Epigenetic Down-Regulation of Sirt 1 via DNA Methylation and Oxidative Stress Signaling Contributes to the Gestational Diabetes Mellitus-Induced Fetal Programming of Heart Ischemia-Sensitive Phenotype in Late Life

Abstract

Rationale: The incidence of gestational diabetes mellitus (GDM) is increasing worldwide. However, whether and how GDM exposure induces fetal programming of adult cardiac dysfunctional phenotype, especially the underlying epigenetic molecular mechanisms and theranostics remain unclear. To address this problem, we developed a late GDM rat model. Methods: Pregnant rats were made diabetic on day 12 of gestation by streptozotocin (STZ). Experiments were conducted in 6 weeks old offspring. Results: There were significant increases in ischemia-induced cardiac infarction and gender-dependent left ventricular (LV) dysfunction in male offspring in GDM group as compared to controls. Exposure to GDM enhanced ROS level and caused a global DNA methylation in offspring cardiomyocytes. GDM attenuated cardiac Sirt 1 protein and p-Akt/Akt levels, but enhanced autophagy-related proteins expression (Atg 5 and LC3 II/LC3 I) as compared to controls. Ex-vivo treatment of DNA methylation inhibitor, 5-Aza directly inhibited Dnmt3A and enhanced Sirt 1 protein expression in fetal hearts. Furthermore, treatment with antioxidant, N-acetyl-cysteine (NAC) in offspring reversed GDM-mediated DNA hypermethylation, Sirt1 repression and autophagy-related gene protein overexpression in the hearts, and rescued GDM-induced deterioration in heart ischemic injury and LV dysfunction. Conclusion: Our data indicated that exposure to GDM induced offspring cardiac oxidative stress and DNA hypermethylation, resulting in an epigenetic down-regulation of Sirt1 gene and aberrant development of heart ischemia-sensitive phenotype, which suggests that Sirt 1-mediated signaling is the potential therapeutic target for the heart ischemic disease in offspring.

Keywords: DNA methylation; GDM; ROS; Sirt 1; heart ischemia-sensitive phenotype.

Figure 1

Effect of GDM on body weight and blood glucose in pregnant rats and their offspring. Pregnant rats were administered with either saline or STZ with 50 mg/kg on 12^th day of gestation. A) Showing the daily blood glucose values in pregnant dams with diabetes induced by injection of STZ (●) on day 12 of gestation and sham control (○). B) Showing the maternal body weight gain values after STZ treatment (●) and sham control (○). C) Showing the blood glucose levels in both male and female offspring of STZ-treated (■) and control (□) groups at 6 weeks-old. D) Showing the body weights in both male and female offspring of STZ-treated (■) and control (□) groups at 6 weeks-old. E) Showing the heart weights in both male and female offspring of STZ-treated (■) and control (□) groups at 6 weeks-old. F) Showing the heart weight (HW)/body weight (BW) ratio in both male and female offspring of STZ-treated (■) and control (□) groups at 6 weeks-old. Values are means ± SE, *p < 0.05 compared to control.

Figure 2

Effect of GDM exposure on ischemia-induced myocardial infarction in offspring. Offspring from each group were subjected heart ischemia as indicated in the Methods Section. 24 hours after heart ischemia, the hearts were isolated and their infarct sizes were determined with 2% TTC staining (A). The bar graph (B) showing percent of left ventricle infarct size (infarct area/risk area x100%) in each offspring group. Data are means ± SEM of animals from each group (n=6-5 in male offspring, n=5-7 in female offspring) were analyzed by 2-way ANOVA. *P < 0.05 versus.

Figure 3

Effect of GDM on heart function in offspring. The echocardiographic data of both the control and STZ exposed groups were examined at 7 days after heart ischemia, as described under Methods Section. (A) A representative echocardiography shows the measurement of LVSd, LVEDd, LVPWd, LVSs, and LVPWs. (B) Percent of ejection fraction (EF), (C) Percent of fractional shortening (FS), and (D) Stroke volume (SV) in both male and female offspring of STZ-treated (■) and control (□) groups at 7^th day after heart ischemia. Data are means ± SEM of animals from each group (n=9-12 in male offspring, n=14-7 in female offspring) were analyzed by 2-way ANOVA. *P < 0.05 versus control.

Figure 4

Effect of GDM on the ROS level in offspring. Heart tissues were isolated from male offspring. (A) ROS levels in the left ventricle (LV) tissues isolated from both control (□) and STZ-treated (■) groups were measured using in vitro ROS/RNS assay kit. (B) NOX1, 2, and 4 protein abundances in the LV tissues isolated from both control (□) and STZ-treated (■) groups were determined by Western blot analysis. Their protein densities were normalized to internal control (GAPDH). (C) After NAC pretreatment, ROS levels in the LV tissues isolated from both control (□) and STZ-treated (■) groups were measured using in vitro ROS/RNS assay kit. Data are means ± SEM (n=4 animals/group). *P < 0.05 vs. control, as determined by Student's t-test.

Figure 5

Effect of GDM on the protein expression of Sirt1 and p-Akt/Akt in male offspring. The hearts were isolated from both control (□) and STZ-treated (■) groups of male offspring. The protein abundance in the LV tissue was determined by Western blot analysis. The Sirt1 protein density (A) and P-Akt/Akt protein density (B) in the LV tissues of the groups which had not treated with NAC. The Sirt1 protein density (C) and P-Akt/Akt protein density (D) in the LV tissues of the groups which had treated with NAC. Data are means ± SEM (n=4 animals/group). *P < 0.05 vs. control, as determined by Student's t-test.

Figure 6

Effect of GDM on autophagy-related protein expression in male offspring. The hearts were isolated from both control (□) and STZ-treated (■) groups of male offspring. The protein abundance in the LV tissue was determined by Western blot analysis. The LC3B-I/II protein density (A), Atg5 protein density (B), and Beclin 1 protein density (C) in the LV tissues of the groups which had not treated with NAC. The LC3B-I/II protein density (D), Atg5 protein density (E), and Beclin 1 protein density (F) in the LV tissues of the groups which had treated with NAC. Data are means ± SEM (n=4 animals/group). *P < 0.05 vs. control, as determined by Student's t-test.

Figure 7

Effect of GDM on the global DNA methylation and DNA methyltransferase (DNMT) protein expression in male offspring. The hearts were isolated from both control (□) and STZ-treated (■) groups of male offspring. Global DNA methylation levels in LV tissues of male offspring, which were without (A) or with (C) NAC treatment, were measured using in 5-mC DNA ELISA kit. The protein abundances of DNMTs in the left ventricle tissues of male offspring, which were without (B) or with (D) NAC treatment, were determined by Western blot analysis. Data are means ± SEM (n=4 animals/group). *P < 0.05 vs. control, as determined by Student's t-test.

Figure 8

Effect of DNA methylation inhibitor on Sirt 1 protein expression in fetal hearts ex vivo. Fetal rat hearts were isolated from gestational day 19 and were cultured in an incubator. After 48 hours of treatment with DNA methylation inhibitor, 5-aza-2'-deoxycytodine (5-Aza, 10 μM) (■) or control vehicle (□). The protein levels of DNMT3a (A) and Sirt 1 (B) in the fetal hearts were determined by Western blot analysis. Data are means ± SEM (n=6 hearts/group). *P < 0.05 vs. control, as determined by Student's t-test.