Chronic hypoxia upregulates DNA methyltransferase and represses large conductance Ca2+-activated K+ channel function in ovine uterine arteries

Abstract

Chronic hypoxia during gestation suppresses large-conductance Ca2+-activated K+ (BKCa) channel function and impedes uterine arterial adaptation to pregnancy. This study tested the hypothesis that chronic hypoxia has a direct effect in upregulating DNA methyltransferase (DNMT) and epigenetically repressing BKCa channel beta-1 subunit (KCNMB1) expression in uterine arteries. Resistance-sized uterine arteries were isolated from near-term pregnant sheep maintained at ∼300 m above sea level or animals acclimatized to high-altitude (3,801 m) hypoxia for 110 days during gestation. For ex vivo hypoxia treatment, uterine arteries from normoxic animals were treated with 21.0% O2 or 10.5% O2 for 48 h. High-altitude hypoxia significantly upregulated DNMT3b expression and enzyme activity in uterine arteries. Similarly, ex vivo hypoxia treatment upregulated DNMT3b expression and enzyme activity that was blocked by a DNMT inhibitor 5-aza-2'-deoxycytidine (5-Aza). Of importance, 5-Aza inhibited hypoxia-induced hypermethylation of specificity protein (SP) 1 binding site at the KCNMB1 promoter and restored transcription factor binding to the KCNMB1 promoter, resulting in the recovery of KCNMB1 gene expression in uterine arteries. Furthermore, 5-Aza blocked the effect of hypoxia and rescued BKCa channel activity and reversed hypoxia-induced decrease in BKCa channel-mediated relaxations and increase in myogenic tone of uterine arteries. Collectively, these results suggest that chronic hypoxia during gestation upregulates DNMT expression and activity, resulting in hypermethylation and repression of KCNMB1 gene and BKCa channel function, impeding uterine arterial adaptation to pregnancy.

Keywords: hypoxia; DNA methylation; BKCa channel; myogenic tone; uterine arteries.

Figure 1.

High-altitude hypoxia increased DNMT expression and activity in uterine arteries. Uterine arteries were isolated from low-altitude (control) and high-altitude pregnant sheep. (A) DNMTs protein abundance determined with western blot. (B) DNMT activity determined with a DNMT activity assay kit. Data are means ± SEM from five animals of each group. *P < 0.05, high altitude vs. control.

Figure 2.

Ex vivo hypoxic treatment increased DNMT expression and activity in uterine arteries. Uterine arteries were isolated from low-altitude pregnant animals and were treated ex vivo with 21.0% O₂ (control) and 10.5% O₂ (hypoxia) for 48 h in the absence or presence of 5-Aza (10.0 μM). (A) DNMTs protein abundance determined with western blot. (B) DNMT activity determined with a DNMT activity assay kit. Data are mean ± SEM from five animals of each group. *P < 0.05, hypoxia vs. control. ^#P < 0.05, +5-Aza vs. –5-Aza.

Figure 3.

5-Aza blocked hypoxia-induced hypermethylation of KCNMB1 promoter in uterine arteries. Uterine arteries were isolated from low-altitude pregnant animals and were treated ex vivo with 21.0% O₂ (control) and 10.5% O₂ (hypoxia) for 48 h in the absence or presence of 5-Aza (10.0 μM). (A) Methylation of Sp1_-380 binding site at the KCNMB1 promoter determined with quantitative methylation-specific PCR. (B) ERα and Sp1 binding to Sp1_–380 site determined with ChIP assays. Data are mean ± SEM from five and six animals for quantitative methylation-specific PCR and ChIP, respectively. *P < 0.05, hypoxia vs. control.

Figure 4.

5-Aza restored hypoxia-induced KCNMB1 gene repression in uterine arteries. Uterine arteries were isolated from low-altitude pregnant animals and were treated ex vivo with 21.0% O₂ (control) and 10.5% O₂ (hypoxia) for 48 h in the absence or presence of 5-Aza (10.0 μM). (A) BK_Ca channel beta-1 subunit (BKβ1) mRNA abundance determined with real-time RT-PCR. (B) BK_Ca channel beta-1 subunit (BKβ1) protein abundance determined with western blot. Data are means ± SEM from six and five animals for mRNA and protein measurements, respectively. *P < 0.05, hypoxia vs. control.

Figure 5.

5-Aza recovered hypoxia-induced reduction of BK_Ca channel activity in uterine arteries. Uterine arteries were isolated from low-altitude pregnant animals and were treated ex vivo with 21.0% O₂ and 10.5% O₂ for 48 h in the absence (A) or presence (B) of 5-Aza (10.0 μM). Arterial myocytes were freshly isolated from uterine arteries, and BK_Ca channel current density was determined in the absence and presence of tetraethylammonium (TEA, 1.0 mM) as described in Materials and Methods. Data are mean ± SEM of 7 to 10 cells from five animals of each group. *P < 0.05, 10.5% O₂ vs. 21.0% O₂.

Figure 6.

5-Aza reversed hypoxia-induced reduction of BK_Ca channel-mediated relaxations of uterine arteries. Uterine arteries were isolated from low-altitude pregnant animals and were treated ex vivo with 21.0% O₂ and 10.5% O₂ for 48 h in the absence (A) or presence (B) of 5-Aza (10.0 μM). Uterine arteries were contracted with serotonin (1 μM) followed by additions of NS1619. Data are mean ± SEM of tissues from six to eight animals of each group. *P < 0.05, 10.5% O₂ vs. 21.0% O₂.

Figure 7.

5-Aza inhibited hypoxia-induced increase in pressure-dependent myogenic tone of uterine arteries. Uterine arteries were isolated from low-altitude pregnant animals and were treated ex vivo with 21.0% O₂ and 10.5% O₂ for 48 h in the absence (A) or presence (B) of 5-Aza (10.0 μM). Pressure-dependent myogenic tone was determined. Data are mean ± SEM of tissues from five animals of each group. *P < 0.05, 10.5% O₂ vs. 21.0% O₂.