GRP78 effectively protect hypoxia/reperfusion-induced myocardial apoptosis via promotion of the Nrf2/HO-1 signaling pathway

Abstract

Myocardial infarction is a major cause of death worldwide. Despite our understanding of the pathophysiology of myocardial infarction and the therapeutic options for treatment have improved substantially, acute myocardial infarction remains a leading cause of morbidity and mortality. Recent findings revealed that GRP78 could protect myocardial cells against ischemia reperfusion injury-induced apoptosis, but the exact function and molecular mechanism remains unclear. In this study, we aimed to explore the effects of GRP78 on hypoxia/reperfusion (H/R)-induced cardiomyocyte injury. Intriguingly, we first observed that GRP78 overexpression significantly protected myocytes from H/R-induced apoptosis. On mechanism, our work revealed that GRP78 protected myocardial cells from hypoxia/reperfusion-induced apoptosis via the activation of the Nrf2/HO-1 signaling pathway. We observed the enhanced expression of Nrf2/HO-1 in GRP78 overexpressed H9c2 cell, while GRP78 deficiency dramatically antagonized the expression of Nrf2/HO-1. Furthermore, we found that blocked the Nrf2/HO-1 signaling by the HO-1 inhibitor zinc protoporphyrin IX (Znpp) significantly retrieved H9c2 cells apoptosis that inhibited by GRP78 overexpression. Taken together, our findings revealed a new mechanism by which GRP78 alleviated H/R-induced cardiomyocyte apoptosis in H9c2 cells via the promotion of the Nrf2/HO-1 signaling pathway.

Keywords: GRP78; HO-1; Nrf2; apoptosis; myocardial hypoxia/reperfusion.

Figure 1

Adenovirus infection mediated GRP78 overexpressing in H9c2 cells. (a) Typical photomicrographs illustrated immunoflourescence staining in H9c2 cells in control, H/R 36 hr, ad‐GRP78 + H/R 36 hr and ad‐NON + H/R 36 groups. GRP78, GFP, and DAPI were used as markers for GRP78 protein expression, adenovirus expression, and nuclei, respectively. (b) Western blot showed the protein level of GRP78 in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells. (c) Quantitative analysis of GRP78 protein expression related to Figure 1b. (d) Quantitative analysis of GRP78 mRNA expression by qRT‐PCR in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells. DAPI, 4′,6‐diamidino‐2‐phenylindole; GFP, green fluorescence protein; mRNA, messenger RNA; qRT‐PCR, quantitative real‐time polymerase chain reaction

Figure 2

Overexpression of GRP78 in H9c2 significantly protected myocytes from hypoxia‐induced apoptosis. (a) Cell growth in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells measured by CCK‐8. (b) Representative EdU staining in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells was shown. (c) Quantitative analysis of EdU positive cells related to Figure 2b. (d) Apoptosis of H9c2 cells assessed by flow cytometry to detect FITC‐Annexin V staining. (e) The relative apoptosis ratio was calculated with Annexin V‐positive apoptotic cells. N = 5 per group, Data are mean ± SEM, ****p < .0001, N.S represent no significance; (f) Western blot showed caspase3 cleavage in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells. (g) Western blot showed caspase3 cleavage, BCL‐2, and BAX expression in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells. (h) Quantitative analysis of caspase3 cleavage, BCL‐2, and BAX expression in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells related to Figure 2g. CCK‐8, cell counting kit 8; EdU, 5‐ethynyl‐2'‐deoxyuridine; SEM standard error of mean

Figure 3

GRP78 overexpression activates the Nrf2/HO‐1 signaling pathway. (a) Representative immunoblot of NRF2 and HO‐1 expression is shown in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr four groups and GAPDH was used as a loading control. (b) Bar graph shows the analysis of Nrf2 and HO‐1 protein level in the four groups, and Nrf2/HO‐1 were augmented by GRP78 overexpression after H/R 36 hr as an inducer. (c) Quantitative analysis of mRNA expression of NRF2 and HO‐1 in control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells. (d) A representative of immunofluorescence with NRF2, GFP, DAPI in the control, H/R 36, ad‐GRP78 + H/R 36, and ad‐NON + H/R 36 hr cells. (e) Western blot showed the GRP78, NFR2, and HO‐1 expression in control and GRP78 knockdown cells. (f) Western blot showed the GRP78, NFR2, and HO‐1 expression in control, GRP78 knockout, and GRP78 knockout with GRP78 putting back cells, respectively. (g) Quantitative analysis of mRNA expression of NRF2 and HO‐1 in control, GRP78 knockout, and GRP78 knockout with GRP78 putting back cells, respectively. DAPI, 4′,6‐diamidino‐2‐phenylindole; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger RNA; SEM standard error of mean

Figure 4

Nrf2/HO‐1 mediated the effect of GRP78 on the apoptotic pathway. (a) A representative immunoblot of HO‐1 in ad‐GRP78 + H/R 36 and ad‐GRP78 + H/R 36 hr + Znpp two groups and GAPDH was used as a loading control. (b) The bar graph showed the analysis of HO‐1 mRNA level in the ad‐GRP78 + H/R 36 hr and ad‐GRP78 + H/R 36 hr + Znpp cells. (c) A representative of immunofluorescence with TUNEL, GFP, DAPI in the ad‐GRP78 + H/R 36 hr and ad‐GRP78 + H/R 36 hr+ Znpp cells. (d) Apoptosis of ad‐GRP78 + H/R 36 hr and ad‐GRP78 + H/R 36 hr + Znpp cells was assessed by flow cytometry to detect annexin V‐FITC staining, and relative apoptosis ratio was calculated with Annexin V‐positive apoptotic cells in this two groups. (e) Representative immunoblot of apoptotic proteins is showed in the two groups and GAPDH was used as a loading control, and bar graph showed western blot analysis for the level of Bax, Bcl‐2, and cleaved caspase 3, N = 5 per group, Data are mean ± SD, ****p < .0001. DAPI, 4′,6‐diamidino‐2‐phenylindole; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; GFP, green fluorescence protein; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; SD, standard deviation