Ischemia/hypoxia inhibits cardiomyocyte autophagy and promotes apoptosis via the Egr-1/Bim/Beclin-1 pathway.

Figures

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  Figure 1.. Alteration of Egr-1 expression impacted the mRNA levels of Bim and Beclin-1 in cardiomyocytes under IH condition. The expressions of Egr-1 (A), Bim (B) and Beclin-1 (C) mRNA levels were determined by RT-qPCR. n ≥ 3 for each group; *P < 0.05 and **P < 0.01 compared with the control group; ΔP < 0.05 and ΔΔP < 0.01 compared with the IH group; ∇P < 0.05 and ∇∇P < 0.01 compared with the IH+shRNA group. Con: control; IH: ischemia/hypoxia; NC: negative control; shRNA: Egr-1 shRNA; 3-MA: 3-methyladenine.

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  Figure 2.. Egr-1 inhibited the expression of autophagy associated proteins in IH-exposed cardiomyocytes. Representative western blot images show the target bands of Egr-1/Bim/Beclin-1 (A) and LC3/P62 (E) in six groups. The expressions of Egr-1 (B), Bim (C), Beclin-1 (D), LC3II (F) and P62 (G) were quantitated by normalization to GAPDH. N ≥ 3 for each group; *P < 0.05 and **P < 0.01, compared with the control group; ΔP < 0.05 and ΔΔP < 0.01 compared with the IH group; ∇P < 0.05 and ∇∇P < 0.01 compared with the IH+shRNA group. Con: control; IH: ischemia/hypoxia; NC: negative control; shRNA: Egr-1 shRNA; 3-MA: 3-methyladenine.

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  Figure 3.. Autophagic vacuoles were reduced after Egr-1 over-expression and increased following Egr-1 knockdown in IH-exposed cardiomyocytes. Autophagic vacuoles were measured by TEM, and the representative images are shown with red arrows indicating autophagolysosomes or autophagosomes. Magnification 30,000 ×. Con: control; IH: ischemia/hypoxia; NC: negative control; shRNA: Egr-1 shRNA; 3-MA: 3-methyladenine.

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  Figure 4.. Autophagic flux in IH-exposed rat cardiomyocytes with manipulated Egr-1 expression measured by confocal microscope. The representative images show the fluorescence of GFP and mRFP in cardiomyocytes, and quantifications on the numbers of the autolysosomes (white) and autophagosomes (black) per cells in 6 groups. Magnification 400 ×. N ≥ 3 for each group; *P < 0.05 compared with the control group; ΔP < 0.05 compared with the IH group; ∇P < 0.05 compared with the IH+shRNA group. Con: control; IH: ischemia/hypoxia; NC: negative control; shRNA: Egr-1 shRNA; 3-MA: 3-methyladenine.

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  Figure 5.. Egr-1 suppression protected cardiomyocytes from IH-induced injury. (A): The levels of LDH leakage from cardiomyocytes in the indicated six groups were quantitated; (B & C): the levels of Cleaved-caspase 3 protein expression in the indicated 6 groups were detected by western blots; (D & E): the apoptosis levels of cardiomyocytes in the indicated six groups were measured by flow cytometry. N ≥ 3 for each group; *P < 0.05 and **P < 0.01 compared with the control group; ΔP < 0.05 and ΔΔP < 0.01 compared with the IH group; ∇P < 0.05 and ∇∇P < 0.01 compared with the IH+shRNA group. Con: control; IH: ischemia/hypoxia; NC: negative control; shRNA: Egr-1 shRNA; 3-MA: 3-methyladenine.

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  Figure 6.. The molecular mechanism of inhibiting autophagy and promoting apoptosis through the Egr-1/Bim/Beclin-1 pathway in IH-exposed cardiomyocyte. IH inhibited autophagy and promoted apoptosis in rat cardiomyocte by the signaling pathway, which up-regulated the expression of Egr-1, and then activated Bim and suppressed Beclin-1. Based on the results in this study, IH-induced cardiomyocyte autophagy and apoptosis may be associated with the Egr-1/Bim/Beclin-1 pathway. IH: ischemia/hypoxia; →: promotion; ⊥: suppression.