Role of DNA methylation in perinatal nicotine-induced development of heart ischemia-sensitive phenotype in rat offspring

Abstract

Background and purpose: Maternal cigarette smoking increases the risk of cardiovascular disease in offspring. Recently, we have demonstrated that perinatal nicotine exposure alters heart development and increases heart susceptibility to ischemia/reperfusion (I/R) injury in rat offspring. The present study tested the hypothesis that DNA methylation plays a key role in the nicotine-induced development of heart ischemia-sensitive phenotype in offspring.

Experimental approach: Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from gestational day 4 until postnatal day 10. After birth, the postnatal offspring were treated with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza) or saline from postnatal day 3 to day 10. Experiments were conducted in 1 month old offspring.

Key results: Perinatal nicotine increased I/R-induced left ventricular (LV) injury, and decreased post-ischemic recovery of the LV function and coronary flow rate in both male and female offspring. Nicotine differentially increased DNMT3a expression and global DNA methylation levels in LV tissues. Treatment with 5-Aza inhibited nicotine-induced an increase in DNMT3a and global DNA methylation, and blocked the nicotine-induced increase in I/R injury and dysfunction in the heart. In addition, nicotine attenuated protein kinases C_ε and large-conductance Ca(2+)-activated K(+) (BKca) channel β1 subunit protein abundances in the heart, which were reversed by 5-Aza treatment.

Conclusions and implications: The present findings provide novel evidence that the increased DNA methylation plays a causal role in nicotine-induced development of heart ischemic sensitive phenotype, and suggest a potential therapeutic target of DNA demethylation for the fetal programming of heart ischemic disease later in life.

Keywords: DNA methylation; heart ischemia-sensitive phenotype; perinatal nicotine.

Figure 1. 5-Aza reversed perinatal nicotine-induced decrease in post-ischemic recovery of LV function in male offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, LVDP (A, E), dp/dt_max (B, F), dp/dt_min (C, G), CF (D, H) of isolated hearts were determined after subjected to 30 min of ischemia and 30 min of reperfusion in Langendorff apparatus. Data are means ± SEM (n = 5-7 animals/group from different litter). * P < 0.05 vs control.

Figure 2. 5-Aza reversed perinatal nicotine-induced decrease in post-ischemic recovery of LV function in female offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, LVDP (A, E), dp/dt_max (B, F), dp/dt_min (C, G), CF (D, H) of isolated hearts were determined after subjected to 30 min of ischemia and 30 min of reperfusion in Langendorff apparatus. Data are means ± SEM (n = 5-6 animals/group from different litter). * P < 0.05 vs control.

Figure 3. 5-Aza prevented perinatal nicotine-induced increase in myocardial infarction in offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, LVEDP of isolated hearts in the absence (A, D) or presence (B, E) of 5-Aza were determined after subjected to 30 min of ischemia and 30 min of reperfusion in Langendorff apparatus, and infarct size (C, F) of isolated hearts was determined by TTC staining. Data are means ± SEM (n = 5-6 animals/group from different litter). * P < 0.05 vs control.

Figure 4. 5-Aza abrogated perinatal nicotine-induced heightened global DNA methylation levels in the LV tissues of male offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, the global DNA methylation was qualified by 5-mC ELISA in the left ventricle tissues isolated from male offspring. Data are means ± SEM (n = 4-6 animals/group from different litter). * P < 0.05 vs control. ^# P < 0.05 vs -5-Aza.

Figure 5. 5-Aza restored perinatal nicotine-induced increase in the expression of DNMT3a in the LV tissues of male offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, the protein abundances of DNMT1 (A, D), DNMT3a (B, E), and DNMT3b (C, F) were determined in the left ventricle tissues isolated from male offspring. Data are means ± SEM (n = 4 animals/group from different litter). * P < 0.05 vs control.

Figure 6. 5-Aza restored perinatal nicotine-induced repression of PKCε in the LV tissues of male offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, the protein abundance of PKCε in the absence (A) and in the presence (B) of 5-Aza treatment was determined in the left ventricle tissues isolated from male offspring. Data are means ± SEM (n = 4 animals/group from different litter). * P < 0.05 vs control.

Figure 7. 5-Aza abolished perinatal nicotine-induced decrease in the expression of BKca channel β1 subunit in the LV tissues of male offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, the protein abundances of BKca channel β1 subunit (B, D) and α subunit (A, C) were determined in the left ventricle tissues isolated from male offspring. Data are means ± SEM (n = 4 animals/group from different litter). * P < 0.05 vs control.

Figure 8. Localization of BKca channel β1 subunit in the LV tissues of male offspring

Immunofluorescence staining of heart section with α-sarcomeric actin (A, green), α-smooth muscle actin (C, green), BKca channel β1 subunit (B and D, red). Nuclei were stained with DAPI (A and C, blue). BKca channel β1 subunit was detected in the coronary vascular walls (B, D).

Figure 9. Effects of 5-Aza on perinatal nicotine-mediated changes of caspase activities in the LV tissues of male offspring

Pregnant rats were treated with saline (control) or nicotine, and pups were administered with 5-Aza (i.p. 1mg/kg/day) on postnatal day 3, 7 and 10 after delivered. At one month of age, caspase activities (A, B, C) were determined in the left ventricle tissues isolated from male offspring. Data are means ± SEM (n = 4-7 animals/group from different litter). * P < 0.05 vs control.