The potential role of calnexin in the activation of cardiac fibroblasts_Journal of Biomedical Engineering

Authors：

TIAN Geer ¹ , ZHAO Mingyue ¹ , ZHOU Junteng ² , QUAN Yue ¹ , WU Wenchao ¹ ,  LIU Xiaojing ^1,2

1. Laboratory of Cardiovascular Diseases, Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;
2. Department of Cardiology, West China Hospital, Sichuan University, Chengdu 610041, P.R.China;

Corresponding author：

LIU Xiaojing, Email: liuxq@scu.edu.cn

Keywords：

calnexin; transverse aortic constriction; cardiac fibrosis; cardiac fibroblast activation; endoplasmic reticulum stress

DOI：

10.7507/1001-5515.202001052

Video：

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Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca²⁺ homeostasis, and Ca²⁺ signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts in vivo, and the in vitro cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

Citation： TIAN Geer, ZHAO Mingyue, ZHOU Junteng, QUAN Yue, WU Wenchao, LIU Xiaojing. The potential role of calnexin in the activation of cardiac fibroblasts. Journal of Biomedical Engineering, 2020, 37(3): 450-459. doi: 10.7507/1001-5515.202001052 Copy

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2.	Ranjan P, Kumari R, Verma S K. Cardiac fibroblasts and cardiac fibrosis: Precise role of exosomes. Front Cell Dev Biol, 2019, 7: 318.
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4.	Horckmans M, Bianchini M, Santovito D, et al. Pericardial adipose tissue regulates granulopoiesis, fibrosis, and cardiac function after myocardial infarction. Circulation, 2018, 137(9): 948-960.
5.	Farris S D, Don C, Helterline D, et al. Cell-specific pathways supporting persistent fibrosis in heart failure. J Am Coll Cardiol, 2017, 70(3): 344-354.
6.	Nomura S, Satoh M, Fujita T, et al. Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure. Nat Commun, 2018, 9(1): 4435.
7.	Chen S, Zhang Y, Lighthouse J K, et al. A novel role of cyclic nucleotide phosphodiesterase 10A in pathological cardiac remodeling and dysfunction. Circulation, 2020, 141(3): 217-233.
8.	Xia P, Wang S, Xiong Z, et al. The ER membrane adaptor ERAdP senses the bacterial second messenger c-di-AMP and initiates anti-bacterial immunity. Nat Immunol, 2018, 19(2): 141-150.
9.	Song S, Tan J, Miao Y, et al. Crosstalk of ER stress-mediated autophagy and ER-phagy: Involvement of UPR and the core autophagy machinery. J Cell Physiol, 2018, 233(5): 3867-3874.
10.	Park S, Lim W, Bazer F W, et al. Apigenin induces ROS-dependent apoptosis and ER stress in human endometriosis cells. J Cell Physiol, 2018, 233(4): 3055-3065.
11.	Shih Y C, Chen C L, Zhang Y, et al. Endoplasmic reticulum protein TXNDC5 augments myocardial fibrosis by facilitating extracellular matrix protein folding and redox-sensitive cardiac fibroblast activation. Circ Res, 2018, 122(8): 1052-1068.
12.	Yao Y, Lu Q, Hu Z, et al. A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure. Nat Commun, 2017, 8(1): 133.
13.	Pinkaew D, Chattopadhyay A, King M D, et al. Fortilin binds IRE1alpha and prevents ER stress from signaling apoptotic cell death. Nat Commun, 2017, 8(1): 18.
14.	Misaka T, Murakawa T, Nishida K, et al. FKBP8 protects the heart from hemodynamic stress by preventing the accumulation of misfolded proteins and endoplasmic reticulum-associated apoptosis in mice. J Mol Cell Cardiol, 2018, 114: 93-104.
15.	Hou X, Fu M, Cheng B, et al. Galanthamine improves myocardial ischemia-reperfusion-induced cardiac dysfunction, endoplasmic reticulum stress-related apoptosis, and myocardial fibrosis by suppressing AMPK/Nrf2 pathway in rats. Ann Transl Med, 2019, 7(22): 634.
16.	Li J, Zhao Y, Zhou N, et al. Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in diabetes mellitus by inhibiting endoplasmic reticulum stress. J Diabetes Res, 2019, 2019: 7869318.
17.	Binder P, Wang S, Radu M, et al. Pak2 as a novel therapeutic target for cardioprotective endoplasmic reticulum stress response. Circ Res, 2019, 124(5): 696-711.
18.	Jung J, Eggleton P, Robinson A, et al. Calnexin is necessary for T cell transmigration into the central nervous system. JCI Insight, 2018, 3(5): e98410.
19.	Ryan D, Carberry S, Murphy A C, et al. Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer. J Transl Med, 2016, 14(1): 196.
20.	Fan Y, Simmen T. Mechanistic connections between endoplasmic reticulum (ER) redox control and mitochondrial metabolism. Cells, 2019, 8(9): 1071.
21.	Nakao H, Seko A, Ito Y, et al. PDI family protein ERp29 recognizes P-domain of molecular chaperone calnexin. Biochem Biophys Res Commun, 2017, 487(3): 763-767.
22.	Lynes E M, Bui M, Yap M C, et al. Palmitoylated TMX and calnexin target to the mitochondria-associated membrane. EMBO J, 2012, 31(2): 457-470.
23.	Lakkaraju A K, Abrami L, Lemmin T, et al. Palmitoylated calnexin is a key component of the ribosome-translocon complex. EMBO J, 2012, 31(7): 1823-1835.
24.	Budd G. On diseases of the liver. 2nd ed. London: John Churchill, 1852.
25.	Xin Y, Wu W, Qu J, et al. Inhibition of mitofusin-2 promotes cardiac fibroblast activation via the PERK/ATF4 pathway and reactive oxygen species. Oxid Med Cell Longev, 2019, 2019: Article ID 3649808.
26.	Xu S, Xiao Y, Zeng S, et al. Piperlongumine inhibits the proliferation, migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Inflamm Res, 2018, 67(3): 233-243.
27.	Guo Y, Gupte M, Umbarkar P, et al. Entanglement of GSK-3beta, beta-catenin and TGF-beta1 signaling network to regulate myocardial fibrosis. J Mol Cell Cardiol, 2017, 110: 109-120.
28.	Olgar Y, Ozdemir S, Turan B. Induction of endoplasmic reticulum stress and changes in expression levels of Zn²⁺-transporters in hypertrophic rat heart. Mol Cell Biochem, 2018, 440(1-2): 209-219.
29.	Senft D, Ronai Z A. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci, 2015, 40(3): 141-148.
30.	Yuan Y, Zhang Y, Han X, et al. Relaxin alleviates TGFbeta1-induced cardiac fibrosis via inhibition of Stat3-dependent autophagy. Biochem Biophys Res Commun, 2017, 493(4): 1601-1607.
31.	Liu X, Shan X, Chen H, et al. Stachydrine ameliorates cardiac fibrosis through inhibition of angiotensin Ⅱ/transformation growth factor beta1 fibrogenic axis. Front Pharmacol, 2019, 10: 538.
32.	Khalil H, Kanisicak O, Prasad V, et al. Fibroblast-specific TGF-beta-Smad2/3 signaling underlies cardiac fibrosis. J Clin Invest, 2017, 127(10): 3770-3783.

1. Li Y, Li Z, Zhang C, et al. Cardiac fibroblast-specific activating transcription factor 3 protects against heart failure by suppressing MAP2K3-p38 signaling. Circulation, 2017, 135(21): 2041-2057.
2. Ranjan P, Kumari R, Verma S K. Cardiac fibroblasts and cardiac fibrosis: Precise role of exosomes. Front Cell Dev Biol, 2019, 7: 318.
3. Liu T, Wen H, Li H, et al. Oleic acid attenuates Ang II (angiotensin II)-induced cardiac remodeling by inhibiting FGF23(fibroblast growth factor 23) expression in mice. Hypertension, 2020, 75(3): Hypertensionaha119.14167.
4. Horckmans M, Bianchini M, Santovito D, et al. Pericardial adipose tissue regulates granulopoiesis, fibrosis, and cardiac function after myocardial infarction. Circulation, 2018, 137(9): 948-960.
5. Farris S D, Don C, Helterline D, et al. Cell-specific pathways supporting persistent fibrosis in heart failure. J Am Coll Cardiol, 2017, 70(3): 344-354.
6. Nomura S, Satoh M, Fujita T, et al. Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure. Nat Commun, 2018, 9(1): 4435.
7. Chen S, Zhang Y, Lighthouse J K, et al. A novel role of cyclic nucleotide phosphodiesterase 10A in pathological cardiac remodeling and dysfunction. Circulation, 2020, 141(3): 217-233.
8. Xia P, Wang S, Xiong Z, et al. The ER membrane adaptor ERAdP senses the bacterial second messenger c-di-AMP and initiates anti-bacterial immunity. Nat Immunol, 2018, 19(2): 141-150.
9. Song S, Tan J, Miao Y, et al. Crosstalk of ER stress-mediated autophagy and ER-phagy: Involvement of UPR and the core autophagy machinery. J Cell Physiol, 2018, 233(5): 3867-3874.
10. Park S, Lim W, Bazer F W, et al. Apigenin induces ROS-dependent apoptosis and ER stress in human endometriosis cells. J Cell Physiol, 2018, 233(4): 3055-3065.
11. Shih Y C, Chen C L, Zhang Y, et al. Endoplasmic reticulum protein TXNDC5 augments myocardial fibrosis by facilitating extracellular matrix protein folding and redox-sensitive cardiac fibroblast activation. Circ Res, 2018, 122(8): 1052-1068.
12. Yao Y, Lu Q, Hu Z, et al. A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure. Nat Commun, 2017, 8(1): 133.
13. Pinkaew D, Chattopadhyay A, King M D, et al. Fortilin binds IRE1alpha and prevents ER stress from signaling apoptotic cell death. Nat Commun, 2017, 8(1): 18.
14. Misaka T, Murakawa T, Nishida K, et al. FKBP8 protects the heart from hemodynamic stress by preventing the accumulation of misfolded proteins and endoplasmic reticulum-associated apoptosis in mice. J Mol Cell Cardiol, 2018, 114: 93-104.
15. Hou X, Fu M, Cheng B, et al. Galanthamine improves myocardial ischemia-reperfusion-induced cardiac dysfunction, endoplasmic reticulum stress-related apoptosis, and myocardial fibrosis by suppressing AMPK/Nrf2 pathway in rats. Ann Transl Med, 2019, 7(22): 634.
16. Li J, Zhao Y, Zhou N, et al. Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in diabetes mellitus by inhibiting endoplasmic reticulum stress. J Diabetes Res, 2019, 2019: 7869318.
17. Binder P, Wang S, Radu M, et al. Pak2 as a novel therapeutic target for cardioprotective endoplasmic reticulum stress response. Circ Res, 2019, 124(5): 696-711.
18. Jung J, Eggleton P, Robinson A, et al. Calnexin is necessary for T cell transmigration into the central nervous system. JCI Insight, 2018, 3(5): e98410.
19. Ryan D, Carberry S, Murphy A C, et al. Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer. J Transl Med, 2016, 14(1): 196.
20. Fan Y, Simmen T. Mechanistic connections between endoplasmic reticulum (ER) redox control and mitochondrial metabolism. Cells, 2019, 8(9): 1071.
21. Nakao H, Seko A, Ito Y, et al. PDI family protein ERp29 recognizes P-domain of molecular chaperone calnexin. Biochem Biophys Res Commun, 2017, 487(3): 763-767.
22. Lynes E M, Bui M, Yap M C, et al. Palmitoylated TMX and calnexin target to the mitochondria-associated membrane. EMBO J, 2012, 31(2): 457-470.
23. Lakkaraju A K, Abrami L, Lemmin T, et al. Palmitoylated calnexin is a key component of the ribosome-translocon complex. EMBO J, 2012, 31(7): 1823-1835.
24. Budd G. On diseases of the liver. 2nd ed. London: John Churchill, 1852.
25. Xin Y, Wu W, Qu J, et al. Inhibition of mitofusin-2 promotes cardiac fibroblast activation via the PERK/ATF4 pathway and reactive oxygen species. Oxid Med Cell Longev, 2019, 2019: Article ID 3649808.
26. Xu S, Xiao Y, Zeng S, et al. Piperlongumine inhibits the proliferation, migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Inflamm Res, 2018, 67(3): 233-243.
27. Guo Y, Gupte M, Umbarkar P, et al. Entanglement of GSK-3beta, beta-catenin and TGF-beta1 signaling network to regulate myocardial fibrosis. J Mol Cell Cardiol, 2017, 110: 109-120.
28. Olgar Y, Ozdemir S, Turan B. Induction of endoplasmic reticulum stress and changes in expression levels of Zn²⁺-transporters in hypertrophic rat heart. Mol Cell Biochem, 2018, 440(1-2): 209-219.
29. Senft D, Ronai Z A. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci, 2015, 40(3): 141-148.
30. Yuan Y, Zhang Y, Han X, et al. Relaxin alleviates TGFbeta1-induced cardiac fibrosis via inhibition of Stat3-dependent autophagy. Biochem Biophys Res Commun, 2017, 493(4): 1601-1607.
31. Liu X, Shan X, Chen H, et al. Stachydrine ameliorates cardiac fibrosis through inhibition of angiotensin Ⅱ/transformation growth factor beta1 fibrogenic axis. Front Pharmacol, 2019, 10: 538.
32. Khalil H, Kanisicak O, Prasad V, et al. Fibroblast-specific TGF-beta-Smad2/3 signaling underlies cardiac fibrosis. J Clin Invest, 2017, 127(10): 3770-3783.

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The potential role of calnexin in the activation of cardiac fibroblasts

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