Attenuation of miR-34a protects cardiomyocytes against hypoxic stress through maintenance of glycolysis

Figures

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  Figure 1. Cardiomyocytes are more sensitive to hypoxia under low glucose condition (A) Rat cardiac cell line, H9c2 (left) was treated with hypoxia for 0, 12, 24, 48, or 72 h. The expressions of Hif-1α were measured by Western blot. The expressions of Hif-1α from rat primary cardiomyocytes isolated from hypoxia-treated rats (right) were analyzed by Western blot. β-actin was the internal control. The relative expressions of Hif-1 α were quantitated using ImageJ software. (B) H9c2 cells were treated with hypoxia for 0, 12, 24, 48, or 72 h. Oxygen consumption was analyzed. (C) H9c2 cells were treated with or without low glucose condition for 0, 12, 24, 48, or 72 h, cell viability was analyzed by MTT assay. (D) H9c2 cells were treated with or without low oxygen condition for 0, 12, 24, 48 or 72 h, cell viability was analyzed by MTT assay. (E) H9c2 cells were treated with normoxia, hypoxia alone or low glucose plus low oxygen conditions for 0, 12, 24, 48, or 72 h, cell viability was analyzed by MTT assay. Data are shown as the means ± S.E.M.; *P<0.05; **P<0.01; ***P<0.001.

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  Figure 2. Glucose metabolism in H9c2 cells are altered under hypoxic conditions H9c2 cells were treated with low oxygen condition for 0, 12, 24, 48, or 72 h, the glucose uptake (A) and lactate product (B) were measured. (C) IHC staining of Hexokinase 2 and LDHA in rat heart tissues under 0, 3, or 14 days’ hypoxia treatments. Data are shown as the means ± S.E.M.; *P<0.05; **P<0.01; ***P<0.001. IHC; immunohistochemistry

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  Figure 3. MiR-34a is adaptively regulated by hypoxia (A) H9c2 cells were treated with low oxygen conditions for 0, 12, 24, 48, or 72 h, the expressions of miR-34a were measured by qRT-PCR. (B) Rats were exposed to hypoxia using hypoxia chamber for 0, 3, or 14 days. Primary rat cardiomyocytes were isolated for measurement of miR-34a expressions by qRT-PCR. (C) H9c2 cells were treated with Oxamate for 48 h, followed by treatments with low oxygen conditions for 0, 48, or 72 h, the expressions of miR-34a were measured by qRT-PCR. (D) H9c2 cells were transfected with control siRNA or siLDHA for 48 h, followed by the treatments with low oxygen condition for 0, 48, or 72 h, the expressions of miR-34a were measured by qRT-PCR. The expressions of LDHA were measured by Western blot. U6 and β-actin was used as internal controls. Data are shown as the means ± S.E.M.; *P<0.05; **P<0.01; ***P<0.001.

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  Figure 4. MiR-34a suppresses glucose metabolism in cardiomyocytes (A) H9c2 cells were transfected with control mimic or miR-34a mimic for 48 h, the expressions of miR-34a were measured by qRT-PCR. (B) H9c2 cells were transfected with control mimic or miR-34a mimic for 48 h, the glucose uptake and (C) lactate product were measured. (D) H9c2 cells were transfected with control inhibitor or miR-34a inhibitor for 48 h, the expressions of miR-34a were measured by qRT-PCR. (E) H9c2 cells were transfected with control inhibitor or miR-34a inhibitor for 48 h, the glucose uptake and (F) lactate product were measured. Data are shown as the means ± S.E.M.; *P<0.05; **P<0.01; ***P<0.001.

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  Figure 5. MiR-34a directly targets LDHA in cardiomyocytes (A) Potential miR-34a target was predicted from TargetScan.org. The position 406–413 of LDHA 3′-UTR contains putative binding sites for miR-34a. (B) Conserved miR-34a binding sites on LDHA 3′-UTR in multiple species. (C) H9c2 cells were transfected with 50 nM control mimic or miR-34a mimic for 48 h. Cell lysates were prepared for Western blotting analysis. β-actin was used as a loading control. (D) H9c2 cells were cotransfected with control mimic or miR-34a mimic with luciferase reporter plasmids with wild-type 3′-UTR or mutant 3′-UTR of LDHA using Lipofectamine 2000 reagent for 48 h. Cells were harvested and the luciferase activities were measured by a dual luciferase reporter assay. Results are expressed as relative LUC activity (firefly LUC/Renilla LUC). (E) IHC staining of LDHA in rat heart tissues with low or high miR-34a expression. (F) The correlation analysis of LDHA mRNAs and miR-34a from 15 rat heart tissues. Experiments were performed in triplicate. Data are shown as the means ± S.E.M.; **P<0.01.

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  Figure 6. Inhibition of miR-34a attenuates the hypoxia-induced cardiomyocytes dysfunction through restoration of glucose metabolism (A) H9c2 cells were transfected with control inhibitor or miR-34a inhibitor for 48 h, cells were treated with hypoxia at 0, 48, or 72 h. The glucose uptake, (B) lactate product, and (C) cell viability were measured. (D) H9c2 cells were transfected with control siRNA, miR-34a inhibitor, or miR-34a inhibitor plus siLDHA for 48 h, followed by the detection of LDHA expression by Western blot. β-actin was the loading control. (E) H9c2 cells were transfected with miR-34a inhibitor or miR-34a inhibitor plus siLDHA for 48 h, cells were then exposed to hypoxia for 0, 48, or 72 h. Cell viability was measured by MTT assay. Data are shown as the means ± S.E.M.; *P<0.05; **P<0.01; ***P<0.001.