MicroRNA-210 mediates hypoxia-induced repression of spontaneous transient outward currents in sheep uterine arteries during gestation

Abstract

Hypoxia during pregnancy is a major contributor to the pathogenesis of preeclampsia and intrauterine growth restriction. Our recent studies revealed that pregnancy-induced uterine vascular adaptation depended on the enhanced Ca²⁺ spark/spontaneous transient outward current (STOC) coupling and hypoxia during gestation diminished this adaption. In the present study, we test the hypothesis of a mechanistic link of microRNA-210 (miR-210) in hypoxia-impaired Ca²⁺ spark/STOC coupling in uterine arteries. Pregnant ewes acclimatized to high altitude (3,801 m) hypoxia for ~110 days significantly increased circulation levels of miR-210 in both the ewe and her fetus. Treatment of uterine arteries from high-altitude animals with the antagomir miR-210-LNA recovered hypoxia-repressed STOCs in pregnant ewes and restored the hormonal regulation of STOCs in non-pregnant animals. In uterine arteries from low-altitude control animals, miR-210 mimic suppressed STOCs in pregnant ewes and inhibited the hormonal regulation of STOCs in non-pregnant animals. Mechanistically, miR-210 directly targeted and downregulated type 2 ryanodine receptor and large-conductance Ca²⁺-activated K⁺ channel β1 subunit, resulting in significant decreases in Ca²⁺ sparks and STOCs in uterine arteries. In addition, miR-210 indirectly decreased STOCs by targeting ten-eleven translocation methylcytosine dioxygenase. Together, the present study revealed a mechanistic link of miR-210 in hypoxia-induced repression of Ca²⁺ spark/STOC coupling in uterine arteries during gestation, providing novel insights into the understanding of pregnancy complications associated with hypoxia and the potential therapeutic targets.

Summary

The present study demonstrates that elevated miR-210 is necessary and sufficient to inhibit Ca²⁺ spark/STOC coupling in uterine arterial adaptation to gestational hypoxia by downregulating RyR2, BK_Ca β1 subunit and TET1. The study provides new insights into the understanding of fundamental mechanisms underlying programming of vascular dysfunction caused by gestational hypoxia, impacting on maternal cardiovascular health and developmental plasticity.

Graphical Abstract

Introduction

Hypoxia is a common and severe stress to an organism’s homeostatic mechanisms, and hypoxia during gestation is associated with significantly increased incidence of maternal complications of preeclampsia, adversely impacting fetal development and increasing subsequent risk for cardiovascular and metabolic disease.¹ Pregnant women at high altitude have a two- to four-fold increase in the incidence of preeclampsia, which is often proceeded by a decrease in uteroplacental blood flow.^2–5 Extensive human and animal studies have revealed a causative role of increased uterine vascular resistance and placental hypoxia in preeclampsia and intrauterine growth restriction (IUGR) associated with hypoxia in pregnancy. In an animal model of pregnant sheep acclimatized to high altitude hypoxia, we have found that pregnant ewes are similar to humans in that they have an increase in uterine vascular resistance and an elevation in maternal systemic blood pressure.^{6, 7} Yet, much remains unknown of the mechanisms underlying maternal cardiovascular adaptation to long-term hypoxia during gestation. It should be noted that the impact of hypoxia on the uterine circulation is complex and contradictory observations have been reported in humans and animals. Reduced uterine artery resistance index was also observed in high-altitude human pregnancy and in a rat model of IUGR induced by chronic hypoxia,^{8, 9} which may represent a compensatory mechanism. Furthermore, nature selection has enabled native Andeans to have greater uterine blood flow during pregnancy than European women at high altitude and protect them from hypoxia-associated IUGR.¹⁰

Hypoxia via HIF1α robustly upregulates microRNA-210 (miR-210), which together with HIF1α are master regulators of the cellular hypoxic response.^11–17 MiR-210 is specifically induced by HIF1α, but not HIF2α.^{18, 19} Mature miR-210 of 22 nt is highly homologous across species and is identical among the human, sheep and rodent. Of importance, an increase in miR-210 appears to be a common feature in preeclampsia and gestational hypoxia. Thus, elevated placental expression of miR-210 and circulating miR-210 levels have been demonstrated in both preeclampsia and pregnancy at high altitude in pregnant women.^20–27 We have demonstrated significant increases in HIF1α and miR-210 in uterine arteries in pregnant sheep acclimatized to high altitude hypoxia.^{6, 28} It should be emphasized that in addition to miR-210, many other miRs in the uteroplacental unit are also altered in preeclampsia/IUGR and contribute to the pathogenesis of these pregnancy complications.²⁹

Spontaneous transient outward currents (STOCs) at physiological membrane potentials of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ, as well as arterial blood pressure.^30–40 We demonstrated in sheep that pregnancy significantly increased STOCs in uterine arteries.⁴¹ Of importance, our recent study revealed that high altitude hypoxia suppressed this pregnancy-induced upregulation of STOCs in uterine arteries,⁴² identifying an exciting and novel regulatory target in programming of uterine vascular malfunction in response to gestational hypoxia. STOCs are mediated by the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel that is mainly activated by highly localized and concentrated Ca²⁺ release events through the opening of ryanodine receptors (RyRs) in the sarcoplasmic reticulum, termed Ca²⁺ sparks.^{30, 39} 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.⁴³ All three subtypes of RyRs, RyR1–3, exist in vascular smooth muscle cells. Our recent studies demonstrated that pregnancy significantly increased the expression of all three RyR subtypes in uterine arteries and enhanced the co-localization of RyR2 with the β1 subunit of BK_Ca channel in the uterine artery.^{41, 44} In addition, we demonstrated that high altitude hypoxia suppressed pregnancy-induced upregulation of RyR2 in uterine arteries ⁴² and knockdown of RyR2 with siRNAs significantly decreased Ca²⁺ spark frequency and suppressed STOCs in uterine arteries.⁴⁴

A question of great relevance is whether and to what extent the elevation of miR-210 mediates the dysregulation of Ca²⁺ spark/STOC coupling in programming of uterine vascular malfunction in response to gestational hypoxia. In the present study, we revealed a mechanistic link of miR-210 in hypoxia-induced repression of Ca²⁺ spark/STOC coupling in uterine arteries during gestation by targeting both type 2 ryanodine receptor and BK_Ca β1 subunit, resulting in significant decreases in Ca²⁺ sparks and STOCs in uterine arteries. These findings provide novel insights into the mechanistic understanding of pregnancy complications associated with hypoxia and the potential therapeutic targets.

Methods

The authors declare that all supporting data are available within the article.

Animal model and tissue preparation

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 euthanized via intravenous injection of 15 mL T-61 solution (Hoechst-Rousel, Somervile, NJ), according to American Veterinary Medical Association guidelines.

Time-dated pregnant and non-pregnant sheep (Ovis aries) were obtained from Nebeker Ranch (~300 m above sea level, ewe PaO₂: ~102 mm Hg), Lancaster, CA. For high-altitude hypoxic treatment, non-pregnant and pregnant ewes when they were able to be confirmed pregnancy with ultrasound around 30 days of gestation were transported to and maintained at the White Mountain Research Station (altitude of 3,801 m, ewe PaO₂: ~60 mm Hg), White Mountain, Bishop, CA. After the treatment, near-term (~142–145 days of gestation) pregnant and non-pregnant ewes were transported to Loma Linda University where they were studied. This high-altitude sheep “model” has been well-established and extensively studied over the past three decades. As we reported in early studies, although this model did not result in pre-term labor or overtly fetal growth restriction, per se, the high-altitude fetuses showed moderate arterial hypoxemia, significantly elevated mean arterial blood pressure and ~25% decreased combined cardiac output. In addition, high-altitude hypoxia had profound effects on maternal uterine vascular and placental adaptation to pregnancy, and on developmental plasticity of cerebrovascular, neural, cardiac, pulmonary, endocrine, metabolic and other systems.¹ Uterine arteries were isolated from non-pregnant and pregnant ewes bearing either singleton or twin fetuses. For pregnant ewes with a singleton fetus, uterine arteries were isolated from the uterine horn bearing the fetus. 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. The resistance-sized uterine artery segments (~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). For tissue culture, uterine arteries were placed in a culture dish containing 5 mL of phenol red-free DMEM supplemented with 1% charcoal-stripped fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin and incubated at 37 °C in humidified incubators for 48 hours, as described previously.^{45, 46} For the hormonal treatment, uterine arteries from non-pregnant sheep were incubated in the presence of vehicle DMSO or 17β-estradiol (E2β, Sigma, St. Louis, MO) (0.3 nmol/L) plus progesterone (P4, Sigma) (100.0 nmol/L), which mimicked the circulating levels of these two hormones in pregnancy, as reported previously.^{45, 46} Following the treatment, the subsequent experiments were conducted in the absence of vehicle or hormones.

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).⁴¹ 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 (ThermoFisher, Waltham, Massachusetts) dissolved in DMSO along with 0.1% pluronic F127 (ThermoFisher) for 2 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 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

Arterial smooth muscle cells were enzymatically dissociated from resistance-sized uterine arteries as described previously.⁴¹ 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 smooth muscle cells 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.⁴¹ 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. 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. The currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA/pF).

Measurement of plasma miR-210

Plasma miRs were purified using miRNeasy serum/plasma kit (Qiagen, Germantown, MD) according to the manufacturers’ instructions. MiR-210 was subsequently measured using miScript II RT kit (Qiagen) following the manufacturers’ instructions. Relative gene expression of miR-210 was calculated by 2^−ΔΔCT method.⁴⁷

Transfection of microRNA mimic and antagomir

Tissue transfection was conducted as described previously.^{6, 48} Briefly, the mixtures of miR-210 mimic (Qiagen), miR-210-LNA (Qiagen), or AllStars negative controls (Qiagen), with HiPerfect transfection reagent (Qiagen) and Opti-MEM 1 (ThermoFisher) were prepared and incubated for ~30 minutes at room temperature. The mixtures were subsequently added to DMEM containing 1% charcoal-stripped fetal bovine serum in a 6-well plate (final concentration of 100 nmol/L) maintained in an incubator at 37 °C for 48 hours.

Knockdown of genes encoding ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and BK_Ca β1 subunit

To knockdown TET1 or BK_Ca β1 subunit encoding gene KCNMB1, resistance-sized uterine arteries of pregnant sheep were transfected with TET1 or KCNMB1 siRNAs (Dharmacon Inc, Lafayette, CO), as described previously.⁴⁹ Target or scrambled siRNAs were mixed with HiPerfect Transfection Reagent (Qiagen) and Opti-MEM I (ThermoFisher) for ~30 minutes at room temperature and subsequently added into DMEM/F12 supplemented with 1% charcoal-stripped fetal bovine serum. Tissues were incubated in the medium containing target or scrambled siRNAs at a final concentration of 100 nmol/L in an incubator at 37 °C for 48 hours. Our previous studies showed that siRNAs of TET1 and KCNMB1 successfully knocked down TET1 and KCNMB1, respectively, in uterine arteries.⁴⁹

Luciferase reporter assay

Luciferase reporter assay was performed as previously described.⁶ A 249-bp 3′UTR segment of ovine KCNMB1 mRNA harboring the mature miR-210 target was cloned into the pmirGLO vector (Promega) between XhoI (5′) and Xba I (3′). This final construct was designated pmiR-XBX. For luciferase assays, uterine arterial smooth muscle cells were transfected with pmiR-XBX or pmirGLO (for the vector control) together with miR-210 mimic or negative control (Qiagen) using X-tremeGENE HP DNA transfection reagent (Roche, Indianapolis, IN) following the manufacturer’s instructions. The cells were processed using the Dual-Luciferase Reporter Assay System (Promega) 48 hours after transfection.

RNA-induced silencing complex-immunoprecipitation (RISC-IP) assay

RISC-IP assay was performed as previously described.⁵⁰ Uterine arterial smooth muscle cells were transfected 100 nmol/L miR-210 mimic or negative control (Qiagen) by using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were harvested 24 hours after transfection and washed in ice-cold PBS followed by complete lysis buffer (Active Motif) at 4 °C for 10 minutes. RISC-IP of the lysate was conducted using the miRNA Target IP Kit (Active Motif) according to the manufacturer’s instructions. The RNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, Fisher Scientific) once, chloroform once and precipitated and resuspended in RNase-free water. The precipitated RNA was subjected to RT-qPCR using primers specific for the sheep RyR2 3′-UTR. β-actin was used as an internal control. The relative abundance of RyR2 transcript pulled down by Ago1/2/3 antibody was calculated by the 2^−ΔΔCT method and is presented as the fold induction relative to the control. Primers used were 5′-CACACACATGCACACACACACACTC (forward) and 5’-TGGCAGTTCACAACATCAGCAAG (reverse) for RyR2 and 5′-GCAGGTCATCACCATCGGCAAT (forward) and 5’-ACCGTGTTGGCGTAGAGGTCCT (reverse) for β-actin.

Western immunoblotting

Protein abundance of RyR2 in uterine arteries was measured as described previously.⁴¹ 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 (1:1000 dilution) against RyR2 (AB9080, EMD Millipore, Billerica, MA). The specificity of RyR2 antibody was confirmed previously.⁴¹ 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).

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 ANOVA or t test where appropriate.

Results

High altitude pregnancy elevated miR-210 in maternal and fetal circulations

High-altitude pregnancy elevated uteroplacental miR-210.^{6, 27} A human study also revealed that circulating miR-210 was increased in high-altitude pregnancy.⁵¹ We then examined whether high altitude hypoxia altered circulating miR-210 in pregnant sheep. We found that pregnant ewes acclimatized to high altitude hypoxia were similar to humans in that they had a significant increase in miR-210 in both maternal and fetal circulations (Figure 1).

Figure 1. High-altitude pregnancy increased circulating miR-210.

Levels of miR-210 in the maternal and fetal circulations of normoxic control and high-altitude hypoxia were determined. Data are means ± SEM of 5 animals of each group; independent-samples t-test; *P < 0.05, high-altitude hypoxia versus normoxic control.

MiR-210-LNA reversed hypoxia-induced suppression of STOCs in uterine arteries

Our recent study demonstrated that high altitude hypoxia suppressed pregnancy-induced upregulation of STOCs in uterine arteries.⁴² In addition, we showed a significant increase in miR-210 in uterine arteries in pregnant sheep acclimatized to high altitude hypoxia, which was blocked by transfecting the miR-210 inhibitor miR-210-LNA.⁴⁸ To reveal a causal effect of elevated miR-210 on repressed STOCs in uterine arteries of high-altitude pregnant animals, we examined whether inhibition of endogenous miR-210 by miR-210-LNA could reverse the effect of hypoxia on STOCs in uterine arteries. To this end, uterine arteries of high-altitude pregnant animals were transfected with negative control or miR-210-LNA under 10.5% O₂. Compared to negative control, miR-210-LNA markedly increased STOC frequency and amplitude in uterine arteries of high-altitude pregnant animals (Figures 2A and 2B). In addition, miR-210-LNA shifted STOC occurrence to a more negative membrane potential (from −20 mV to −40 mV), similar to that observed in normoxic control animals.

Figure 2. MiR-210-LNA reversed the effect of hypoxia on STOCs in uterine arteries of high-altitude animals.

A and B, Uterine arteries from high-altitude pregnant sheep were transfected with 100 nmol/L miR-210-LNA or negative control (Neg Ctr) under 10.5% O₂ for 48 hours and STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 5 animals in each group. *P < 0.05, miR-210-LNA versus Neg Ctr. C - F, Uterine arteries from high-altitude nonpregnant sheep were transfected with Neg Ctr (C, D) or 100 nmol/L miR-210-LNA (E, F) under 10.5% O₂ for 48 hours, in the presence of vehicle control (Ctr) or 17β-estradiol (E₂β 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L). STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 5 animals in each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, E₂β/P4 versus Ctr.

MiR-210-LNA restored hormonal regulation of STOCs in uterine arteries

Steroid hormones play a pivotal role in the adaptation of uterine circulation during pregnancy. Previously, we demonstrated that the ex vivo treatment of uterine arteries from nonpregnant animals with estrogen and progesterone significantly increased both STOC frequency and amplitude.⁴¹ This hormonal regulation of STOCs was rescinded in uterine arteries of non-pregnant animals acclimatized to high altitude hypoxia. Thus, in contrast to that observed in normoxic control animals, the ex vivo hormonal treatment of uterine arteries from high altitude non-pregnant sheep failed to affect STOC frequency and amplitude (Figures 2C and 2D). Of importance, blockade of miR-210 with miR-210-LNA restored the hormonal regulation of STOCs in uterine arteries of non-pregnant animals acclimatized to high altitude hypoxia (Figures 2E and 2F).

MiR-210 mimic imitated the effect of gestational hypoxia and suppressed STOCs in uterine arteries

We next determine whether and to what extent miR-210 mimic has a direct effect in suppressing STOCs in uterine arteries. Uterine arteries from normoxic control pregnant sheep were treated with miR-210 mimic and STOCs were measured after the treatment. As shown in Figures 3A and 3B, miR-210 treatment shifted STOC occurrence to a more positive membrane potential (from −40 mV to −20 mV) and significantly suppressed both STOC frequency and amplitude. Indeed, the effect of miR-210 imitated that of high-altitude hypoxia, and STOCs recorded in uterine arteries of control animals in the presence of miR-210 were not significantly different from those observed in uterine arteries of pregnant ewes acclimatized to high altitude hypoxia (Figures 2A and 2B). In addition, miR-210 inhibited the ex vivo hormonal regulation of STOCs in uterine arteries. Uterine arteries from control non-pregnant animals were treated ex vivo with estrogen and progesterone under 21.0% O₂ for 48 hours in the absence or presence of miR-210 mimic. In the absence of miR-210 mimic, the hormonal treatment significantly increased both STOC frequency and amplitude (Figures 3C and 3D). In contrast, in the presence of miR-210 mimic, uterine arteries responded like the tissues from no-pregnant ewes acclimatized to high altitude hypoxia and lost the effect of ex vivo hormonal regulation of STOCs (Figures 3E and 3F).

Figure 3. MiR-210 mimic inhibited STOCs in uterine arteries of normoxic control animals.

A and B, Uterine arteries from normoxic control pregnant sheep were transfected with 100 nmol/L miR-210 mimic (miR-210) or negative control (Neg Ctr) under 21.0% O₂ for 48 hours and STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 5 animals in each group. *P < 0.05, miR-210 versus Neg Ctr. C - F, Uterine arteries from normoxic control nonpregnant sheep were transfected with Neg Ctr (C, D) or 100 nmol/L miR-210 (E, F) under 21.0% O₂ for 48 hours, in the presence of vehicle control (Ctr) or 17β-estradiol (E₂β 0.3 nmol/L)/progesterone (P4; 100.0 nmol/L). STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 6 animals in each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, E₂β/P4 versus Ctr.

MiR-210 directly targeted and downregulated RyR2 and Ca²⁺ sparks in uterine arteries

STOCs are manifestations of RyR-mediated Ca²⁺ sparks. Although all three subtypes of RyRs exist in vascular smooth muscle cells, their function in the regulation of Ca²⁺ sparks may be different. Our recent study identified an important role of RyR2 and demonstrated that knockdown of RyR2 with siRNAs significantly decreased Ca²⁺ sparks and suppressed STOCs in uterine arteries.⁴⁴ In addition, we showed that high altitude hypoxia suppressed pregnancy-induced upregulation of RyR2 in uterine arteries.⁴² We thus investigated the effect of miR-210 in the regulation of RyR2 and Ca²⁺ sparks in uterine arteries. We identified a potential miR-210 complementary binding site in RyR2 3′-UTR using the sequence alignment program MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html) (Figure 4A). We then determined whether RyR2 is genuinely targeted and regulated by miR-210. RISC-IP assay revealed that RyR2 mRNA was enriched by ~2-fold in miR-210-transfected uterine arteries of pregnant animals compared to negative control-treated tissues (Figure 4A), indicating that RyR2 is a direct target of miR-210. Moreover, miR-210 treatment significantly downregulated RyR2 protein abundance in uterine arteries compared to negative control (Figure 4B).

Figure 4. MiR-210 mimic downregulated RyR2 and Ca²⁺ sparks in uterine arteries.

A. RyR2 is a bona fide target of miR-210 in uterine arteries. Schematic representation of alignment of the predicted miR-210 binding site to ovine RyR2 3′UTR is shown. Uterine arterial smooth muscle cells were treated with 100 nmol/L miR-210 mimic (miR-210) or negative control (Neg Ctr) for 24 hours and RISC-IP analysis of abundance of RyR2 3′UTR pulled down by Ago1/2/3 antibody was determined after the treatment. B - D. Uterine arteries from normoxic control pregnant sheep were treated with 100 nmol/L miR-210 or Neg Ctr under 21.0% O₂ for 48 hours. RyR2 protein abundance (B), the percentage of myocytes with Ca²⁺ sparks (C) and Ca²⁺ spark frequency (sparks/μm/s) (D) were determined after the treatment. Data are means ± SEM of 5 animals in each group; independent-samples t-test; *P < 0.05, miR-210 versus Neg Ctr.

We next determined whether miR-210 altered Ca²⁺ sparks in uterine arteries. In uterine arteries of pregnant animals transfected with negative control, Ca²⁺ sparks were observed in 67.2 ± 5.1% of myocytes. MiR-210 significantly reduced Ca²⁺ spark-firing myocytes to 46.8 ± 4.8 (Figure 4C), representing a ~30% decrease. Moreover, miR-210 also lowered Ca²⁺ spark frequency by ~50% (Figure 4D). Other Ca²⁺ spark firing characteristics such as amplitude (F/F0), width (FWHM) and duration (FDHM) in uterine arteries were not significantly altered by miR-210 (data not shown). Of interest, the effects of miR-210 on Ca²⁺ sparks are similar to those observed in the previously study, showing ~30% decrease in Ca²⁺ spark-firing myocytes and ~50% reduction in Ca²⁺ spark frequency in uterine arteries of pregnant ewes acclimatized to high altitude hypoxia.⁴²

MiR-210 downregulated BK_Ca β1 subunit in uterine arteries

BK_Ca β1 subunit residing in the close proximity to RyRs within spatial boundaries of the Ca²⁺ microdomain functions as a Ca²⁺ sensor and is critical for the Ca²⁺ spark/STOC coupling.^{37, 52–54} Pregnancy increased the co-localization of RyR2 with the β1 subunit in the uterine artery.⁴⁴ Potential miR-210 binding sequences were identified in the 3′-UTR of ovine β1 subunit encoding gene KCNMB1. As shown in Figure 5A, a co-transfection of pmirGLO-XBX containing a 249-bp 3′-UTR segment of ovine KCNMB1 harboring the miR-210 target sites and miR-210 mimic produced a significant decrease in the luciferase activity, demonstrating a direct interaction of miR-210 with the 3′-UTR of KCNMB1. This is consistent with the previous finding showing that miR-210 mimic downregulated the β1 subunit in uterine arteries.⁶ Of importance, knockdown of the β1 subunit with siRNAs significantly reduced both STOC frequency and amplitude (Figures 5B and 5C), recapitulating the effect of miR-210 on STOCs in uterine arteries (Figures 3A and 3B).

Figure 5. MiR-210 mimic downregulated BK_Ca β1 subunit encoding gene KCNMB1 and STOCs in uterine arteries.

A, Schematic representation of alignment of the predicted miR-210 binding site to ovine KCNMB1 3′-UTR is shown. Uterine arterial smooth muscle cells were co-transfected with the constructs containing ovine KCNMB1 3′-UTR (pmiR-XBX) in the presence of 100 nmol/L miR-210 mimic (miR-210) or negative control (scramble) for 48 hours. Luciferase assays of various pmirGLO reporters were measured after the treatment. Data are means ± SEM from 8-12 assays; one-way ANOVA with the post hoc Bonferroni test; *P < 0.05, pmirGLO-XBX + miR-210 versus pmirGLO-XBX + scramble. B and C, Uterine arteries of normoxic control pregnant sheep were treated with scramble control (Ctr siRNA) or KCNMB1 siRNAs for 48 hours and STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 5 animals in each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, KCNMB1 siRNA versus Ctr siRNA.

Involvement of TET1 in miR-210-mediated regulation of STOCs in uterine arteries

TETs regulate active DNA demethylation. TET1 is a target of miR-210 and it was downregulated by miR-210 in uterine arteries.⁶ We thus determined the role of TET1 in the regulation of STOCs in uterine arteries. Uterine arteries from pregnant ewes were transfected with TET1 siRNAs and scramble controls. As shown in Figures 6A and 6B, TET1 siRNAs treatment significantly decreased both STOC frequency and amplitude in uterine arteries. To examine whether TET was involved in miR-210-LNA-mediated enhancement of STOCs in uterine arteries of pregnant animals acclimatized to high altitude hypoxia (Figures 2A and 2B), we examine the effect of miR-210-LNA on STOCs in the uterine arteries in the presence of a TET inhibitor, fumarate. As shown in Figures 6C and 6D, fumarate annulled the effect of miR-210-LNA to restore STOCs in uterine arteries of pregnant animals acclimatized to high altitude hypoxia.

Figure 6. Effect of TET1 on STOCs in uterine arteries.

A and B, Uterine arteries of normoxic control pregnant sheep were treated with scramble control (Ctr siRNA) or TET1 siRNAs for 48 hours and STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 5 animals in each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, TET1 siRNA versus Ctr siRNA. C and D, Uterine arteries from high-altitude pregnant sheep were treated with 100 nmol/L miR-210-LNA or negative control (Neg Ctr) in the presence of 3 mmol/L monoethyl fumarate (MEF) under 10.5% O₂ for 48 hours. STOC frequency and amplitude were measured after the treatment. Data are means ± SEM of 5 animals in each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test.

Discussion

The present study provides novel evidence of a mechanistic link of miR-210 in the hypoxia-induced repression of Ca²⁺ spark/STOC coupling in uterine arteries during gestation by targeting both type 2 ryanodine receptor and BK_Ca β1 subunit, resulting in significant decreases in Ca²⁺ sparks and STOCs that are of fundamental importance in programming of uterine vascular malfunction in response to gestational hypoxia. The major findings are: 1) hypoxia during gestation elevated miR-210 in maternal and fetal circulations, 2) inhibition of endogenous miR-210 by miR-210-LNA reversed hypoxia-induced suppression of STOCs in uterine arteries in pregnant ewes acclimatized to gestational hypoxia, 3) miR-210-LNA restored the ex vivo hormonal regulation of STOCs in uterine arteries of non-pregnant animals exposed to high altitude hypoxia, 4) miR-210 mimic imitated the effect of gestational hypoxia and suppressed STOCs in uterine arteries of pregnant ewes, and inhibited the ex vivo hormonal regulation of STOCs in uterine arteries of non-pregnant ewes, 5) miR-210 directly targeted and downregulated RyR2 and Ca²⁺ sparks in uterine arteries, 6) miR-210 downregulated BK_Ca β1 subunit resulting in a decrease in STOCs of uterine arteries, 7) TET1 was involved in the miR-210-mediated inhibition of STOCs in uterine arteries in response to gestational hypoxia.

Extensive human and animal studies have revealed a causative role of increased uterine vascular resistance and placental hypoxia in preeclampsia and IUGR associated with hypoxia in pregnancy.¹ In an animal model of pregnant sheep acclimatized to high altitude hypoxia, we have found that pregnant ewes are similar to humans in that they have an increase in uterine vascular resistance and elevation in maternal systemic blood pressure.^{6, 7} Although much remains unknown of the mechanisms underlying maternal cardiovascular adaptation to long-term hypoxia during gestation, hypoxia via HIF1α robustly upregulates miR210, which together with HIF1α are master regulators of the cellular hypoxic response. Our previous studies demonstrated that HIF1α and miR-210 were significantly increased in uterine arteries in pregnant sheep acclimatized to high altitude hypoxia.^{6, 28} In the present study, we showed that pregnant ewes of high-altitude hypoxia had a significant increase in miR-210 in both maternal and fetal circulations. Previous studies in humans revealed that increased miR-210 is a common feature in preeclampsia and gestational hypoxia, showing elevated placental expression of miR-210 and circulating miR2-10 levels in both preeclampsia and pregnancy at high altitude in pregnant women.^20–27 Similarly, hypoxia-induced miR-210 was found to be a major contributor to the development of pulmonary hypertension.^{55, 56} In addition to being biomarkers, recent evidence of endocrine and disease-modifying extracellular microRNAs in the circulation expanded our understanding of the systemic physiology of miRNA activity in the vasculature and beyond. It has been shown that miR-223 in the circulation is absorbed by vascular smooth muscle cells to regulate proliferation, migration and apoptosis by targeting insulin-like growth factor 1 receptor encoding gene IGF1R.⁵⁷ In addition, it has been demonstrated that chronic hypoxia upregulates circulating miR-210 originated from the bone marrow, which has long-range tissue uptake and endocrine activity, acting on pulmonary arteries to promote pulmonary hypertension.⁵⁸ Of importance, mature miR-210 of 22 nt is highly homologous across species and is identical among the human, sheep and rodent, suggesting a translational potential of miR-210 in the preclinical study.

The finding that inhibition of endogenous miR-210 by miR-210-LNA reversed hypoxia-induced suppression of STOCs in uterine arteries revealed a causal effect of elevated miR-210 on repressed STOCs in uterine arteries of high-altitude pregnant animals. STOCs at physiological membrane potentials of vascular smooth muscle cells are a key determinant of vascular myogenic tone and blood flow in an organ, as well as arterial blood pressure. STOCs produce K⁺ efflux mediated by BK_Ca channels, and cause membrane hyperpolarization resulting in vascular smooth muscle cell relaxation. We demonstrated recently in sheep that pregnancy significantly increased STOCs in uterine arteries,⁴¹ and hypoxia during gestation suppressed this pregnancy-induced upregulation of STOCs in uterine arteries.⁴² In addition, the previous study showed that miR-210 antagomir miR-210-LNA blocked the hypoxia-induced increase of miR-210 in uterine arteries of pregnant sheep.⁴⁸ In the present study, we found that miR-210-LNA shifted STOC occurrence to a more negative membrane potential (from −20 mV to −40 mV) and markedly increased STOC frequency and amplitude in uterine arteries of high-altitude pregnant animals. Indeed, the blockade of miR-210 restored STOCs in uterine arteries of high-altitude pregnant ewes to those seen in normoxic control animals. In addition, miR-210-LNA recovered the ex vivo hormonal regulation of STOCs in uterine arteries of animals exposed to high altitude hypoxia. Steroid hormones play a pivotal role in the adaptation of uterine circulation during pregnancy. Previously, we showed that the ex vivo treatment of uterine arteries from nonpregnant animals with estrogen and progesterone significantly increased both STOC frequency and amplitude,⁴¹ and this hormonal regulation of STOCs was rescinded in uterine arteries of non-pregnant animals exposed to high altitude hypoxia.⁴² The present finding that inhibition of miR-210 with miR-210-LNA restored the ex vivo hormonal regulation of STOCs in uterine arteries of non-pregnant animals acclimatized to high altitude hypoxia revealed a mechanism of elevated miR-210 in hypoxia-induced repression of hormonal regulation of uterine arterial adaptation. Further evidence of a mechanistic link of miR-210 in the hypoxia-mediated effect on STOCs in uterine arteries comes from the present finding that miR-210 mimic had a direct effect in suppressing STOCs in uterine arteries. Indeed, the effect of miR-210 recapitulated that of high-altitude hypoxia, and STOCs recorded in uterine arteries of control pregnant animals in the presence of miR-210 mimic were not significantly different from those observed in uterine arteries of pregnant ewes acclimatized to high altitude hypoxia. In addition, miR-210 imitated the effect of hypoxia and inhibited the ex vivo hormonal regulation of STOCs in uterine arteries. In the presence of miR-210 mimic, uterine arteries responded in the same manner as those from non-pregnant ewes acclimatized to high altitude hypoxia, losing the effect of hormonal regulation of STOCs. Taken together, these findings provide evidence that elevated miR-210 is necessary and sufficient in mediating hypoxia-induced repression of STOCs in uterine arterial adaptation during pregnancy.

STOCs are activated by Ca²⁺ sparks. The functional coupling between Ca²⁺ sparks and STOCs is highly efficient that virtually a one-to-one relationship exists between Ca²⁺ sparks and STOCs. In animal models, abnormal Ca²⁺ spark/STOC coupling has been associated with cardiovascular dysfunction associated with hypertension, subarachnoid hemorrhage, shock, diabetes, obesity, and Duchenne muscular dystrophy.^{31, 32, 40, 59–63} Ca²⁺ sparks are mediated by RyRs. All 3 subtypes of RyRs, RyR1, RyR2, and RyR3 are expressed in vascular smooth muscle cells. Although both RyR1 and RyR2 constitute independent Ca²⁺ sparks, RyR2 is primarily located in the subplasmalemmal region of the endoplasmic reticulum^{53, 64} and is the predominant mediator of Ca²⁺ sparks in vascular smooth muscle.^{44, 65, 66} We showed that RyR2 knockdown decreased both Ca²⁺ sparks and STOCs in uterine arteries.⁴⁴ In addition, we demonstrated that RyR2 played a key role in the regulation of Ca²⁺ sparks in uterine arterial adaptation to pregnancy, and that high-altitude hypoxia suppressed pregnancy-induced upregulation of RyR2 and Ca²⁺ sparks in uterine arteries.^{41, 42, 44} Of interest, the present study revealed that RyR2 is a direct target of miR-210. We showed that miR-210 mimic treatment significantly reduced RyR2 protein abundance in uterine arteries, which is consistent with the finding that gestational hypoxia suppressed pregnancy-induced RyR2 expression in uterine arteries.⁴² Of importance, miR-210 mimic recapitulated the effect of gestational hypoxia and reduced Ca²⁺ spark-firing myocytes and Ca²⁺ spark frequency to the levels observed in uterine arteries of pregnant ewes acclimatized to high altitude hypoxia.⁴² These findings suggest that increased miR-210 is sufficient in the downregulation of RyR2 and the reduction in Ca²⁺ sparks in uterine arterial response to hypoxia during pregnancy.

RyR2 is colocalized with the BK_Ca channel in vascular smooth muscle cells.⁶² Our recent study revealed that pregnancy significantly increased the co-localization of RyR2 with BK_Ca β1 subunit in the uterine artery.⁴⁴ This spatial organization of close proximity facilitates the β1 subunit to sense Ca²⁺ sparks and promote Ca²⁺ spark/STOC coupling, which is proportional to the size of RyR/BK_Ca cluster.^{32, 62, 67} The BK_Ca β1 subunit plays a vital role in determining the coupling efficiency of Ca²⁺ sparks and STOCs, which is dynamically regulated under various physiological and pathophysiological conditions. In addition to RyR2, we identified that the BK_Ca β1 subunit is also a target of miR-210 by demonstrating a direct interaction of miR-210 with the 3′-UTR of KCNMB1. Our previous studies showed that miR-210 and hypoxia during gestation decreased BK_Ca channel β1 protein abundance in uterine arteries, which was restored by miR-210-LNA.^{6, 48} The present finding that knockdown of the β1 subunit recapitulated the effect of miR-210 and suppressed both STOC frequency and amplitude in uterine arteries revealed a causal role of miR-210-meidated downregulation of BK_Ca β1 subunit in hypoxia-induced repression of STOCs in uterine arteries.

In addition to directly targeting and downregulating RyR2 and the β1 subunit, the present study also revealed the involvement of TET1 in miR-210-mediated regulation of STOCs in uterine arteries. TETs regulate active DNA demethylation. Previously, we demonstrated that gestational hypoxia-induced miR-210 directly targeted and downregulated TET1, leading to KCNMB1 hypermethylation and the repression of β1 subunit in uterine arteries.^{6, 48} In addition to the β1 subunit, hypermethylation of RyR2 promoter was found to be associated with reduced RyR2 expression.⁶⁸ Thus, miR-210 targeting TET1 provides an additional mechanism in the downregulation of RyR2 and the β1 subunit and the inhibition of Ca²⁺ spark/STOC coupling in uterine arteries. Given that a miR can target many transcripts, it is not surprising that miR-210 interacts with TET1, RyR2 and KCNMB1. Similarly, miR-29b was also found to directly target both TET1 and KCNMB1.^{69, 70} The finding that miR-210 downregulates TET is highly intriguing because TET via its effect of DNA demethylation may regulate the expression of many other genes, including RyR2 and KCNMB1 in uterine arteries. Of interest, the downregulation of uteroplacental TETs also occurred in preeclampsia.^71–73 Thus, it is likely that the impairment of STOCs in uterine arteries of pregnant animals acclimatized to high-altitude hypoxia is conferred in part by miR-210-induced TET1 downregulation and the resultant repression of RyR2 and KCNMB1. This notion is supported by the observations that 1) the suppressed STOCs by miR-210 and hypoxia were simulated by TET1 knockdown with siRNAs, 2) the restoration of STOCs by miR-210-LNA in uterine arteries of gestational hypoxia was offset by the TET inhibitor fumarate.

Perspective

Human and animal studies have revealed a causative role of increased uterine vascular resistance and placental hypoxia in preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia. Pregnant women at high altitude have a two- to four-fold increase in the incidence of preeclampsia, which is often proceeded by a decrease in uteroplacental blood flow. Similar to humans, we have shown that pregnant sheep at high altitude have an increase in uterine vascular resistance and elevation in maternal systemic blood pressure. Our recent study revealed a regulatory target of repressed STOCs in programming of uterine vascular malfunction in response to gestational hypoxia. The present study provides evidence of a mechanistic link of miR-210 in hypoxia-mediated impairment of Ca²⁺ spark/STOC coupling in uterine arteries during gestation in an animal model of pregnant ewes acclimatized to high-altitude hypoxia. We demonstrated that elevated miR-210 is necessary and sufficient in inhibiting STOCs in uterine arterial adaptation to gestational hypoxia. MiR-210 directly targeted and downregulated RyR2 and BK_Ca β1 subunit, repressing Ca²⁺ sparks and STOCs in uterine arteries. In addition, miR-210 indirectly decreased STOCs by targeting TET1. Of importance, overexpression of HIF-1α-responsive miR-210 and downregulation of TET1 and RyR2 have been constantly detected in the circulation and/or uteroplacental tissues in preeclampsia and IUGR.^{27, 74–77} Thus, the present study provides new insights into the understanding of fundamental mechanisms underlying programming of vascular dysfunction caused by gestational hypoxia, impacting on maternal cardiovascular health and developmental plasticity. Given that miR-210 is a master regulator of cellular hypoxic response, and STOCs are fundamentally important in regulating vascular tone and pressure in virtually all vascular beds, the present study has a broad impact in the comprehensive understanding of molecular mechanisms in the regulation of vascular function and hemodynamics. Although the focus of the present study is to investigate miR-210-mediated regulation of STOCs in uterine arterial smooth muscle, the endothelium also plays an important role in the adaptation of uterine circulation during pregnancy^78–80 and endothelial dysfunction contributes to the pathogenesis of preeclampsia/IUGR.⁸¹ The role of miR-210 in regulating endothelial function remains an intriguing area of further investigation. Indeed, a recent study demonstrated that circulating miR-210 contributes to the development of hypoxia-induced pulmonary hypertension by targeting endothelial mitochondrial function.⁵⁸ Although the present study highlights the potential of developing miR-210-targeting therapies for pregnancy complication associated with hypoxia, it should be noted that miR-210 also participates in proliferation, angiogenesis and other cellular processes.⁸² To be beneficial, the miR-210-targeting therapy should be efficiently delivered to the target tissue/cells and possess high potency and limited cytotoxicity/off-target effects.

Novelty and Significance.

What Is New?

• Hypoxia during gestation elevates miR-210 in maternal and fetal circulations.

• Inhibition of miR-210 reverses hypoxia-induced suppression of STOCs in uterine arteries.

• MiR-210-LNA inhibits the effect of hypoxia and restores hormonal regulation of STOCs.

• MiR-210 mimic recapitulates the effect of gestational hypoxia and suppresses STOCs.

• MiR-210 inhibits the hormonal regulation of STOCs in uterine arteries.

• MiR-210 directly targets and downregulates RyR2 and Ca²⁺ sparks in uterine arteries.

• MiR-210 downregulates BK_Ca β1 subunit resulting in a decrease in STOCs.

• TET1 is involved in the miR-210-mediated inhibition of STOCs in uterine arteries.

What Is Relevant?

• The present study reveals a mechanistic link of miR-210 in hypoxia-induced repression of Ca²⁺ spark/STOC coupling in uterine arteries during gestation and provides novel insights into the understanding of pregnancy complications associated with hypoxia and potential therapeutic targets.