Abstract
This investigation was under taken to explore probable mechanisms and signal pathways involved in cytotoxicity induced by bacterial endotoxin lipopolysaccharide (LPS). Herein, we selected muscle precursor C2C12 myoblasts as representative cells to test effect of calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) on LPS induced inflammation and apoptosis. In order to rule out the toxicity of endotoxin, mouse myoblasts were exposed to various concentrations of LPS and viability of cells and morphology were assessed using CCK-8 assay and simple microscopy respectively. Apoptotic cell death was examined by fluorescence microscope at regular time intervals. Additionally, LPS induced apoptosis in C2C12 cells were determined by mRNA expression of µ-calpain, caspase-3 and tumor necrosis factor alpha (TNF-α) and were quantified by qRT-PCR. Our results point out that LPS stimulation produced dose dependent toxicity in muscle precursor cells. Pre-treatment with a calpain inhibitor can significantly attenuate LPS-induced inflammation/apoptosis. Results of present research determined that mRNA expression of aforesaid genes was amplified (p < 0.05) in LPS stimulated C2C12 cells, whereas a noticeable drop off in mRNA expression of these genes was observed when pre-exposed with calpain inhibitor PD150606. Our study has outlined the current understanding regarding the connection between µ-calpain and caspase-3 in skeletal muscle wasting and as a result provides suitable choice for designing promising chemotherapeutic system for muscle illness and atrophy.
Similar content being viewed by others
References
Li H, Malhotra S, Kumar A (2008) Nuclear factor-kappa B signaling in skeletal muscle atrophy. J Mol Med 86(10):1113–1126. doi:10.1007/s00109-008-0373-8
Ventadour S, Attaix D (2006) Mechanisms of skeletal muscle atrophy. Curr Opin Rheumatol 18(6):631–635. doi:10.1097/01.bor.0000245731.25383.de
Eley HL, Tisdale MJ (2007) Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation. J Biol Chem 282(10):7087–7097. doi:10.1074/jbc.M610378200
Callahan LA, Supinski GS (2009) Sepsis-induced myopathy. Crit Care Med 37(10):S354–S367. doi:10.1097/CCM.0b013e3181b6e439
Nozaki K, Das A, Ray SK, Banik NL (2010) Calpain inhibition attenuates intracellular changes in muscle cells in response to extracellular inflammatory stimulation. Exp Neurol 225(2):430–435. doi:10.1016/j.expneurol.2010.07.021
Sharifi AM, Hoda FE, Noor AM (2010) Studying the effect of LPS on cytotoxicity and apoptosis in PC12 neuronal cells: role of Bax, Bcl-2, and Caspase-3 protein expression. Toxicol Mech Methods 20(6):316–320. doi:10.3109/15376516.2010.486420
Frost RA, Nystrom GJ, Lang CH (2002) Lipopolysaccharide regulates proinflammatory cytokine expression in mouse myoblasts and skeletal muscle. Am J Physiol Regul Integr Comp Physiol 283(3):R698–R709. doi:10.1152/ajpregu.00039.2002
Frost RA, Nystrom GJ, Lang CH (2004) Lipopolysaccharide stimulates nitric oxide synthase-2 expression in murine skeletal muscle and C2C12 myoblasts via Toll-like receptor-4 and c-Jun NH2-terminal kinase pathways. Am J Physiol Cell Physiol 287(6):C1605–C1615. doi:10.1152/ajpcell.00010.2004
Jortay J, Senou M, Delaigle A, Noel L, Funahashi T, Maeda N, Many MC, Brichard SM (2010) Local induction of adiponectin reduces lipopolysaccharide-triggered skeletal muscle damage. Endocrinology 151(10):4840–4851. doi:10.1210/en.2009-1462
Smith IJ, Lecker SH, Hasselgren P-O (2008) Calpain activity and muscle wasting in sepsis. Am J Physiol-Endoc M 295(4):E762–E771. doi:10.1152/ajpendo.90226.2008
Tissier S, Lancel S, Marechal X, Mordon S, Depontieu F, Scherpereel A, Chopin C, Neviere R (2004) Calpain inhibitors improve myocardial dysfunction and inflammation induced by endotoxin in rats. Shock 21(4):352–357. doi:10.1097/00024382-200404000-00010
Pollack JR, Witt RC and Sugimoto JT (2003) Differential effects of calpain inhibitors on hypertrophy of cardiomyocytes. Biochem Hypertrophy Heart Fail 47–50. doi: 10.1023/A:1025413428259
Chatterjee PK, Todorovic Z, Sivarajah A, Mota-Filipe H, Brown PA, Stewart KN, Mazzon E, Cuzzocrea S, Thiemermann C (2005) Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury. Biochem Pharmacol 69(7):1121–1131. doi:10.1016/j.bcp.2005.01.003
Del Bello B, Moretti D, Gamberucci A, Maellaro E (2007) Cross-talk between calpain and caspase-3/-7 in cisplatin-induced apoptosis of melanoma cells: a major role of calpain inhibition in cell death protection and p53 status. Oncogene 26(19):2717–2726. doi:10.1038/sj.onc.1210079
Ali MA, Stepanko A, Fan X, Holt A, Schulz R (2012) Calpain inhibitors exhibit matrix metalloproteinase-2 inhibitory activity. Biochem Biophys Res Commun 423(1):1–5. doi:10.1016/j.bbrc.2012.05.005
Li X, Luo R, Jiang R, Meng X, Wu X, Zhang S, Hua W (2013) The role of the Hsp90/Akt pathway in myocardial calpain-induced caspase-3 activation and apoptosis during sepsis. BMC Cardiovasc Disord 13:8. doi:10.1186/1471-2261-13-8
Li X, Luo R, Chen R, Song L, Zhang S, Hua W, Chen H (2014) Cleavage of IkappaBalpha by calpain induces myocardial NF-kappaB activation, TNF-alpha expression, and cardiac dysfunction in septic mice. Am J Physiol Heart Circ Physiol 306(6):H833–H843. doi:10.1152/ajpheart.00893.2012
Li X, Li Y, Shan L, Shen E, Chen R, Peng T (2009) Over-expression of calpastatin inhibits calpain activation and attenuates myocardial dysfunction during endotoxaemia. Cardiovasc Res 83(1):72–79. doi:10.1093/cvr/cvp100
Xiaoping L, Rong L, Ruizhen C, Lang L (2011) Calpain induces TNF-α expression and cardiac dysfunction by IκB/NF-κB system in septic mice. Heart 97(Suppl 3):A132. doi:10.1136/heartjnl-2011-300867.383
Amna T, Hassan MS, Sheikh FA, Lee HK, Seo KS, Yoon D, Hwang IH (2013) Zinc oxide-doped poly (urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering. Appl Microbiol Biotechnol 97(4):1725–1734. doi:10.1007/s00253-012-4353-0
Russell ST, Tisdale MJ (2009) Mechanism of attenuation by beta-hydroxy-beta-methylbutyrate of muscle protein degradation induced by lipopolysaccharide. Mol Cell Biochem 330(1–2):171–179. doi:10.1007/s11010-009-0130-5
Amna T, Hassan MS, Shin WS, Van Ba H, Lee HK, Khil MS, Hwang IH (2013) TiO2 nanorods via one-step electrospinning technique: a novel nanomatrix for mouse myoblasts adhesion and propagation. Colloid Surf B 101(1):424–429. doi:10.1016/j.colsurfb.2012.06.012
Amna T, Van Ba H, Vaseem M, Hassan MS, Khil MS, Hahn YB, Lee HK, Hwang IH (2013) Apoptosis induced by copper oxide quantum dots in cultured C2C12 cells via caspase 3 and caspase 7: a study on cytotoxicity assessment. Appl Microbiol Biotechnol 97(12):5545–5553. doi:10.1007/s00253-013-4724-1
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(900):2002–2007. doi:10.1093/nar/29.9.e45
Eley Helen L, Russell Steven T, Tisdale MJ (2008) Mechanism of attenuation of muscle protein degradation induced by tumor necrosis factor-α and angiotensin II by β-hydroxy-β-methylbutyrate. Am J Physiol Endocrinol Metab 295(6):E1417–E1426. doi:10.1152/ajpendo.90567.2008
Huang J, Forsberg NE (1998) Role of calpain in skeletal-muscle protein degradation. Proc Natl Acad Sci 95:12100–12105. doi:10.1073/pnas.95.21.12100
Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270(5639):725–727. doi:10.1038/270725a0
Lee JW, Bae CJ, Choi YJ, Kim SI, Kwon YS, Lee HJ, Kim SS, Chun W (2014) 3,4,5-Trihydroxycinnamic acid inhibits lipopolysaccharide (LPS)-induced inflammation by Nrf2 activation in vitro and improves survival of mice in LPS-induced endotoxemia model in vivo. Mol Cell Biochem 390(1–2):143–153. doi:10.1007/s11010-014-1965-y
Supinski GS, Callahan LA (2007) Free radical-mediated skeletal muscle dysfunction in inflammatory conditions. J Appl Physiol 102(5):2056–2063. doi:10.1152/japplphysiol.01138.2006
Van Ba H, Inho H (2013) Significant role of mu-calpain (CANP1) in proliferation/survival of bovine skeletal muscle satellite cells. Vitro Cell Dev Biol Anim 49(10):785–797. doi:10.1007/s11626-013-9666-5
Supinski GS, Wang W, Callahan LA (2009) Caspase and calpain activation both contribute to sepsis-induced diaphragmatic weakness. J Appl Physiol 107(5):1389–1396. doi:10.1152/japplphysiol.00341.2009
Nozaki K, Das A, Ray SK, Banik NL (2011) Calpeptin attenuated apoptosis and intracellular inflammatory changes in muscle cells. J Neurosci Res 89(4):536–543. doi:10.1002/jnr.22585
Talbert EE, Smuder AJ, Min K, Kwon O-S, Powers SK (2012) Inhibition of calpain or caspase-3 protects against immobilization-induced muscle atrophy. FASEB J 26(10):75–77
Talbert EE, Smuder AJ, Min K, Kwon OS, Powers SK (2013) Calpain and caspase-3 play required roles in immobilization-induced limb muscle atrophy. J Appl Physiol 114(10):1482–1489. doi:10.1152/japplphysiol.00925.2012
Nelson WB, Smuder AJ, Hudson MB, Talbert EE, Powers SK (2012) Cross-talk between the calpain and caspase-3 proteolytic systems in the diaphragm during prolonged mechanical ventilation. Crit Care Med 40(6):1857–1863. doi:10.1097/CCM.0b013e318246bb5d
Mohanty TR, Park KM, Pramod AB, Kim JH, Choe HS, Hwang IH (2010) Molecular and biological factors affecting skeletal muscle cells after slaughtering and their impact on meat quality: a mini-review. J Muscle Foods 21(1):51–78. doi:10.1111/j.1745-4573.2009.00167.x
Acknowledgments
This work was supported by a grant from the Rural Development Administration, Republic of Korea (No. PJ010170) and the Next Generation Biogreen 21(No. PJ011101). This research was partly supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Project no. 2014R1A1A2007175). Dr Touseef Amna acknowledges the research Grant from NRF.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Ke Shang, Junfeng Zhang and Touseef Amna have contributed equally to this work.
Rights and permissions
About this article
Cite this article
Shang, K., Zhang, J., Amna, T. et al. Attenuation of cellular toxicity by calpain inhibitor induced by bacterial endotoxin: a mechanistic study using muscle precursor cells as a model system. Mol Biol Rep 42, 1281–1288 (2015). https://doi.org/10.1007/s11033-015-3869-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-015-3869-7