Altered placental ion channel gene expression in preeclamptic high-altitude pregnancies

Abstract

High-altitude (>2,500 m) residence increases the risk of pregnancy vascular disorders such as fetal growth restriction and preeclampsia, each characterized by impaired placental function. Genetic attributes of highland ancestry confer relative protection against vascular disorders of pregnancy at high altitudes. Although ion channels have been implicated in placental function regulation, neither their expression in high-altitude placentas nor their relationship to high-altitude preeclampsia has been determined. Here, we measured the expression of 26 ion-channel genes in placentas from preeclampsia cases and normotensive controls in La Paz, Bolivia (3,850 m). In addition, we correlated gene transcription to maternal and infant ancestry proportions. Gene expression was assessed by PCR, genetic ancestry evaluated by ADMIXTURE, and ion channel proteins localized by immunofluorescence. In preeclamptic placentas, 11 genes were downregulated (ABCC9, ATP2A2, CACNA1C, KCNE1, KCNJ8, KCNK3, KCNMA1, KCNQ1, KCNQ4, PKD2, and TRPV6) and two were upregulated (KCNQ3 and SCNN1G). KCNE1 expression was positively correlated with high-altitude Amerindian ancestry and negatively correlated with non-high altitude. SCNN1G was negatively correlated with African ancestry, despite minimal African admixture. Most ion channels were localized in syncytiotrophoblasts (Cav1.2, TRPP2, TRPV6, and Kv7.1), whereas expression of Kv7.4 was primarily in microvillous membranes, Kir6.1 in chorionic plate and fetal vessels, and MinK in stromal cells. Our findings suggest a role for differential placental ion channel expression in the development of preeclampsia. Functional studies are needed to determine processes affected by these ion channels in the placenta and whether therapies directed at modulating their activity could influence the onset or severity of preeclampsia.

Keywords: ancestry; high altitude; ion channel; placenta; preeclampsia.

Graphical abstract

Figure 1.

mRNA expression of Ca²⁺-activated, ATP-sensitive and voltage-gated K⁺ channel families in placentas from normotensive and preeclamptic women. mRNA expression of large-conductance Ca²⁺-activated α and β1 subunits (KCNMA1 and KCNMB1), intermediate-conductance Ca²⁺-activated (KCNN4), ATP-sensitive Kir6.1, Kir6.2 and sulfonylurea receptor (KCNJ8, KCNJ11 and ABCC9) K⁺ channels (A), voltage-gated Kv7.1, Kv7.3, Kv7.4, and Kv7.5, regulatory subunits MinK and MirP4 (KCNQ1, KCNQ3, KCNQ4, KCNQ5, KCNE1, and KCNE5), Kv subtype 9.3 (KCNS3) and two-pore domain (KCNK3) K⁺ channels (B) in placentas from normotensive controls (Norm, black circles) and preeclamptic (PE, gray circles) women. Data are expressed as ΔC_t values. Each circle represents individual values. Error bars are means ± SD. Statistical significance was determined by unpaired Student’s t test or Mann–Whitney U test and annotated as follows: *P < 0.05 and **P < 0.01. A secondary analysis to determine mRNA expression differences by PE status after adjusting for variation in gestational age and infant sex was performed using univariate general linear models with significance indicated as follows: †P < 0.05.

Figure 2.

mRNA expression of Na⁺ and Ca²⁺ channels, transient receptor potential, mechanosensitive and Cl^- channels in placentas from normotensive and preeclamptic women. mRNA expression of endothelial Na⁺ channels subtypes α, β and γ (SNN1A, SNN1B, and SNN1G), voltage-gated Ca²⁺ channel subtype 1.2 (CACNA1C), sarcoplasmic reticulum Ca²⁺ ATPase (ATP2A2) and plasma membrane Ca²⁺ ATPase (ATP2B1) (A), and transient receptor potential vanilloid subtypes 1, 5, and 6 (TRPV1, TRPV5, and TRPV6), transient receptor potential polycystic 2 (PKD2), piezo type mechanosensitive component 1 (PIEZO1), and Cl^- intracellular channel 3 (CLIC3) (B) in placentas from normotensive controls (Norm, black circles) and preeclamptic (PE, gray circles) women, expressed as ΔC_t values. Each circle represents individual values. Error bars are means ± SD. Statistical significance was determined by unpaired Student’s t test or Mann–Whitney U test and annotated as follows: *P < 0.05 and **P < 0.01. A secondary analysis to determine mRNA expression differences by PE status after adjusting for variation in gestational age and infant sex was performed using univariate general linear models with significance indicated as follows: †P < 0.05.

Figure 3.

Immunofluorescence detection of Ca²⁺-permeable channels in placentas from normotensive and preeclamptic women at high altitude. Placentas from normotensive or preeclamptic women were co-stained for the endothelial cell marker (CD-31, red) and either Cav1.2 (A and B, green), TRPP2 (C and D, green) or TRPV6 (E and F, green). Nuclei were stained with DAPI (blue). CPv, chorionic plate vessels; STB, syncytiotrophoblast; Fc, fetal capillaries. Bars are 50 µm.

Figure 4.

Immunofluorescence detection of K⁺ channels in placentas from normotensive and preeclamptic women at high altitude. Placentas from normotensive or preeclamptic women were co-stained for the endothelial cell marker (CD-31, red) and either Kir6.1 (A and B, green), Kv7.1 (C and D, green), or Kv7.4 (E and F, green). Nuclei were stained with DAPI (blue). CPv, chorionic plate vessels; Fc, fetal capillaries; STB, syncytiotrophoblast; MVM, microvillous membrane. Bars are 50 µm.

Figure 5.

Immunofluorescence detection of MinK subunit in placentas from normotensive and preeclamptic women at high altitudes. Placentas from normotensive (A and C) or preeclamptic (B and D) women were co-stained for the endothelial cell marker (CD-31, red, A and B) or fibroblast marker (FSP1, red, C and D) and MinK (green). Nuclei were stained with DAPI (blue). Orange arrows in indicate MinK-positive stromal cell staining. CPv, chorionic plate vessels; STB, syncytiotrophoblast. Bars in A and B are 50 µm, bars in C and D are 10 µm.