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Abstract
M yocardial infarction (MI) is the common cause of cardiomyocyte apoptosis and hypoxia alone is sufficient to induce apoptosis of cardiomyocytes. In hearts, autophagy might play important roles in hypoxia-mediated cardioprotection or myocardial damage effects. To date, hypoxia-inducible factor-1α (HIF-1α) transcriptional factor and BH3-only bcl-2 family protein (BNIP3) are known to play fundamental roles in adaptive or death process in response to hypoxia. In addition, hypoxia can induce insulin-like growth factor binding protein 3 (IGFBP- 3) to block the IGF1R/PI3K/Akt survival pathway. Therefore, we would like to investigate the molecular mechanism and the interaction of IGFBP-3, HIF-1α and BNIP3 in hypoxia-induced cell injury of H9c2 cardiomyoblast cells. Moreover, 17β-Estradiol (E2) has been reported recently to prevent cardiac apoptosis via estrogen receptors (ERs).
Previous studies have ever revealed the novel cardioprotective role of ER β in myocardial ischemia. Therefore, our studies aim to reveal the regulatory mechanism of ER β on hypoxia-induced cell death. Heart- derived H9c2 cells were incubated in normoxic or hypoxic (<1% oxygen) conditions for 24 h after ER β overexpression. Results showed the hypoxia primarily caused HIF-1α expression highly increase, then activated downstream genes such as BNIP3 and IGFBP-3, and further triggered autophagic and apoptotic pathways. However, all phenomena were recovered by E2/ER β overexpression. E2/ER β overexpression also further promoted the cardiac survival pathway related proteins, p-IGF1R and p-Akt activation. Taken together, ER β exerts the protective effect through repressed hypoxia-inducible BNIP3 and IGFBP-3 levels to restrain the hypoxia-induced autophagy and apoptosis effects in H9c2 cardiomyoblast cells.
Results
Fig. 1. Effects of hypoxia on (A) the mRNA expression of HIF-1 α, IGFBP-3 and BNIP3 was measured by RT-PCR, (B) the protein level of HIF-1 α, IGFBP-3 and BNIP3, (D) the phosphorylation of IGF-1R and Akt, and (C) HIF-1 α nuclear translocation were measured by Western blot in H9c2 cardiomyoblast cells.
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Fig. 2. Role of HIF-1 α in the IGFBP-3, BNIP3 and cleaved caspase-3 expression under normoxia and hypoxia. (A) H9c2 cells were treated with 5-30 μM HIF-1 inhibitor and were exposed to hypoxia for 24 h. (B) Rat cardiomyoblasts were transient transfected with 1-15 μg HIF-1 α plasmid under normoxia for 24 h.
Fig. 5. Role of BNIP3 in the LC3 and cleaved caspase-3 protein level under normoxia were measured by Western blot. (A and B) H9c2 cardiomyoblast cells were transient transfected with 1-15 μg BNIP3 plasmid for 24 h.
Fig. 8. Effects of ER β overexpression by transient transfection under hypoxia on (A) the protein level of HIF-1 α, IGFBP-3 and BNIP3, (B) the phosphorylation of Akt, Bad, and (C) the autophagy expression of LC3-II/LC3-I were measured by Western blot in H9c2 cardiomyoblast cells.
Fig. 3. Effects of hypoxia on (A) apoptosis was detected by annexin-V/PI, (B) the basal autophagosome formation was detected by GFP-LC3 puncta, and (C) the protein level of LC3, cytochrome c and cleaved caspase-3 was detected by Western blot. Data are presented as the mean ± SD (n=3). **P<0.01 vs.
normoxia-treated cells. #P<0.05 and ##P<0.01 vs. hypoxia-treated cells.
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Fig. 4. Effects of autophagy inhibitor (3-MA), siRNA such as Atg-5 and Beclin-1 and autophagy inducer (rapamycin) under hypoxia for 24 h on apoptosis were measured by TUNEL assay (A) and were measured by Western blot (B and C).
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Fig. 6. Immunohistochemical analysis for HIF-1 α and transcriptional targets in sections from the human cardiovascular tissue with myocardial infarction disease. Immunohistochemical analysis, with the indicated antibodies, of serial sections of representative lesions: a normal tissue, an acute infraction, a granulation tissue and a myocardial scar. Final magnifications: × 200.
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Fig. 7. Effects of ER β on the phosphorylation of IGF1R and Akt survival pathway were measured by Western blot in Rat cardiomyoblast cells. (A) H9c2 cells were transient transfected with 1-15 μg ER β plasmid for 24 h. (B) Tet-on ER β H9c2 cells were treated with 0.5-2 μg/ml doxcyclin (Dox) for 24 h or stimulated with 1 μg/ml Dox in time course to overexpress ER β.
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Fig. 9. Effects of ER β overexpression under hypoxia on the protein level of (A) Atg7, Atg5, Bax, Bak, Bcl-2, cytochrome c, cleaved caspase 9, and (B) HIF-1 α, IGFBP-3 and BNIP3 were measured by Western blot in Tet-on ER β H9c2 cells.
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Summary
Fig. 9. Hypoxia could stabilize HIF-1 α protein accumulation to trigger downstream IGFBP-3 and BNIP3. And then IGFBP-3 could block the IGF1R survival pathway to further enhance cell apoptosis. And BNIP3 could activate caspase 3 to induce apoptosis through LC3-dependent autophagy induction in H9c2 cardiomyoblast cells. However, under hypoxia ER β could activate the IGF- 1R survival pathway and totally abolish hypoxia related proteins such as HIF-1 α, IGFBP3 and BNIP3 to inhibit cell apoptosis indicated that ER β has a protection role in H9c2 cardiomyoblast cells.
