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N-myc downstream-regulated gene 4, up-regulated by tumor necrosis factor-α and nuclear factor kappa B, aggravates cardiac ischemia/reperfusion injury by inhibiting reperfusion injury salvage kinase pathway

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Abstract

N-myc downstream-regulated gene 4 (NDRG4) is expressed weakly in heart and has been reported to modulate cardiac development and QT interval duration, but the role of NDRG4 in myocardial ischemia/reperfusion (I/R) injury remains unknown. In the present study, we analyzed the expression as well as potential function of cardiac NDRG4 and investigated how NDRG4 expression is regulated by inflammation. We found that NDRG4 was weakly expressed in cardiomyocytes and that its expression increased significantly both in I/R injured heart and in hypoxia-reoxygenation (H/R) injured neonatal rat ventricular myocytes (NRVMs). The increased NDRG4 expression aggravated myocardial I/R injury by inhibiting the activation of the reperfusion injury salvage kinase (RISK) pathway. Forced over-expression of NDRG4 inhibited RISK activation and exacerbated injury not only in I/R injured heart, but also in H/R treated NRVMs, whereas short hairpin RNA (shRNA)-mediated knock-down of NDRG4 enhanced RISK activation and attenuated injury. Upon injury, myocardial NDRG4 expression was induced by tumor necrosis factor-α (TNF-α) through nuclear factor kappa B (NF-κB), and we found that pre-treatment with inhibitors of either TNF-α or NF-κB blocked NDRG4 expression as well as I/R injury in vivo and H/R injury in vitro. Our study indicates that up-regulation of NDRG4 aggravates myocardial I/R injury by inhibiting activation of the RISK pathway, thereby identifying NDRG4 as a potential therapeutic target in I/R injury.

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    Authors and Affiliations

    1. Department of Physiology, Fourth Military Medical University, Xi’an, 710032, China

      Yuan Xing

    2. Department of International Medical, China-Japan Frindship Hospital, Beijing, 100029, China

      Bin Tang

    3. Institute of Orthopaedics, Xi’jing Hospital, Fourth Military Medical University, Xi’an, 710032, China

      Chao Zhu

    4. Department of Histology and Embryology, Fourth Military Medical University, Xi’an, 710032, China

      Wei Li, Zhen Li, Jie Zhao, Chu-chao Zhu & Yuan-qiang Zhang

    5. Department of Interventional Radiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, 710038, China

      Wei-dong Gong & Zhi-qun Wu

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    Correspondence to Zhi-qun Wu, Chu-chao Zhu or Yuan-qiang Zhang.

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    This work was supported by the National Natural Science Foundation of China, NSFC (Nos. 31371216 and 31171154 to YQ Z), and by the Program of China Scholarship Council (No. (2014)3026 to CC Z).

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    Y. Xing, B. Tang and C. Zhu contributed equally to this work.

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    395_2015_519_MOESM1_ESM.tif

    Figure S1 The effect of NDRG4 knockdown on I/R-induced myocardial injury. (A) Measurement of cardiac function using echocardiography was performed at 24 h following reperfusion to assess left ventricular ejection fraction (LVEF) in hearts with adenovirus-packaged shRNA-NC or shRNA-ND4. (*P < 0.05 vs. sham) (B) Serum was collected at 24-h reperfusion to analyze the total LDH level in each group. (n = 6 per group, *P < 0.05 vs. sham, # P < 0.05 vs. shRNA-NC + I/R). Values are presented as mean ± SEM. (C) Quantitation of caspase 3 activation results. (n = 5 per group, *P < 0.05 vs. Con + shRNA-NC group; # P < 0.05 vs. H/R + shRNA-NC group). (D, E) Flow cytometry assay results indicating the apoptosis rate of NRVMs with or without NDRG4 knockdown after H/R. The percentage of apoptotic cells in H/R-treated cardiomyocytes without NDRG4 knockdown is 13.9%, whereas the percentage of apoptotic cells in H/R-treated myocytes with NDRG4 knockdown is 6.3%. (n = 3 per group, *P < 0.05 vs. Con + shRNA-NC group; # P < 0.05 vs. H/R + shRNA-NC group). Data are presented as the mean ± SEM. (TIFF 5952 kb)

    395_2015_519_MOESM2_ESM.tif

    Figure S2 The effect of NDRG4 over-expression on I/R-induced myocardial injury. (A) Measurement of cardiac function using echocardiography was performed at 24 h following reperfusion in hearts with adenovirus-packaged Adv-NC or Adv-ND4. LVEF showed no significant difference. (B) Serum was collected at 24-h reperfusion to analyze the total LDH level in each group. (n = 6 per group, *P < 0.05 vs. Adv-NC + I/R). (C) Representative immunoblot image and quantitation of caspase 3 activation results. (n = 4 per group, *P < 0.05 vs. Con + Adv-NC group; # P < 0.05 vs. H/R + Adv-NC group). (D, E) Flow cytometry assay results showing the apoptosis rate of NRVMs with or without NDRG4 over-expression after H/R. The percentage of apoptotic cells in H/R-treated cardiomyocytes without NDRG4 over-expression is 13.3% whereas the percentage of apoptotic cells in H/R-treated cardiomyocytes with NDRG4 over-expression is 31.9%. (n = 3 per group, *P < 0.05 vs. Con + Adv-NC group; # P < 0.05 vs. H/R + Adv-NC group). Data are presented as the mean ± SEM. (TIFF 6319 kb)

    395_2015_519_MOESM3_ESM.tif

    Figure S3 NDRG4 regulated myocardial I/R injury through inhibiting RISK pathway. (A) Representative immunoblot image of pAkt and pERK levels in NDRG4 knockdown NRVMs subjected to H/R with vehicle, LY294002 (10 μM) and PD98059 (10 μM)), respectively. (B) Quantitation of Akt activation results. (C) Quantitation of ERK activation results. (n = 5 per group, *P < 0.05 vs. shRNA-NC group; # P < 0.05 vs. LY294002 and PD98059 + shRNA-NC group). (D) Representative immunoblot image of pAkt and pERK levels in inhibitors treated NRVMs subjected to H/R with NDRG4 over-expression. (E) Quantitation of Akt activation results. (F) Quantitation of ERK activation results. (n = 5 per group, *P < 0.05 vs. Adv-NC group; # P < 0.05 vs. LY294002 and PD98059 + Adv-NC group). Data are presented as the mean ± SEM. (TIFF 7102 kb)

    395_2015_519_MOESM4_ESM.tif

    Figure S4 Determination of TNF-α level in vivo and in vitro. (A) TNF-α expression at various time points of reperfusion in the heart. (B) Myocardial I/R-stimulated serum TNF-α levels in vivo. Blood samples were collected at the end of reperfusion and detected by ELISA test. (n = 5 per group, *P < 0.05, compared to that observed in the sham group). (C) TNF-α expression increased in NRVMs subjected to H/R. NRVMs were subjected to hypoxia for 4 h and reoxygenation at three different time points (2, 4 and 6 h). Western blot analysis of TNF-α expression in lysates of NRVMs. (D) TNF-α level in the supernatant of H/R-treated cardiomyocytes. (n = 5 per group, *P < 0.05, compared to control group). Data are presented as the mean ± SEM. (TIFF 7240 kb)

    395_2015_519_MOESM5_ESM.tif

    Figure S5 TNF-α inhibitor pre-treatment inhibited NF-κB expression in both I/R heart and H/R NRVMs. (A) Representative immunoblot images showing the expression of NF-κB in I/R-injured hearts with pre-treatment of Et (100 μM). (B) Quantitation of NF-κB expression in the I/R experiments. (n = 5 per group, *P < 0.05, compared to sham group, # P < 0.05 vs. I/R 2 h group). (C) Representative immunoblot image of NF-κB in H/R-treated NRVMs with or without Et pre-treatment. (D) Quantitation of NF-κB expression in the H/R experiments. (n = 5 per group, *P < 0.05, compared to control group, # P < 0.05 vs. H/R 2 h group). (TIFF 6716 kb)

    395_2015_519_MOESM6_ESM.tif

    Figure S6 NF-κB inhibitor pre-treatment inhibited apoptosis in I/R heart and H/R NRVMs. (A, B) The number of TUNEL-positive cells decreased in I/R-injured hearts with PDTC pre-treatment. (n = 5 per group, *P < 0.05, compared to I/R group). Data are presented as the mean ± SEM. (Scare bar 50 μm) (C) Representative immunoblot images and quantitation of caspase 3 activation in I/R-injured hearts with pre-treatment of PDTC (100 μM). (n = 4 per group, *P < 0.05 vs. sham group; # P < 0.05 vs. I/R 2 h group). Data are presented as the mean ± SEM (D) Representative immunoblot images and quantitation of caspase 3 activation in. H/R injured NRVMs with pre-treatment of PDTC (100 μM). (n = 4 per group, *P < 0.05 vs. control group; # P < 0.05 vs. H/R 2 h group). (TIFF 6391 kb)

    395_2015_519_MOESM7_ESM.tif

    Figure S7 NF-κB inhibitor pre-treatment inhibited TNF-α-induced RISK activation in NRVMs with H/R treatment. (A) Representative immunoblot image of RISK activation in H/R-treated NRVMs with or without PDTC pre-treatment. (B) Quantitation of Akt activation. (C) Quantitation of ERK activation. (n = 4 per group, *P < 0.05 vs. Con + vehicle group; # P < 0.05 vs. I/R + vehicle group). (D) Representative immunoblot image showing RISK activation in TNF-α-treated NRVMs with or without PDTC pre-treatment. (E) Quantitation of Akt activation. (F) Quantitation of ERK activation. (n = 4 per group, *P < 0.05 vs. Con group; # P < 0.05 vs. TNF-α 2 h group). (TIFF 10595 kb)

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    Xing, Y., Tang, B., Zhu, C. et al. N-myc downstream-regulated gene 4, up-regulated by tumor necrosis factor-α and nuclear factor kappa B, aggravates cardiac ischemia/reperfusion injury by inhibiting reperfusion injury salvage kinase pathway. Basic Res Cardiol 111, 11 (2016). https://doi.org/10.1007/s00395-015-0519-0

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