Ryanodine receptor subtypes regulate Ca²⁺ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

Abstract

Aims

Our recent study demonstrated that increased Ca²⁺ sparks and spontaneous transient outward currents (STOCs) played an important role in uterine vascular tone and haemodynamic adaptation to pregnancy. The present study examined the role of ryanodine receptor (RyR) subtypes in regulating Ca²⁺ sparks/STOCs and myogenic tone in uterine arterial adaptation to pregnancy.

Methods and results

Uterine arteries isolated from non-pregnant and near-term pregnant sheep were used in the present study. Pregnancy increased the association of α and β1 subunits of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and enhanced the co-localization of RyR1 and RyR2 with the β1 subunit in the uterine artery. In contrast, RyR3 was not co-localized with BK_Ca β1 subunit. Knockdown of RyR1 or RyR2 in uterine arteries of pregnant sheep downregulated the β1 but not α subunit of the BK_Ca channel and decreased the association of α and β1 subunits. Unlike RyR1 and RyR2, knockdown of RyR3 had no significant effect on either expression or association of BK_Ca subunits. In addition, knockdown of RyR1 or RyR2 significantly decreased Ca²⁺ spark frequency, suppressed STOCs frequency and amplitude, and increased pressure-dependent myogenic tone in uterine arteries of pregnant animals. RyR3 knockdown did not affect Ca²⁺ sparks/STOCs and myogenic tone in the uterine artery.

Conclusion

Together, the present study demonstrates a novel mechanistic paradigm of RyR subtypes in the regulation of Ca²⁺ sparks/STOCs and uterine vascular tone, providing new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Graphical Abstract

1. Introduction

To accommodate the demand of foetal growth during pregnancy, uterine vascular tone reduces substantially and uterine blood flow increases dramatically.¹ Uterine vascular adaptation has been extensively studied, and yet the mechanisms underlying this phenomenon remain not fully understood. Previous studies have demonstrated that upregulation of the β1 subunit (BK_Ca β1) of the large-conductance Ca²⁺-activated K⁺ channel and enhanced channel function in uterine arteries are essential for reduced uterine vascular tone and increased uterine blood flow.^2–7 Under physiological conditions, the activity of BK_Ca channels in vascular smooth muscle cells is stimulated by ryanodine receptor (RyR)-mediated Ca²⁺ sparks and exists in the form of spontaneous transient outward currents (STOCs). The K⁺ efflux carried by STOCs promotes vascular smooth muscle cell membrane hyperpolarization and subsequent closure of L-type voltage-gated Ca²⁺ (Ca_V1.2) channels, leading to vasorelaxation.⁸ The functional coupling between RyRs and BK_Ca channels is highly efficient that virtually a one-to-one relationship exists between Ca²⁺ sparks and STOCs.⁹ The regulation of vascular tone by the Ca²⁺ spark-STOC coupling has been frequently observed in vascular beds of cerebral and mesenteric circulations.^10–14 We have recently revealed that pregnancy attenuates myogenic tone in uterine arteries by promoting Ca²⁺ spark-STOC coupling in uterine arteries, providing a novel mechanism underlying the uterine vascular adaptation.¹⁵ RyRs consist of three subtypes: RyR1, RyR2, and RyR3. All of them are expressed in smooth muscle cells.¹⁶ Consistently, uterine arteries also express all RyR subtypes.¹⁵ Both RyR1 and RyR2 have been shown to contribute to the generation of Ca²⁺ sparks.^17–19 However, RyR3 was found either not to initiate Ca²⁺ sparks or to negatively regulate Ca²⁺ spark generation.^17,20 The sarcoplasmic reticulum runs parallel with the plasma membrane and they stay close to each other. This structural arrangement permits a close interaction between RyRs in the sarcoplasmic reticulum membrane and BK_Ca channels in the plasma membrane. It is estimated that activation of the BK_Ca channel by Ca²⁺ requires the channel being located within 10–30 nm of RyRs.²¹ Interestingly, a Ca²⁺ microdomain created by Ca²⁺ sparks may cover a radius of ∼200 nm.²² BK_Ca β1 residing within the spatial boundaries of the Ca²⁺ microdomain could then function as a Ca²⁺ sensor and transmit the Ca²⁺ signal to the BK_Ca channel, leading to increased BK_Ca channel activity.^23,24 Importantly, the spatial organization of RyRs and BK_Ca channels is critical for the Ca²⁺ spark-STOC coupling.^25,26

Our recent study revealed that all three subtypes of RyRs were upregulated in uterine arteries during pregnancy, and the upregulation of RyRs conferred pregnancy-induced increases in Ca²⁺ spark activity and STOCs and reduction in uterine arterial myogenic tone.¹⁵ A question of great importance that needs to be addressed is which subtype(s) of RyRs and its spatial organization with the BK_Ca channel contribute to the enhanced Ca²⁺ spark-STOC coupling in uterine arteries in pregnancy. Herein, we present evidence that pregnancy promotes co-localization of RyR1 and RyR2 with BK_Ca β1, leading to the upregulation of Ca²⁺ spark activity and STOCs and attenuation of myogenic tone in uterine arteries. Moreover, RyR1 and RyR2 also play a role in regulating the expression of BK_Ca β1and its association with the BK_Ca channel α subunit (BK_Ca α) in uterine arteries. These findings provide novel insights into the mechanisms underlying the physiological adaptation of uterine vasculature during pregnancy.

2. Methods

2.1 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. Tissue collection was carried out under anaesthesia. After tissue collection, animals were killed via intravenous injection of 15 mL T-61 solution (Hoechst-Rousel, Somervile, NJ, USA), according to American Veterinary Medical Association guidelines.

Uterine arteries were harvested from non-pregnant or near-term (∼142–145 days of gestation) pregnant sheep.²⁷ Animals were anaesthetized with intravenous injection of propofol (2 mg/kg) followed by intubation, and anaesthesia was maintained on 1.5–3.0% isoflurane balanced in O₂ throughout the surgery. An incision was made in the abdomen, and the uterus was exposed. Resistance-sized uterine arteries (∼150–200 µm in diameter) were used in all experiments. Arteries 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). To knockdown RyRs, resistance-sized uterine arteries of pregnant sheep were transfected with RyR siRNAs (Dharmacon Inc., Lafayette, CO, USA) as described previously.⁶ Target or scrambled siRNAs were mixed with HiPerfect Transfection Reagent (Qiagen) and Opti-MEM I (ThermoFisher) for 30–45 min at room temperature and subsequently added into DMEM/F12 supplemented with 1% charcoal-stripped foetal bovine serum. Tissues were incubated with the medium containing target or scrambled siRNAs (control siRNA, the final concentration of 200 nmol/L) in an incubator at 37°C for 48 h.

2.2 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, USA). The mRNA abundance of RyRs was measured with real-time polymerase chain reaction (PCR) using iQ SYBR Green Supermix (Bio-Rad), as described previously.²⁸ 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. Ribosomal protein L4 (RPL4) content in uterine arteries was not altered by pregnancy¹⁵ and was used for normalization of the abundance of mRNAs. PCR was performed in triplicate, and threshold cycle numbers (C_T), generated by CFX connect Real-time System (Bio-Rad), were averaged for each sample. The RNA quality was assessed by a Nanodrop Spectrophotometer (ThermoFisher) by determining A260/A280, A260/A230 values. The relative gene expression of the gene of interest (GOI) was calculated by the modified $2_{\mathrm{T}}^{-\Delta \Delta \mathrm{C}}$ method.²⁹

2.3 Immunoblotting

Protein abundance of RyR1, RyR2, and RyR3 in uterine arteries was measured as described previously.⁵ Briefly, tissues were homogenized in a lysis buffer followed by centrifugation at 4°C for 10 min at 10 000 g, and the supernatants were collected. Samples with equal proteins were loaded onto NuPAGE 3–8% Tris-Acetate Protein Gels (Thermo Fisher Scientific, Waltham, MA, USA) and were separated by electrophoresis at 150 V for 1 H. Proteins were then transferred onto nitrocellulose membranes. After blocking non-specific binding sites by dry milk, membranes were incubated with primary antibodies (1:1000 dilution) against rabbit polyclonal RyR1 (8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (AB9082, EMD Millipore), mouse monoclonal BK_Ca channel α, or mouse monoclonal BK_Ca channel β1 (Santa Cruz Biotechnology, Dallas, TX, USA). 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, USA). The target protein abundance was normalized to the abundance of β-actin as a protein loading control, whose abundance in uterine arteries was not altered by pregnancy.¹⁵

2.4 Ca²⁺ spark measurements

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).³⁰ The endothelium was mechanically disrupted by gently pulling a silver wire across the intimal surface of the uterine arterial segments five 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, MA, USA) dissolved in DMSO along with 0.1% pluronic F127 (Thermo Fisher) for 1–1.5 h at room temperature. Tissues were then washed for 30 min 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 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 the morphological examination of live preparations. Line scans were analysed 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.

2.5 Measurement of STOCs

Vascular smooth muscle cells were enzymatically dissociated from resistance-sized uterine arteries as described previously.¹⁵ Briefly, uterine arteries were minced and incubated (37°C, 10 min) 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: (i) a 60-min incubation in low-Ca²⁺ HEPES-buffered PSS (37°C) containing 1.5 mg/mL papain (Worthington Biochemical; Lakewood, NJ, USA), 1.5 mg/mL dithiothreitol (MilliporeSigma, St. Louis, MO, USA), and 1.5 mg/mL bovine serum albumin (MilliporeSigma); and (ii) a 60-min 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 h 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 described previously.¹⁵ 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 vascular smooth muscle cells were used for recording. Micropipettes were pulled from borosilicate glass and had resistances of 2–5 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 analysed with Mini Analysis program (Synaptosoft, Leonia, NJ, USA) with a threshold for detection set at 10 pA. The currents were normalized to cell capacitance and expressed as picoampere per picofarad (pA/pF).

2.6 Measurement of pressure-dependent myogenic tone

The pressure-dependent myogenic tone of resistance-sized uterine arteries was measured as described previously.⁵ Briefly, the arterial segments (∼150 μm diameter) were mounted and pressurized in an organ chamber (Living Systems Instruments, Burlington, VT, USA). 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 min to allow vessel diameter to stabilize before the measurement. Ca²⁺-free PSS contains zero Ca²⁺ and 3 mM EGTA. PSS was allowed to pass through the lumen of the pressurized vessels before the detection of myogenic tone, and the 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⁺.

2.7 Immunofluorescence staining and Duolink proximity ligation assay

The co-localization of RyRs and BK_Ca channel was examined using immunofluorescent staining as described previously with modifications.³¹ After immersion in optimal cutting temperature compound, uterine arteries were sectioned at a thickness of 8 µm using a Leica cryostat (Leica, Buffalo Grove, IL, USA). Uterine arterial slices were blocked in 5% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature (RT) for 1 h, and then incubated with primary antibodies, rabbit polyclonal MaxiKβ antibody (1:200; sc-33608, Santa Cruz Biotechnology), rabbit polyclonal RyR1 antibody (1:100; 8153, Cell Signaling, Danvers, MA, USA), rabbit polyclonal RyR2 (1:100; ARR-002, Alomone Labs, Israel), rabbit polyclonal RyR3 (1:100; AB9082, EMD Millipore), mouse monoclonal BK_Ca channel α (1:200; sc-374142), or mouse monoclonal BK_Ca channel β1 (1:200; sc-377023) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight. After washing with phosphate-buffered saline (PBS), sections were incubated with corresponding fluorescent secondary antibodies (1:1000, Thermo Fisher Scientific) at RT for 1 h, and then mounted and coverslipped using fluorescent mounting media with a classic nuclear counterstain DAPI (diamidino-2-phenylindole) (VECTOR LABORATORIES, INC., Burlingame, CA, USA). All slices were scanned with a Zeiss LSM 710 confocal microscopy (Zeiss, Oberkochen, Germany). Images were acquired using a z-stack of 1.0 µm intervals. Image stacks were analysed using NIH Image J software.

Proximity ligation assay (PLA) was performed using the Duolink in situ kit (MilliporeSigma, St. Louis, MO, USA) according to the manufacturer’s instructions. After pre-incubation with a blocking agent for 60 min, cryo-fixed samples were incubated overnight with the primary antibodies against RyR1 or RyR2 (1:300) and BK_Ca β1 (1:300). The PLUS and MINUS PLA probes were diluted (1:5 in the Duolink^® Antibody Diluent) and applied to the slides, followed by incubation for 2 h in a pre-heated humidity chamber at 37°C. Unbound PLA probes were removed by washing. The samples were incubated in the ligation solution consisting of Duolink Ligation stock (1:5) and Duolink Ligase (1:40) for 30 min at 37°C. Detection of the amplified probe was done with the Duolink Detection Kit. Duolink Detection stock was diluted at 1:5 and applied for 100 min at 37°C. After final washing, coverslips were mounted onto the slides using a minimal volume of Duolink^® in situ mounting medium containing DAPI nuclear stain. Image acquisition was carried out on a Zeiss LSM 710 confocal microscopy. Maximum projection images were analysed using ImageJ (National Institutes of Health, Bethesda, MD, USA) to quantify PLA punctate signals. Images were smoothed, and a threshold to distinguish signal from background fluorescence was applied equally to all images and the number of puncta quantified using the ‘Analyze Particles’ macro with the exclusion criteria of the size of objects being higher than 5 µm.³²

2.8 Co-immunoprecipitation

The co-immunoprecipitation experiments were performed with Pierce™ Co-Immunoprecipitation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, 20 µg of the monoclonal BK_Ca α antibody (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated with the delivered resin and covalently coupled. The antibody-coupled resin was incubated with 200 µL of the whole sheep uterine artery protein lysates overnight at 4°C. The resin was washed, and the protein complexes bound to the antibody were eluted. Subsequently, western blot analyses were performed. BK_Ca α and BK_Ca β1 protein levels in the immunoprecipitates were analysed by monoclonal mouse antibodies against BK_Ca α and BK_Ca β1 (Santa Cruz Biotechnology) and immunoblotting.

2.9 Statistical analysis

Data were expressed as means ± SEM obtained from the number of experimental animals. Data were analysed with GraphPad Prism (GraphPad Software, San Diego, CA, USA). Differences were evaluated for statistical significance (P < 0.05) by one-way analysis of variance (ANOVA) with the post hoc Bonferroni/Dunn test or independent-samples t-test where appropriate.

3. Results

3.1 Pregnancy increased the co-localization of RyR1 and RyR2 with BK_Ca β1 in uterine arteries

BK_Ca β1 is the primary sensor of the BK_Ca channel to Ca²⁺ sparks mediated by RyRs.³³ Hence, we first determined the spatial organization of BK_Ca β1 and RyRs in uterine arteries using immunofluorescence and confocal microscopy. Figure 1 shows immunofluorescence confocal images of BK_Ca β1 and RyRs expression in uterine arteries of non-pregnant and pregnant sheep. Apparently, pregnancy increased the expression of BK_Ca β1 and all three subtypes of RyRs in the tunica media of uterine arteries (Figure 1A), consistent with western blot findings in our previous studies.^5,15 Moreover, pregnancy also promoted immunofluorescence co-localization of BK_Ca β1 with RyR1 and RyR2, respectively. In contrast, the co-localization of BK_Ca β1 with RyR3 was negligible and not affected by pregnancy. The interactions between BK_Ca β1 and RyR1/RyR2 were further examined using the PLA assay. As shown in Figures1B andC, pregnancy significantly increased the close co-localization between BK_Ca β1 and RyR1/RyR2.

Figure 1.

Pregnancy increased co-localization of RyR1/RyR2 with BKCa channel in uterine arteries. (A) Representative confocal immunofluorescence images from five replicates show the co-localization of RyRs and BK_Ca channels in uterine arteries. Uterine arteries of non-pregnant (NUA) and pregnant (PUA) animals were stained with antibodies against the β1 subunit of BK_Ca channel (BK_Ca β1, red) and RyR1, RyR2, or RyR3 (green). Merged images show the co-localization of RyR1 or RyR2 with BK_Ca β1 (in yellow). The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (B) PLA assay to confirm the co-localization of RyR1/RyR2 and BK_Ca β1 in uterine arteries. (C) Quantification of the PLA signals. Images from five independent replicates were analysed. The nuclear region was stained with DAPI and shown in blue. Scale bar: 50 µm. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, PUA vs. NUA.

3.2 Knockdown of RyRs functionally impaired Ca²⁺ sparks and STOCs in uterine arteries

Our previous study revealed that pan-inhibition of RyRs with ryanodine inhibited the Ca²⁺ spark-STOC coupling in uterine arteries of pregnant sheep.¹⁵ To determine the functional importance of individual RyR subtypes, we examined Ca²⁺ sparks and STOCs in uterine artery vascular smooth muscle cells following knockdown of each RyR subtype using siRNAs. As shown in Supplementary material online, Figure S1, the expression levels of mRNA and protein of each RyR1, RyR2, and RyR3 in uterine arteries were significantly reduced by corresponding RyR siRNAs. Figure 2A illustrates representative line-scan confocal images of Ca²⁺ sparks in control siRNA and RyR siRNA-treated uterine arteries from pregnant sheep loaded with the Ca²⁺ indicator Fluo-4. The percentage of Ca²⁺ spark firing smooth muscle cells in uterine arteries was not significantly altered by the knockdown of RyRs (Figure 2B). However, the ability of membrane depolarization with 30 mmol/L K⁺ to stimulate Ca²⁺ spark frequency was significantly decreased by the knockdown of RyR1 and RyR2, respectively, but not significantly affected by RyR3 knockdown (Figure 2C). The other Ca²⁺ spark parameters such as amplitude (F/F₀), width (FWHM), and duration (FDHM) were not or slightly altered by knocking down RyRs in uterine arterial smooth muscle cells (Supplementary material online, Figure S2).

Figure 2.

Knockdown of RyR1/RyR2 decreased Ca²⁺ sparks in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Ca²⁺ sparks in uterine arteries were measured 48 h later. (A) Representative line-scan images of Fluo-4AM loaded uterine arteries showing Ca²⁺ sparks recorded before and after the sequential application of 30 mmol/L K⁺ (30 K) following siRNA treatments. (B) Percentage of Ca²⁺ spark-firing vascular smooth muscle cells. (C) Ca²⁺ spark frequency. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

We then examined the contribution of individual RyRs in the regulation of STOCs in uterine arteries of pregnant sheep. Figure 3A shows representative tracing of STOCs in vascular smooth muscle cells isolated from uterine arteries treated with either scramble control siRNA or RyR siRNAs at a holding potential of 10 mV. In agreement with the actions of RyR siRNAs on Ca²⁺ sparks, RyR1 and RyR2, but not RyR3, knockdown suppressed STOCs. The occurrence of STOCs in RyR1/RyR2-knockdown uterine artery vascular smooth muscle cells was shifted to a much more positive membrane potential (from −40 mV to −20 mV), approximating the phenotype of non-pregnant animals, as demonstrated previously.¹⁵ Moreover, knockdown of RyR1 or RyR2 significantly reduced both STOC frequency (Figure 3B) and amplitude (Figure 3C) at membrane potentials between −30 and 10 mV. For example, STOC frequency and amplitude at 10 mV were decreased by 44.7 ± 5.5% and 60.1 ± 4.4% in RyR1-knockdown uterine artery vascular smooth muscle cells, and by 57.2 ± 1.7% and 71.2 ± 2.3% in RyR2-knockdown uterine artery vascular smooth muscle cells, respectively. In contrast, RyR3 knockdown did not significantly alter STOC frequency and amplitude (Figure3B andC). Treatments with control siRNA or RyR siRNAs apparently did not alter the inhibitory effect of BK_Ca channel inhibitor iberiotoxin on STOCs. Similar to the previous findings in vascular smooth muscle cells from freshly harvested uterine arteries,¹⁵ iberiotoxin produced a progressive inhibition of STOCs in smooth muscle cells of siRNA-treated uterine arteries (Supplementary material online, Figure S3).

Figure 3.

Knockdown of RyR1/RyR2 suppressed STOCs in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. STOCs were measured in uterine artery vascular smooth muscle cells 48 hours later. (A) Representative traces showing STOCs at holding potentials of 10 mV in uterine artery vascular smooth muscle cells. (B) STOC frequency. (C) STOC amplitude. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. control siRNA.

3.3 Ryr1 and RyR2 knockdown increased pressure-dependent myogenic tone in uterine arteries

Increased Ca²⁺ sparks and STOCs are of critical importance in pregnancy-induced attenuation in uterine arterial myogenic tone.¹⁵ We thus investigated the functional importance of individual RyR subtypes in regulating pressure-dependent myogenic tone in uterine arteries of pregnant animals by knocking down individual RyR subtypes. Representative traces and averaged data of the myogenic response were shown in Supplementary material online, Figure S4 and Figure 4A–C, respectively. As shown in Figure 4, knockdown of RyR1 or RyR2 significantly increased pressure-dependent myogenic tone (Figure 4D). Consistent with the lack of effect on Ca²⁺ sparks and STOCs, knockdown of RyR3 had no significant effect on uterine arterial myogenic tone (Figure 4D). Control siRNA had no effect on myogenic tone compared to the untreated control (Figure 4D). Furthermore, treatment with control siRNA did not alter the effect of iberiotoxin on increasing uterine arterial myogenic tone (Supplementary material online, Figure S5). Our previous study demonstrated that iberiotoxin or RyR blockade with ryanodine increased myogenic tone of uterine arteries from pregnant sheep, and combined action of iberiotoxin and ryanodine did not produce additive effects on myogenic tone, as compared to the response induced by each alone.¹⁵ Similar findings were obtained in the present study. Iberiotoxin had no additive effect on increased myogenic tone induced by knockdown of RyR1 or RyR2, respectively (Supplementary material online, Figure S5). In contrast to pregnant animals, treatments of uterine arteries of non-pregnant animals with RyR siRNAs had no significant effect on myogenic tone (Supplementary material online, Figure S6).

Figure 4.

Knockdown of RyR1/RyR2 increased myogenic tone in uterine arteries of pregnant sheep. Uterine arteries of pregnant animals were treated with scramble control siRNAs or siRNAs for RyR1, RyR2, and RyR3, respectively. Pressure-dependent myogenic tone was measured in uterine arteries 48 h later. (A–C) Changes of lumen diameters of uterine arteries in response to increases in intravascular pressure. (D) Data summary showing the percentage myogenic tone in RyR siRNA-treated uterine arteries. Data are means ± SEM from five animals of each group; repeated measures ANOVA with the post hoc Bonferroni/Dunn test; *P < 0.05, RyR siRNAs vs. untreated control or control siRNA.

3.4 Ryrs regulated BK_Ca β1 expression and its association to BK_Ca α subunit in uterine arteries

The BK_Ca channel in smooth muscle cells is heteromeric of the pore-forming α subunit and auxiliary β1 subunit.^34,35 The β1:α subunit stoichiometry is dynamically regulated under various physiological and pathophysiological conditions and plays a vital role in regulating the BK_Ca channel activity in vascular smooth muscle. As shown in Figure 5A, co-immunoprecipitation and western blot demonstrated that pregnancy increased the association of BK_Ca α and BK_Ca β1 in uterine arteries. Co-localization of BK_Ca α and BK_Ca β1 examined by immunofluorescence confocal microscopy revealed that knockdown of RyR1 or RyR2, but not RyR3, decreased the co-localization of BK_Ca α and BK_Ca β1 (Figure 5B). This was further confirmed by co-immunoprecipitation and western blot, as illustrated in Figure 5C. Of interest, knockdown of either RyR1 or RyR2 in uterine arteries of pregnant sheep markedly reduced protein abundance of BK_Ca β1 when compared to the scramble control siRNA treatment (Figure6A andB). In contrast, the protein expression of BK_Ca α was not altered by RyR1/RyR2 knockdown (Figure6A andB). Unlike RyR1 or RyR2, RyR3 knockdown altered neither BK_Ca α nor BK_Ca β1 protein expression (Figure 6C).

Figure 5.

Knockdown of RyR1/RyR2 reduced the interaction of BK_Ca channel α and β1 subunits in uterine arteries. (A) Representative immunoblots from five replicates show co-immunoprecipitation of BK_Ca channel α and β1 subunits in uterine arteries of non-pregnant (NUA) and pregnant (PUA) sheep. Uterine arteries were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively, for 48 h. IgG was used as a control to show antibody specificity. (B) Representative confocal immunofluorescence images from five replicates show the co-localization of BK_Ca channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNAs or RyR siRNAs treatments. The arteries were stained with antibodies against α (green) and β1 (red) subunits. Merged images show in yellow. The nuclear region was stained with DAPI and shows in blue. Scale bar: 100 µm. (C) Representative immunoblots from five replicates show co-immunoprecipitation of BK_Ca channel α and β1 subunits in uterine arteries of pregnant sheep after control siRNA or RyR siRNAs treatments. β-Actin blots showing equal total protein lysates (input).

Figure 6.

Knockdown of RyR1/RyR2 downregulated the expression of BK_Ca channel β1 subunit in uterine arteries of pregnant animals. Uterine arteries of pregnant sheep were treated with scramble control siRNA or siRNAs for RyR1, RyR2, and RyR3, respectively. Protein abundance of BK_Ca channel α and β1 subunits was measured by western blot in uterine arteries 48 h after the treatment. (A) RyR1 siRNAs treatment. (B) RyR2 siRNAs treatment. (C) RyR3 siRNAs treatment. Data are means ± SEM from five animals of each group; independent-samples t-test; *P < 0.05, RyR siRNAs vs. control siRNA.

4. Discussion

In the present study, we examined the contribution of individual RyR subtypes to pregnancy-induced increase in the Ca²⁺ spark-STOC coupling and uterine vascular adaptation. The major findings are: (i) pregnancy increased the co-localization of BK_Ca β1 and RyR1/RyR2 in uterine arteries; (ii) RyR3 was not co-localized with BK_Ca β1; (iii) RyR1 or RyR2, but not RyR3, knockdown impaired Ca²⁺ sparks/STOCs and increased myogenic tone in uterine arteries; (iv) pregnancy augmented the association of BK_Ca α and BK_Ca β1 in uterine arteries, which was suppressed by RyR1/RyR2, but not RyR3, knockdown; (v) knockdown of RyR1/RyR2 deceased BK_Ca β1 but not BK_Ca α expression in uterine arteries.

Owing to its large conductance and high density, the BK_Ca channel plays a critical role in regulating vascular smooth muscle cell membrane potential and vascular tone.³⁶ Although the BK_Ca channel can be activated by either voltage or Ca²⁺, its activity in smooth muscle cells is primarily regulated by Ca²⁺ sparks mediated by RyRs under physiological conditions.³⁷ The binding affinity (Kd) of Ca²⁺ for the BK_Ca channel in smooth muscle cells is ∼20 μmol/L at the physiological membrane potential around −40 mV. A single Ca²⁺ sparks can rise [Ca²⁺]_i to 10–30 μmol/L, and this Ca²⁺ microdomain covers a radius of ∼200 nm.^22,38 It should be noted that [Ca²⁺]_i decreases sharply when it travels away from the Ca²⁺ source.^21,25 This requires proper spatial organization of the BK_Ca channel and RyRs for the BK_Ca channel to sense the Ca²⁺ signal within the Ca²⁺ microdomain. The BK_Ca channel is a heteromeric assembly of the pore-forming BK_Ca α and auxiliary BK_Ca β1 in smooth muscle cells and the association of BK_Ca β1 to BK_Ca α significantly increases the channel activity by enhancing channel’s Ca²⁺ sensitivity.^33,39 BK_Ca α co-localized with RyR1/RyR2 in airway and cerebral arterial vascular smooth muscle cells.^25,40 To our knowledge, the present study is the first to show the co-localization of BK_Ca β1 and RyR1/RyR2 in uterine artery vascular smooth muscle cells using immunofluorescence and confocal microscopy and proximity ligation assay that detects protein–protein interaction within 40 nm. It remains to be determined whether the co-localization of RyRs with BK_Ca β1 may alter the association between BK_Caα and BK_Caβ1 subunits. Similar to the dynamic association of BK_Ca β1 and BK_Ca α subunits in vascular smooth muscle cells,⁴¹ it is likely that the co-localization of BK_Ca β1 with RyR1/RyR2 in uterine artery vascular smooth muscle cells also undergoes a dynamic process. It is currently not known whether the co-localization of RyR1/RyR2 with BK_Ca β1 may alter the half-life of BK_Ca β1. Such a spatial proximity keeps BK_Ca β1 in close contact with RyRs and enables BK_Ca channel to be activated by Ca²⁺ sparks, facilitating the functional coupling between Ca²⁺ sparks and STOCs. A disruption of this spatial organization may impair the coupling. For example, an increase in the distance between sarcoplasmic reticulum and plasma membranes by microtubule depolymerization using nocodazole reduced the number of close contacts between BK_Ca α and RyR2 in rat cerebral arteries, leading to almost abolition of STOCs.²⁶ Whether and to what extent SR distance from the plasma membrane is altered during pregnancy remain to be determined. Only a small fraction of BK_Ca channels and RyRs are co-localized in smooth muscle cells.^25,40 Interestingly, pregnancy significantly increased the co-localization of BK_Ca β1 and RyR1/RyR2, suggesting potentially increased contact sites between these proteins, which may account for the enhanced Ca²⁺ spark-STOC coupling in uterine arteries of pregnant animals, as demonstrated in our previous study.¹⁵ Among other mechanisms, the increased co-localization of BK_Ca β1 and RyR1/RyR2 is probably due to their upregulation in uterine arteries.^5,15 Previous studies demonstrated that BK_Caβ1 and RyR1/RyR2 mRNA and protein levels in uterine arteries increased in a parallel manner during pregnancy,^5,15 suggesting an increase in transcription. It also remains possible that pregnancy may alter half-lives of these proteins, which may contribute to the increase in their abundance in uterine arteries.

The close co-localization of RyRs and BK_Ca channels allows BK_Ca β1 to function as a vital relay that functionally couples the Ca²⁺ sparks to STOCs. The integrity of the coupling machinery is essential for signal transduction. Knockdown of RyR1/RyR2, which could interrupt the coupling of RyRs and BK_Ca channels, impaired the Ca²⁺ spark-STOC coupling as evidenced by blunted 30 mmol/L K⁺-stimulated Ca²⁺ spark activity and diminished STOCs in uterine artery vascular smooth muscle cells. These findings are in agreement with previous observations.^17–19,42 The finding that knockdown of RyR1/2 had no significant effect on the proportion of Ca²⁺ spark-firing smooth muscle cells, but reduced Ca²⁺ spark frequency is intriguing and suggests that the primary action of RyR knockdown is to reduce Ca²⁺ spark frequency within the cells (which hence decreases the Ca²⁺ spark-STOC coupling), but not the number of firing cells. This is consistent with the notion that gene knockdown only reduces, but not silences, the gene of interest in the cells. In contrast to RyR1/RyR2, RyR3 was not co-localized with BK_Ca β1 in uterine arteries. Similarly, RyR3 and BK_Ca α were not co-localized in airway smooth muscle cells.²⁵ Functionally, RyR3 knockdown did not alter either Ca²⁺ sparks or STOCs. This is in line with observations reported by Mironneau et al.,¹⁷ but not by Löhn et al. and Matsuki et al.^20,43 The finding that RyR1/RyR2, but not RyR3 was co-localized with BK_Ca β1 is intriguing. Several previous studies demonstrated a distinct spatial distribution of RyRs in smooth muscle cells.^25,44–46 In general, RyR2 is predominantly located in the subplasmalemmal region and RyR3 is in the perinuclear region, whereas RyR1 is located in both subplasmalemmal and perinuclear regions. This distinct spatial distribution of RyRs is likely to contribute to the differential interaction of RyRs and BK_Ca channels. The aberrant expression of BK_Ca β1 could also impact the Ca²⁺ spark-STOC coupling in vascular smooth muscle cells. The coupling was attenuated and boosted by BK_Ca β1 deficiency and BK_Ca β1 overexpression, respectively.^23,33,47

We previously demonstrated that enhanced Ca²⁺ spark-STOC coupling in the uterine arteries during pregnancy was associated with upregulation of all three subtypes of RyRs.¹⁵ Physiologically, the enhanced Ca²⁺ spark-STOC coupling during pregnancy was found to promote uterine vascular adaptation by attenuating uterine arterial myogenic tone.¹⁵ However, the roles of individual RyRs in the pregnancy-induced enhancement of Ca²⁺ spark-STOC coupling remain elusive. Interestingly, in the present study, we demonstrated that knockdown of RyR1/RyR2, but not RyR3, in uterine arteries of pregnant animals repressed the Ca²⁺ spark-STOC coupling and increased uterine arterial myogenic tone. The finding that knockdown of RyR1/2 has little effect on myogenic tone in uterine arteries of non-pregnant animals is not surprising, and indeed is consistent with the previous finding that the pan-inhibition of ryanodine receptors with ryanodine had no significant effect on myogenic tone in uterine artery of non-pregnant sheep.¹⁵ Apparently, repressing RyR1/RyR2 in uterine arteries of pregnant animals using the RNA interference tool produced a phenotype resembles uterine arteries of non-pregnant animals. The finding that siRNAs knockdown RyR1 and RyR2 about 40% at the mRNA level but about 60% of protein abundance is not surprising. Indeed, it is not uncommon that changes in mRNA abundance may not always precisely correlate to changes in protein abundance due to post-transcriptional, translational, and protein degradation regulations.^48–50 Although downregulation of RyRs had no significant effect on Ca²⁺ spark firing smooth muscle cells, knockdown of RyR1 and RyR2 significantly decreased Ca²⁺ spark frequency, STOC frequency and amplitude, and increased myogenic tone in uterine arteries. Thus, the present study established a cause-and-effect relationship between RyR1/RyR2 and the Ca²⁺ spark-STOC coupling in the regulation of uterine arterial myogenic tone during pregnancy. Although RyR3 is upregulated in uterine arteries during pregnancy,¹⁵ its physiological relevance is currently unclear. Both RyR1 and RyR2 participate in generating Ca²⁺ sparks in smooth muscle cells.^18,19,51 Arterial stiffness is affected by vascular smooth muscle cell tone.⁵² It is not surprising that knockdown of RyR1 or RyR2 affected arterial stiffness via altering myogenic tone in uterine arteries. The apparent redundancy of increasing both RyR1 and RyR2 in uterine arteries during pregnancy is likely to provide a protective mechanism to ensure the adaptation of Ca²⁺ dynamics and myogenic tone in uterine arteries and the optimization of uterine blood flow during pregnancy. Whether and to what extent RyR1 and RyR2 may produce additive or synergistic effect in the regulation of Ca²⁺ sparks remains to be determined. Together, this study revealed an explicit contribution of RyR1/RyR2 in mediating enhanced Ca²⁺-release events that resulted in reduced vascular tone in the uterine circulation during pregnancy.

The association of BK_Ca β1 to BK_Ca α is essential for the channel to regulate arterial vascular smooth muscle cell membrane potential, contractility, and blood pressure.^23,33,35 It is not surprising that pregnancy increased the association of BK_Ca β1 to BK_Ca α in the uterine artery in the present study. However, it is somewhat surprising that RyR1/RyR2 knockdown promoted BK_Ca β1 downregulation and decreased the association of BK_Ca α and BK_Ca β1 in uterine arteries of pregnant animals. These observations are puzzling at first glance. However, digging into Ca²⁺ sparks’ action could help unfold this mysterious issue. RyR-mediated Ca²⁺ sparks in vascular smooth muscle cells have been shown to counter vasoconstriction via BK_Ca channel-mediated hyperpolarization of the vascular smooth muscle cell membrane and closing Ca_V1.2.^10,11 It is conceivable that RyR1/RyR2 knockdown would diminish the inhibitory effect of Ca²⁺ sparks on Ca_V1.2 in uterine artery vascular smooth muscle cells, thus leading to increased Ca²⁺ influx through Ca_V1.2. The regulation of gene expression by Ca_V1.2 activity is a well-established phenomenon and frequently involves transcription factors such as cAMP-response element binding protein and nuclear factor of activated T-cells (NFAT).⁵³ Intriguingly, in an animal model of hypertension, in vivo administration of angiotensin II activated NFATc3 via stimulating Ca_V1.2-mediated Ca²⁺ influx.⁵⁴ The activation of NFATc3 consequently led to downregulation of BK_Ca β1 in vascular smooth muscle cells. Significantly, RyR inhibition with ryanodine enhanced transcriptional activities of NFAT in skeletal muscle fibres and potentiated UTP-induced NFATc3 nuclear accumulation in cerebral arteries.^55,56 It is possible that RyR1/RyR2 knockdown induces the activation of NFATc3, which confers the downregulation of BK_Ca β1 in uterine arteries. The diminished BK_Caβ1 abundance in uterine arterial smooth muscle probably then contributed to the reduced association of BK_Ca α and BK_Ca β1. To our knowledge, this study is the first to provide evidence that RyR1/RyR2 participate in the regulation of BK_Ca β1 expression. These findings suggest that RyRs play an important role in maintaining BK_Ca β1 homeostasis and the dynamic interaction of BK_Ca α and BK_Ca β1 in the uterine artery in pregnancy.

Adequate uterine vascular adaptation is essential for a successful pregnancy. We recently demonstrated an important role of RyR-mediated Ca²⁺ sparks and BK_Ca channel-mediated STOCs in this adaptation.¹⁵ In the present study, we revealed (i) enhanced close co-localization between RyR1/RyR2 and BK_Ca β1 in uterine arteries during pregnancy and (ii) a cause-and-effect relationship of RyR1/RyR2 in the enhanced Ca²⁺ spark-STOC coupling and attenuated uterine arterial myogenic tone in uterine arteries of pregnant animals. Moreover, our findings also suggest the regulatory divergence of RyRs on the Ca²⁺ spark-STOC cascade at two levels: direct regulation of Ca²⁺ sparks and indirect regulation of BK_Ca β1 expression and the dynamic interaction of BK_Ca α and BK_Ca β1 as a consequence of altered Ca²⁺ sparks. Together, these novel findings provide new mechanistic insights into pregnancy-induced uterine vascular adaptation.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Funding

This work was supported by National Institutes of Health Grants (HD083132, HL128209, and HL137649 to L.Z.).

 

Time for primary review: 32 days

 

Translational perspective

A successful pregnancy requires adequate uterine vascular adaptation. The present study demonstrates that pregnancy-induced RyR1 and RyR2 upregulation in uterine arteries exerts their regulatory role on uterine arterial myogenic tone via impacting the Ca²⁺ spark-STOC coupling, BK_Ca β1 expression, and association of BK_Ca β1 and BK_Ca α. Our findings reveal a crucial role of the RyRs-BK_Ca channel partnership in mediating pregnancy-induced uterine vascular adaptation. Thus, our findings provide valuable insights into understanding the mechanisms underlying uterine vascular adaptation in pregnancy.