Asparaginyl endopeptidase (AEP) regulates myocardial apoptosis in response to radiation exposure via alterations in NRF2 activation

Abstract

Radiation-induced heart disease (RIHD) leads to myocardial dysfunction and metabolic abnormalities in patients treated with thoracic irradiation which restricts the long-term survival benefits of radiotherapy. There is no specific or effective manner of intervention currently available. Asparaginyl endopeptidase (AEP) plays a pivotal role in the maintenance of cellular functions through regulating proteolytic cleavage as peptidase enzyme. We aimed to investigate the role of unique cardiac AEP in cardiac function by modulating key signaling elements in the myocardium. The murine heart was exposed to a single dose of 14 Gy radiation. Cellular signaling and apoptosis was analyzed in human and rat cardiomyocytes treated with various doses of radiation, we observed expression of AEP was increased by immunohistochemical staining in murine heart exposed to radiation. The AEP production along with its increased level of mRNA expression was associated with increased doses of radiation (0, 2, 5, 10 Gy) in cardiomyocytes. The myocardial cells transfected with AEP overexpression showed overall cellular viability enhancement, DNA damage inhibition, the foci formation of γ-H2AX suppressed and DNA repair enhancement significantly after radiation exposure. Small interfering RNA-mediated AEP knockdown was with reduced cardiomyocyte viability, elevated apoptotic rate, increased γ-H2AX foci formation and inhibited DNA repair as well after irradiation. After radiation exposure of 10 Gy, the expression of AEP increased in P53 overexpressing cardiomyocytes and decreased in the P53 knockdown cells, indicates that radiation-induced expression of AEP might be regulated by P53. Moreover, treatments with either AEP overexpression or knockdown showed enhanced NRF2 activity in the nuclear or suppressed NRF2 expression in the cytoplasm of myocardial cells after irradiation, respectively, defined a possible regulatory effect of AEP associated with diminished NRF2 translocation and activation by radiation exposure, including impair myocardium and myocardial apoptosis. These findings suggest that increased levels of AEP in failing myocardium after irradiation is mediated by P53 and regulate a novel pathway that involves NRF2 activation. AEP is essential for maintaining cellular redox homeostasis of cardiac function.

Keywords: Radiation-induced myocardial injury; asparaginyl endopeptidase (AEP); irradiation; nuclear factor erythroid 2-related factor 2 (NRF2).

Figure 1

Effect of irradiation on cardiac tissue and function. The whole heart of mice was irradiated with a single dose of 14 Gy using 6-MV X-ray. The representative photomicrographs (× 20, scale bar = 100 μm) of left ventricular sections with hematoxylin and eosin (HE)-staining were shown in mice treated with irradiation (IR) and without irradiation (Control) at day 21 after irradiation (A). The Left ventricular systolic function was tested by M-mode Echocardiography in mice treated with irradiation (IR) and without irradiation (Control) at day 21 after irradiation (B); The systolic function parameter of ejection fraction (EF) and fractional shortening (FS) were measured in the IR and Control group (C and D). Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups. NS = not significantly (P≥0.05).

Figure 2

Increased AEP expression in cardiomyocytes after irradiation. The whole heart of mice was irradiated with a single dose of 14 Gy using 6-MV X-ray. Cardiac tissue was sampled at day 21 after irradiation. Three representative photomicrographs (× 20, scale bar = 100 μm) of left ventricular sections with immunohistochemical staining for AEP were shown in mice treated with irradiation (IR) and without irradiation (Control) (A and B). Escalated irradiation dose of 0 Gy, 2 Gy, 5 Gy and 10 Gy was delivered to cardiomyocytes. Cells were collected at 24th hour after irradiation. The RT-PCR was used to determine the relative mRNA expression level of AEP (C and D). Western blot analysis was used to determine the AEP protein expression after irradiation in normal control group after irradiation (E and F). Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups. ***P < 0.0001, **P < 0.001, *P < 0.01 vs. Normal Control group treated with sham irradiation.

Figure 3

Construction of AEP overexpressing and knockdown cardiomyocytes. Lentivirus transfection was used to transfer AEP overexpressing or knockdown plasmid into the cardiomyocytes to build AEP overexpressing or knockdown cell lines using AC16 cells and H9C2 cells. The RT-PCR was used to determine the relative mRNA expression level of AEP in AC16 cells (A and C) and H9C2 cells (B and D), respectively. Western blot analysis was used to confirm the effect of expression of AEP overexpression and knockdown in AC16 cells (E) and H9C2 cells (F) with or without irradiation. Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups.

Figure 4

Overexpression of AEP enhanced cardiomyocyte survival after irradiation. Escalated irradiation dose of 0 Gy, 2 Gy, 5 Gy and 10 Gy was delivered to AEP overexpressing cells. Cell viability was evaluated by CCK-8 assay (A and D). Myocardium apoptosis was quantified with flow cytometry. The apoptosis rate was presented as the percentage of cells with FITC-Annexin V positive/PI negative in (B and E). The representative photomicrographs of flow cytometry detecting myocardium apoptosis were shown in (C and F). Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups. ***P < 0.0001, **P < 0.001, *P < 0.01 vs. Normal Control group at every dose point.

Figure 5

Knockdown of AEP reduced cardiomyocyte survival after irradiation. Escalated irradiation dose of 0 Gy, 2 Gy, 5 Gy and 10 Gy was delivered to cardiomyocytes. Cell viability was evaluated by CCK-8 assay (A and D). Myocardium apoptosis was quantified with flow cytometry (B and E). The representative photomicrographs of flow cytometry were shown in (C and F). Each value represents the mean ± SD of three determinations. Student’s t-test was used to detect the difference between groups. ***P < 0.0001, **P < 0.001, *P < 0.01 vs. Normal Control group at every dose point. NS = not significantly (P≥0.05).

Figure 6

AEP reduced DNA damage significantly in cardiomyocyte after irradiation. Escalated irradiation dose of 0 Gy, 2 Gy, 5 Gy and 10 Gy was delivered to cardiomyocytes. Cells were collected at one hour after irradiation. The representative images of γ-H2AX staining of AC16 and H9C2 cells was showed in the left column as in (A-D); Quantitative γ-H2AX staining assays was showed in the corresponding row as in (E-H). Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups. ***P < 0.0001, **P < 0.001, *P < 0.01 compared with normal control group, NS = not significantly (P≥0.05).

Figure 7

AEP promotes the DNA repair after irradiation. Radiation dose of 10 Gy was delivered to AEP overexpressing or knocked down H9C2 cells as well as control cells. Cells were collected at 24th hour after irradiation. Western blot analyses were conducted to examine the protein expression of BRCA1, ATM, ATR, Ku80. Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups.

Figure 8

AEP alters NRF2 activity in cardiomyocytes upon irradiation. Escalated irradiation dose of 0 Gy, 2 Gy, 5 Gy and 10 Gy was delivered to AEP overexpressing and knockdown cardiomyocytes. Cells were collected at 24th hour after irradiation. Western blot analyses of NRF2 expression in the cytoplasm and nuclear were conducted, respectively (A-D). RT-PCR was also conducted to determine the relative mRNA expression of NRF2 as shown in (E-H). Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups. ***P < 0.0001, **P < 0.001, *P < 0.01 vs. sham irradiation in normal control group, ###P < 0.0001, ##P < 0.001, #P < 0.01 vs. sham irradiation in AEP overexpressing and knockdown cells.

Figure 9

P53 regulates the expression of AEP after radiation. Lentivirus transfection was used to transfer P53 overexpressing or knockdown plasmid into the cardiomyocytes to build P53 overexpressing or knockdown cell lines using AC16 cells and H9C2 cells. The RT-PCR was used to determine the relative mRNA expression level of P53 in AC16 cells (A and B) and H9C2 cells (E and F). Irradiation dose of 10 Gy was delivered to cardiomyocytes. Cells were collected at 24th hour after irradiation. RT-PCR was also conducted to determine the relative mRNA expression of AEP as shown in (C, D, G and H). Western blot analyses of AEP expression were conducted, as shown in (I and J). Each value represents the mean ± S.E.M of three determinations. Student’s t-test was used to detect the difference between groups.