High altitude differentially modulates potassium channel-evoked vasodilation in pregnant human myometrial arteries

Abstract

High-altitude (>2500 m or 8200 ft) residence reduces uterine artery blood flow during pregnancy, contributing to an increased incidence of preeclampsia and intrauterine growth restriction. However, not all pregnancies are affected by the chronic hypoxic conditions of high-altitude residence. K⁺ channels play important roles in the uterine vascular adaptation to pregnancy, promoting a reduction in myogenic tone and an increase in blood flow. We hypothesized that, in pregnancies with normal fetal growth at high altitude, K⁺ channel-dependent vasodilation of myometrial arteries is increased compared to those from healthy pregnant women at a lower altitude (~1700 m). Using pharmacological modulation of two K⁺ channels, ATP-sensitive (K_ATP) and large-conductance Ca²⁺-activated (BK_Ca) K⁺ channels, we assessed the vasodilation of myometrial arteries from AGA pregnancies in women living at high or low altitudes. In addition, we evaluated the localization of these channels in the myometrial arteries using immunofluorescence. Our results showed an endothelium-dependent increase in K_ATP-dependent vasodilation in myometrial arteries from high vs. low altitude, whereas vasodilation induced by BK_Ca activation was reduced in these vessels. Additionally, K_ATP channel co-localization with endothelial markers was reduced in the high-altitude myometrial arteries, which suggested that the functional increase in K_ATP activity may be by mechanisms other than regulation of channel localization. These observations highlight an important contribution of K⁺ channels to the human uterine vascular adaptation to pregnancy at high altitude serving to maintain normal fetal growth under conditions of chronic hypoxia.

Graphical Abstract

We assessed the role of BK_Ca and K_ATP channels in the vasodilation of myometrial arteries from healthy pregnant women residing at low (<1700 m) and high altitudes (>2500 m). High-altitude residence increased the sensitivity of K_ATP channels to the blocker glibenclamide in an endothelium dependent manner. Conversely, the sensitivity of the BK_Ca channel blocker TEA was reduced in myometrial arteries from high-altitude women in a smooth muscle-dependent manner. Our findings highlight a possible mechanism of maternal vascular adaptation to pregnancy under conditions of chronic hypoxia to maintain a healthy pregnancy.

INTRODUCTION

To sustain pregnancy, the maternal systemic and especially uteroplacental circulation undergoes significant changes that serve to raise uterine artery blood flow (Ford, 1982). Important contributors to this increase in uterine blood flow are the dilation and growth of maternal uterine vessels, including myometrial (MyoA), arcuate, and main uterine arteries (Palmer et al., 1992; Cipolla & Osol, 1994; Keyes et al., 1997; Mu & Adamson, 2006). Pregnancy complications such as preeclampsia and intrauterine growth restriction (IUGR) are characterized by a lesser rise in uterine artery blood flow (Konje et al., 2003), impaired uterine vasodilation (Ong et al., 2003; Sweeney et al., 2008; Lorca et al., 2020), and reduced vascular remodeling (Lyall et al., 2013). Studies have also found women residing at high altitude (HA, >2500 m) during pregnancy have a reduced rise in the uterine artery blood flow (Julian et al., 2008) and impaired acetylcholine vasodilatory responses in MyoA (Lorca et al., 2019) as compared to women from lower altitudes (LA, ~1700 m). Although HA residence increases the risk of developing preeclampsia and IUGR (Lichty et al., 1957; Jensen & Moore, 1997; Palmer et al., 1999), not all HA mothers develop preeclampsia, nor are all their babies born small. MyoA from IUGR pregnancies have reduced vasodilatory responses to bradykinin and acetylcholine (Ong et al., 2003; Lorca et al., 2020). Bradykinin and acetylcholine act mainly through triggering endothelial signaling pathways, such as nitric oxide and prostaglandins, which in turn promote smooth muscle dilation by increasing cyclic guanosine monophosphate levels and/or reducing intracellular Ca²⁺ (Nelson et al., 1998; Nelson et al., 2000; Lorca et al., 2019). In the smooth muscle cells, Ca²⁺ levels are regulated by a fine balance between Ca²⁺ entry, predominantly through voltage-gated L-type Ca²⁺ channels, and the release of Ca²⁺ from sarcoplasmic reticulum stores (Nelson et al., 1990). K⁺ channels are important regulators of vasoreactivity by reducing Ca²⁺ entry, enhancing vasodilatory responses, and opposing vasoconstriction (Nelson & Quayle, 1995).

Ca²⁺-activated (BK_Ca) and ATP-sensitive (K_ATP) K⁺ channels are expressed in vascular beds, where they hyperpolarize the plasma membrane, reducing intracellular Ca²⁺ levels and promoting vasodilation (Nelson & Quayle, 1995; Brayden, 2002; Korovkina & England, 2002). Both channels are key determinants of the vasodilatory responses in the uterine vasculature (Keyes et al., 1998; Rosenfeld et al., 2009; Xiao et al., 2010; Hu et al., 2011; Lorca et al., 2018). For instance, BK_Ca activity is increased in the uterine artery during ovine and mouse pregnancy to augment uterine artery diameter and blood flow (Rosenfeld et al., 2001; Hu et al., 2011; Lorca et al., 2018). Similarly, K_ATP channels stimulate uterine artery vasodilation during pregnancy (Keyes et al., 1998; Xiao et al., 2010).

BK_Ca and K_ATP channel vasodilatory responses are reduced by HA hypoxia in pregnant ovine uterine arteries, leading to increased myogenic tone (Xiao et al., 2010; Hu et al., 2012). However, the effect of HA on the K⁺ channel-dependent human MyoA vasodilation is unknown. Here we assessed the vasodilatory responses to BK_Ca and K_ATP channel openers in MyoA from HA or LA residents with appropriate for gestational age (AGA) pregnancies and whether HA-associated differences were due to endothelial or smooth muscle responses. We hypothesized that MyoA from healthy, AGA pregnancies at HA compared to those at LA had similar, or perhaps even increased, BK_Ca and K_ATP channel activity, contributing to the maintenance of a normal pregnancy rise in uterine artery blood flow and fetal growth under conditions of chronic hypoxia.

METHODS

Ethical approval.

Pregnant women scheduled for elective Cesarean delivery provided written informed consent before being included in the study. All studies were performed following the standards set by the Declaration of Helsinki and approved by the Catholic Health Initiative Institute for Research and Innovation Institutional Review Board (1310) and the University of Colorado Multiple Institutional Review Board (14–2178).

Subjects.

Subjects resided at 2797 ± 160 m (HA, Summit County and surrounding areas, CO, n = 17) or at 1692 ± 118 m (LA, Denver, CO, n = 21) for the duration of their pregnancy. LA subjects were recruited at the University of Colorado Hospital (Aurora, CO), and HA subjects were recruited at Centura Health High Country Healthcare (Frisco, CO). Inclusion criteria were maternal age 18–45 years, singleton AGA pregnancy (>10^th percentile weight at birth for gestational age and sex) (Kiserud et al., 2017), pre-pregnant body mass index less than 30 kg/m², absence of IUGR or preeclampsia diagnoses during the current pregnancy, no known risk factors for IUGR (i.e., diabetes, chronic hypertension, smoking), and no fetal congenital anomalies. Additionally, all women participating in the study were planning elective, non-laboring Cesarean delivery under spinal anesthesia at term (37–40 weeks of gestation) at the University of Colorado Hospital (Aurora, CO) or St. Anthony’s Summit Medical Center (Frisco, CO). Birth weight percentiles were calculated using World Health Organization’s fetal growth charts (Kiserud et al., 2017). Demographic data, health history, and obstetric information from all subjects were obtained from medical records and by questionnaire.

Sample collection.

After delivery of the baby and placenta, a full section of myometrial biopsy was obtained from the upper lip of the lower uterine segment. Myometrial tissue was immediately rinsed in ice-cold phosphate saline buffer (PBS, Thermo Fisher Scientific, Waltham, MA), a 0.5 cm³ section was fixed in 4% paraformaldehyde (PFA, Affymetrix, Cleveland, OH), and the rest stored in PBS at 4°C until experiments were performed as detailed below. HA specimens were stored in ice-cold PBS during the approximately two-hour transport to the University of Colorado Anschutz Medical Campus for study. Tissues obtained from the University of Colorado Hospital were stored in ice-cold PBS for an equivalent period. MyoA vasoreactivity is intact after such storage times (Eckman et al., 2012; Lorca et al., 2019; Lorca et al., 2020), and transport from HA to LA does not affect altitude-related changes in vasoreactivity (Chang et al., 2009).

Wire myography.

MyoA vascular responses were studied in a wire myograph as described previously (Lorca et al., 2019; Lorca et al., 2020). Briefly, isolated MyoA were mounted in a small-vessel wire myograph (Multi Wire Myograph System 610M, DMT-USA, Ann Arbor, MI), where they were normalized to an internal diameter of 0.9 of L_13.3kPa in a chamber containing oxygenated (95% O₂/5% CO₂) and warmed (37°C) Krebs buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, and 11 mM D-glucose). After at least 45 minutes of equilibration, MyoA were constricted with 60 mM KCl to establish viability; vessels failing to constrict at least 1 mN (estimated as an appropriate signal-to-noise ratio for tension measurement) were excluded from further study. MyoA were pre-constricted with 10 μM phenylephrine (PE, Sigma-Aldrich, St. Louis, MO), a concentration previously found to induce submaximal constriction in MyoA (Lorca et al., 2019). MyoA were then superfused with increasing concentrations of the K_ATP channel opener pinacidil (0.1–100 μM, Cayman Chemical, Ann Arbor, MI) in the absence or presence of the K_ATP channel blocker glibenclamide (30 μM, Tocris Bioscience, Bristol, UK), or with the BK_Ca channel opener NS-11021 (0.1–100 μM, Alomone Labs, Jerusalem, Israel) in the absence or presence of the BK_Ca channel blocker tetraethylammonium (TEA, 1 mM, Thermo Fisher Scientific). These concentrations of glibenclamide and TEA have been shown to selectively block K_ATP and BK_Ca channels in mesenteric and uterine arteries (Standen et al., 1989; Hu et al., 2011; Hu et al., 2012). To assess the contributions of the endothelium, the endothelial cell layer was mechanically disrupted by rolling a hair through the vessel lumen; loss of endothelial function was judged as < 30% relaxation to 100 nM bradykinin (Thermo Fisher Scientific). Due to the limited number of vessels per patient as well as vessels that failed to constrict to PE or that had incomplete endothelium removal, not all the curves could be performed for each patient sample. Pinacidil and NS-11021 relaxation are shown as a percentage of PE contraction. The area under the curve (AUC), half-maximal inhibitory concentration (IC₅₀) and maximal response (E_max) were calculated using GraphPad Prism 9 software (San Diego, CA).

Immunofluorescence.

The myometrial samples were fixed in 4% PFA for at least 24 hours at room temperature and then embedded in paraffin. Sections (5 μm) were adhered to slides, deparaffinized with xylenes, and rehydrated. Slides were treated with an antigen retrieval solution (10 mM Tris-base, 1 mM EDTA, 0.1% Tween-20, pH 9.0) and then blocked overnight at 4°C with 10% normal goat serum (S-1000, Vector Laboratories, Newark, CA) in PBS with 0.1% Tween-20 (PBS-T). For endothelial cell staining, slides were incubated with mouse monoclonal anti-CD31 antibody (IgG1, 1:50, #3528, Cell Signaling Technology, Danvers, MA) followed by a specific secondary antibody (anti-IgG1, Alexa Fluor® 680 [115–605-205]; Jackson ImmunoResearch, West Grove, PA). For smooth muscle staining, a mouse monoclonal anti-smooth muscle α-actin (α-SMA) antibody was used (IgG2a, 1:50, M085129–2, Agilent Technologies, Santa Clara, CA) followed by a specific secondary antibody (anti-IgG2a, Alexa Fluor® 594 [115–295-206]; Jackson ImmunoResearch). These slides were co-stained with either rabbit polyclonal anti-Kir6.1 (1:50, OAAB08559, Aviva Systems Biology, San Diego, CA), anti-Kir6.2 (1:50, APC-020, Alomone Labs), or anti-BK_Ca α subunit (1:50, APC-021, Alomone Labs) followed by a specific secondary antibody (anti-rabbit, FITC [111–546-144]; Jackson ImmunoResearch). Primary antibodies were incubated overnight at 4°C, secondary antibodies were incubated for 90 minutes at room temperature. Slides were washed with PBS-T, mounted, and imaged with a fluorescent microscope (Olympus, Waltham, MA). Quantitative analysis of co-localization was performed by creating a mask on the vessel wall and calculating Pearson’s correlation coefficients (Dunn et al., 2011) between Kir6.1, Kir6.2, or BK_Ca-α and CD31 or α-SMA within each mask using SlideBook 6 software (Intelligent Imaging Innovations, Denver, CO). Average Pearson’s correlation coefficients of four vessels per subject were used for analysis.

Statistical Analyses.

Sample sizes are shown as the number of subjects. For the vasoreactivity experiments, values were averaged from 1–3 vessels from the same subject under the same conditions. The effects of drug treatment or altitude were determined using two-way ANOVA followed by Sidak’s multiple comparisons. Immunofluorescence analyses were compared between altitudes by parametric t test or non-parametric Mann-Whitney U test. Maternal and newborn characteristics and immunofluorescence data were analyzed by parametric t test, non-parametric Mann-Whitney U test, or chi-square analysis as needed. All statistical analyses were performed using GraphPad 9 software. Birth weights adjusted for gestational age at the time of delivery and infant sex were compared between groups using analysis of covariance (ANCOVA) within SPSS v26 (IBM, Chicago, IL). A two-tailed p-value < 0.05 was considered significant.

RESULTS

Maternal and newborn characteristics.

Maternal age, height, pre-gravid body mass index, parity, and ethnicity were similar between LA and HA groups (Table 1). Mean arterial pressure at admission also did not differ between groups. Although gestational age was slightly shorter in HA than in LA, the difference was clinically insignificant (Table 1). All newborns were considered AGA and had similar birth weight percentiles (Table 1); however, birth weight was 8.7% lower for HA babies compared to LA (p = 0.0113), even after adjusting for gestational age and newborn sex (p = 0.040, Table 1). Other newborn characteristics such as length, head circumference, ponderal index, sex, and APGAR scores were not different between altitudes (Table 1).

Table 1.

Maternal and newborn characteristics

Characteristic	LA (n = 21)	HA (n = 17)	p-value
Maternal
Age at delivery (y)	33.2 ± 4.4	34.1 ± 4.9	0.5686
Height (cm)	166.9 ± 6.3	164.9 ± 6.0	0.2778
Pre-gravid BMI (kg/m2)	24.8 ± 3.1	24.6 ± 2.8	0.7984
Parity (no. of live births)	1.9 ± 0.6	2.1 ± 0.9	0.4913
MAP (mm Hg)	88.9 ± 8.5	87.0 ± 7.2	0.4488
Ethnicity (% of Hispanic)	19	29	0.4549
Newborn
Gestational age (weeks)	39.3 ± 0.3	38.9 ± 0.7	0.0135
Birth weight (g)	3591 ± 370	3277 ± 329	0.0113
Adjusted birth weight§ (g)	3571 ± 79	3303 ± 88	0.040
Birth weight (percentile)	59.0 ± 27.3	41.6 ± 26.2	0.0545
Length (cm)	50.6 ± 2.2	49.8 ± 2.1	0.2451
Head circumference (cm)	35.0 ± 2.3	35.0 ± 1.4	0.3462
Ponderal Index	2.8 ± 0.2	2.7 ± 0.3	0.2931
Sex (% female)	52	35	0.2922
APGAR, 1 min	7.7 ± 1.3	7.9 ± 0.2	0.6633
APGAR, 5 min	8.6 ± 0.6	8.8 ± 0.4	0.2422

All values are means ± SD, except for adjusted birth weight, which is expressed as the estimated marginal mean ± SEM; n = number of subjects. Birth weights were adjusted for gestational age at the time of delivery and newborn sex, then compared between groups using analysis of covariance (ANCOVA). BMI, body mass index; MAP, mean arterial pressure at admission.

K_ATP channel-elicited vasodilation of MyoA

The K_ATP channel opener pinacidil evoked similar vasodilation of MyoA from LA and HA women in intact (+ endothelium) and endothelium-denuded (− endothelium) vessels (Figure 1, Table 2). Yet the K_ATP channel blocker glibenclamide diminished the pinacidil-dependent vasodilatory responses in intact HA MyoA but not LA (Figure 1A and B, Table 2). Moreover, removal of endothelial layer eliminated the inhibitory effect of glibenclamide in vessels from both altitudes (Figure 1C and D, Table 2). These results suggest that K_ATP channel activity is augmented in HA MyoA and that this increase depends on the presence of the endothelium.

Figure 1. K_ATP-evoked vasodilation is increased in high- (HA) vs low-altitude (LA) MyoA in an endothelium-dependent manner.

A and C, K_ATP opener pinacidil concentration-response curves in MyoA from women living at LA (closed symbols) or HA (open symbols), calculated as percentage of phenylephrine (PE, 10 μM) response in the absence (black symbols) or presence (blue symbols) of the K_ATP blocker glibenclamide (Glib, 30 μM). Vessels were intact (A, + endothelium) or endothelium-denuded (C, − endothelium). Symbols are mean values ± SEM, the number of women is in parentheses. B and D show area under the curve (AUC) analyses for data in A and C, respectively. * p = 0.0351 by two-way ANOVA.

Table 2.

Vasodilator parameters in human myometrial arteries from low- (LA) and high-altitude (HA) women.

	IC50 (μM)		Emax (%)
Treatment	LA	HA	LA	HA
+ endothelium
Pinacidil	2.4 [0.1–16.0] (8)	4.8 [0.9–24.4] (6)	0.6 [−1–26] (8)	13.4 [−77–36] (6)
Pinacidil + Glib	18.9 [2.3–62.1] (8)	52.8 [12.1–110] (7)a	0.8 [−60–10] (8)	61 [31–116] (7)b
− endothelium
Pinacidil	4.8 [0.9–110] (6)	1.0 [0.1–4.4] (5)	4 [−22–43] (6)	6 [−5–16] (5)
Pinacidil + Glib	8.0 [0.005–106] (7)	1.6 [1.1–25.5] (5)	−7 [−30–36] (7)	6 [−18–26] (5)
+ endothelium
NS-11021	12.9 [1–87] (6)	16.5 [9.1–51.3] (5)	13 [−17–45] (6)	2 [−33–10.7] (5)
NS-11021 + TEA	12.6 [4.8–22.8] (4)	70.3 [12.6–76.5] (5)	7 [−22–20] (4)	13 [−1–35] (5)
− endothelium
NS-11021	7.8 [1.0–11.2] (8)	18.5 [2.4–47.2] (8)	0.04 [-44–14] (8)	1 [−9–15] (8)
NS-11021 + TEA	27.3 [19.7–78.1] (5)c	14.4 [0.9–58.0] (5)	36 [−8–89] (5)d	4 [−4–20] (5)

Values are median [range] (number of subjects). TEA, tetraethylammonium; Glib, glibenclamide.

^ap = 0.0036 and

^bp = 0.0074 vs Pinacidil alone

^cp = 0.0178 and

^dp = 0.0106 vs NS-11021 alone.

BK_Ca channel-dependent vasodilation of MyoA

No differences were observed in the BK_Ca channel opener NS-11021 vasodilation of MyoA from LA and HA women in both intact (+ endothelium) and endothelium-denuded (− endothelium) vessels (Figure 2, Table 2). In intact vessels, vasodilatory responses to NS-11021 were unaffected by BK_Ca channel blocking with TEA at both altitudes (Figure 2A and B, Table 2). In contrast, the vasodilatory responses to NS-11021 were significantly reduced in denuded MyoA at LA but not at HA (Figure 2C and D, Table 2). These results suggest that BK_Ca-dependent vasodilation is reduced in HA MyoA compared to LA in an endothelium-independent manner.

Figure 2. BK_Ca-elicited vasodilation is reduced in high- (HA) vs low-altitude (LA) MyoA in an endothelium-independent manner.

A and C, BK_Ca opener NS-11021 concentration-response curves in MyoA from women living at LA (closed symbols) or HA (open symbols), calculated as percentage of phenylephrine (PE, 10 μM) response in the absence (black symbols) or presence (red symbols) of the BK_Ca blocker TEA (1 mM). Vessels were intact (A, + endothelium) or endothelium-denuded (C, − endothelium). Symbols are mean values ± SEM, the number of women is in parentheses. B and D show area under the curve (AUC) analyses for data in A and C, respectively. * p = 0.0170 by two-way ANOVA.

BK_Ca and K_ATP channel localization in myometrial vessels

We analyzed the localization of K_ATP and BK_Ca channels within myometrial vessels by immunofluorescence. Our analyses showed that the pore-forming subunits of K_ATP channels, Kir6.1 and Kir6.2, co-localized with markers of endothelial and smooth muscle cells (Figure 3A–D). A quantitative analysis of co-localization was used by measuring Pearson’s correlation coefficients (Dunn et al., 2011). These analyses showed a reduction in the co-localization of Kir6.1 and the endothelial cell marker CD31 in HA vs LA myometrial tissue (Figure 3B), whereas no differences were found in the dual staining of Kir6.2 and endothelium or smooth muscle among altitudes (Figure 3D). We also assessed the staining of the pore-forming α subunits of BK_Ca channels. BK_Ca channels were co-localized in both smooth muscle and endothelial layers of myometrial vessels; we did not find differences between LA and HA groups in this analysis (Figures 3E and F). These observations suggest that the co-localization of these channels in endothelial or smooth muscle layers of the myometrial vessels cannot explain the altitude-dependent functional changes in BK_Ca and K_ATP channel vasodilation, thus, other regulatory mechanisms may contribute to the altered activity of these channels.

Figure 3. K_ATP and BK_Ca channels localization in myometrial vessels from women residing at low (LA) and high altitude (HA) during pregnancy.

Immunofluorescence detection and analysis of Kir6.1 (A and B), Kir6.2 (C and D), or BK_Ca α subunit (E and F), in myometrium from LA and HA women. Specific antibodies were used for each channel subunit and detected with secondary anti-rabbit antibodies (left panels). Specific antibodies for α-SMA or CD31 were used to identify smooth muscle (SMC) or endothelial cells (EC), respectively. Merged images show all markers combined and nuclear staining with DAPI (blue). Scale bars = 10 μm. Pearson’s correlation coefficients were calculated using Slidebook software. ** p = 0.0015 by Unpaired t test.

DISCUSSION

Residence at HA during pregnancy has been associated with diminished rise in uterine artery blood flow and impaired MyoA vasodilatory responses, reducing fetal growth (Julian et al., 2008; Lorca et al., 2019). However, not all HA pregnancies develop fetal growth restriction. Here we observed that, in MyoA from term pregnant AGA pregnancies, HA residence affected the activity of two K⁺ channels, BK_Ca and K_ATP, in opposite ways. The sensitivity of K_ATP channel blocker in reducing vasodilatory responses was increased in MyoA from HA compared to LA women, and this increase required the presence of the endothelium. Conversely, the sensitivity of BK_Ca channel blocker to decrease vasodilatory responses was reduced in MyoA from HA women in comparison to their LA counterparts, and this reduction was endothelium independent. These observations suggest that more K_ATP channels are active and/or available for blocking at HA, whereas fewer BK_Ca channels are active and accessible to blockers in HA. Furthermore, we found that HA was associated with a reduced K_ATP channel localization in the endothelium, which did not follow our functional results, suggesting that the altitudinal differences in K_ATP channel activity are likely not due to increased K_ATP channel density in the vascular endothelium.

K_ATP channel activity is enhanced during pregnancy in ways that serve to reduce uteroplacental vascular resistance and facilitate high blood flow (Keyes et al., 1998). Similarly, in vitro studies in ovine uterine arteries showed that the activation of K_ATP channels induces greater vasodilator response in vessels from pregnant than non-pregnant animals (Xiao et al., 2010). Furthermore, non-pregnant human uterine arteries vasodilate in response to K_ATP channel openers (Nelson et al., 1993). These observations highlight a well-conserved role of K_ATP channels in the uterine vasculature. Supporting this, we found that the K_ATP activation elicited vasodilation in MyoA from healthy, pregnant women. Prior studies indicate that the pregnancy-induced increase in K_ATP channel activity is primarily localized in the uterine and coronary vascular beds (Keyes et al., 1998), suggesting that treatment with selective K_ATP channel openers could be a potential therapy for pregnancies complicated by IUGR and/or preeclampsia.

Our results support a role for BK_Ca in the pregnancy-mediated vasodilation of human uterine vasculature, as has been shown in pregnant animal models (Rosenfeld et al., 2001; Rosenfeld et al., 2005; Hu et al., 2011; Lorca et al., 2018) and human studies (Li et al., 2020). Several mechanisms have been described to increase BK_Ca channel activity during pregnancy. The pore-forming BK_Ca α, and its auxiliary subunits β1, γ1, and γ3, are expressed in the uterine vascular smooth muscle (Nagar et al., 2005; Rosenfeld et al., 2009; Lorca et al., 2018; Li et al., 2020). These subunits increase the channel activity, and further the expression of the β1 and γ1 subunits is increased during pregnancy (Rosenfeld et al., 2009; Hu et al., 2011; Lorca et al., 2018). In ovine pregnancy, the reduced uterine myogenic tone was accompanied by increased β1 subunit expression and enhanced the Ca²⁺ sensitivity of BK_Ca channels (Hu et al., 2011). Furthermore, the BK_Ca channel blocker TEA has been shown to decrease basal uterine blood flow, inhibit K⁺ currents, and increase pressure-dependent vascular tone in pregnant sheep (Rosenfeld et al., 2001; Rosenfeld et al., 2005; Hu et al., 2011). Increased BK_Ca activity during pregnancy has also been shown in mice, and this increase seems to be related to the presence of the BK_Ca γ1 subunit (Lorca et al., 2018).

Chronic hypoxia has been described to reduce the effects of the K_ATP channel opener diazoxide in pregnant uterine arteries (Xiao et al., 2010), suggesting that a decrease in K_ATP channel activity underlies, in part, the increased myogenic tone of the uterine artery under chronic hypoxic conditions. Chronic hypoxia also decreases BK_Ca channel currents and vasodilation of the uterine artery in pregnant sheep (Xiao et al., 2014). Several mechanisms have been proposed for the diminished BK_Ca channel activation in the uterine vasculature of chronic hypoxic animal models. For instance, protein kinase C, a known inhibitor of BK_Ca channel activity in the vasculature (Minami et al., 1993; Barman, 1999), is decreased during pregnancy (Magness et al., 1991; Farley & Ford, 1992) and upregulated in the uterine artery under chronic hypoxic conditions leading to a reduction in BK_Ca activity (Xiao et al., 2014). Additionally, a hypoxia-dependent increase in DNA methyltransferase activity and expression of microRNA 210 has been proposed to induce hypermethylation of the β1 gene promoter which represses the expression of the β1 subunit, resulting in reduced BK_Ca-dependent currents and vasodilation of the uterine artery (Hu et al., 2017a; Hu et al., 2017b; Hu et al., 2018). Our results align with the described HA-dependent reduction in BK_Ca activity. However, our findings contrast with the reduction in K_ATP channel activity from the sheep studies, as we observed that K_ATP-dependent vasodilation was increased in MyoA under chronic hypoxic conditions. This discrepancy suggests that the hypoxia-induced regulation of these channels in the uterine vasculature may be unique to human beings and highlights the importance of conducting human, as well as experimental animal studies under conditions of chronic hypoxia. In addition, our observation that co-localization of K_ATP and BK_Ca with endothelium or smooth muscle did not align with the altitudinal differences in K_ATP and BK_Ca vasodilatory responses and supports the idea that other mechanisms, such as posttranslational modifications (i.e., protein kinase C phosphorylation) or reduced association with auxiliary subunits, rather than channel localization in a specific cell type, may explain the functional changes in channel activity at HA. Future studies are needed in human HA populations to determine mechanisms underlying the decreased BK_Ca and increased K_ATP channel activity in uterine vasculature and whether these processes are also present in vascular complications of pregnancy, such as IUGR and preeclampsia.

Although the present study was limited to assessing the vasodilator role of K⁺ channels in myometrial arteries at high altitude, it is possible that non-vasodilatory effects of K_ATP channels may contribute to the maintenance of fetal growth. For instance, K_ATP channels play important roles in integrating glucose availability to insulin secretion in pancreatic beta cells (Akrouh et al., 2009). Studies in pregnant women at high altitude and pregnant mice under hypoxic conditions have shown a hypoxia-dependent reduction of the pregnancy-induced secretion of insulin (Krampl et al., 2001; Maatta et al., 2018), which is triggered by a decrease in insulin resistance under these conditions. Thus, it may be possible that the high altitude increase of K_ATP channels activation contributes to the decrease in insulin secretion, which, in turn, may lead to a normalization of fetal growth by augmenting nutrient availability to the fetus.

Our findings showing decreased BK_Ca and increased K_ATP vasodilation within the human uterine vasculature may be due to ion channel redundancy, a mechanism by which a reduction in ion channel activity is compensated by increasing the activity of another ion channel. Comparable compensatory mechanisms have been described for other vascular beds. Studies in coronary arteries from spontaneously hypertensive rats have shown that an inwardly rectifying K⁺ channel current is increased and partially compensates for the reduced vasorelaxation by endothelial small- and intermediate-conductance Ca²⁺-activated K⁺ channels, SK3 and SK4 (Kim et al., 2020). Similarly, in subcutaneous arteries of diabetic patients, the impaired endothelium-dependent vasorelaxations due to a reduction in nitric oxide bioavailability are partially compensated by an increase in endothelium-derived hyperpolarization factor responses (Mokhtar et al., 2016). Furthermore, endothelial dysfunction in hypertensive human patients has been shown to reduce endothelium-dependent vasorelaxation, which seems to be compensated by an ouabain-sensitive bradykinin response (Taddei et al., 1999). Likewise, our previous studies have shown that HA residence had differing effects on the MyoA vasodilatory responses to acetylcholine- and AMP-activated protein kinase (AMPK) in healthy, pregnant women. Specifically, we showed that the enhanced MyoA AMPK-dependent vasodilation in HA vs. LA women may be compensatory for the reduced acetylcholine-dependent vasodilation and served to help maintain normal fetal growth (Lorca et al., 2020). Thus, the increased K_ATP channel MyoA vasodilation under chronic hypoxic conditions may also be compensatory for the reduction in BK_Ca channel activity and serve to maintain normal uteroplacental blood flow and fetal growth.

A limitation of our study is the relatively small difference in elevation (~1100 m) between our LA and HA sites. We acknowledge that this elevation difference may not be enough to unveil the full spectrum of effects associated with pregnancy at HA. Nonetheless, as noted above, we have reported decreased MyoA vasodilatory responses to acetylcholine, increased responses to AMPK activation and reduced birth weight across this altitude range (Lorca et al., 2019; Lorca et al., 2020). Our study also focused on AGA pregnancies, thus, studies of K_ATP and BK_Ca channels in MyoA from IUGR and/or preeclamptic pregnancies as well as in vessels from women residing across a greater altitude gradient are needed.

In summary, we found different MyoA vasodilatory responses to blocking BK_Ca and K_ATP channel during pregnancy based on altitude of residence. We propose the larger K_ATP channel vasodilatory response in MyoA is a mechanism to maintain normal blood flow and healthy human pregnancy under hypoxic conditions. There is a complex network of factors underlying the vasodilatory sensitivity of uteroplacental resistance arteries. This affords the chance to test the efficacy of drugs aimed at selectively altering one set of such factors as treatment for pregnancy disorders characterized by uteroplacental ischemia, especially given the lack of progress in developing such treatments for these truly life-threatening conditions. Future studies will aim at elucidating the mechanisms underlying the increased K_ATP channel vasodilatory responses in MyoA from HA women, which could be relevant for the identification of therapeutic targets to prevent or alleviate vascular-related pregnancy complications such as IUGR or preeclampsia.

Key points summary.

• High-altitude (>2500 m or 8200 ft) residence reduces uterine blood flow during pregnancy and fetal growth. Animal models of high altitude/chronic hypoxia suggest that these reductions are partially due to reduced vascular K⁺ channel responses, such as those elicited by large conductance Ca²⁺-activated (BK_Ca) and ATP-sensitive (K_ATP) K⁺ channel activation.

• We found that women residing at high versus low altitude during pregnancy showed diminished myometrial artery vasodilatory responses to endothelium-independent BK_Ca channel activation but greater responses to endothelium-dependent K_ATP channel activation.

• Our observations indicate that K_ATP channels play an adaptive role in maintaining myometrial artery vasodilator sensitivity under chronic hypoxic conditions during pregnancy. Thus, K_ATP channels represent potential therapeutic targets for augmenting uteroplacental blood flow and, in turn, preserving fetal growth in cases of uteroplacental hypoperfusion.

Biography

Sahand Fallahi received his B.S. in Biochemistry and Mathematics from Regis University in 2020. He is currently a professional research assistant in the Department of Obstetrics and Gynecology at the University of Colorado Anschutz Medical Campus. His research interests focus on the study of maternal vascular function in association with fetal growth.

Data availability statement

The data that support the observations of this study are available from the corresponding author upon reasonable request.