Nuclear Lactate Dehydrogenase A Resists Cardiomyocyte Cell Cycle Arrest Induced by Oxidative Stress

Figures

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  Figure 1. Nuclear LDHA is increased in the remaining cardiomyocytes after MI injury. C57BL/6 J mice MI models were constructed via permanent ligation of the left anterior descending branch. TTC staining (A) and serum LDH detection (B) were performed to verify cardiac damage. Heart cross-sections were stained with Masson’s trichrome (scale bar = 200 µm) to examine fibrosis and stained with H&E (scale bar = 50 µm) to detect the infiltration of inflammatory cells (C). IF staining of Ki67 (red, scale bar = 50 µm) and LDHA (red, scale bar = 20 µm) were performed to observe the cell cycle re-entry of cardiomyocytes (green) and nuclear translocation of LDHA in the infarct boundary region after MI injury, and white arrows indicate double-stained positive cardiomyocytes (D). The level of ROS production and oxidative damage to DNA were detected by DHE staining (scale bar = 50 µm) and 8-OHdG fluorescence probe (scale bar = 50 µm), respectively (E). Data are expressed as mean ± SEM, and compared by Student’s t-test. **** p < 0.0001 (n = 6).

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  Figure 2. Effects of hypoxia on cardiomyocyte proliferation and glycolysis in vitro. Primary mouse cardiomyocytes were obtained from C57BL/6 J mice (1–2 days). Cardiomyocytes were exposed to hypoxia (10%, 7%, 5%, 3%, and 1%) for 12 h. The effects of hypoxia on glycolysis were evaluated by western blot analysis of GLUT1 and HK2, while Ki67, p-H3, Aurora B, and PCNA were used to evaluate the proliferation potential of cardiomyocytes. Histograms represent protein ratios normalized to β-actin (A). Double IF staining of cTnT and EDU further confirmed cardiomyocyte cell cycle re-entry (scale bar = 50 µm), and white arrows indicate double-stained positive cardiomyocytes (B). For glycolysis inhibition, cells were incubated with 2-DG (5 mM). The western blot detected the indicators of cardiomyocyte proliferation (p-H3 and PCNA) under 5% oxygen concentration along with 2-DG treatment. Histograms represent protein ratios normalized to β-actin (C). Data are expressed as mean ± SEM. Differences between two groups were analyzed with Student’s t-test. One-way ANOVA was used to analyze the multiple groups. *** p < 0.001, **** p < 0.0001 (n = 6).

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  Figure 3. Nuclear LDHA is upregulated in response to severe hypoxia-induced oxidative damage of cardiomyocytes. Cardiomyocytes were exposed to hypoxia (10%, 5%, and 1%) for 12 h. ROS production in cardiomyocytes was detected by DHE staining (scale bar = 50 µm) (A). The redox state in cardiomyocytes under hypoxia was assessed via the GSH/GSSG ratio (B). The localization and quantification of LDHA in cardiomyocytes were detected by double IF staining of cTnT and LDHA (scale bar = 20 µm) (C). Data are expressed as mean ± SEM. Differences between two groups were analyzed with Student’s t-test. One-way ANOVA was used to analyze the multiple groups. *** p < 0.001, **** p < 0.0001 (n = 6).

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  Figure 4. NAC treatment promotes cardiomyocyte cell cycle re-entry while preventing LDHA nuclear translocation. Cardiomyocytes were pre-treated with NAC (5 mM) for 4 h, and then exposed to 1% oxygen concentration for 12 h. ROS production in cardiomyocytes was detected by DHE staining (scale bar = 50 µm) (A). Western blot detected the indicators of cardiomyocyte proliferation (p-H3 and PCNA). Histograms represent protein ratios normalized to β-actin (B). Double IF staining of Cardiac Troponin T (cTnT) and Ki67 further confirmed cardiomyocyte cell cycle re-entry (scale bar = 100 µm), and white arrows indicate double-stained positive cardiomyocytes (C). The effects of NAC treatment on total LDHA and nuclear LDHA in cardiomyocytes under severe hypoxia were evaluated via western blot (D). Data are expressed as mean ± SEM, and compared by Student’s t-test. **** p < 0.0001, no significant difference (ns, p > 0.05) (n = 6).

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  Figure 5. ROS represses mild hypoxia-induced cardiomyocyte cell cycle re-entry, accompanied by LDHA nuclear translocation. Berberine chloride is an alkaloid that induces ROS generation. Cardiomyocytes were pre-stimulated with Berberine chloride (10 µM) for 12 h, and then exposed to 5% oxygen concentration for another 12 h. ROS production in cardiomyocytes was detected by DHE staining (scale bar = 50 µm) (A). Western blot detected the indicators of cardiomyocyte proliferation (p-H3 and PCNA). Histograms represent protein ratios normalized to β-actin (B). Double IF staining of cTnT and p-H3 further confirmed cardiomyocyte cell cycle re-entry (scale bar = 50 µm), and white arrows indicate double-stained positive cardiomyocytes (C). The effects of ROS on total LDHA and nuclear LDHA in cardiomyocytes under mild hypoxia were evaluated via western blot (D). Data are expressed as mean ± SEM, and compared by Student’s t-test. *** p < 0.001, **** p < 0.0001, no significant difference (ns, p > 0.05) (n = 6).

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  Figure 6. Inhibition of LDHA nuclear translocation aggravates oxidative stress and cardiomyocyte cell cycle arrest under severe hypoxia. For LDHA nuclear translocation inhibition, cardiomyocytes were pre-treated with importazole (40 µM) for 2 h, and then exposed to a 1% oxygen concentration for 12 h. Western blot confirmed the inhibition of LDHA nuclear translocation mediated by importazole (A). The effect of nuclear LDHA suppression on oxidative stress was detected by DHE staining (scale bar = 50 µm) (B). Real-time PCR analysis was performed to evaluate the expression of antioxidant genes (C). The western blot detected the indicators of cardiomyocyte proliferation (p-H3 and PCNA). Histograms represent protein ratios normalized to β-actin (D). Double IF staining of cTnT and EDU further confirmed cardiomyocyte cell cycle re-entry (scale bar = 50 µm), and white arrows indicate double-stained positive cardiomyocytes (E). Data are expressed as mean ± SEM, and compared by Student’s t-test. **** p < 0.0001, no significant difference (ns, p > 0.05) (n = 6).