A novel endoplasmic reticulum stress‑induced apoptosis model using tunicamycin in primary cultured neonatal rat cardiomyocytes

Shen,Mingzhi; Wang,Lin; Guo,Xiaowang; Xue,Qiao; Huo,Cong; Li,Xing; Fan,Li; Wang,Xiaoming

doi:10.3892/mmr.2015.4040

A novel endoplasmic reticulum stress‑induced apoptosis model using tunicamycin in primary cultured neonatal rat cardiomyocytes

Authors:
- Mingzhi Shen
- Lin Wang
- Xiaowang Guo
- Qiao Xue
- Cong Huo
- Xing Li
- Li Fan
- Xiaoming Wang
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Affiliations: Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China, Department of Cardiology, Hainan Branch of PLA General Hospital, Sanya, Hainan 572013, P.R. China, Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, P.R. China
Published online on: July 3, 2015 https://doi.org/10.3892/mmr.2015.4040
Pages: 5149-5154

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Abstract

Endoplasmic reticulum (ER) stress is key in the development of cardiovascular diseases. However, there is a lack of a systemic ER stress‑induced cardiomyocyte apoptosis model. In the present study, primary cultured neonatal rat cardiomyocytes were exposed to tunicamycin. Cell viability was determined by an MTT assay, and cell damage was detected by a lactose dehydrogenase assay. Flow cytometry was used and the activity of caspase‑3 was analyzed in order to measure apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to examine the expression of glucose‑regulated protein 78‑kDa (GRP78) and C/EBP homologous protein (CHOP). As a result, tunicamycin significantly increased cardiomyocyte injury, which occurred in a time- and concentration‑dependent manner. In addition, tunicamycin treatment resulted in apoptosis of cardiomyocytes. Molecularly, tunicamycin (100 ng/ml) increased the levels of GRP78 and CHOP 6 h after administration. In addition, GRP78 and CHOP reached maximum mRNA and protein levels 24 h after administration. In conclusion, the results implicate that the tunicamycin‑induced ER stress‑induced apoptotic model was successfully constructed in cultured neonatal rat cardiomyocytes. A 100 ng/ml concentration of tunicamycin was selected, and MTT, LDH release and flow cytometry assay was at 72 h. In addition, GRP78 and GRP94 were detected 24 h following administration. The results of the present study indicate a novel experimental basis for the investigation of ERS-induced cardiac apoptosis.

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1	Millott R, Dudek E and Michalak M: The endoplasmic reticulum in cardiovascular health and disease. Can J Physiol Pharmacol. 90:1209–1217. 2012. View Article : Google Scholar : PubMed/NCBI
2	Peralta C and Brenner C: Endoplasmic reticulum stress inhibition enhances liver tolerance to ischemia/reperfusion. Curr Med Chem. 18:2016–2024. 2011. View Article : Google Scholar : PubMed/NCBI
3	Ding W and Zhang X, Huang H, Ding N, Zhang S, Hutchinson SZ and Zhang X: Adiponectin protects rat myocardium against chronic intermittent hypoxia-induced injury via inhibition of endoplasmic reticulum stress. PLoS One. 9:e945452014. View Article : Google Scholar : PubMed/NCBI
4	Gomes-Alves P, Couto F, Pesquita C, Coelho AV and Penque D: Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response. Biochim Biophys Acta. 1804:856–865. 2010. View Article : Google Scholar : PubMed/NCBI
5	Farrukh MR, Nissar UA, Afnan Q, et al: Oxidative stress mediated Ca (2+) release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells. J Dermatol Sci. 75:24–35. 2014. View Article : Google Scholar : PubMed/NCBI
6	Zhang K and Kaufman RJ: From endoplasmic-reticulum stress to the inflammatory response. Nature. 454:455–462. 2008. View Article : Google Scholar : PubMed/NCBI
7	Fu HY, Okada K, Liao Y, et al: Ablation of C/EBP homologous protein attenuates endoplasmic reticulum-mediated apoptosis and cardiac dysfunction induced by pressure overload. Circulation. 122:361–369. 2010. View Article : Google Scholar : PubMed/NCBI
8	Yang L, Zhao D, Ren J and Yang J: Endoplasmic reticulum stress and protein quality control in diabetic cardiomyopathy. Biochim Biophys Acta. 1852:209–218. 2014. View Article : Google Scholar : PubMed/NCBI
9	Groenendyk J, Sreenivasaiah PK, Kim do H, Agellon LB and Michalak M: Biology of endoplasmic reticulum stress in the heart. Circ Res. 107:1185–1197. 2010. View Article : Google Scholar : PubMed/NCBI
10	Avery J, Etzion S, DeBosch BJ, et al: TRB3 function in cardiac endoplasmic reticulum stress. Circ Res. 106:1516–1523. 2010. View Article : Google Scholar : PubMed/NCBI
11	Liu D, Zhang M and Yin H: Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis. Int J Neurosci. 123:155–162. 2013. View Article : Google Scholar
12	Thuerauf DJ, Marcinko M, Gude N, Rubio M, Sussman MA and Glembotski CC: Activation of the unfolded protein response in infarcted mouse heart and hypoxic cultured cardiac myocytes. Circ Res. 99:275–282. 2006. View Article : Google Scholar : PubMed/NCBI
13	Endo H, Murata K, Mukai M, Ishikawa O and Inoue M: Activation of insulin-like growth factor signaling induces apoptotic cell death under prolonged hypoxia by enhancing endoplasmic reticulum stress response. Cancer Res. 67:8095–8103. 2007. View Article : Google Scholar : PubMed/NCBI
14	Erbay E, Babaev VR, Mayers JR, et al: Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis. Nat Med. 15:1383–1391. 2009. View Article : Google Scholar : PubMed/NCBI
15	Hotamisligil GS: Endoplasmic reticulum stress and atherosclerosis. Nat Med. 16:396–399. 2010. View Article : Google Scholar : PubMed/NCBI
16	Myoishi M, Hao H, Minamino T, et al: Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome. Circulation. 116:1226–1233. 2007. View Article : Google Scholar : PubMed/NCBI
17	Glembotski CC: Endoplasmic reticulum stress in the heart. Circ Res. 101:975–984. 2007. View Article : Google Scholar : PubMed/NCBI
18	Kim I, Xu W and Reed JC: Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 7:1013–1030. 2008. View Article : Google Scholar : PubMed/NCBI
19	Dickhout JG, Carlisle RE and Austin RC: Interrelationship between cardiac hypertrophy, heart failure and chronic kidney disease: endoplasmic reticulum stress as a mediator of pathogenesis. Circ Res. 108:629–642. 2011. View Article : Google Scholar : PubMed/NCBI
20	van Empel VP, Bertrand AT, Hofstra L, Crijns HJ, Doevendans PA and De Windt LJ: Myocyte apoptosis in heart failure. Cardiovasc Res. 67:21–29. 2005. View Article : Google Scholar : PubMed/NCBI
21	Shen M, Wang L, Yang G, et al: Baicalin protects the cardiomyocytes from ER stress-induced apoptosis: inhibition of CHOP through induction of endothelial nitric oxide synthase. PLoS One. 9:e883892014. View Article : Google Scholar : PubMed/NCBI
22	Liu B, Niu L, Shen MZ, et al: Decreased astroglial monocarboxylate transporter 4 expression in temporal lobe epilepsy. Mol Neurobiol. 50:327–338. 2014. View Article : Google Scholar : PubMed/NCBI
23	Fu HY, Minamino T, Tsukamoto O, et al: Overexpression of endoplasmic reticulum-resident chaperone attenuates cardiomyocyte death induced by proteasome inhibition. Cardiovasc Res. 79:600–610. 2008. View Article : Google Scholar : PubMed/NCBI
24	Liu B, Wang L, Shen LL, et al: RNAi-mediated inhibition of presenilin 2 inhibits glioma cell growth and invasion and is involved in the regulation of Nrg1/ErbB signaling. Neuro Oncol. 14:994–1006. 2012. View Article : Google Scholar : PubMed/NCBI
25	Zhou S, Yin X, Zheng Y, et al: Metallothionein prevents intermittent hypoxia-induced cardiac endoplasmic reticulum stress and cell death likely via activation of Akt signaling pathway in mice. Toxicol Lett. 227:113–123. 2014. View Article : Google Scholar : PubMed/NCBI
26	Yang Y, Li C, Xiang X, et al: Ursolic acid prevents endoplasmic reticulum stress-mediated apoptosis induced by heat stress in mouse cardiac myocytes. J Mol Cell Cardiol. 67:103–111. 2014. View Article : Google Scholar : PubMed/NCBI
27	Qi X, Vallentin A, Churchill E and Mochly-Rosen D: DeltaPKC participates in the endoplasmic reticulum stress-induced response in cultured cardiac myocytes and ischemic heart. J Mol Cell Cardiol. 43:420–428. 2007. View Article : Google Scholar : PubMed/NCBI
28	Okada K, Minamino T, Tsukamoto Y, et al: Prolonged endoplasmic reticulum stress in hypertrophic and failing heart after aortic constriction: possible contribution of endoplasmic reticulum stress to cardiac myocyte apoptosis. Circulation. 110:705–712. 2004. View Article : Google Scholar : PubMed/NCBI
29	Lin JH, Li H, Yasumura D, et al: IRE1 signaling affects cell fate during the unfolded protein response. Science. 318:944–949. 2007. View Article : Google Scholar : PubMed/NCBI