Gestational hypoxia inhibits pregnancy-induced upregulation of Ca²⁺ sparks and spontaneous transient outward currents in uterine arteries via heightened endoplasmic reticulum/oxidative stress

Abstract

Hypoxia during pregnancy profoundly affects uterine vascular adaptation and increases the risk of pregnancy complications including preeclampsia and fetal intrauterine growth restriction. We recently demonstrated that increases in Ca²⁺ sparks and spontaneous transient outward currents (STOCs) played an essential role in pregnancy-induced uterine vascular adaptation. In the present study, we hypothesize that gestational hypoxia suppresses Ca²⁺ sparks/STOCs coupling leading to increased uterine vascular tone via enhanced endoplasmic reticulum (ER)/oxidative stress. Uterine arteries were obtained from non-pregnant and near-term pregnant sheep residing in low altitude or acclimatizing to high altitude (3,801 m) hypoxia for~110 days. High-altitude hypoxia suppressed pregnancy-induced upregulation of ryanodine receptor 1 (RyR1) and 2 (RyR2) protein abundance, Ca²⁺ sparks and STOCs in uterine arteries. Inhibition of Ca²⁺ sparks/STOCs with the RyR inhibitor ryanodine significantly increased pressure-dependent myogenic tone in uterine arteries from low-altitude normoxic pregnant animals, but not those from high-altitude hypoxic pregnant animals. Gestational hypoxia significantly increased ER/oxidative stress in uterine arteries. Of importance, the hypoxia-mediated suppression of Ca²⁺ sparks/STOCs and increase in myogenic tone in uterine arteries of pregnant animals were reversed by inhibiting ER/oxidative stress. Of great interest, the impaired sex hormonal regulation of STOCs in high-altitude animals was annulled by scavenging reactive oxygen species (ROS) but not by inhibiting ER stress. Together, the findings reveal the differential mechanisms of ER and oxidative stresses in suppressing Ca²⁺ sparks/STOCs and increasing myogenic tone of uterine arteries in hypoxia during gestation, providing new insights into the understanding of pregnancy complications associated with hypoxia.

Graphical Abstract

Introduction

Hypoxia is one of the most frequent and severe stresses to an organism’s homeostatic mechanisms, and hypoxia during gestation has profound adverse effects on both maternal and fetal health, impacting developmental plasticity.¹ Gestational hypoxia is believed to be a major cause of pregnancy complications such as preeclampsia and intrauterine growth restriction (IUGR).² Not surprising, high-altitude pregnancy is associated with significantly increased incidence of preeclampsia and IUGR.^{3, 4} Both human and animal studies have revealed a causative role of increased uterine vascular resistance and lowered uterine blood flow in preeclampsia and IUGR.¹ In an animal model of pregnant sheep acclimatized to high altitude, we demonstrated that high-altitude hypoxia suppressed pregnancy-induced uterine arterial adaptation and increased uterine vascular resistance and maternal systemic blood pressure.⁵ In general, the basic mechanisms whereby an organism adapts to high-altitude, long-term hypoxia are elusive, and much remains unknown of the molecular mechanisms underlying uterine vascular maladaptation to long-term hypoxia during gestation.

Endoplasmic reticulum (ER)/oxidative stress plays an important role in pregnancy homeostasis and complications, and ER/oxidative stress in uteroplacental tissues is a common feature of high altitude pregnancy, preeclampsia and IUGR.^6–12 It has been demonstrated that ER/oxidative stress plays a pivotal role in vascular dysfunction in systemic hypertension as well as in pulmonary hypertension.^13–17 Our recent study demonstrated a mechanism of Ca²⁺ sparks and STOCs in the regulation of uterine myogenic tone underlying uterine vascular adaptation to pregnancy.¹⁸ Specifically, pregnancy increased Ca²⁺ sparks firing myocytes and Ca²⁺ sparks frequency via upregulation of ryanodine receptors (RyRs) in uterine arteries. In addition, pregnancy promoted Ca²⁺ sparks/STOCs coupling and enhanced STOCs frequency and amplitude, which was mediated by the action of steroid hormones. Furthermore, blockade of Ca²⁺ sparks/STOCs inhibited pregnancy/hormone-induced attenuation of uterine arterial myogenic tone. A question of great relevance is whether and to what extent gestational hypoxia alters Ca²⁺ sparks/STOCs in an ER/oxidative stress-dependent manner in programming of uterine vascular maladaptation in pregnancy complications. Herein, we provide evidence that high-altitude hypoxia during gestation suppresses pregnancy-induced upregulation of RyR-induced Ca²⁺ sparks and STOCs and increases pressure-dependent myogenic tone in uterine arteries in part due to heightened ER/oxidative stress, providing mechanistic insights into uterine vascular maladaptation in pregnancy complications associated with hypoxia.

Methods

The authors declare that all supporting data are available within the article [and its online supplementary files].

Tissue preparation and treatment

All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Loma Linda University and followed the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After tissue collection, animals were killed via intravenous injection of 15 mL T-61 solution (Hoechst-Rousel, Somervile, NJ), according to American Veterinary Medical Association guidelines.

Uterine arteries were harvested from non-pregnant or near-term (~142–145 days of gestation) pregnant sheep (Ovis aries) carrying singleton or twin maintained at low altitude (~300 m above sea level, PaO₂:~102 mm Hg) or exposed to high-altitude hypoxia (3,801 m, PaO₂:~60 mm Hg) starting from 30 days of gestation for 110 days, as described in previous studies.¹⁹ Animals were anesthetized with intravenous injection of propofol (2 mg/kg) followed by intubation, and anesthesia was maintained on 1.5% to 3.0% isoflurane balanced in O₂ throughout the surgery. An incision was made in the abdomen and the uterus was exposed. Resistance-sized uterine artery segments (~150–200 μm in diameter) were isolated and removed without stretching and placed into a physiological salt solution (PSS) containing (in mmol/L) 130.0 NaCl, 10.0 HEPES, 6.0 glucose, 4.0 KCl, 4.0 NaHCO₃, 1.8 CaCl₂, 1.2 MgSO₄, 1.18 KH₂PO₄, and 0.025 EDTA (pH 7.4). Similarly sized uterine arteries from non-pregnant and pregnant animals were used. For the ex vivo tissue culture, uterine arteries were incubated in 5 mL phenol red-free DMEM with 1% charcoal-stripped FBS, 100 U/mL penicillin and 100 μg/mL streptomycin for 48 hours at 37 °C in a humidified incubator with 5% CO₂/95% air in the presence of vehicle controls or the following inhibitors or hormones: ER stress inhibitors, tauroursodeoxycholic acid (TUDCA, 1.0 or 3.0 mmol/L), GSK2606414 (1 μmol/L); reactive oxygen species (ROS) scavengers, N-acetylcysteine (1.0 mmol/L), EUK-134 (20.0 μmol/L); steroid hormones, 17β-estradiol (0.3 nmol/L, Sigma) and progesterone (100.0 nmol/L, Sigma), as described previously^{19, 20} The concentrations of 17β-estradiol and progesterone chosen are physiologically relevant as observed in ovine pregnancy,²¹ which have been shown to exhibit direct genomic/epigenomic effects on expression of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel β1 subunit and RyRs as well as pressure-dependent myogenic tone in the uterine arteries.^{18–20, 22, 23}

Measurement of Ca²⁺ sparks

Ca²⁺ sparks were measured in endothelium-denuded uterine arteries loaded with the Ca²⁺ sensitive dye Fluo-4 AM and using a Zeiss LSM 710 NLO laser scanning confocal imaging workstation on an inverted microscope platform (Zeiss Axio Observer Z1, Thornwood, NY).¹⁸ The endothelium was mechanically disrupted by gently pulling a silver wire across the intimal surface of the uterine arterial segments 5 times, with confirmation by visual analysis of the preparations on the confocal microscope after loading the tissue with Fluo-4. Arterial segments were incubated with 10 μmol/L Fluo-4 AM (Thermo Fisher, Waltham, Massachusetts) dissolved in DMSO along with 0.1% pluronic F127 (Thermo Fisher) for 1–1.5 hours at room temperature. Tissues were then washed for 30 minutes to allow dye esterification and then cut into linear strips. The arterial segments were pinned to Sylgard blocks and placed in an open bath imaging chamber (Warner Instruments, Hamden, CT) mounted on the confocal imaging stage. Cells were illuminated at 488 nm with a krypton argon laser and the emitted light was collected using a photomultiplier tube. Line scans were imaged at 529 frames s⁻¹ with the emission signal recorded at 493–622 nm. The acquisition period for Ca²⁺ spark recordings was 18.9 s. The resultant pixel size ranged from 0.021 to 0.1 μm per pixel. To ensure that sparks within the cell were imaged, the pinhole was adjusted to provide an imaging depth of 2.5 μm. This depth is roughly equivalent to the width of 50% of the cell based on morphological examination of live preparations. Line scans were analyzed using Sparklab 4.2.1 to characterize Ca²⁺ spark parameters such as frequency (sparks/μm/s), amplitude (F/F₀), spatial size (the full width at half maximum, FWHM) and duration (the full duration at half maximum, FDHM). The fractional fluorescence intensity was calculated as F/F₀ = F - baseline/F₀ - baseline, where baseline is the intensity from a region of interest with no cells, F is the fluorescence intensity for the region of interest, and F₀ is the fluorescence intensity during a period from the beginning of the recording when there was no Ca²⁺ activity.

Measurement of STOCs

Uterine arterial smooth muscle cells (SMCs) were enzymatically dissociated from resistance-sized uterine arteries as described previously.¹⁸ Briefly, uterine arteries were minced and incubated (37 °C, 10 minutes) in low-Ca²⁺ HEPES-buffered physiological salt (PSS) solution containing (in mmol/L) 140.0 NaCl, 5.0 KCl, 0.1 CaCl₂, 1.2 MgCl₂, 10.0 HEPES, and 10.0 glucose (pH 7.4). Vessels were then exposed to a two-step digestion process that involved: 1) a 60-minute incubation in low-Ca²⁺ HEPES-buffered PSS (37 °C) containing 1.5 mg/ml papain (Worthington Biochemical; Lakewood, NJ), 1.5 mg/ml dithiothreitol (MilliporeSigma, St. Louis, MO), and 1.5 mg/ml bovine serum albumin (MilliporeSigma); and 2) a 60-minute incubation in low-Ca²⁺ HEPES-buffered PSS (37 °C) containing 1.5 mg/ml collagenase IV (Worthington) and 1.5 mg/ml bovine serum albumin (MilliporeSigma). Following the enzyme treatment, tissues were washed with low Ca²⁺ HEPES-buffered PSS. Single SMCs were released by gently inverting the tube(s) containing low Ca²⁺ HEPES-buffered PSS and digested tissues several times. The cells were kept at 4°C and experiments were conducted within 6 hours of cell isolation. STOCs were recorded in the whole-cell configuration of the perforated patch-clamp technique using an EPC 10 patch-clamp amplifier with Patchmaster software (HEKA, Lambrecht/Pfalz, Germany) at room temperature as previously described.¹⁸ Briefly, cell suspension drops were placed in a recording chamber, and adherent cells were continuously superfused with HEPES-buffered PSS containing (in mmol/L) 140.0 NaCl, 5.0 KCl, 1.8 CaCl₂, 1.2 MgCl₂, 10.0 HEPES, and 10.0 glucose (pH 7.4). Only relaxed and spindle-shaped myocytes were used for recording. Micropipettes were pulled from borosilicate glass and had resistances of 2 to 5 megaohm (mΩ) when filled with the pipette solution containing (in mmol/L) 140.0 KCl, 1.0 MgCl₂, 5.0 Na₂ATP, 5.0 EGTA, 10.0 HEPES (pH 7.2) with 250 μg/ml amphotericin B. CaCl₂ was added to bring free Ca²⁺ concentrations to 100 nmol/L as determined using WinMAXC software (Chris Patton, Stanford University). Membrane currents were recorded while the cells were held at steady membrane potentials between −50 and 10 mV in 10 mV-increments. STOCs were analyzed with Mini Analysis program (Synaptosoft, Leonia, NJ) with a threshold for detection setting at 10 pA. To facilitate comparison of STOC amplitude in differently sized myocytes, the currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA/pF).

Measurement of pressure-dependent myogenic tone

Pressure-dependent myogenic tone of resistance-sized uterine arteries was measured as described previously.^{19, 20} Briefly, the arterial segments (~150 μm diameter) were mounted and pressurized in an organ chamber (Living Systems Instruments, Burlington, VT). The intraluminal pressure was controlled by a servo-system to set transmural pressures, and arterial diameter was recorded using the SoftEdge Acquisition Subsystem (IonOptix LLC, Milton, MA, USA). After the equilibration period, the intraluminal pressure was increased in a stepwise manner from 10 to 100 mmHg in 10-mmHg increments, and each pressure was maintained for 5 minutes to allow vessel diameter to stabilize before the measurement. Ca²⁺-free PSS contains zero Ca²⁺ and 3 mmol/L EGTA. PSS was allowed to pass through the lumen of the pressurized vessels before detection of myogenic tone, and myogenic tone was measured under the static flow. The passive pressure-diameter relationship was conducted in Ca²⁺-free PSS to determine the maximum passive diameter. The following formula was used to calculate the percentage of pressure-dependent tone at each pressure step: % tone = (D1 − D2)/D1 × 100, where D1 is the passive diameter in Ca²⁺-free PSS, and D2 is the active diameter with normal PSS in the presence of extracellular Ca²⁺.

Real-time RT-PCR

Total RNA was isolated using TRIzol reagent (Thermo Fisher) and subjected to reverse transcription with iScript cDNA Synthesis system (Bio-Rad, Hercules, CA). To remove the possible genomic DNA contamination from RNA, the total RNA preparation was treated with RNase-free DNase I (1 U/μg RNA, Thermo Scientific) at 37 °C for 30 min. This was followed by phenol/chloroform/isoamyl alcohol extraction, and isopropanol precipitation of RNA. The washed and dried RNA pellets were then reconstituted in nuclease free water and used in real-time PCR. The mRNA abundance of RyRs was measured with real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad), as described previously.¹⁸ Primers used were 5′-CAGAGGGGGAAAAAGAGGAC-3′ (forward) and 5′-ACGGTGCTGTAGCTCTTGGT-3′ (reverse) for RyR1, 5′-TGAGGCTCACAGGCTTTTCT-3′ (forward) and 5′-ATGCAGGGGATACAGGTTTG-3′ (reverse) for RyR2, and 5′-TAAAGTATGGGCCCGAAGTG-3′ (forward) and 5′-TTTCATTTCTGCTGCCTGTG-3′ (reverse) for RyR3. PCR was performed in triplicate, and relative abundance of target mRNA was normalized to sheep ribosomal protein L4 which was not altered by pregnancy in uterine arteries.¹⁸

Western immunoblotting

Protein abundance of RyRs, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and eukaryotic initiation factor 2α (eIF2α), phospho-PERK, and phospho-eIF2α in uterine arteries was measured as described previously.¹⁸ Briefly, tissues were homogenized in a lysis buffer followed by centrifugation at 4 °C for 10 minutes at 10,000g, and the supernatants were collected. Samples with equal proteins were loaded onto 4–12% PAGEr™ Gold Gels (Lonza, Allendale, NJ) and were separated by electrophoresis at 100 V for 2–2.5 hours. Proteins were then transferred onto nitrocellulose membranes. After blocking nonspecific binding sites by dry milk, membranes were incubated with primary antibodies (1:300–1000 dilution) against RyR1 (8153, Cell Signaling, Danvers, MA), RyR2 (AB9080, EMD Millipore, Billerica, MA), RyR3 (AB9082, EMD Millipore), PERK (3192, Cell Signaling), eIF2α (9722S, Cell Signaling), phospho-PERK (3179S, Cell Signaling), or phospho-eIF2α (9721S, Cell Signaling). The specificities of RyR antibodies were confirmed previously.¹⁸ After washing, membranes were incubated with secondary horseradish peroxidase–conjugated antibodies. Proteins were visualized with enhanced chemiluminescence reagents, and blots were exposed to Hyperfilm. Results were quantified with the Kodak electrophoresis documentation and analysis system and Kodak ID image analysis software (Kodak, Rochester, NY).

Chemicals

TUDCA (sodium salt), N-acetylcysteine, estrogen, and progesterone were purchased from MilliporeSigma, GSK2606414 and ryanodine from Tocris (Minneapolis, MN), and EUK-134 from Cayman Chemical (Ann Arbor, MI). Stocks of TUDCA and N-acetylcysteine were prepared in deionized H₂O, whereas stocks of estrogen, progesterone, GSK2606414, EUK-134 and ryanodine were prepared in DMSO. Stocks were then diluted to the appropriate concentration in corresponding experimental solutions and used immediately. An equal volume of vehicle was also used to serve as a control.

Statistical analysis

Data were expressed as means ± SEM obtained from the number of experimental animals. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Differences were evaluated for statistical significance (P < 0.05) by two-way ANOVA and two-way repeated measures ANOVA with the post hoc Bonferroni test or the t test where appropriate.

Results

High-altitude hypoxia suppressed pregnancy-induced increase in Ca²⁺ sparks and upregulation of RyR1 and RyR2 expression.

Ca²⁺ sparks were measured in uterine arterial SMCs as described previously.¹⁸ Figure S1 illustrate representative line-scan confocal images of Ca²⁺ sparks in uterine arteries from non-pregnant and pregnant sheep residing in low altitude or acclimatizing to high altitude hypoxia for~110 day. As shown in Figure 1A, pregnancy increased the percentage of myocytes that were firing Ca²⁺ sparks in uterine arteries of both low-altitude normoxic and high-altitude hypoxic animals. However, pregnancy produced a significantly less increase in firing myocytes of uterine arteries from high-altitude animals than those in low-altitude animals (Figure 1A). In addition, pregnancy significantly increased Ca²⁺ spark frequency (sparks/μm/s) in uterine arterial SMCs. Of importance, pregnancy-induced increases in Ca²⁺ spark frequency were significantly reduced in uterine arteries from high altitude animals (Figure 1B), as compared with those observed in low altitude animals. On the other hand, the spatial and temporal Ca²⁺ spark firing characteristics including amplitude (F/F₀), width (FWHM) and duration (FDHM) in uterine arterial SMCs were not significantly changed by pregnancy in both low- and high-altitude animals (Figure S2).

Figure 1. High-altitude hypoxia inhibited pregnancy-induced increases in Ca²⁺ sparks and RyR expression in uterine arteries.

Ca²⁺ sparks were measured in en-face endothelium-denuded uterine arteries. A and B, Pregnancy-induced increases in the percentage of myocytes firing Ca²⁺ sparks (A) and Ca²⁺ spark frequency (sparks/μm/s) (B) in uterine arteries from non-pregnant (UA_NP) and pregnant (UA_P) sheep in low- and high-altitude animals. Data are means ± SEM of 6 animals in each group. C and D, mRNA (C) and protein (D) expression of ryanodine receptors (RyRs) in uterine arteries of high-altitude animals. Data are means ± SEM of 5 animals in each group. Panels A, B were analyzed with two-way ANOVA, and C, D with the t test. *P < 0.05, UA_P versus UA_NP; ^#P < 0.05, high altitude versus low altitude.

All three subtypes of RyRs, RyR1, RyR2 and RyR3 are expressed in vascular SMCs, and RyR1 and RyR2 are the major isoforms that constitute independent Ca²⁺ sparks.^24–26 Previously, we demonstrated that pregnancy significantly increased the expression of all three subtypes of RyRs in uterine arteries (Figure S3).¹⁸ We thus determined whether and to what extent high altitude hypoxia during gestation altered pregnancy-induced upregulation of RyRs expression in uterine arteries. As shown in Figure 1C, pregnancy significantly increased mRNA abundance of RyR1 and RyR3, but not RyR2, in uterine arteries from high altitude animals. Of interest, pregnancy only increased protein expression of RyR3, but not RyR1 or RyR2, in uterine arteries of high-altitude animals (Figure 1D).

High-altitude hypoxia downregulated pregnancy-induced increase in STOCs and inhibited the regulation of myogenic tone by Ca²⁺ sparks/STOCs.

We then examined the effect of gestational hypoxia on STOCs in uterine arterial SMCs. Representative STOC tracings are illustrated in Figure S4. In low-altitude normoxic animals, STOCs in uterine arterial myocytes of pregnant animals occurred at a much more negative membrane potential (−50 mV) as compared with that in myocytes of non-pregnant animals (−20 mV), and pregnancy significantly increased STOC frequency and amplitude in uterine arteries (Figures 2A and 2C). Compared to the normoxic animals, gestational hypoxia significantly repressed pregnancy-induced increases in STOC frequency and amplitude in uterine arteries (Figures 2B and 2D). As shown in Figures 2A to 2D, STOCs in uterine arterial myocytes of pregnant animals at high altitude were shifted to a much more positive membrane potential and occurred around −30 mV. Of interest, STOCs in uterine arterial myocytes of pregnant animals at high altitude became similar to that observed in low-altitude normoxic non-pregnant animals (Figures 2A to 2D).

Figure 2. High-altitude hypoxia suppressed pregnancy-induced increases in STOCs and annulled the regulation of myogenic tone by ryanodine in uterine arteries.

A and B, STOC frequency in uterine arteries of low- (LA, A) and high-altitude (HA, B) non-pregnant (UA_NP) and pregnant (UA_P) animals. C and D, STOC amplitude in uterine arteries of low- (LA, C) and high-altitude (HA, D) non-pregnant (UA_NP) and pregnant (UA_P) animals. Data are means ± SEM of 5–6 animals in each group. E and F, Pressure-dependent myogenic tone was determined in uterine arteries from low- (LA, E) and high-altitude (HA, F) pregnant (UA_P) animals in the absence (Ctr) or presence of ryanodine (Ry, 30 μmol/L). Data are means ± SEM of 5 animals in each group. Data were analyzed by two-way repeated measures ANOVA with the post hoc Bonferroni test. A, C: *P < 0.05, LA-UA_P versus LA-UA_NP. B, D: HA-UA_P versus HA-UA_NP. E and F: *P < 0.05, Ry versus Ctr.

To explore whether hypoxia had a direct effect in the suppression of STOCs, we treated uterine arteries from low-altitude pregnant animals ex vivo with hypoxia (10.5% O₂) for 48 hours. As shown in Figure S5, myocytes from tissues treated ex vivo with hypoxia had significantly reduced STOC frequency (Figure S5A) and amplitude (Figure S5B) when compared to those observed in tissues treated with 21.0% O₂, indicating a direct effect of hypoxia on the inhibition of STOCs in uterine arteries observed in high-altitude animals.

Our previous study in low-altitude normoxic animals demonstrated that inhibition of Ca²⁺ sparks/STOCs with the RyR inhibitor ryanodine significantly increased pressure-dependent myogenic tone in uterine arteries of pregnant sheep and eliminated pregnancy-induced attenuation of uterine arterial myogenic response.¹⁸ We thus investigated the functional significance of hypoxic-suppressed Ca²⁺ sparks/STOCs by assessing myogenic tone before and after blocking RyRs with ryanodine (30 μmol/L) in uterine arteries from pregnant sheep acclimatized to high-altitude hypoxia. In contrast to the finding that ryanodine significantly increased myogenic tone in low-altitude pregnant animals (Figure 2E), the inhibition of RyR had no significant effect on pressure-dependent myogenic tone of uterine arteries from pregnant animals acclimatized to high-altitude hypoxia during gestation (Figure 2F).

High altitude hypoxia increased ER stress in uterine arteries of pregnant animals.

Hypoxia triggers ER stress and ER stress in uteroplacental tissues is a common feature of preeclampsia, IUGR and high-altitude pregnancy.^{6, 8, 27–29} We then examined whether gestational hypoxia at high altitude increased ER stress in uterine arteries. As shown in Figure 3A, the abundance of ER stress markers phosphorylated PERK and eIF2α (phospho-PERK and phospho-eIF2α) in uterine arteries were significantly greater in high-altitude than those in low-altitude animals, indicating enhanced ER stress in uterine arteries in response to chronic hypoxia during gestation.

Figure 3. High-altitude hypoxia increased endoplasmic reticulum (ER) stress in uterine arteries and inhibition of ER stress alleviated hypoxia-induced effects on STOCs, Ca²⁺ sparks and myogenic tone.

A, The ratio of phosphorylated protein/total protein of PERK and eIF2α in uterine arteries from low-(LA) and high-altitude (HA) pregnant (UA_P) animals. Data are means ± SEM of 3 animals in each group. B to E, Uterine arteries from pregnant sheep acclimatized to high-altitude hypoxia were treated ex vivo in the absence (Ctr) or presence of 1 mmol/L tauroursodeoxycholic acid (TUDCA) under 10.5% O₂ for 48 hours. STOC frequency and amplitude (B and C), Ca²⁺ sparks (D) and myogenic tone (E) were measured following the treatment. Data are means ± SEM of 4–6 animals of each group. Panels A and D were analyzed by the t test. A: *P < 0.05, HA-UA_P versus LA-UA_P; D: *P < 0.05, TUDCA versus Ctr. Panels B, C, E were analyzed by two-way repeated measures ANOVA with the post hoc Bonferroni test. *P < 0.05, TUDCA versus Ctr.

Inhibition of ER stress alleviated hypoxia-induced effects on Ca²⁺ sparks/STOCs and myogenic tone in uterine arteries.

The co-occurrence of elevated ER stress, suppressed Ca²⁺ sparks/STOCs and increased myogenic tone in uterine arteries of high-altitude pregnant animals suggests that gestational hypoxia-induced ER stress may attribute to the maladaptation of uterine vascular function. We then determined whether ER stress played a causative role in the development of uterine vascular maladaptation induced by gestational hypoxia. To this end, uterine arteries from high-altitude pregnant animals were treated ex vivo with an ER stress inhibitor, TUDCA (1 mmol/L) under hypoxia (10.5% O₂) for 48 hours. As shown in Figure S6, TUDCA treatment apparently increased STOCs in uterine arteries of high-altitude pregnant animals. Indeed, TUDCA treatment significantly increased both STOC frequency and amplitude (Figures 3B and 3C). Moreover, the occurrence of STOCs was shifted to a more negative membrane potential (e.g., from −30 mV to −40 mV) following TUDCA treatment. Furthermore, TUDCA treatment significantly increased Ca²⁺ spark frequency, without affecting percentage of Ca²⁺ spark-firing myocytes (Figure 3D) and other spark parameters such as amplitude, width and duration (Figure S7). Importantly, TUDCA treatment significantly reduced uterine arterial myogenic tone of high-altitude pregnant animals (Figure 3E), to the level similar to that of low-altitude pregnant animals shown in Figure 2E. Thus, the inhibition of ER stress in uterine arteries from high-altitude pregnant animals produced a functional phenotype similar to that of low-altitude pregnant animals. Additionally, ex vivo hypoxia-induced suppression of STOC frequency and amplitude in uterine arteries of low-altitude pregnant animals was also reversed by TUDCA (Figures S8A and S8B). Our previous study revealed that myogenic tone in uterine arteries of low-altitude pregnant animals was elevated by ex vivo hypoxic treatment.³⁰ Consistent with its impact on STOCs, TUDCA treatment also attenuated ex vivo hypoxia-induced increase in myogenic tone in uterine arteries of low-altitude pregnant animals (Figure S8C).

The increased levels of both phospho-PERK and phospho-eIF2α in uterine arteries of high-altitude pregnant animals suggest that the PERK pathway of ER stress was activated by gestation hypoxia. We then examined whether and to what extent a selective PERK inhibitor, GSK2606414 would reverse the effects of gestational hypoxia on STOCs in uterine arteries. In a way similar to TUDCA, GSK2606414 treatment also significantly increased both STOC frequency and amplitude in uterine arteries of high-altitude pregnant animals (Figures 4A and 4B) and attenuated myogenic tone (Figure 4C).

Figure 4. Inhibition of the PERK pathway relieved hypoxia-induced effects on STOCs and myogenic tone in uterine arteries of high-altitude pregnant animals.

Uterine arteries from pregnant sheep acclimatized to high-altitude hypoxia were treated ex vivo in the absence (Ctr) or presence of 1 μmol/L GSK2606414 under 10.5% O₂ for 48 hours. STOC frequency (A) and amplitude (B) and myogenic tone (C) were measured following the treatment. Data are means ± SEM of 5 animals of each group. Data were analyzed by two-way repeated measures ANOVA with the post hoc Bonferroni test. *P < 0.05, GSK2606414 versus Ctr.

Inhibition of ROS rescued gestational hypoxia-induced suppression of STOCs.

ER and oxidative stresses are closely linked events.^31–33 We previously demonstrated that oxidative stress played an important role in high-altitude hypoxia-mediated repression of BK_Ca channel activity and the increase of myogenic tone in uterine arteries of pregnant animals.^{30, 34, 35} We thus determined whether inhibiting oxidative stress affected gestational hypoxia-induced suppression of STOCs in uterine arteries. Uterine arteries obtained from high-altitude pregnant sheep were treated ex vivo with the antioxidant N-acetylcysteine (1 mmol/L) or synthetic superoxide dismutase/catalase mimetic EUK-134 (20 μmol/L) under hypoxia (10.5% O₂) for 48 hours, and STOCs were measured. N-acetylcysteine (Figures 5A and 5B) or EUK-134 (Figures 5C and 5D) treatment significantly increased STOCs frequency and amplitude. Interestingly, after N-acetylcystein or EUK-134 treatment, STOCs of uterine arteries from high-altitude hypoxic pregnant sheep functioned similarly to those from low-altitude normoxic pregnant animals shown in Figure 2A and 2C.

Figure 5. Effect of antioxidants on STOCs in uterine arteries of high-altitude pregnant animals.

A and B, STOC frequency (A) and amplitude (B) in uterine arteries from high-altitude pregnant animals treated ex vivo in the absence (Ctr) or presence of 1 mmol/L N-acetylcysteine (NAC) under 10.5% O₂ for 48 hours. C and D, STOC frequency (C) and amplitude (D) in uterine arteries from high-altitude pregnant animals treated ex vivo in the absence (Ctr) or presence of 20 μmol/L EUK-134 under 10.5% O₂ for 48 hours. Data are means ± SEM of 5 animals of each group. Data were analyzed by two-way repeated measures ANOVA with the post hoc Bonferroni test. *P < 0.05, NAC or EUK-134 versus Ctr.

ER and oxidative stresses differentially affected sex hormonal regulation of STOCs and myogenic tone in uterine arteries.

Sex steroid hormones play an important role in uterine vascular adaptation in pregnancy.³⁶ This hormonal regulation was diminished by gestational hypoxia and oxidative stress associated with hypoxia.^{19, 34, 37} We thus investigated the role of ER and oxidative stresses in the impaired hormonal regulation of STOCs in uterine arteries in hypoxia during pregnancy. As reported previously,¹⁸ sex hormones significantly increased both STOC frequency and amplitude in uterine arteries from low-altitude non-pregnant sheep (Figure S9A and S9B). In contrast, the hormonal treatment was unable to increase STOCs in uterine arteries from non-pregnant sheep acclimatized to high altitude hypoxia (Figure S9C and S9D). To determine roles of ER/oxidative stress in the suppression of hormonal regulation of STOCs during hypoxia, uterine arteries of high-altitude non-pregnant animals were treated with vehicle or estrogen/progesterone in the presence of an ER stress inhibitor TUDCA or a ROS scavenger N-acetylcysteine under 10.5% O₂. Inhibition of oxidative stress with N-acetylcysteine rescued the hormonal effect, in which steroid hormones upregulated STOC frequency and amplitude in uterine arteries of non-pregnant animals exposed to high altitude hypoxia (Figure 6A and 6B). In contrast, despite its effectiveness in alleviating hypoxia-induced effects on STOCs in uterine arteries of pregnant animals, TUDCA up to 3 mmol/L was unable to restore the effect of sex hormones in the upregulation of STOCs in high altitude hypoxic animals (Figure 6C and 6D). Consistently, TUDCA failed to recover the hormonal effect on myogenic tone of uterine arteries from high-altitude animals (Figure 6E).

Figure 6. Effect of ROS scavenging and ER stress inhibition on hormonal regulation of STOCs and myogenic tone in uterine arteries of high altitude non-pregnant animals.

A and B, STOC frequency (A) and amplitude (B) in myocytes of uterine arteries from high-altitude non-pregnant animals treated ex vivo with 17β-estradiol (E₂β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) or vehicle control (Ctr) in the presence of 1 mmol/L N-acetylcysteine (NAC) under 10.5% O₂ for 48 hours. C and D, STOC frequency (C) and amplitude (D) in myocytes of uterine arteries from high-altitude non-pregnant animals treated ex vivo with 17β-estradiol (E₂β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) or vehicle control (Ctr) in the presence of 3 mmol/L tauroursodeoxycholic acid (TUDCA) under 10.5% O₂ for 48 hours. E, Myogenic tone in uterine arteries from high-altitude non-pregnant animals treated ex vivo with 17β-estradiol (E₂β; 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L) or vehicle control (Ctr) in the presence of 3 mmol/L TUDCA under 10.5% O₂ for 48 hours. Data are means ± S.E.M. from 4–5 animals of each group. Data were analyzed by two-way repeated measures ANOVA with the post hoc Bonferroni test. *P < 0.05, E₂β/P4 versus Ctr.

Discussion

The present study provides evidence that gestational hypoxia suppresses Ca²⁺ sparks/STOCs in uterine arteries in part due to heightened ER/oxidative stress, revealing mechanistic insights into uterine vascular maladaptation in pregnancy complications associated with hypoxia. The major findings are (1) pregnancy-induced upregulation of RyR1/2 protein expression, Ca²⁺ sparks and STOCs in uterine arteries was inhibited by gestational hypoxia, (2) the regulation of myogenic tone by Ca²⁺ sparks/STOCs was diminished in uterine arteries of high-altitude pregnant animals, (3) gestational hypoxia increased ER/oxidative stress in uterine arteries, (4) inhibiting the ER stress PERK pathway alleviated gestational hypoxia-induced effects in the suppression of Ca²⁺ sparks/STOCs and increase of pressure-dependent myogenic tone in uterine arteries of pregnant animals, but failed to restore the effect of sex hormones in the regulation of STOCs and myogenic tone in high altitude hypoxic animals, (5) unlike the findings of ER stress, inhibition of oxidative stress recovered the hormonal effect in the upregulation of STOCs in high-altitude hypoxic animals and rescued gestational hypoxia-induced suppression of STOCs in uterine arteries.

STOCs at physiological membrane potentials (~ −40 mV) of vascular smooth muscle cells (VSMCs) fundamentally regulate vascular myogenic tone and blood flow in an organ as well as arterial pressure.^{38, 39} STOCs produce K⁺ efflux and cause membrane hyperpolarization, resulting in VSMC relaxation. STOCs are mediated by BK_Ca channels that sense RyR-mediated Ca²⁺ sparks. Ca²⁺ sparks and STOCs are tightly coupled in that the appearance of a Ca²⁺ spark almost always transiently activates a group of BK_Ca channels.⁴⁰ Uterine vascular adaptation during pregnancy involves increases in both the percentage of VSMCs with Ca²⁺ sparks and the Ca²⁺ spark frequency in uterine arteries.¹⁸ The finding that high altitude hypoxia significantly reduced pregnancy-induced increases in the percentage of myocytes with Ca²⁺ sparks and Ca²⁺ spark frequency without altering other spatiotemporal properties of Ca²⁺ sparks suggests that gestational hypoxia predominantly suppresses the effectiveness of pregnancy in promoting Ca²⁺ spark frequency in uterine arteries. Chronic hypoxia appears to be a general suppressor of Ca²⁺ spark frequency in VSMCs and the inhibition was also observed in mesenteric and pulmonary arterial myocytes.^{41, 42} Moreover, subarachnoid hemorrhage also reduced Ca²⁺ spark frequency without affecting amplitude in cerebral arterial myocytes.⁴³

Ca²⁺ sparks are mediated by coordinated activation of RyRs. Pregnancy at low altitude upregulated the expression of all three RyR subtypes, RyR1, RyR2 and RyR3, in uterine arteries.¹⁸ In the present study, we demonstrated that gestational hypoxia averted pregnancy-induced upregulation of RyR2 mRNA and protein abundance, but blocked RyR1 protein upregulation without affecting its mRNA abundance. The inconsistence of hypoxia-induced changes between RyR1 mRNA and protein abundance is probably due to changes in posttranscriptional modifications, mRNA stability, or translational efficiency, etc.^{44, 45} The global translation suppression due to activation of the PERK pathway could also account for this discrepancy. Moreover, microRNAs (miRs) may play a role in the hypoxia-induced effect by targeting RyR1 mRNA and downregulating its translation. Indeed, high-altitude hypoxia during gestation significantly increased miR-210, which played an important role in suppressing uterine vascular adaptation to pregnancy.^{5, 46} Sheep RyR1 mRNA 3’UTR possesses a less-than-perfect binding motif for the seed sequences of miR-210, revealing a possible regulatory target of miR-210 for future investigation. Consistent with the present study, chronic hypoxia also decreased RyR expression in fetal chicken heart.⁴⁷ Moreover, RyR1/2 repression was demonstrated in cultured syncytiotrophoblasts from preeclamptic placentas.⁴⁸ Among the three RyR subtypes, RyR1 and especially RyR2 contribute to generating Ca²⁺ sparks⁴⁹ Various copies of RyR1 and/or RyR2 form discrete clusters in the sarcoplasmic reticulum of SMCs and function as Ca²⁺ sparks discharge sites.^{26, 43, 50} Of importance, Ca²⁺ spark frequency is dependent on the size of RyR1/RyR2 clusters.^{50, 51} Subarachnoid hemorrhage-induced RyR2 repression reduced Ca²⁺ sparks discharge sites in cerebral arterial myocytes, resulting in subsided Ca²⁺ spark activity.⁴³ Our previous study in normoxic animals demonstrated that pregnancy-upregulated RyRs played an important role in increased Ca²⁺ sparks/STOCs in uterine arteries.¹⁸ The inhibition of pregnancy-induced RyR1 and RyR2 upregulation in hypoxic animals could reduce number and/or size of Ca²⁺ spark discharge sites in uterine arterial myocytes, consequently reducing Ca²⁺ spark activity. Although pregnancy-induced upregulation of RyR3 in the uterine artery was not altered by gestational hypoxia, RyR3 apparently did not contribute to the regulation of Ca²⁺ sparks, STOCs and myogenic tone in uterine arteries.⁵²

RyRs in the sarcoplasmic reticulum and BK_Ca channels in the plasmalemma are localized in close proximity to form functional units of the Ca²⁺ microdomain.⁵³ BK_Ca channels sense Ca²⁺ sparks to generate STOCs, and Ca²⁺ spark frequency is a major determinant of STOCs frequency and amplitude²⁴. Increased Ca²⁺ spark activity augmented STOCs frequency and amplitude in myocytes of cerebral and mesenteric arteries.^{54, 55} A functional link between Ca²⁺ sparks and STOCs in uterine arteries was established in our previous study.¹⁸ In the present study, we demonstrated the co-occurrence of reduced Ca²⁺ spark frequency and diminished STOCs in uterine arterial myocytes of high-altitude pregnant animals. Thus, aberrant Ca²⁺ sparks triggered by gestational hypoxia apparently impaired the Ca²⁺ spark/STOC coupling in uterine arteries. Intriguingly, ex vivo hypoxia treatment of uterine arteries of low-altitude animals simulated the effect of high-altitude hypoxia on STOCs in uterine arteries, suggesting a direct effect of hypoxia on the coupling mechanism. The BK_Ca channel β1 subunit was selectively downregulated in uterine arteries by gestational hypoxia and in cultured human and rodent arterial myocytes by chronic hypoxia.^{37, 56} As the β1 subunit is a pivotal facilitator to relay the signal of Ca²⁺ sparks to BK_Ca channel activation,⁵⁷ the BK_Ca channel dysfunction owing to altered stoichiometry of β1 and α subunits could also contribute to the suppressed STOCs in uterine arteries of high-altitude pregnant animals. In addition, the Ca²⁺ spark/STOC coupling only occurred at much more depolarized membrane potentials in uterine arteries of pregnant animals acclimatized to high altitude hypoxia. Thus, the Ca²⁺ spark-STOC coupling in uterine arteries of high-altitude pregnant animals became more like that observed in low-altitude non-pregnant animals,¹⁸ suggesting that gestational hypoxia inhibits pregnancy-induced uterine vascular adaptation.

The Ca²⁺ sparks/STOC coupling is an important mechanism to regulate vascular myogenic tone.³⁸ This regulatory mechanism also existed in uterine arteries of low-altitude pregnant animals, showing that inhibition of Ca²⁺ sparks/STOCs with RyR inhibitor ryanodine significantly increased pressure-dependent myogenic tone in uterine arteries of pregnant sheep and eliminated pregnancy-induced attenuation of uterine arterial myogenic response.¹⁸ In contrast, inhibiting Ca²⁺ spark activity with ryanodine was unable to alter uterine arterial myogenic tone of high-altitude pregnant animals, which is likely attributed to the downregulation of RyR1 and RyR2. Similar findings were obtained in cerebral arteries, showing that RyR2 downregulation conferred by subarachnoid hemorrhage resulted in the inability of Ca²⁺ sparks to regulate myogenic tone.⁴³ Likewise, inhibition of BK_Ca channels with tetraethylammonium was without effect on uterine arterial myogenic tone of high-altitude pregnant animals.³⁷ Indeed, disruption of the Ca²⁺ spark/STOC coupling by genetic deletion of the BK_Ca channel β1 subunit ablated the regulation of myogenic tone by ryanodine and iberiotoxin in cerebral arteries.⁵⁸ Our previous and present findings suggest that the incapability for the Ca²⁺ spark/STOC coupling to regulate myogenic tone following exposure to gestational hypoxia probably involves dysfunctions of both RyRs and BK_Ca channels. This diminished negative feedback mechanism is consistent with increased uterine vascular resistance and decreased uterine blood flow in high-altitude pregnancy^{5, 59} and in animal models of gestational hypoxia or preeclampsia.^{60, 61}

Hypoxia is a major trigger of ER/oxidative stress.^{27, 62} Heightened uteroplacental ER/oxidative stress were observed in preeclampsia, IUGR and high-altitude pregnancy^{6, 8, 28, 30, 34, 63, 64}and in rodent models of gestational hypoxia.^65–67 Among three branches of unfolded protein response (UPR) associated with ER stress,⁶⁸ the PERK signaling pathway is frequently activated by hypoxia.^{13, 67, 69, 70} Consistently, activation of the PERK branch was demonstrated in human placentas during gestation at high altitude⁶ and in uterine arteries of pregnant sheep acclimatized to high altitude hypoxia in the present study. Once activated, PERK phosphorylates eIF2α leading to reduced global translation.⁷¹ ER stress with activated PERK pathway plays an important role in vascular dysfunction in systemic hypertension and in pulmonary hypertension.^13–17 Activation of the PERK branch was shown to downregulate BK_Ca channel β1 subunit in VSMCs.⁷² Thus, gestational hypoxia-promoted PERK signaling may contribute to the downregulation of RyR1/2 receptors and BK_Ca channel β1 subunit in uterine arteries, leading to impaired Ca²⁺ spark/STOC coupling. As expected, a causative role of ER stress in the maladaptation of uterine vascular function was established by the findings that ER stress inhibition with TUDCA increased Ca²⁺ spark frequency, improved Ca²⁺ spark/STOC coupling and reduced myogenic tone in uterine arteries of high-altitude animals and in ex vivo hypoxia-treated uterine arteries of low-altitude animals. Similarly, elevated myogenic tone in coronary arteries of spontaneously hypertensive rats was attenuated by TUDCA.⁷³ Inhibition of ER stress also prevented/reversed chronic hypoxia-induced pulmonary hypertension in mice by reducing pulmonary vascular resistance, pulmonary artery remodeling, and right ventricular hypertrophy.^{13, 15}

ER stress and oxidative stress are closely associated and interreacted.^31–33 ROS could either arise in response to ER stress or trigger ER stress.³¹ Our previous studies revealed that the downregulation of BK_Ca channel β1 subunit and increased uterine arterial myogenic tone in high-altitude pregnant animals was attributed to hypoxia-induced increases in NADPH oxidase (Nox) 2 expression and ROS production, which could be ameliorated by apocynin and N-acetylcysteine.^{30, 34} Interestingly, Nox2 was a critical element linking ER stress to oxidative stress.^{74, 75} ER stress induction with tunicamycin increased Nox2 expression and Nox activity in primary endothelial cells from coronary arteries.⁷⁵ Importantly, Nox2 deficiency protects mice against ER stress-induced renal dysfunction.⁷⁴ In addition, p47phox^−/− (a cytosolic regulatory subunit of Noxs) mice attenuated ER stress-induced vascular dysfunction.⁷⁵ Moreover, hypoxia-induced ROS increased eIF2α phosphorylation by activating the PERK pathway.⁷⁶ Intriguingly, the antioxidant treatment functioned similarly to ER inhibition and restored the ability of pregnancy to upregulate STOCs in uterine arteries from high-altitude animals, suggesting a link between ER and oxidative stresses in uterine arteries from animals acclimatized to high-altitude hypoxia. Importantly, both ER and oxidative stresses promoted FOXO3a-dependent BK_Ca channel β1 degradation in VSMCs.^{72, 77} Thus, ER/oxidative stress could interdependently suppress the Ca²⁺ spark/STOC coupling, leading to increased uterine arterial myogenic tone. Significantly, supplementation with the antioxidant resveratrol increased both uterine artery blood flow velocity and fetal growth in a preeclamptic rodent model.⁷⁸ Moreover, maternal supplement with vitamins C and/or E in high-altitude pregnant sheep and hypoxic pregnant rats also increased birth weight.^{65, 79} In a rodent model, N-acetylcysteine diminished cadmium-induced placental ER stress and fetal growth restriction.⁸⁰ Therefore, albeit remaining a debate, scavenging ROS presents a potential therapeutic strategy for improving both uterine hemodynamics and fetal growth in high altitude, preeclamptic and IUGR pregnancies.

Although the primary goal of UPR in response to ER stress is to restore ER protein homeostasis by adjusting protein biosynthesis and folding capacity, it also plays a role in regulating gene expression. A recent study comparing the RNA transcription profile in response to tunicamycin-induced ER stress and H₂O₂ treatment in Hela cells revealed that only ~10% of the altered transcription (either up- or down-regulated) were shared by ER and oxidative stresses.⁸¹ It is not surprising that ER and oxidative stresses could also act divergently to impact uterine vascular function. Indeed, we observed that scavenging ROS, but not inhibition of ER stress, enabled estrogen/progesterone to upregulate STOCs in uterine arteries of high-altitude non-pregnant animals. Thus, in response to ER and oxidative stresses induced by gestational hypoxia, hormonal regulation of the Ca²⁺ spark/STOC coupling machinery in uterine arteries was selectively disrupted by oxidative stress in high altitude animals. This is consistent with our previous findings that N-acetylcysteine restored steroid hormone-mediated up-regulation of both BK_Ca channel activity and BK_Ca channel-mediated relaxation in uterine arteries under gestational hypoxia.^{30, 35} Our previous studies demonstrated that the estrogen-induced upregulation of BK_Ca channel β1 subunit and probably RyRs in uterine arteries was mediated by the estrogen receptor α (ERα).^{18, 82} The promoter of ERα encoding gene ESR1 in uterine arteries of non-pregnant animals was hypermethylated and pregnancy promoted demethylation leading to increased ESR1 expression.^{23, 83} The DNA methylation-demethylation cycle is maintained by DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs) in uterine arteries.^{23, 84} Hypoxia-inducible factor 1 (HIF1) is the primary factor mediating the adaptive responses to hypoxia. HIF1α levels in uterine arteries of both high-altitude non-pregnant and pregnant animals increased by ~2-fold compared to low-altitude partners.³⁴ Hypoxia with the participation of ROS stabilizes HIF1α, which is dimerized with HIF1β to regulate of target gene expression by binding to hypoxia-responsive element (HRE) in the promoter.⁸⁵ Interestingly, DNMT1 and DNMT3b are HIF1-dependent and were upregulated in response to hypoxia.^{84, 86} In addition, TET activity was suppressed by oxidative stress.⁸⁷ Furthermore, the expression of TET1 was suppressed by miR-210 in uterine arteries.⁵ The expression of miR-210 is regulated by HIF1α and was upregulated in uterine arteries of both high-altitude non-pregnant and pregnant animals.⁵ Thus, relieving oxidative stress would destabilize HIF1α and boost the demethylation of ESR1 promoter to facilitate the estrogen signaling. In contrast, inhibition of ER stress increased HIF1α expression.⁸⁸ Consistently, TUDCA was unable to facilitate activating the estrogen signaling in uterine arteries of non-pregnant hypoxic animals. Consequently, ER and oxidative stresses exhibited different effects on hormonal regulation of the Ca²⁺ spark/STOC machinery expression. The hormonal regulation of BK_Ca channel β1 expression in uterine arteries started early in pregnancy.⁸⁹ Our results suggest a temporal order of ER/oxidative stresses actions in the dysfunction of uterine circulation in pregnancy complications. Oxidative stress apparently acted in early stage of pregnancy to suppress the hormonal regulation of the Ca²⁺ spark/STOC coupling machinery, whereas both ER and oxidative stresses also directly impaired the mediators of Ca²⁺ sparks and STOCs throughout pregnancy. Together, both the convergent and divergent actions of ER and oxidative stresses could contribute to uterine vascular maladaptation conferred by gestational hypoxia.

Perspective

Pregnancy at high altitude is associated with increased incidence of preeclampsia and IUGR, and the maladaptation of uterine vascular function contributes to pregnancy complications associated with gestational hypoxia.¹ Previously, we demonstrated in an animal model of pregnant sheep acclimatized to high altitude that gestational hypoxia suppressed pregnancy-induced uterine arterial adaptation and increased uterine vascular resistance and maternal systemic blood pressure.⁵ In the present study, we find that hypoxia during gestation diminishes pregnancy-induced upregulation of RyRs, leading to decreased Ca²⁺ sparks and STOCs and increased myogenic tone and vascular resistance of uterine arteries. Of importance, the finding demonstrates that inhibition of ER and oxidative stresses alleviates hypoxia-induced effects on Ca²⁺ sparks/STOCs and myogenic tone in uterine arteries, revealing a novel mechanism of ER and oxidative stresses in phenotypic programming of attenuated Ca²⁺ sparks/STOCs and heightened vascular resistance in the maladaptation of uterine circulation in high-altitude pregnancy. The finding of divergent actions of ER and oxidative stresses in the regulation of hormonal effect on uterine arteries during hypoxia is highly intriguing and suggests different mechanisms that deserve further investigation. Thus, the present study provides mechanistic insights into the understanding of pregnancy complications of preeclampsia and IUGR associated with gestational hypoxia.

Novelty and Significance.

What Is New?

• High altitude hypoxia during gestation suppresses pregnancy-induced upregulation of RyR1/RyR2 protein abundance, Ca²⁺ sparks and STOCs in uterine arteries.

• Consequently, aberrant Ca²⁺ sparks and STOCs induced by gestational hypoxia impairs pregnancy-induced attenuation of uterine arterial myogenic tone.

• Gestational hypoxia increases ER/oxidative stress in uterine arteries.

• Inhibition of ER/oxidative stress alleviates gestational hypoxia-induced effects on Ca²⁺ sparks/STOCs and myogenic tone in uterine arteries.

• Alleviating oxidative stress but not ER inhibition restores the hormonal effect in the upregulation of STOCs in uterine arteries during hypoxia.

What Is Relevant?

• The present study reveals novel effects of both convergent and divergent actions of ER and oxidative stresses impacting on Ca²⁺ sparks and STOCs in phenotypic programming of heightened uterine vascular resistance in high-altitude pregnancy and provides mechanistic insights and potential therapeutic targets in pregnancy complications associated with gestational hypoxia.

Summary.

The present study demonstrates that high altitude hypoxia during gestation inhibits pregnancy-mediated uterine arterial adaptation by suppressing Ca²⁺ sparks/STOCs and enhancing myogenic tone. Of importance, it reveals novel mechanisms of both convergent and divergent actions of ER and oxidative stresses impacting on Ca²⁺ sparks and STOCs in phenotypic programming of heightened uterine vascular resistance in high-altitude pregnancy and provides mechanistic insights and potential therapeutic targets in pregnancy complications associated with gestational hypoxia.