EFFECTS OF MECHANICAL STIMULATION ON EXPRESSION OF AUTOANTIGENS IN MYOBLASTS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Rong , LIU Xinghui , HUANG Weiyi , ZENG Huijun , SHI Dandan , CAO Biao , LIAO Hua.

Department of Anatomy, Southern Medical University, the Key Laboratory of Construction and Detection of Guangdong Province, Guangzhou Guangdong, 510515, P.R.China. Corresponding author: LIAO Hua, E-mail: hua-liao@163.com;

Corresponding author：

Keywords：

Mechanical stimulation; Myoblasts; Autoantigen Idiopathic inflammatory myopathies

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Objective To explore the effects of mechanical stimulation on the expression of autoantigens in myoblasts. Methods According to different processing methods, C2C12 cells were divided into the experimental group and control group; the experimental group was divided into 4 subgroups: 2-, 4-, and 6-day and 1-day stretch groups. In 2-, 4-, and 6-day stretch groups, mechanical loading was added on the C2C12 cells at a stretching frequency of 0.25 Hz and cellular deformation amplitude of 10%, 2 hours a day for 2, 4, and 6 days respectively by Flexercell 5000 strain unit, and at a stretching frequency of 1 Hz and cellular deformation amplitude of 15% for 1 hour in 1-day stretch group. In the control group, the cells were routinely cultured for 1, 2, 4, and 6 days (1-, 2-, 4-, and 6-day control). The cells were observed by inverted phase contrast microscope. The cell proliferation was detected by flow cytometry; the expressions of autoantigens were detected by Western blot method, including the Ku/the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), U1-70 (A part of ATP-dependent DNA helicase II), histidyl tRNA synthetase (HRS), and Mi-2 (reconfigurable components deacetylase complexes of NuRD). Results The exfoliated cells were found in 1-day stretch group, but no exfoliated cell was seen in the control group for 1-day culture. The cells proliferated more obviously in 2-day stretch group than in the control group for 2-day culture; cell differentiation was found in 4-day stretch group, and cell fusion in 6-day stretch group, which were similar to those in the control group for 4- and 6-day culture. After single stretching, cell apoptosis was found in 1-day stretch group, showing no significant difference in the relative DNA proliferation index (DPI) when compared with DPI of control group for 1-day culture (t=0.346, P=0.747). After cyclic stretching, DPIs of 2- and 4- day stretch groups were significantly increased when compared with those of the control group for 2- and 4-day culture (P lt; 0.05), but no significant difference was found between control group for 6-day culture and 6-day stretch group (t=1.191, P=0.303). Compared with the control group for 2-day culture, the relative protein expression of autoantigens (DNA-Pkcs, Mi-2, HRS, and U1-70) in 2-day stretch group decreased significantly (P lt; 0.05), but no significant difference was found between control group for 4-day culture and 4-day stretch group (P gt; 0.05). The relative protein expressions of autoantigens in 4-day stretch group significantly increased when compared with those of 2-day stretch group (P lt; 0.05), but the relative protein expressions of autoantigens in the control group for 4-day culture significantly decreased when compared with those of the control group for 2-day culture (P lt; 0.05). Conclusion Short-term mechanical stimulation can inhibit the expressions of autoantigens in myoblasts, but with the time prolonging, cell differentiation and fusion and adaptation to mechanical stimulation would result in diminished inhibitory effect.

Citation： CHEN Rong,LIU Xinghui,HUANG Weiyi,ZENG Huijun,SHI Dandan,CAO Biao,LIAO Hua.. EFFECTS OF MECHANICAL STIMULATION ON EXPRESSION OF AUTOANTIGENS IN MYOBLASTS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(9): 1128-1133. doi: Copy

1.	Mammen AL. Dermatomyositis and polymyositis: clinical presentation, autoantibodies, and pathogenesis. Ann N Y Acad Sci, 2010, 1184: 134-153.
2.	Dimachkie MM. Idiopathic inflammatory myopathies. J Neuroimmunol, 2011, 231(1-2): 32-42.
3.	Wiesinger GF, Quittan M, Graninger M, et al. Benefit of 6 months long-term physical training in polymyositis/dermatomyositis patients. Br J Rheumatol, 1998, 37(12): 1338-1342.
4.	Spector SA, Lemmer JT, Koffman BM, et al. Safety and efficacy of strength training in patients with sporadic inclusion body myositis. Muscle Nerve, 1997, 20(10): 1242-1248.
5.	Habers GE, Takken T. Safety and efficacy of exercise training in patients with an idiopathic inflammatory myopathy—a systematic review. Rheumatology (Oxford), 2011, 50(11): 2113-2124.
6.	Alexanderson H, Lundberg IE. Exercise as a therapeutic modality in patients with idiopathic inflammatory myopathies. Curr Opin Rheumatol, 2012, 24(2): 201-207.
7.	Tatsumi R. Mechano-biology of skeletal muscle hypertrophy and regeneration: possible mechanism of stretch-induced activation of resident myogenic stem cells. Anim Sci J, 2010, 81(1): 11-20.
8.	Tatsumi R, Wuollet AL, Tabata K, et al. A role for calcium-calmodulin in regulating nitric oxide production during skeletal muscle satellite cell activation. Am J Physiol Cell Physiol, 2009, 296(4): C922-C929.
9.	Tatsumi R, Allen RE. Active hepatocyte growth factor is present in skeletal muscle extracellular matrix. Muscle Nerve, 2004, 30(5): 654-658.
10.	Hasselgren PO, Alamdari N, Aversa Z, et al. Corticosteroids and muscle wasting: role of transcription factors, nuclear cofactors, and hyperacetylation. Curr Opin Clin Nutr Metab Care, 2010, 13(4): 423-428.
11.	Abe S, Rhee S, Iwanuma O, et al. Effect of mechanical stretching on expressions of muscle specific transcription factors MyoD, Myf-5, myogenin and MRF4 in proliferated myoblasts. Anat Histol Embryol, 2009, 38(4): 305-310.
12.	邱小忠, 李小娜, 陈维毅, 等. 周期性机械拉伸对C2C12成肌细胞增殖的影响. 中国临床解剖学杂志, 2006, 24(2): 183-185.
13.	Tan J, Kuang W, Jin Z, et al. Inhibition of NFkappaB by activated c-Jun NH2 terminal kinase 1 acts as a switch for C2C12 cell death under excessive stretch. Apoptosis, 2009, 14(6): 764-770.
14.	Soltow QA, Lira VA, Betters JL, et al. Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts. J Muscle Res Cell Motil, 2010, 31(3): 215-225.
15.	Kook SH, Lee HJ, Chung WT, et al. Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK. Mol Cells, 2008, 25(4): 479-486.
16.	Casciola-Rosen L, Nagaraju K, Plotz P, et al. Enhanced autoantigen expression in regenerating muscle cells in idiopathic inflammatory myopathy. J Exp Med, 2005, 201(4): 591-601.
17.	Mammen AL, Casciola-Rosen LA, Hall JC, et al. Expression of the dermatomyositis autoantigen Mi-2 in regenerating muscle. Arthritis Rheum, 2009, 60(12): 3784-3793.
18.	Chakkalakal JV, Michel SA, Chin ER, et al. Targeted inhibition of Ca2+/calmodulin signaling exacerbates the dystrophic phenotype in mdx mouse muscle. Hum Mol Genet, 2006, 15(9): 1423-1435.
19.	Wehling M, Spencer MJ, Tidball JG. A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol, 2001, 155(1): 123-131.
20.	Mizunoya W, Upadhaya R, Burczynski FJ, et al. Nitric oxide donors improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm. Am J Physiol Cell Physiol, 2011, 300(5): C1065-C1077.
21.	Sugiura T, Kawaguchi Y, Soejima M, et al. Increased HGF and c-Met in muscle tissues of polymyositis and dermatomyositis patients: beneficial roles of HGF in muscle regeneration. Clin Immunol, 2010, 136(3): 387-399.

1. Mammen AL. Dermatomyositis and polymyositis: clinical presentation, autoantibodies, and pathogenesis. Ann N Y Acad Sci, 2010, 1184: 134-153.
2. Dimachkie MM. Idiopathic inflammatory myopathies. J Neuroimmunol, 2011, 231(1-2): 32-42.
3. Wiesinger GF, Quittan M, Graninger M, et al. Benefit of 6 months long-term physical training in polymyositis/dermatomyositis patients. Br J Rheumatol, 1998, 37(12): 1338-1342.
4. Spector SA, Lemmer JT, Koffman BM, et al. Safety and efficacy of strength training in patients with sporadic inclusion body myositis. Muscle Nerve, 1997, 20(10): 1242-1248.
5. Habers GE, Takken T. Safety and efficacy of exercise training in patients with an idiopathic inflammatory myopathy—a systematic review. Rheumatology (Oxford), 2011, 50(11): 2113-2124.
6. Alexanderson H, Lundberg IE. Exercise as a therapeutic modality in patients with idiopathic inflammatory myopathies. Curr Opin Rheumatol, 2012, 24(2): 201-207.
7. Tatsumi R. Mechano-biology of skeletal muscle hypertrophy and regeneration: possible mechanism of stretch-induced activation of resident myogenic stem cells. Anim Sci J, 2010, 81(1): 11-20.
8. Tatsumi R, Wuollet AL, Tabata K, et al. A role for calcium-calmodulin in regulating nitric oxide production during skeletal muscle satellite cell activation. Am J Physiol Cell Physiol, 2009, 296(4): C922-C929.
9. Tatsumi R, Allen RE. Active hepatocyte growth factor is present in skeletal muscle extracellular matrix. Muscle Nerve, 2004, 30(5): 654-658.
10. Hasselgren PO, Alamdari N, Aversa Z, et al. Corticosteroids and muscle wasting: role of transcription factors, nuclear cofactors, and hyperacetylation. Curr Opin Clin Nutr Metab Care, 2010, 13(4): 423-428.
11. Abe S, Rhee S, Iwanuma O, et al. Effect of mechanical stretching on expressions of muscle specific transcription factors MyoD, Myf-5, myogenin and MRF4 in proliferated myoblasts. Anat Histol Embryol, 2009, 38(4): 305-310.
12. 邱小忠, 李小娜, 陈维毅, 等. 周期性机械拉伸对C2C12成肌细胞增殖的影响. 中国临床解剖学杂志, 2006, 24(2): 183-185.
13. Tan J, Kuang W, Jin Z, et al. Inhibition of NFkappaB by activated c-Jun NH2 terminal kinase 1 acts as a switch for C2C12 cell death under excessive stretch. Apoptosis, 2009, 14(6): 764-770.
14. Soltow QA, Lira VA, Betters JL, et al. Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts. J Muscle Res Cell Motil, 2010, 31(3): 215-225.
15. Kook SH, Lee HJ, Chung WT, et al. Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK. Mol Cells, 2008, 25(4): 479-486.
16. Casciola-Rosen L, Nagaraju K, Plotz P, et al. Enhanced autoantigen expression in regenerating muscle cells in idiopathic inflammatory myopathy. J Exp Med, 2005, 201(4): 591-601.
17. Mammen AL, Casciola-Rosen LA, Hall JC, et al. Expression of the dermatomyositis autoantigen Mi-2 in regenerating muscle. Arthritis Rheum, 2009, 60(12): 3784-3793.
18. Chakkalakal JV, Michel SA, Chin ER, et al. Targeted inhibition of Ca2+/calmodulin signaling exacerbates the dystrophic phenotype in mdx mouse muscle. Hum Mol Genet, 2006, 15(9): 1423-1435.
19. Wehling M, Spencer MJ, Tidball JG. A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol, 2001, 155(1): 123-131.
20. Mizunoya W, Upadhaya R, Burczynski FJ, et al. Nitric oxide donors improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm. Am J Physiol Cell Physiol, 2011, 300(5): C1065-C1077.
21. Sugiura T, Kawaguchi Y, Soejima M, et al. Increased HGF and c-Met in muscle tissues of polymyositis and dermatomyositis patients: beneficial roles of HGF in muscle regeneration. Clin Immunol, 2010, 136(3): 387-399.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF MECHANICAL STIMULATION ON EXPRESSION OF AUTOANTIGENS IN MYOBLASTS

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