Piezo1-modified mice

All animal use was authorized by the University of Leeds Animal Ethics Committee and The Home Office, UK. All animals were maintained in GM500 individually ventilated cages (Animal Care Systems), except during telemetry recordings, at 21 °C 50–70% humidity, light/dark cycle 12/12 h on RM1 diet (SpecialDiet Services, Witham, UK) ad libitum and bedding of Pure’o Cell (Datesand, Manchester, UK). Genotypes were determined using real-time PCR with specific probes designed for each gene (Transnetyx, Cordova, TN). C57BL/6 J mice with Piezo1 gene flanked with LoxP sites (Piezo1flox) were described previously7. To generate tamoxifen (TAM) inducible disruption of Piezo1 gene in the endothelium, Piezo1flox mice were crossed with mice expressing cre recombinase under the Cadherin5 promoter (Tg(Cdh5-cre/ERT2)1Rha and inbred to obtain Piezo1flox/flox/Cdh5-cre mice. TAM (Sigma-Aldrich) was dissolved in corn oil (Sigma-Aldrich) at 20 mg ml−1. Mice were injected intra-peritoneal with 75 mg kg−1 TAM for 5 consecutive days and studies were performed 10–14 days later. Piezo1flox/flox/Cdh5-cre mice that received TAM injections are referred to as Piezo1ΔEC. Piezo1flox/flox littermates (lacking Cdh5-cre) that received TAM injections were the controls (control genotype). For experiments, mice were males aged 12–16 weeks, except for telemetry (14–18-week old) and femoral artery injury (18–22-week old).

Analysis of Piezo1 deletion

Samples (about 10 mm3) of liver, lung, aorta, femoral artery and mesenteric artery were digested overnight at 37 °C in a lysis buffer containing 10 mM Tris pH 7.4, 50 mM EDTA, 1 % SDS; 5 µg ml−1 proteinase K (Sigma-Aldrich). Samples were then vortexed and 400 µl of phenol/chloroform/isoamyl (Sigma-Aldrich) was added. Tubes were mixed by inverting/shaking 20 times every 15 min for 1 h then centrifuged at 13,000×g for 15 min at room temperature. Four-hundred microliter of the top layer was transferred to a new tube followed by an addition of 440 µl of isopropanol and 40 µl of 3 M NaCl. Tubes were mixed by gentle inverting and left to stand for 1 h at room temperature. DNA was pelleted by centrifugation at 13,000×g for 30 min at room temperature. The supernatant was discarded and the pellet was washed with 70% ethanol, briefly air dried and resuspended in 60 µl of TE buffer. DNA was amplified using 12.5 µl Bioline MyTaq Red Mix, 0.5 µl of DNA solution, 1 µM of each primer. Sequences of PCR primers are specified in Supplementary Table 1. PCR was 95 °C for 5 min; 32 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min. PCR products were electrophoresed on 2 % agarose gels containing SYBR safe (Roche) at 80 V for 45 min.

Mouse liver endothelial cells

Mouse liver sinusoidal endothelial cells were isolated using an immunomagnetic separation technique. A whole mouse liver was minced using 2 scalpel blades and resuspended in a dissociation solution consisting of 9 ml 0.1 % collagenase II, 1 ml 2.5 U ml−1 dispase, 1 µM CaCl 2 and 1 μM MgCl 2 in Hanks Buffer solution. The tissue-dissociation mix was incubated at 37 °C for 50 min in a MACSMix Tube Rotator (Miltenyi Biotech) to provide continuous agitation. At the end of enzymatic digestion the sample was passed through 100 and 40 μm cell strainers to remove any undigested tissue. Cells were washed twice in PEB buffer consisting of Phosphate Buffered Saline (PBS), EDTA 2 mM and 0.5% Bovine Serum Albumin (BSA), pH 7.2. The washed pellets were resuspended in 1 ml of PEB buffer and 200 µl of dead cell removal paramagnetic microbeads per 1 × 107 cells (Miltneyi Biotec) at room temperature for 15 min. After incubation the cells were passed through an LS column prepared with 1 × binding buffer (Miltenyi Biotec) in a magnetic field (MiniMACS Separator, Miltenyi Biotec). The eluate was then incubated with 20 ml red blood cell lysis buffer consisting of 0.206 g Tris base, 0.749 g NH 4 Cl in 100 ml PBS pH to 7.2. Cells were washed again in PEB buffer, and the pellet was resuspended in 1 ml PEB buffer and 30 µl CD146 microbeads (Miltenyi Biotec) at 4 °C for 15 min under continuous agitation. After incubation this solution was passed through an MS column prepared with PEB buffer. CD146 positive cells were retained in the column and CD146 negative cells passed through as eluate. CD146 positive cells were washed through with warm EGM-2 media and the CD146 selection process was repeated a second time. After a second purification cells were plated and grown in a 5% CO 2 incubator at 37 °C. Media were changed at 12 h and then every 24 h until confluent.

Piezo1 inducible cell line

Piezo1-GFP7 was used as a PCR template to clone human Piezo1 coding sequence into pcDNA™4/TO between HindIII and EcoRI restriction sites. Piezo1 was amplified as two fragments using the following primers: (HindIII-Piezo1-Fw: AATAAGCTTATGGAGCCGCACGTG and BamHI-Int.Piezo1-Rv: AATGGATCCCCCTGGACTGTCG) and (BamHI-Int.Piezo1-Fw: AATGGATCCTCCCCGCCACGGA and EcoRI-Piezo1-Rv: AATGAATTCTTACTCCTTCTCACGAGT). The two fragments were fused using BamHI restriction site, resulting in the full length Piezo1 coding sequence with the c4182a silent mutation. T-RExTM-293 cells were transfected with pcDNA4/TO-Piezo1 using Lipofectamine 2000 (Thermo Fisher Scientific). Subsequently cells were treated with 10 μg ml−1 blasticidin and 200 μg ml−1 zeocin (Invitrogen, Thermo Fisher Scientific) to select stably transfected cells. Single cell clones were isolated and analysed individually. Expression was induced by treating the cells for 24 h with 10 ng ml−1 tetracycline (Sigma-Aldrich) and analysed by quantitative RT-PCR and western blot.

Fura-2 Ca2+ measurements

Intracellular Ca2+ was measured using the ratiometric Ca2+ indicator dye fura-2. Experiments were performed on confluent cells in a 96-well plate. Cells in each well were incubated with 50 μl fura-2 AM loading solution for 1 h at 37 °C. The loading solution consisted of 2 µM fura-2 AM and 0.01% pluronic acid in Standard Bath Solution (Ca2+-SBS) consisting of 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl 2 , 1.5 mM CaCl 2 , 8 mM d-glucose and 10 mM HEPES (pH 7.4). After 1 h the loading solution was removed and 100 μl of Ca2+-SBS was added to each well and left at room temperature for 30 min. A compound plate was prepared at twice the final concentration tested in Ca2+-SBS. The FlexStation II384 was set to add 80 μl of the compound solution to each well on the test plate containing 80 μl of Ca2+-SBS. Baseline fluorescence ratios were recorded before addition of the compound solution to the cell plate after 60 s, with regular recordings thereafter for a total of 5 min.

Thallium FluxOR measurements

Cells were plated at 80–90% confluence in 96-well plates 24 h prior to recordings (5 × 104 Human Embryonic Kidney (HEK) T-REx cells; 1.92 × 104 Human Umbilical Vein Endothelial Cells (HUVECs)). HEK T-Rex cells were from Thermo Fisher Scientific (catalogue #R71007) and HUVECs were from Lonza (catalogue #CC-2519). HEK T-Rex cells were validated to be tetracycline-responsive (as expected) (Supplementary Fig. 6). HUVECs were validated by positive staining with anti-CD31 antibody, response to vascular endothelial growth factor, and alignment of the cells to shear stress; human nucleotide sequences were detected, confirming human origin. To measure thallium (Tl+) influx, cells were loaded with the FluxORTM dye for 1 h at room temperature, transferred to assay buffer and stimulated with a Tl+ containing K+-free solution as per the manufacturer’s instructions (Molecular Probes). Measurements were made on a fluorescence plate reader (FlexStation II384). FluxOR was excited at 485 nm and emitted light collected at 520 nm, measurements expressed as a ratio increase over baseline (F/F 0 ), with vehicle (DMSO) values subtracted from Yoda1 values at each time point (∆F/F 0 ). Rates of increase in fluorescence intensity were determined between 7.5 and 35 s after injection of Yoda1 (∆F/F 0 /ms).

Arterial contraction studies

Animals were culled by cervical dislocation according to Schedule 1 procedure approved by the UK Home Office. Mesenteric arcades were dissected out and placed immediately into ice-cold Krebs solution (125 mM NaCl, 3.8 mM KCl, 1.2 mM CaCl 2 , 25 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , 1.5 mM MgSO 4 , 0.02 mM EDTA and 8 mM d-glucose, pH 7.4). Second-order mesenteric, saphenous or carotid arteries were cleaned of fat and connective tissue under a dissection microscope. Segments of 1 mm length were mounted in an isometric wire myograph system (Multi Wire Myograph System, 620 M), bathed with Krebs solution warmed at 37 °C and gassed with 95% O 2 /5% CO 2 then stretched stepwise radially to their optimum resting level to an equivalent transmural pressure of 100 mm Hg and equilibrated for 1 h prior to experiments. For studies of luminal flow in second-order mesenteric artery, vessel segments were mounted on glass cannulas in a pressure myograph (Model 110p, Danish Myo Technology A/S, Denmark). Flow was generated by increasing the pressure difference (ΔP) between inflow and outflow without change in the absolute intraluminal pressure. The outer arterial diameter was monitored using a CCD camera (DMX41 AU02, Imaging Source Europe, Germany) and recorded with MyoView II software. Arteries were only used for investigation if they constricted in response to phenylephrine (PE) and dilated in response to acetylcholine (ACh).

Blood pressure measurements

Conscious long-term recordings of arterial blood pressure (mean, systolic and diastolic) were achieved via a radiotelemetry probe (model TA11PA-C10, Data Sciences International). Adult male mice (14–18-week old) were anaesthetised with isoflurane (5% induction 1.5% maintenance) in 95% O 2 and body temperature maintained via a heating pad. The probe catheter was advanced, via the left carotid artery, into the ascending aorta. The body of the transmitter was placed in a subcutaneous pocket along the left flank. A period of at least 14 days was allowed for recovery from surgery before the start of experimental recordings. TAM treatment started 4 days after probe implantation and recordings started 10 days after the last TAM injection. Mice were housed singly in cages and synchronized to a light–dark cycle of 12:12 h with lights on at 06:00 h. Cages were positioned over receivers connected to a computer system for data recording. Blood pressure waveforms and parameters were analysed using DSI analysis package, Dataquest ART 4.1. Continuous 24 h recordings were begun 3 days following singular housing and obtained over a 7 day period. During the recording period animals were allowed free access to a voluntary running wheel.

Freshly isolated mesenteric endothelial cells

Endothelial cells were freshly isolated from second-order branches of mouse mesenteric arteries as described previously14. Briefly, dissected second-order mesenteric arteries were enzymatically digested in dissociation solution (126 mM NaCl, 6 mM KCl, 10 mM Glucose, 11 mM HEPES, 1.2 mM MgCl 2 , 0.05 mM CaCl 2 , with pH adjusted to 7.2) containing 1 mg ml−1 collagenase Type IA (Sigma-Aldrich, Dorset, UK) for 14 min at 37 °C and then triturated gently to release the endothelial cells on a glass coverslip.

Patch-clamp electrophysiology

Membrane potential was measured using the perforated whole-cell configuration of the patch-clamp technique in current clamp mode with an Axopatch-200A amplifier (Axon Instruments, Inc.) equipped with Digidata 1440 A and pCLAMP 10.6 software (Molecular Devices, Sunnyvale, CA, USA) at room temperature. Outside-out membrane patch recordings were made using the same equipment but in voltage-clamp mode. Endothelial cells and endothelium were bathed in a solution consisting of 135 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose and 10 mM HEPES (pH 7.4). Heat-polished patch pipettes with tip resistances between 3 and 5 MΩ were used. For membrane potential recordings, amphotericin B (Sigma-Aldrich) was used as the perforating agent, added in the pipette solution composed of 145 mM KCl, 1 mM MgCl 2 , 0.5 mM EGTA and 10 mM HEPES (pH 7.2). For application of fluid flow, endothelium or membrane patches were manoeuvred to the exit of a capillary tube with tip diameter of 350 μm, out of which ionic (bath) solution flowed at rates specified in the main text and figure legends. Calculation of shear stress (τ ω ) was achieved using the Hagen-Poiseuille formula31 (τ ω = 4μ Q/πR 3) where μ is dynamic viscosity, Q is flow rate and R is radius of the capillary tube.

Echocardiography

Animals were maintained under steady-state isofluorane anaesthesia and placed on a heated platform with ECG and respiration monitoring. Core temperature was measured using a rectal probe (Indus Instruments) and maintained at 37.5 °C throughout recording. Echocardiography was performed using a Vevo2100 high resolution, pre-clinical in vivo ultrasound system (VisualSonics) with the MS-550D transducer at 40 MHz frequency and 100% power. Imaging was performed on a layer of aquasonic gel after the pre-cordial skin had been clipped and de-epliated with cream (Veet). Parasternal long-axis view (PLAX) images were obtained in EKV mode (set at 1000 Hz for recording) over the entire cardiac cycle. The left ventricular area was traced in end-distole (LVAd) and end-systole (LVAs) and used to derive the ejection fraction (EF) with the Vevo LAB cardiac package software. The investigator performing sonography was blinded to the genotype of the animals. Transverse EKV recordings were also obtained over the abdominal aorta just below the diaphragm using the same settings as described for the heart. These images were evaluated in the VevoVasc software package to determine vessel distensibility. Maximal anteroposterior aortic diameter (from inner wall to inner wall) was measured in the same images in systole and diastole using Vevo LAB general imaging package software.

Retina vasculature staining and analysis

Retinas were dissected from eyes after fixation in 4% paraformaldehyde in PBS for 4 h at room temperature, then stored overnight at 4 °C in permeabilisation and blocking buffer (PBS; 0.5% triton; 1% BSA; 0.01% sodium deoxycholate; 0.02% sodium azide; 0.1 mM CaCl 2 ; 0.1 mM MgCl 2 ; 0.1 mM MnCl 2 ). Retinal vasculature was then stained overnight at 4 °C with isolectin B4 Alexa Fluor 488 conjugate (Molecular Probes, Thermo Fisher), diluted 1:100 in PBLEC buffer (PBS; 1% triton; 0.1 mM CaCl 2 ; 0.1 mM MgCl 2 ; 0.1 mM MnCl 2 ). Retinas were washed with 0.25% Triton in PBS, then flat-mounted on slides with ProLong Gold (Molecular Probes, Thermo Fisher). Confocal microscopy (LSM 880, Zeiss) was used to image retinas, with analysis blinded to genotype conducted using ImageJ software (NIH, Bethesda, MD). Distal arterial diameter was analysed 1500 μm from the optic disc in the largest branch of each artery emanating from the disc. Capillary area was determined in regions of interest, which excluded arteries and veins, using the threshold function and fractional area measurement.

Femoral injury

Experiments were carried out on 18–22-week-old male mice. Femoral injury was performed 12–16 days after the last TAM injection. Mice were anesthetized with isoflurane (1.5–2%) before a small incision was made in the mid-thigh and extended. Having carefully isolated the femoral artery, an arteriotomy was made in the saphenous artery using iris scissors (World-Precision Instruments, Sarasota, FL) and a 0.014-inch-diameter angioplasty guidewire with tapered tip (Hi-Torque Cross-It 200XT, Abbott-Vascular, IL) was introduced. The guidewire was advanced 1.5 cm in to the femoral artery, and three passages performed per mouse, resulting in complete endothelial denudation. The guidewire was removed completely and a suture tightened rapidly immediately distal to the bifurcation of the femoral artery. The skin was closed with a continuous suture. Animals received peri-operative analgesia with buprenorphine (0.25 mg kg−1 s.c.). Mice were anesthetized at 5 days after wire injury and 50 μl of 0.5% Evans blue dye injected into the inferior vena cava. The mice were perfused/fixed with 4% paraformaldehyde in PBS before the femoral arteries were collected. The vessels were opened longitudinally. The areas stained and unstained in blue were measured in a 5 mm injured segment beginning 5 mm distal to the aortic bifurcation, and the percentage areas were calculated using ImagePro Plus 7.0 software (Media Cybernetics, Bethesda, MD).

Running wheel analysis

Mice were individually housed and had free access to a running wheel. Custom-built hardware and software allowed detailed characteristics of running activity to be recorded for each animal. A mouse was considered to be active when there were ≥ 2 revolutions of the running wheel during each 1 min recording period. This equates to ≈10% of the mean dark cycle velocity of running for control animals (0.34 m/s). Continuous periods of activity (bouts) were defined as activity seen in two or more consecutive minutes. One Piezo1ΔEC mouse showed complete inactivity for the first 3 days and was excluded from the analysis along with its control genotype pair.

Cell and tissue staining

Epididymal fat pad and liver tissues were fixed for 48 h in 4% PFA at 4 °C prior to processing on a Leica ASP 200 and embedding in CellWax (Cellpath) on a Leica EG1150H embedding station. Sections of 4 μm were cut on a Leica RM2235 microtome onto Plus Frost slides (Solmedia) and allowed to dry at 37 °C overnight prior to staining. Slides were de-waxed in xylene and rehydrated in ethanol. H&E was performed by staining in Mayer’s Haematoxylin for 2 min and eosin for 2 min. Slides were imaged on an Aperio AT2 (Leica Biosystems) high definition digital pathology slide scanner with a maximal magnification of ×20. Tissue processing and imaging were performed at Section of Pathology and Tumour Biology, Leeds Institute of Cancer and Pathology. For CD31 immunofluorescence, cells were fixed on coverslips in 4% paraformaldehyde and permeablised with 0.1% TritonX-100 at room temperature. Cells were blocked with donkey serum for 30 min to prevent non-specific binding. Cells were then incubated with 1% BSA in PBS containing rabbit anti-mouse CD31 (1:50, Abcam ab28364). Following incubation with primary antibody, cells were washed in PBS and incubated with Alexa Fluor 488-conjugated affinipure donkey anti-rabbit IgG (1:300, Jackson Immuno Research Laboratories) for 45 min at room temperature. Cells were mounted with Prolong Gold Antifade Reagent containing DAPI (Invitrogen) and visualised using a LSM 880 confocal microscope (Zeiss).

RNA isolation and quantitative PCR

Total RNA was isolated using a standard TriReagent protocol and treated with DNAse (TURBO DNA-free, AM1907M, Ambion). An aliquot was used for cDNA synthesis using a High Capacity RNA-to-cDNA kit (Applied Biosystems, UK) containing Oligo-dT and random primers. Real-time PCR was performed using Roche Fast Start SYBR Green I on a Lightcycler2 with Lightcycler 3.5 software or using Roche 480 SYBR Green I on a Lightcycler480II with Lightcycler 1.5.62 software. DNA amplification was for 35 cycles with an initial 10 min at 95 °C followed by 10 s at 95 °C, 6 s at 55 °C and 14 s at 72 °C. Primers were used at 0.5 μM. Sequences of PCR primers are specified in Supplementary Table 1. The specificity of PCR was verified by reactions without RT (-RT) and by melt-curve analysis. PCR cycle crossing-points (CP) were determined by fit-points methodology. Relative abundance of target RNA was calculated from (E18sCp)/(EtargetCp). All quantitative PCR reactions were performed in duplicate and the data averaged to generate one value per experiment.

Data analysis

Genotypes of mice were always blinded to the experimenter and mice were studied in random order determined by the genotype of litters. Data were generated in pairs (control mice and Piezo1ΔEC mice) and data sets compared statistically by independent t-test without assuming equal variance. Paired t-tests were used when comparing data before and after application of flow or a substance to the same membrane patch or cell. Statistical significance was considered to exist at probability (P) < 0.05 (* < 0.05, ** < 0.01, *** < 0.001). Where data comparisons lack an asterisk, they were not significantly different. The number of independent experiments (mice or independent cell cultures) is indicated by n. For multi-well assays or multiple cell on coverslip studies, the number of replicates is indicated by N. Descriptive statistics are shown as mean ± s.e.m. unless indicated as mean ± s.d. (standard deviation). Origin Pro software was used for data analysis and presentation.

Data availability

All relevant data are available from the authors upon reasonable request.