A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy, and Heart Failure

Supplemental Digital Content is available in the text.

for pain relief. All animals were singly housed and allowed to recover for 7-10 days before recording baseline blood pressure for three 3 nights and 2 days in a quiet room. Blood pressure, heart rate activity was monitored at 10 min intervals, for duration of 2 min using the DSI software (DSI Dataquest A.R.T.) and data analysed in Microsoft Excel and GraphPad Prism 5. Data are represented as an average of 6h measurement.
All mice received osmotic pumps containing AngII (1.1mg/kg/day) and at day 7 following AngII infusion when mice showed a significant increase in blood pressure from baseline, they received daily treatment of vehicle or αAnalogue (50nmol/kg, s.c., Novo Nordisk) at Day 7 to Day 14 of AngII infusion ( Figure 6).
At the end of the study (Day 15), mice were briefly anaesthetised with isoflurane (Abbott Laboratories) in 2l/min O 2 and blood was collected via cardiac puncture. Mice were culled by cervical dislocation and organs (heart and aorta) were harvested for post-analysis. Heart, lung, kidney and spleen from each mouse was excised, dried on filter paper and weighed. Organs' weights were normalised to body weight and tibia length.

Cardiac hypertrophy murine model
Mice were surgically subjected to pressure-overload induced-cardiac hypertrophy and heart failure as previously described. 11 Briefly, suprarenal aortic constriction was performed in anesthetised mice (2% isoflurane carried in O 2 , Abbott Laboratories), where the abdominal aorta was ligated to the width of a 28G needle (0.36mm) using suture thread Ethilon Sutures,Ethicon) in between the superior mesenteric and celiac bifurcation. Sham surgeries were performed in the same manner, excluding the constriction. Post-surgery analgesia was provided in the form of buprenorphine (50 µg/kg, i.m., Vetergesic). Following surgery, mice were treated with daily with vehicle (0.219M Mannitol, 5% HPCD, 1.6% ammonium acetate at pH 6.5) or αAnalogue 6 (50nmol/kg, s.c., Novo Nordisk) for 5 weeks.
Body weight, food and water intake were measured throughout the study. At the end of week 5, mice were briefly anaesthetised with isoflurane (Abbott Laboratories) in in 2l/min O 2 and blood was collected via cardiac puncture. Mice were culled by cervical dislocation and organs were harvested for postanalysis. Heart, lung, kidney and spleen from each mouse was excised and weighed. Organs' weights were normalised to body weight and tibia length.

Echocardiography
Anesthesia in mice was induced in a chamber with 5% isoflurane for 1 min, afterwards maintained with 1.5% on a heated platform. Imaging was performed 10 min after induction using a Vevo 2100 Imaging System with a 40-MHz linear probe (Visualsonics, Canada), as previously described. 11,12 Cardiac dimensions as well as systolic function were assessed using M-mode imaging in long axis views. The relative wall thickness (RWT) in diastole was calculated as follows: RWT = (septal wall thickness + posterior wall thickness) / left ventricular diameter. Data analysis was performed with Vevo®2100 software v.1.2.1 (Visualsonics).

Light aversion assay
The light aversion assay is based on rodents' natural preference for darker areas compared to lighter areas 13,14 and was used to study photophobia in rodents, a common symptom of migraine. 15 The apparatus consisted of two equal compartments: one-half (20 (d) x 20 (w) x 14 (h) cm) black plexiglass, with a black lid on top, the remaining half was transparent to enable behavioural assessment and recording. Mice were able to move freely between the two compartments through a small doorway (5 × 5 cm) in the center of the black plexiglass wall separating the two compartments, as described previously. 14  Mice were trained in the light/dark box under a range of lighting conditions (500-1000 lux) twice for 7 days. Mice that displayed equal preference between light/dark compartments (>40% light preference) were used in our study. Following acclimation in the testing room for 1h (200 lux), baseline recordings (10 min) were obtained, followed by systemic administration of vehicle (0.219M Mannitol, 5% HPCD, 1.6% ammonium acetate at pH 6.5) or αAnalogue (50 nmol/kg, s.c.) or the positive control glyceryl trinitrate (GTN, 320 nmol/kg, i.v.). Behavioural responses were resumed for 10min at 2h following treatment. 16 To evaluate the effects of chronic administration of the αAnalogue (5 weeks), sham mice were assessed   on week 5 for light aversion at 1h following systemic administration of vehicle (0.219M Mannitol, 5% HPCD, 1.6% ammonium acetate at pH 6.5) or αAnalogue (50 nmol/kg, s.c.); behavioural responses were recorded for 10min.
Mouse behaviour was digitally recorded using an IPEVO camera (IPEVO, UK), placed 30cm above the light/dark box and attached to a laptop. Videos were continuously recorded at 30 frames/s and decomposed to individual frames of 1s interval. Each 10min experimental interval was taken to start immediately after the mouse entered the dark chamber. Behavioural experiments were conducted by a blinded observer and analysis was conducted procedurally with a custom ImageJ script, applying foreground extraction to determine the presence of the mouse against the white background of the light box floor across sequential frames. Absolute duration and % dwell time within the light and dark chambers was automatically calculated, as was the number of transitions, interpreted as crossings. Data was represented as the % of time spent in light during the 600s test session.

Glucose tolerance test
Glucose tolerance tests (GTT) were performed following a 6h fast (from 08:30 to 14:30), as previously described. 17 Glucose (1g/kg, Sigma) was injected i.p. and blood glucose levels were monitored for 2h (from 08:30 to 14:30) at the indicated time points after injection with a One Touch Vita glucose meter (Lifescan, UK). GTT was determined at baseline and at Day 14 following daily treatment with the αAnalogue (50nmol/kg, s.c.) or its respective vehicle (0.219M Mannitol, 5% HPCD, 1.6% ammonium acetate at pH 6.5).

Core body temperature measurement using radiotelemetry
Core body temperature and activity were measured using radio-telemetry as described previously. 18 Briefly, animals were anaesthetised by isoflurane (2%, Abbott Laboratories, UK) in 2l/min O 2 , placed on a homeothermic heating pad and buprenorphine (50µg/kg, i.m., Vetergesic, Alstoe, Animal Health, UK) was administered peri-operatively. The abdomen was shaved and scrubbed using surgical scrub and a small ventral midline abdominal incision (< 2 cm) was made to expose the abdominal muscle wall. A ventral incision was made on the abdominal wall and a small volume of (< 80 µl) sterile saline was applied to facilitate the insertion of the radio telemetry transmitter (TA10TA-F10; DSI, St Paul, Minnesota, USA). Following implantation, the abdominal wall and skin were sutured separately using absorbable sutures (Vicryl ® 4.0, Ethicon, Johnson & Johnson, UK). The mice were monitored until ambulatory and were individually housed with food and water ad libitum. Mice were weighed and examined daily, and were allowed a minimum 7 days post-surgical recovery period. Cages containing the telemetered animals were placed on the receiver plates (RPC-1; Data Sciences Incorporated (DSI, Minnesota, USA); radio signals from the implanted transmitters were monitored via a fully automated data acquisition system (DataquestART, version 3.1; DSI, Minnesota, USA). Locomotor activity and core body temperature were monitored at 10 min intervals, for duration of 2 min.

Measurement of gene expression using Real-time reverse transcription polymerase chain reaction
Real-time reverse transcription polymerase chain reaction (RT-qPCR) was carried out as previously described. 3,7,10 Total ribonucleic acid (RNA) was extracted from aorta, heart and kidney tissue using the Qiagen RNeasy Microarray tissue mini Kit (Qiagen), according to manufacturer's instructions. Total RNA (1µg) was then reverse transcribed into cDNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Life technologies Ltd) as per manufacturer's instructions. A thermal cycler (DNA Engine Tetrad 2 Peltier Thermal Cycler) was used for reverse transcription to complementary DNA (cDNA).
Negative RT samples were carried out as a control to exclude possible contamination of genomic DNA.
cDNA was then diluted 1:40 in nuclease-free water for RT-qPCR. Quantitative PCR (qPCR) was then conducted using a SYBR-green-based PCR mix (Sensi-Mix, SYBR-green No ROX; Bioline) and primers from the specific gene of interest pipetted into 100-well gene disks (Qiagen) by an automated robot (CAS1200; Qiagen Corbett), followed by PCR in a Corbett Rotor-gene 6000. Settings were as follows; initial denaturation: 10min at 95°C; cycling: 45 cycles-10 s at 95°C, 15 s at 57°C, and 5s at 72°C; melt: 68-90°C. Samples were subjected to melting curve analysis to confirm amplification specificity. Data were collected as copies/µl and normalised against murine hypoxanthine phosphorribosyltransferase (HPRT), β-2-microglobulin (B 2 M) and β-actin expression using GeNorm3.4 software. A list of primers are summarised in Supplemental Table I.

Quantification of noradrenaline using ELISA
Plasma and renal noradrenaline (NA) content were quantified using a commercially available NA ELISA kit (RE59261; IBL International, Hamburg, Germany), as previously described. 1 Briefly, kidneys were homogenised in phosphate buffered saline (1M), supplemented with protease inhibitors (Roche, Germany). Lysates were cleared by centrifugation at 2,600 g for 15 min at 4°C. NA content was extracted from both plasma and kidney tissue lysate and assessed by standard ELISA technique, according to the manufacturer instructions. The limit of sensitivity was 20 pg/ml and the linearity limit was 8.0 ng/ml. Cross-reactivity to other catecholamines or metabolites was manufacturer tested as <0.02%. Protein concentration of each kidney samples were determined by Bradford dye-binding method (Bio-rad) and noradrenaline content was normalised to mg of protein.

Quantification of IL-6 and TNF-α using ELISA
Briefly, frozen kidney tissues were homogenised in phosphate buffered saline (1M), supplemented with protease inhibitors (Roche, Germany). Lysates were cleared by centrifugation at 2,600 g for 15 min at 4°C. Renal interleukin-6 (IL-6) and Tumour necrosis factor-α (TNF-α) content were quantified using a commercially available ELISA kit (RAB0308 & RAB0477, Sigma, UK), following manufacturer's protocol. Protein concentration of each kidney samples were determined by Bradford dye-binding method (Bio-rad). The final concentrations of both IL-6 and TNF-α were calculated with respect to the total amount of protein (mg).

Histology
Aorta, heart and kidney tissues were fixed in 4% paraformaldehyde (overnight, 4°C), as previously described. 7,19,20 For heart and kidney samples, after routine paraffin wax embedding, transverse sections (6µm) were prepared using a Reichert-Jung 2030 Biocut microtome onto poly-L-lysine slides. For aorta samples, following fixation aorta segments were embedded in optimal cutting temperature compound (OCT, VWR), frozen on dry ice in a cryomould and cryosectioned on a microtome cryostat (6µm, Bright, UK) on Superfrost Plus glass slides. Sections were allowed to dry overnight. Approximately 8-10 transverse sections were obtained from each heart or aorta per mouse for staining.
All slides were imaged using an Olympus Colourview III camera, connected to an Olympus BX51 microscope (x10 magnification). Images were taken using CellSens Dimension viewing software (Version 1.1.3, Olympus). Aortic wall width (medial thickness) were measured (mean of 8 measurements taken from at least 3 sections for each 4 mice/group) using ImageJ analysis software (scale bars, 100µm; NIH, USA).
Heart Staining: Picrosirius Red (0.1% w/v) staining was used to visualise collagen fibres in the heart 20 and fibrosis was assessed blindly. All slides were imaged using a Leica Diaplan microsope (x10 magnification) and images captured using ProgRes Capture Pro viewing software (Version 2.8.8, JENOPTIK) under bright light and circular polarised light according to a modified Junqueira method. 22,23 Cardiac fibrosis was quantified on circular polarised light images using Image J analysis software (scale bars, 200µm; NIH, USA).
Conjugated wheat germ agglutinin (WGA, CF488A, Biotium) staining was applied to outline cardiomyocyte boundaries and quantify cross-sectional area. 20 All slides were imaged using an Olympus Colourview III camera, connected to an Olympus BX51 microscope (x40 magnification).
Images were taken using CellSens Dimension viewing software (Version 1.1.3, Olympus). Myocyte cross-sectional area was employed as an index of cardiac hypertrophy and determined on fibres with circular shapes in a blinded fashion by quantitative image analysis using Image J (scale bars, 20µm; NIH, USA).
Capillaries were immunostained with isolectin B4 (Vector B-1205) and capillary density was quantified as the number of capillaries per mm 2 of LV sections 11 . All slides were imaged using an Olympus Colourview III camera, connected to an Olympus BX51 microscope (x40 magnification). Images were taken using CellSens Dimension viewing software (Version 1.1.3, Olympus). Number of capillaries were manually counted in a blinded fashion by quantitative image analysis using Image J (scale bars, 20µm; NIH, USA).
Images were taken using CellSens Dimension viewing software (Version 1.1.3, Olympus). TUNELpositive myocytes and DAPI-stained nuclei were manually counted by quantitative image analysis using Image J (scale bars, 50µm; NIH, USA).
Kidney Staining: Sections of kidney were stained with Periodic acid-Schiff's (PAS) and counter stained with hematoxylin, as previously described 25 . All slides were imaged using an Olympus Colourview III camera, connected to an Olympus BX51 microscope (x40 magnification). Images were taken using CellSens Dimension viewing software (Version 1.1.3, Olympus). A semi quantitative system was used to grade mesangial expansion by either diffuse (irregular streaks) or segmental (localized to a lobule) type. In each of the experimental animals, 20 glomeruli were examined for mesangial expansion and each glomerulus was graded 0 to 4 corresponding to the percentage of mesangial matrix covering/occupying the urinary space of the Bowman's capsule. Glomeruli were assessed and graded 1-4, as follows: grade 1, involvement of 0-24%; grade 2, 25-29%; grade 3, 50-74% or grade 4; 75-100% of mesangial area to Bowman's capsule (scale bars, 20µm; NIH, USA). Mean glomerular score index was determined for each kidney 26 .
All morphometric measurements were performed in a blinded manner.

Statistical analysis
Data in the manuscript is expressed as mean + SEM. Statistical analysis was performed using a twotailed Student's t-test (2 unpaired groups), one-way or repeated measures two-way analysis of variance (ANOVA) followed by Bonferroni's comparison post hoc test (multiple groups comparison). p<0.05 was considered to represent a significant difference.

Supplemental Tables
Supplemental Table 1 Table 7