SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels

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Animals
All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act (1986) and the Spanish Royal Decree 1201/2005 and 53/2013 (Care and Use of Laboratory Animals), and were approved by local Animal Welfare committees (St George's University and Universidad Complutense de Madrid). Gonadal hormones regulate arterial contractility by modulating the response to vasocontrinctors and altering the response. In order to reduce variability due to the effects of sex hormones on arterial contractility, only male rats were used in the present study. 10-12 weeks-old male Wistar rats (Charles River, UK) were killed by cervical dislocation. Animals were housed in a climatically controlled environment, on a 12h light/dark cycle, with free access to water and standard food ad libitum.

Arteries
Main conduit renal and third order mesenteric arteries were microdissected and cleaned of adherent fat in ice cold Krebs solution as previously described 1 . Arteries were incubated in a 24-well plate containing DMEM/F-12 culture medium (Sigma Aldrich, Dorset, UK) supplemented with 1% penicillin-streptomycin (Sigma Aldrich, Dorset, UK) in a 37°C incubator with 5% CO2 for 16 hours (for myo-inositol/raffinose treatments) or 48 hours (for gene-silencing experiments). In double knockdown experiments, arteries were incubated in the morpholinos transfection mix for 32 hours and then incubated in medium containing either vehicle or raffinose plus myo-inositol for 16 hours.

Isolation of vascular smooth muscle cells
VSMCs were isolated as previously described 1 . Briefly, renal and mesenteric arteries from male Wistar rats were digested with collagenase type IA (2 mg/ml) and protease type X (1 mg/ml); single cells were isolated by gentle mechanical agitation with a Pasteur pipette, and plated on 25 mm diameter coverslips. Cells were allowed to settle for 1 hour at room temperature (RT, 20-22°C) and then used for immunofluorescence experiments. For qPCR experiments, VSMCs were prepared to ensure removal of endothelium as previously described 2 . Briefly, segments of renal artery were longitudinally cut and the lumen was rubbed with a human hair. The arteries were then placed in PBS containing 0.1% Triton X for 1 min followed by three 1-min washes in fresh PBS. The treated arteries were subsequently dispersed to a cell suspension, spun down into a pellet and used for RNA extraction.
Gene knock-down Knockdown of Kv7.4, Kv7.5 and SMIT1 in renal and mesenteric arteries was performed by transfection with morpholino oligonucleotides as described in vessels previously 3 . Targeting and scrambled (control) morpholino oligonucleotides (5 µmol/L; Genetools, Oregon, USA) were mixed in Opti-MEM and transfected using Lipofectamine 2000 (Thermo Fisher, Paisley, UK). Double knockdown of Kv7.4 and Kv7.5 was obtained by incubating both targeting morpholinos in the same mix (5 µmol/L each); control arteries for these experiments were transfected with 10 µmol/L scrambled morpholino. The mix was then added to the arteries incubated with DMEM/ F-12 with 1% penicillin/streptomycin. RNA extraction, reverse transcription and quantitative PCR Total RNA was extracted from arteries using Monarch Total RNA Miniprep Kit (New England Biolabs, Hitchin, UK), according to the manufacturer's instruction, and reverse transcribed to cDNA using Luna Script RT Super Mix ((New England Biolabs, Hitchin, UK). Q-PCR experiments were run in CFX96 Real-Time PCR Detection System (Bio-Rad, Hertfordshire, UK) using the SYBR-Green detection technique and specific primers (see table 1), as previously described 1 . Cycle threshold (Ct) for each gene of interest was calculated using Bio-Rad CFX96 Manager 3.0 software, normalized to two reference genes, namely Ubiqutin C (UBC) and DNA Topoisomerase I (TOP1). Data were expressed using the 2 -ΔCt formula. No template controls and reverse transcriptase-negative controls were run alongside all reactions to assess contamination. Mastermix for the qPCR experiments were purchased from Primerdesign (Southampton, UK). For further information regarding primers, see Supplemental Table I.

Immunofluorescence
VSMCs were fixed with 3% paraformaldehyde (PFA) in PBS solution at RT for 10 min, treated with 0.1 mol/L glycine for 5 min and incubated in blocking solution (PBS containing 0.1% Triton X-100 and 1% bovine serum albumin) for 1 hour at RT. Cells were then incubated overnight at 4°C with a mouse anti-SMIT1 antibody (dilution 1:100, Santa Cruz, Texas, USA) and a rabbit anti-Kv7.4 (dilution 1:200, Abcam, Cambridge, UK) that has been validated in previous studies 4 . Samples were then washed with PBS and incubated for 1 hour with donkey anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 567, respectively (dilution 1:100, Thermo-Fisher, Paisley, UK). All antibodies were diluted in blocking solution. Coverslips were washed with PBS and analysed with a Nikon A1R confocal microscope (Nikon Instruments Europe BV, Amsterdam, Netherlands). Corrected total cell fluorescence (CTCF) was calculated using ImageJ software as elsewhere described 5 . The number of cells analysed is indicated by 'n', whereas 'N' represents the number of animals used.

Patch-clamp recordings
All current recordings were made with an Axopatch 200B and a Digidata 1322A (Axon Instruments, Burlingame, CA, USA). For renal VSMCs, membrane Kv currents were measured using perforated whole-cell voltage clamp by including amphotericin B in the pipette solution (final concentration of 200 μg/ml) at room temperature (21-23ºC). Single renal artery myocytes were enzymatically isolated, as described previously 6 . Freshly isolated VSMCs were superfused with a bath solution containing (in mmol/L): 120 NaCl, 3 NaHCO3, 4.2 KCl, 1.2 KH2PO4, 0.5 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.4) and an internal pipette solution contained (in mmol/L): 110 potassium gluconate, 30 KCl, 0.5 MgCl2, 5 HEPES and 0.1 EGTA (pH 7.2). Kv currents were evoked by the application of 325 ms step pulses to between −90 mV to +50 mV in increments of 10 mV from a holding potential of −70 mV. In order to inhibit Kv1, Kv2, Ca 2+ -activated K + currents and voltage-dependent inward Ca 2+ currents, 4-AP (100 μmol/L), TEA+ (4 mmol/L) and nifedipine (100 nmol/L) were included in the bath solution 6 . The magnitude of the Kv7 component was considered to be the residual current that was sensitive to 10 μmol/L XE991. Current-voltage relationships were constructed by measuring the currents at the end of the pulse. Cell capacitance was calculated from the integral of the capacitive transient current elicited by 10 mV hyperpolarizing pulses from a holding potential of −70 mV. Currents were normalized for cell capacitance and expressed as pA pF-1.
For patch-clamp recording in CHO cells, we used an external solution containing (in mmol/L): 140 NaCl, 4 KCl, 2 CaCl, 1 MgCl2 and 10 HEPES. Patch pipettes with a resistance of 4-12 MΩ were filled with a pipette solution containing (in mmol/L): 110 K gluconate, 30 KCl, 0.5 MgCl2, 5 HEPES and 0.5 EGTA. Cells were held at -60 mV and currents amplitude was monitored by application of a test pulse to 40 mV every 20 s. To generate current-voltage relationships cells were stepped from a holding potential of -60 mV to voltages from -70 to +40 mV for 500 ms at 15 s intervals.

Proximity Ligation Assay
Proximity Ligation Assay (PLA) was used to assess protein-protein interactions as previously described using validated antibodies 1,3 . VSMCs were fixed with 3% PFA for 10 min and permeabilised in PBS-0.1 % Triton X-100 for 5 minutes. The Duolink in situ PLA detection kit (Sigma Aldrich, Dorset, UK) was used according to the manufacturer's protocol. Cells were blocked for 30 min at 37°C in Duolink blocking solution and incubated overnight at 4°C with a combination of two of the following primary antibodies: i) mouse anti-SMIT1 (1:100, Santa Cruz, Texas, USA); ii) rabbit anti-Kv7. probes for 1 hour at 37°C. Hybridised oligonucleotides attached to the probes were ligated for 30 mins at 37°C prior to rolling circle amplification for 100 mins at 37°C. Zstack images were acquired using a Nikon confocal microscope (Nikon Instruments Europe BV, Amsterdam, Netherlands). Analysis of mid-cell xy sections were performed using Image-J software. For PLA in CHO cells, the number of puncta per cell was calculated by dividing the total number of dots by the number of nuclei in the microscopic field.

Statistical analysis
All data are expressed as mean ± S.E.M. One-or two-way ANOVA test followed by a Dunnett's or Tukey's multiple comparisons test, and Student's t-test (paired or unpaired) were used to determine statistical significance between groups, according to the different experiments. Normality and variance of the data were not tested. The significance level for statistic tests was 0.05 (differences were considered statistically significant when p<0.05). Asterisks in the figures indicate the approximate p value given by the test, as described in the figure legends.  Fig 3A-B). The responses to the second incubation of methoxamine before the incubation with ML213 (MO) are also shown.