Reprogramming of miR-181a/DNA methylation patterns contribute to the maternal nicotine exposure-induced fetal programming of cardiac ischemia-sensitive phenotype in postnatal life

Figure 1. Effects of PNE on heart function and ischemia-reperfusion induced heart infarct size. Rats were administered with either saline or nicotine from gestation day 4 until postnatal day 10. The offspring at 3-month-old age from each group were subjected to 45 min of heart ischemia followed by reperfusion. Echocardiographic analysis was obtained from different time periods including baseline, 1 day before I/R, 1 day after I/R, and 7 days after I/R. (A) left ventricular end-diastolic dimension (LVEDD), (B) left ventricular end-systolic dimension (LVESD), (C) percentage of ejection fraction (EF%), (D) percentage of fractional shortening (FS%), (E) representative echocardiograph evaluation of cardiac function from each group (n=3~10 animals/group), *P < 0.05 versus saline control group, as determined by Student's t-test. For the infarction size, the hearts of rats were isolated 24 h after I/R and their infarct sizes in each rat group were determined with 2% TTC staining (F), the graph showing the percentage of left ventricle infarct size (infarct area/ left ventricle area x100%) in each offspring group (n=4~5 animals/group). *P < 0.05 versus saline control group, as determined by…

Figure 2. Effects of PNE on miR-181a and H19 levels in offspring rats. The total RNA samples were isolated from left ventricle tissues of the male offspring from each group and the cardiac miR-181a and H19 levels were determined by qRT-PCR analysis as described in the Material and Methods. (A) miR-181a levels of each group (n=6 animals/group), (B) H19 levels of each group (n=7 animals/group). Data are means ± SEM. The expression levels of miR-181a are normalized by SNOTD61. *P < 0.05 versus saline control group, as determined by Student's t-test and the expression levels of H19 are normalized by GAPDH. *P < 0.05 versus saline control group, #P <0.05 vs. without LNA-miR-181a treatment, as determined by two-way ANOVA test.

Figure 3. Effects of PNE on heart function and ischemia-reperfusion induced heart infarct size after treatment of LNA-miR-181a. Rats were administered with either saline or nicotine from gestation day 4 until postnatal day 10. After treatment with LNA-miR-181a, the offspring at 3-month-old age from each group were subjected to 45 min of heart ischemia followed by reperfusion. Echocardiographic analysis was obtained from different time periods including baseline, 1 day before I/R, 1 day after I/R, and 7 days after I/R. (A) left ventricular end-diastolic dimension (LVEDD), (B) left ventricular end-systolic dimension (LVESD), (C) percentage of ejection fraction (EF%), (D) percentage of fractional shortening (FS%), (E) representative echocardiograph evaluation of cardiac function from each group (n=4~10 animals/group), *P < 0.05 versus saline control group, as determined by Student's t-test. For the infarction size, the hearts of rats were isolated 24 h after I/R and their infarct sizes in each rat group were determined with 2% TTC staining (F), the graph showing percent of left ventricle infarct size (infarct area/ left ventricle area x100%) in each offspring group (n=4 animals/group). *P <…

Figure 4. Effects of PNE on DNA methylation of AT1R and AT2R promoter in male offspring. Left ventricle tissues were freshly isolated from male offspring that had been perinatally exposed to saline and nicotine. DNA sample isolated and methylation levels were determined by methylation specific PCR analysis. (A) Methylation levels in AT1R promoter region. (B) Methylation levels in AT2R promoter region. Data are means ± SEM (n=4~5 animals/group). *P < 0.05 vs. control, #P <0.05 vs. without LNA-miR-181a treatment, as determined by two-way ANOVA test.

Figure 5. Effects of PNE on AT1R and AT2R mRNA and protein expression in male offspring. Left ventricle tissues were freshly isolated from male offspring that had been perinatally exposed to saline and nicotine. The cardiac mRNA levels of AT1R (A) and AT2R (B) were measured by qRT-PCR analysis, the mRNA expression levels of AT1R and AT2R are both normalized by GAPDH. *P < 0.05 vs. saline control group, #P <0.05 vs. without LNA, as determined by two-way ANOVA test. The cardiac protein levels of AT1R (C) and AT2R (D) were determined by Western blot analysis. Their protein densities are normalized as fold of GADPH density. Data are means ± SEM (n=4~5 animals/groups). *P < 0.05 vs. control, as determined by Student's t-test.

Figure 6. Effects of PNE on TGF-β and Smad protein expression in male offspring. Left ventricle tissues were freshly isolated from male offspring that had been perinatally exposed to saline and nicotine. The protein levels of TGF-β isoforms and Smads family were determined by Western blot analysis and were normalized as fold of GAPDH density. (A) TGF-β protein density in the absence of LNA-miR-181a, (B) TGF-β protein density in the presence of LNA-miR-181a, (C) Smads protein density in the absence of LNA-miR-181a, (D) Smads protein density in the presence of LNA-miR-181a. Data are means ± SEM (n=4~5 animals/groups). *P < 0.05 vs. control, as determined by Student's t-test.

Figure 7. Effects of PNE on autophagy protein expression in male offspring. Left ventricle tissues were freshly isolated from male offspring that had been perinatally exposed to saline and nicotine. The protein levels of autophagy-related genes were determined by Western blot analysis and were normalized as fold of GAPDH density. (A) Atg-5 protein density in the absence of LNA-miR-181a, (B) Atg-5 protein density in the presence of LNA-miR-181a, (C) Beclin-1 protein density of each group, (D) p62 protein density of each group, (E) LC3B-I and LC3B-II protein density in the absence of LNA-miR-181a, (F) LC3B-I and LC3B-II protein density in the presence of LNA-miR-181a. Data are means ± SEM (n=4~5 animals/groups). *P < 0.05 vs. control, as determined by Student's t-test.