The Effect Of Cigarettes Smoke Extract On The Expression Of MSTN And MHC In Rat Skeletal Muscle Cells

BackgroundChronic Obstructive Pulmonary Disease (COPD) is common disease which is characterized by persistent limited airflow and accompanied by chronic inflammation reaction of airway and lungs to noxious particles or gas. Patients with acute exacerbation and complications affect overall severity of such disease. The World Bank/World Health Organization (WHO) data show that COPD will become the third cause of death and the fifth economic burden by 2020 around the world. In China, the prevalence of COPD patients over 40 years was 8.2%, the total number of disabled patients was 500-10 million and death was about 1 million per year. COPD has become an important public health problem because of its high prevalence, mortality and huge social burden。Patients with usually have obstructive ventilation dysfunction, they need overcome more resistance and consume more energy to breathe which contributes to excessive work, function decline, weakness of skeletal muscle. Clinically, patients with end-stage COPD suffered shortness of breath, weakness, and must be treated by long-term mechanical-assisted ventilation, and it is difficult to withdraw breathing machine for some patients after invasive mechanical ventilation.COPD is also a kind of systemic diseases which can cause multiple organs impairment. Physical limitation is a common clinical symptom of COPD and also an important factor of lower quality of life. COPD not only affects the lungs, but also causes multiple organs impairment, including skeletal muscle dysfunction (SMD) and other system dysfunction, such as cardiovascular system, nervous system, digestive system etc. The previous studies have shown that impaired lung function contributed to the physical limitation of patients with COPD. But a growing number of recent studies have found that about 40% of COPD patients who had physical limitation did not have severe impaired lung function, but had obvious SMD. COPD patients with SMD would get worse Health-related quality of life. The study had found that the consumption of skeletal muscle was an important predictor of mortality in patients with COPD. Formation mechanism of skeletal muscle dysfunction caused by COPD is very complex, has not yet been fully understood.Severe COPD patients usually have skeletal muscle atrophy, which is associated with decreased pulmonary function, activity, tolerance, health status, and increased mortality. A variety of molecular biology mechanisms may lead to the SMD that has not yet been fully understood.Cigarette smoking is a major risk factor for COPD. Smoking produces a large number of reactive oxygen species (ROS), which can cause oxidative stress. Respiratory system is not only the main target organ of oxidative stress, ROS can distribute into circulation. As the largest tissue, muscle is also a "victim" of oxidative stress. There was evidence to suggest that oxidative stress significantly increased in skeletal muscle for patients with COPD. Myostatin (MSTN) is mainly expressed in skeletal muscle, which is associated with abnormal skeletal muscle growth and function is to inhibit the growth and development of muscles and leads to muscular consumption and atrophy.Myostatin (MSTN) is a member of the bone morphogenetic protein (BMP) family and the TGF-beta superfamily., which inhibits the growth and regulation function of skeletal muscle. MSTN has 375 amino acids. MSTN expresses in skeletal muscle and can distribute into circulation. Existing research showed that as we age, in weight loss and elderly patients with symptoms of skeletal muscle consumption, MSTN in plasma increases due to aging and weight loss. MSTN regulate the skeletal muscle growth negatively mainly through the inhibition of cell cycle process including myoblast proliferation. Patients with COPD skeletal muscle existed muscular consumption, so we speculated that SMD might be associated with increased expression of MSTN in COPD patients. We used western blotting to detect the expression of MSTN protein; the results showed that protein levels of MSTN in the quadriceps significantly increased with CSE.In 1873, skeletal muscle fibers were divided into two types:red muscles fibers and white muscles fibers by Ranuier. White muscles contract faster than red muscles. With the development of technology, the skeletal muscle fiber was divided into Ⅰ, Ⅱ and Ⅱ B through myosin ATPase (ATP-ase) reaction in1970. Then researches further identified the forms of skeletal muscle fibers using the methods of myosin heavy chain (MHC) monoclonal antibody immunohistochemical, in situ hybridization experiment, a single muscle fiber MHC SDS-PAGE electrophoresis and gene cloning techniques. Human skeletal muscle fibers can be further divided into three types:slow contraction fatigue resistant aerobic type (type I), fast contraction fatigue glycolysis type (type Ⅱ A) and fast contraction fatigue resistance aerobic-glycolysis type (type Ⅱ B). Exercise can result in a transition between different adjacent myosin heavy chain subtypes.Cosker et. al found that Ⅰ→Ⅱ A→Ⅱ B transition of myosin heavy chain subtypes existed and accompanied by decline of oxidative metabolism ability in patients with COPD whose skeletal muscle fiber were type ⅡB. Type ⅡB shows decreased endurance, strength and speed. Compared with healthy control of the same age, the strength of skeletal muscle decreased in patients with COPD significantly, especially for the quadriceps muscle. However, anti-oxidation treatment could improve their skeletal muscle endurance. Such study further confirmed the link between oxidative stress and skeletal muscle dysfunction in COPD patients. Therefore, our studies chose newborn SD rat skeletal muscle cells which had strong vitality. We also found that, weight and body diameter increased in patients after quitting smoking. We speculate that in addition to the movement, cigarettes can also cause skeletal muscle type transition.In conclusion, smoking is an important etiology of COPD, oxidative caused by smoking stress can cause DNA oxidative damage and play an important role in the COPD patients with SMD. Myostatin (MSTN) is predominantly expressed in skeletal muscles and plays important roles in inhibiting muscle growth and development and causing muscle consumption. Our study found that the quadriceps cells of rat stimulated by CSE showed increased oxidative damage and MSTN level, suggested that oxidative damage caused by smoking may be associated with elevated MSTN expression, but the specific mechanism is unclear, on the other hand, Ⅰ→Ⅱ A→ⅡB transition was accompanied by declined oxidative metabolism ability. This study aims at further elucidating the pathogenesis of COPD skeletal muscle dysfunction and provide scientific basis for treatment.ObjectiveIn the cellular level, to explore the pathogenesis of MSTN involvement in skeletal muscle dysfunction in COPD, using molecular biologic techniques and physiopathology techniques. To further elucidate the skeletal muscle dysfunction in COPD pathogenesis, and to provide new ideas for its treatment. At the same time, confirmed that the toxic effects of cigarettes on skeletal muscle. Therefore, smoking cessation is effective measures to prevent COPD/emphysema, has important clinical and social value.Materials and Methods:1. Materials:3-5d SD rats were selected, executed by cervical dislocation, and quadriceps femoris muscles were detached and minced in 1mm3. Bank of skeletal muscle in vitro primary cell culture (tissue culture were performed, monolayer cell culture), cell morphology was observed under the microscope. After 3-5 generations of cells differentiation and mature, skeletal muscle cells were confirmed by HE staining and immunofluorescence. And skeletal muscle cells were randomly in four groups:normal control group,2.5% CSE group,5.0% CSE group,10.0% CSE group.2. Mehhods:When 10 generations of cells cultured cells covering 50% flasks, flasks were added containing 0%,2.5%,5%,10% CSE medium, cigarette smoke extract preparation for 30 minutes before use. After 48 hours culture; real-time quantitative PCR MSTN mRNA, the expression of MHC-1, MHC-ⅡA, and MHC-ⅡB mRNA expression, and Western blotting MSTN protein.Results:1. Success in vitro primary cultured SD rat skeletal muscle cells, under microscope, cells were round early, uniform shape, strong refraction, suspended in culture medium; 5-6 h after the start of adherent,24 h after fully adherent and started proliferating cells gradually extended into spindle-shaped, ordered in a certain direction. With increasing cell density, cell gradually regularly arranged in parallel; cultured to 2-3 d cell differentiation peaked, and then gradually began to apoptosis float. HE staining Showed oval nuclei, immunofluorescence examinations showed more than 90% of cells were positive, in line with the phenotypic characteristics of skeletal muscle cells.2. Real-time quantitative PCR results showed that MSTN mRNA relative expression were significant differences among four groups,(P<0.05). MSTN mRNA relative expression were increased significantly in CBE groups including 5.0% group, 10.0% group when compared with control group (P<0.05); The higher concentrations of CSE, and the more mRNA expression of MSTN.3. Real-time quantitative PCR results showed there were significant differences of MHC-Ⅰ mRNA relative expression among four groups.(P<0.05), MHC-Ⅰ mRNA relative expression decreased significantly in 5.0% CSE group or 10.0% CSE group when compared with control group (P<0.05). The higher concentrations of CSE, and the lower mRNA expression of MSTN.4. Real-time quantitative PCR results showed there were significant differences of MHC-1 Ⅱ A mRNA relative expression among four groups.(P<0.05), 2.5% CSE group MHC-Ⅱ A mRNA relative expression decreased significantly in 5.0% CSE group or 10.0% CSE group when compared with control group (P<0.05). The higher concentrations of CSE, and the lower mRNA expression of MHC-Ⅱ A.5. We failed to make the graphics in repeated experiments of real-time quantitative RT-PCR to evaluate MHC-ⅡB mRNA expression.6. Western blotting analysis showed that four MSTN protein group, the difference was statistically significant (P<0.05); of which,5.0% group,10.0% higher than the control group MSTN protein level and 2.5% group, the difference was statistically significant (P<0.05); CSE accompanying elevated concentration, the higher the protein target. With the increase of the concentration of CSE, MSTN protein expression is higher; the difference was statistically significant (P<0.05).Conclusion:1. Cigarette smoking is a risk factor for COPD, CSE can induce skeletal muscle cells damage and elevated mRNA and protein levels of MSTN, which leads to consumption and atrophy of skeletal muscle.The higher CSE concentration, the more sever muscle damage.2. Cigarette smoke extract exposure can lead to MHC-Ⅰ, MHC-ⅡA skeletal muscle fibers declined.