyzer than high-flux (31.0 vs. 18.5 μg/ml). Interestingly, the high-flux dialyzer reduced the serum myostatin by 36%, whereas low-flux dialyzer increased it by 25%. The myostatin was inversely related to age and the use of high-flux dialyzer. Furthermore, the grip strength was negatively related to age, female gender, muscle mass, myostatin levels and hemodialysis, but positively to the use of high-flux dialyzer in linear regression. The risk of low grip strength was 7.6 times higher in those with higher serum myostatin with the adjustment of age, gender, muscle mass, hemodialysis and mode of dialysis in a logistic regression. The mode of dialyzer modulates the blood levels of myostatin. Higher myostatin is associated with lower muscle function. The use of myostatin assay in various clinical settings merits further investigation.
Pompe disease patients had lower serum myostatin and IGF-1 levels than controls before ERT. However, myostatin, IGF-1, and follistatin levels increased 129%, 74%, and 62% after ERT respectively, and have fell into normal ranges. In contrast, these were not changed in the controls during follow-up. At the same time, the percentage of muscle fibers having intracytoplasmic vacuoles decreased 60%, and the quotient of PDMS declined significantly from 95.2 to 79.5. The increase in myostatin and IGF-1 levels may reflect muscle regeneration. They are potential therapeutic biomarkers for Pompe disease.
Discussion
The main contribution of this study included revealing novel signaling pathways modulating myostatin, and establishing myostatin as a biomarker for muscle growth/differentiation in different clinical scenarios. The p38MAPK and JNK in MAPK pathway, and PI3K/Akt/mTOR axis are involved in the activation of myostatin. Dissection of these pathways may help design new therapeutic modality for the treatment of muscle related diseases. We may treat muscle related diseases through myostatin inhibition, satellite cell activation, and muscle hypertrophy. The small molecules including SB203580, LY294002, PD98059, PIK-75, rapamycin, AKT inhibitor VIII, and shRNA plasmids will be employed to inhibit myostatin. On the other hand, HDACI (eg. TSA and valproic acid), anisomycin, and IGF-1 can activate myostatin. There are differentiated myotubes, undifferentiated myoblasts, and quiescent satellite cells at different stages in the whole body. We should perform experiment in animal or human model to establish the role of these molecules in the complex body. Besides, since muscle mass can predict morbidity and mortality in many diseases, we should perform large scale longitudinal studies to see the correlation between myostatin level and morbidity and mortality.
In the future, we will collect the blood samples and anthropometric parameters from elite sportsmen performing weight training, frail patients performing strength training, patients performing acute bout exercise and chronic training, and stroke patients performing rehabilitation. The correlation between serum myostatin and body composition will be sought. We hope to establish the serum myostatin as a new biomarker of systemic muscle atrophy and regeneration.
Subjects
myostatin
histone deacetylase inhibitor
hemodialysis
Pompe’s disease
enzyme-linked immnosorbent assay
Type
thesis
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