Experimental model

Sixteen- to eighteen-week-old male spontaneously hypertensive rats (SHRs, n = 80) were divided into four experimental groups: the control (SHR; n = 20), MLN-4760-treated (SHR + MLN; n = 20), zofenopril-treated (SHR + ZOFE; n = 20), and MLN-4760 and zofenopril-treated groups (SHR + MLN + ZOFE; n = 20). Each experimental group was divided into two separate subgroups: one for the realization of in vivo (echocardiography, integrated blood pressure response (n = 8) and one for in vitro studies (vasoactive responses and biochemical analyses, n = 12). The samples of randomly selected 8–10 rats were used in the study.

The specific ACE2 inhibitor MLN-4760 (MedChemExpress, Monmouth Junction, NJ, USA) was administered using Alzet® miniosmotic pumps (Durect™, Cupertino, CA, USA), Model 2002, with a pumping rate of 0.5 µL/h for 14 days at a dose of 1 mg/kg/day dissolved in 10% dimethyl sulfoxide (DMSO) in isotonic saline (sodium chloride 0.9% Braun intravenous solution for infusion; 308 mOsm/L; 250 mL; B. Braun Melsungen AG, Melsungen, Germany) in the SHR + MLN and SHR + MLN + ZOFE groups. In the SHR and SHR + ZOFE groups, miniosmotic pumps were filled with 10% DMSO in isotonic saline. All pumps were implanted subcutaneously on the dorsum of rats under isoflurane inhalation (2.5-3%) anesthesia in aseptic conditions. The cut was closed by three stitches of braided nonabsorbable silk suture (SMI, St. Vith, Belgium). The SHR + ZOFE and SHR + MLN + ZOFE groups received Zofenopril calcium (AdooQ Bioscience LLC, Irvine, CA, USA) that was administered p.o. (mixed with chow) at a dose of 10 mg/kg/day for 10 days. Treatment with ZOFE started on the 5th day of MLN administration in the SHR + ZOFE and SHR + MLN + ZOFE groups to determine its function in rats in which ACE2 function was disrupted.

General parameters and blood pressure determination

Basal and final body weights (BWs) were determined 1 day before minipump implantation and on the last day of treatment, respectively. The changes in BW in each group were expressed as a percentage of the weight gain during the treatments (Δ BW%). Systolic blood pressure (SBP) was measured using tail-cuff plethysmography (MRBP, IITC Life Science Inc., Los Angeles, CA, USA). The SBP was expressed as the percent difference between the SBP values at the beginning and end of the treatments (Δ SBP%). The basal SBP was measured 3 days before minipump implantation. The measurement of the final blood pressure was performed on Day 12 in subgroups for in vivo studies or Day 13 in subgroups for in vitro studies. Five measurements were performed on each rat, and SBP was calculated as the average of the last four measurements.

In vivo studiesEchocardiography

Transthoracic echocardiography was performed on eight animals per group (the in vivo subgroup of the experiment) at the end of the experiment using a 14-MHz matrix probe (M12 L) coupled with a GE Medical Vivid 7 Dimension System (GE Medical Systems CZ Ltd., Prague, Czech Republic), as described previously [22, 23]. For general anesthesia, a 2.5% inspiratory concentration of isoflurane at a flow rate of 2 L/min during spontaneous breathing was used throughout the protocol. The rat was placed in the supine position on a warming pad (38 °C), and the transthoracic wall was shaved. The body temperature and heart rate were continuously monitored. To assess the systolic function of the left ventricle (LV), the LV end-systolic and end-diastolic internal diameters were measured from the anatomical M-mode images in a long-axis view using the leading-edge method. The left ventricular fractional shortening (LVFS) and ejection fraction (LVEF, using the Teichholz formula) were subsequently determined. To assess the diastolic function of the LV, the diastolic transmitral peak early (E) and late (A) filling velocities were measured from the two-dimensionally guided Doppler spectra of mitral inflow in the apical four-chamber view, and the E/A ratio was subsequently calculated. Tissue Doppler imaging from the apical four-chamber view was used to determine the maximal velocities of the early (Em) and late (Am) diastolic wall movement waves at the level of the septal mitral annulus, and the E/Em ratio was subsequently calculated. Echocardiographic assessment of LV function was performed by an experienced blinded echocardiographer. Measurements were averaged over three consecutive cardiac cycles.

Integrated blood pressure response

The in vivo measurement of blood pressure was performed under inhalation anesthesia using isoflurane (2.5%, O2-1.5 L/min), which was continually maintained during the experiment, the body temperature was constantly kept at 38 °C by a warming pad. For the application of vasoactive substances, the right jugular vein was cannulated, and heparin sulfate (25 IU, 100 µL) was administered to prevent coagulation. Then, a pressure sensor connected to a pressure transducer (FOP-LS-PT9-10, FISO Technologies, Quebec, Canada) was placed in the right carotid artery. Later, the basal BP was left to stabilize within 15–20 min, and the vasoactive compounds dissolved in 100 µL saline were administered into the jugular vein over a 10 s period. The integrated pressure responses were induced by noradrenaline (NA, 1 µg/kg; agonist of adrenergic receptors; Zentiva, Prague, Czech Republic), acetylcholine (Ach, 1 µg/kg; agonist of muscarinic receptors), captopril (10 mg/kg; angiotensin-converting enzyme inhibitor), bismuth(III) subsalicylate (BSC, 0.25 µg/kg; hydrogen sulfide scavenger), and NG-nitro-L-arginine methylester (L-NAME, 30 mg/kg; NO synthase inhibitor). The responses of blood pressure were expressed as the mean arterial BP (MAP). The changes in MAP (ΔMAP in mmHg) stimulated by Ach and NA were measured as the difference between values of maximal response after the addition of the compound (blood pressure increase or decrease) and basal MAP (immediately before the addition of the compound). The responses stimulated by captopril, BSC, and L-NAME, which generated long-lasting responses, were evaluated 10 min after their injection. The response to Ach (1 µg/kg) was realized again after a 15 min pretreatment with L-NAME (30 mg/kg). Stock solutions of all compounds were prepared freshly on the day of the experiments and used within a few hours. The recorded analog signal was digitalized, and DEWESoft 6.6.7 software (DEWETRON, Prague, Czech Republic) was used for data collection and further analysis.

Ex vivo studies

On Day 14 of the treatment, the rats were sacrificed by decapitation after brief CO2 anesthesia. Trunk blood was collected into preprepared heparinized tubes (140 UI/5 mL) and then centrifuged (850× g, 10 min, 4 °C, Centrifuge 5430 R, Eppendorf, Hamburg, Germany). Plasma was aliquoted and stored at -80 °C until the analysis of H2S and angiotensins. Tissue samples (heart, visceral fat - retroperitoneal plus epididymal fat, aortic tissue) were weighed, rapidly frozen in liquid nitrogen and stored at -80 °C for further analyses. The thoracic aorta (TA) was isolated for the in vitro functional study, which was performed on the day of sacrifice.

Determination of H2S concentration in plasma and heart tissue

The H2S concentration in plasma and heart tissue was measured using a methylene blue assay as described in detail previously [24, 25]. Briefly, 75 µL of the plasma samples was added to 0.1 mol/L potassium phosphate buffer (325 µL), and then this mixture was combined with reaction buffer (total volume 500 µL) containing pyridoxal-5-phosphate and L-cysteine. The heart tissue was homogenized with lysis buffer including sodium orthovanadate and protease inhibitor. The protein concentration was determined by the Lowry method. The absorbance was measured at 650 nm via a spectrophotometer (NanoDrop™ 2000/2000c Spectrophotometers, Thermo Fisher Scientific, Waltham, MA USA). All standards and samples were assayed in duplicate using a 96-well plate. The H2S concentration was calculated against a calibration curve of NaHS (3.9–250 µmol/L). The results of plasma H2S concentration are given in µmol/L, and the results of heart tissue are determined as nmol/g of protein.

Analysis of angiotensins

The equilibrium concentration of angiotensin (1–10) (Ang I), angiotensin (1–8) (Ang II), angiotensin (1–7), and angiotensin (1–5) were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in heparinized plasma samples as described previously [26]. The basis of the ex vivo analysis is to determine the established equilibrium concentration between angiotensin peptide formation and elimination, and it considers all plasma RAS soluble factors of angiotensin production/elimination in the condition of RAS activity. Therefore, the total alternative RAS activity can be calculated relying on this analysis as a ratio of (Ang 1–7 + Ang 1–5)/(Ang I + Ang II + Ang 1–7 + Ang 1–5) as well as ACE activity as a ratio of Ang II/Ang I. The renin activity is expressed as sum of Ang I and Ang II concentrations.

Vasoactive responses of thoracic aorta

The segment of thoracic aorta (TA) was removed from the intact aorta beginning 5 mm below the aortic arch and ending before a diaphragm and cut into 5 mm length rings. Setting such a length of the specimen is suitable for the used type of tissue holder and allows optimal and reproducible vasoactive responses to be recorded. The TA was carefully cleaned, its connective and adipose tissue was removed while avoiding impairment of the endothelium. Then, TA was placed vertically fixed between two stainless steel wire triangles, and inserted into a 20-mL organ bath with Krebs solution (oxygenated with 95% O2 and 5% CO2, 37 °C) containing 118 mmol/L NaCl, 5 mmol/L KCl, 25 mmol/L NaHCO3, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 2.5 mmol/L CaCl2, 11 mmol/L glucose, and 0.032 mmol/L Ca-Na2EDTA. The bottom triangles were fixed, and the upper triangles were connected with a sensor of isometric tension (FSG-01, MDE GmbH, Budapest, Hungary), thereby recording the vasoactive changes via an NI USB-6221 AD converter (MDE GmbH, Budapest, Hungary) and S.P.E.L. Advanced Kymograph software (MDE GmbH, Budapest, Hungary). To set the optimal diameter of thoracic aorta segment close to the in vivo values, a series of preliminary experiments was carried out. Different values of resting pretension, from the lowest to the highest, were applied to rings of the thoracic aorta isolated from SHR and the rings were exposed to increasing concentrations of noradrenaline inducing a concentration-dependent contractile response. The value of resting tension (1 g) at which the largest contractile response was repeatedly recorded was applicated for arterial rings used in this study. A resting tension of 1 g was carried out on each TA ring to eliminate any nonspecific stress relaxation between 45 and 60 min until the arteries were stabilized.

Single concentrations of NA (10− 6 mol/L) and ACh (10− 5 mol/L) were added to test the integrity of the arterial wall (the contractile abilities and the integrity of the endothelium). The endothelium-dependent relaxant response was evaluated on the NA-precontracted (10− 6 mol/L) TA rings. Increasing concentrations of exogenous ACh (10− 10-10− 5 mol/L) were cumulatively applied after achieving a stable plateau. The ratio of vasorelaxation was determined as a percentage of the maximal contraction stimulated by NA.

Next, we examined the participation of certain signaling pathways in the vasoactive responses of the TA. To investigate the participation of Mas receptors in vasoactive responses, a selective Mas receptor inhibitor, A-799 trifluoroacetate salt, was used (10− 5 mol/L). To specify the role of the endogenous NO pathway, the rings of TA were incubated with a nonspecific inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME, 10− 5 mol/L). The H2S scavenger bismuth(III) subsalicylate (BSC, 10− 5 mol/L) was used to evaluate the participation of H2S in the vasoactive responses. To determine the effect on endothelium-derived relaxation, all mentioned compounds were acutely incubated for 20 min in the organ bath prior to the addition of NA (10− 6 mol/L) and ACh (10− 10-10− 5 mol/L).

Total NO synthase activity

Total NOS activity was evaluated in 10% of aorta homogenates and 20% of left ventricle homogenates by following the previous instructions [27]. The Quanta Smart Tri-Carb Liquid Scintillation Analyzer (TriCarb, Packard, UK) was used for measuring [3 H]-L-citrulline formation from [3 H]-L-arginine (MP Biochemicals, CA, USA). NOS activity was expressed as picokatal per gram of protein (pkat/g protein).

Superoxide production measurement

The production of superoxide was measured in samples of the left heart ventricle (~ 10–20 mg) and thoracic aorta (~ 5–15 mg, ring segments cleaned of surrounding adipose and connective tissue) using lucigenin-enhanced chemiluminescence. Freshly collected tissue samples (two samples from each tissue) were placed into Eppendorf test tubes with ice-cold modified Krebs-Henseleit (KH) solution (KH concentration in mmol/L: 119 NaCl, 4.7 KCl, 1.17 MgSO4·7H2O, 25 NaHCO3, 1.18 KH2PO4, 0.03 Na2 EDTA, 2.5 CaCl2·2H2O, 5.5 glucose). After all tissue samples were obtained, a freshly prepared stock solution of lucigenin (5 mmol/L, dissolved in ice-cold KH) was prepared. From a freshly prepared lucigenin stock solution, a lucigenin measuring solution was prepared by diluting the stock solution with pneumoxide-bubbled KH (5% CO2 and 95% O2, pH 7.4, temperature 37 °C) to a final concentration of 50 µmol/L. Samples were gradually placed into 1.5 mL Eppendorf tubes containing 1 mL of pneumoxide-bubbled KH solution, and preincubated in the dark at 37 °C for 18 min. At the same time, 1.5 mL Eppendorf test tubes containing 1 mL of lucigenin measuring solution were gradually preincubated under the same conditions.

After preincubation, the tissue samples were transferred into preincubated Eppendorf test tubes containing lucigenin measuring solution, and chemiluminescence was immediately measured in 10 s intervals for 3 min (18 measurements in total) using a GloMax® 20/20 Luminometer (Promega, Southampton, UK). Chemiluminescence was also measured in a blank sample containing lucigenin measurement solution alone. The average chemiluminescence value was calculated from the last 12 measurements for each tissue sample (as well as the blank sample) in doublets. Then, the chemiluminiscence of the blank was subtracted from the chemiluminescence of the tissue samples, and doublets were averaged to obtain the final chemiluminescence level. Superoxide production was expressed as relative chemiluminescence units per mg of tissue sample (RLU/mg).

RNA isolation and determination of gene expression

The gene expression levels of neuronal nitric oxide synthase (Nos1), inducible NOS (Nos2), endothelial NOS (Nos3), and 60 S ribosomal protein RPL10a (Rpl10a) in the aorta were determined by using two-step reverse transcription quantitative polymerase chain reaction (RT-qPCR). The total RNA of the aorta was isolated using the PureZOL™ RNA Isolation Reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. The amount and purity of total isolated RNA was spectrophotometrically quantified at 260/280 and 260/230 nm using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription was performed using 1 µg of total RNA from each sample using an Eppendorf Mastercycler (Hamburg, Germany) and an iScript-Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. Gene-specific primers were designed using the PubMed program (Primer-BLAST) and database (Gene). The DNA sequences and melting temperature of the primers used, the size of the amplicons and the reference numbers of the templates are described in Table 1. The PCRs were conducted in a final volume of 20 µL containing 2 µL of 5-fold diluted template cDNA, 10 µL SsoAdvanced mix (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad, Hercules, CA, USA), 1.5 µL of both forward and reverse primers (Metabion, Germany, 4 µmol/L), and 5 µL RNase free water (Sigma–Aldrich, Germany) in a final volume of 20 µL. The thermal cycling conditions were as follows: (1) 95 °C for 30 s, (2) 40 cycles consisting of (a) 95 °C for 10 s, and (b) an optimal annealing temperature (depending on the selected primer, see Table 1) for 20 s. Finally, melt curves for amplicon analyses were constructed at 60–95 °C, 5 s/1°C. RT‒qPCR was performed using a CFX96 Real-Time PCR (Bio-Rad, Hercules, CA, USA) detection system and evaluated by Bio-Rad CFX Manager software 2.0 (Bio-Rad, Hercules, CA, USA). The expression of each gene was determined in 8 rats. The quantities (Ct values) of target genes were normalized to the quantities (Ct) of the housekeeping gene (Rpl10a). Relative mRNA expression was calculated using the 2−ΔΔCt method. For Ace2 and Mas1, RNA (n = 7–12) was separated from deep-frozen abdominal aorta tissue samples (-80 °C) by application of a Minilys personal homogenizer (Bertin Technologies SAS, Montigny-le-Bretonneux, France) and a RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer´s protocol. Reverse transcription of isolated RNA was performed by a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) by following the manufacturer’s instructions. RT-qPCR was performed in FastStart Universal SYBR Green Master mix solution (Roche, Indianapolis, IN) and a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Rat-specific primer pairs are shown in Table 1. Data from mRNA levels were normalized to the expression of the housekeeping gene TATA box binding protein (Tbp), which was not changed by the treatment.

Table 1 Used primer pairs Western blotting

Protein expression levels of H2S producing enzymes - cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), ACE2, the Mas receptor, and all NOS isoforms were evaluated in the aorta and LV by Western blot analysis. First, tissue samples were homogenized and then centrifuged at G 17 608,5 and at 4 °C for 20 min. For homogenization, lysis buffer containing 0.05 mmol/L Tris and protease inhibitor cocktail was used. A Lowry assay was used to calculate the protein concentrations of the supernatant [28]. The equal amounts of protein (50 µg) were subjected to SDS-PAGE using 12% gels and transferred to nitrocellulose membranes. 5% nonfat milk in TBST (Tris-buffer solution, pH 7.6, containing 0.1% Tween-20) was used to block the membranes for 1 h at room temperature. After washing the membranes in TBST, the following primary antibodies were applied: rabbit polyclonal anti-endothelial NOS, anti-neuronal NOS, anti-GAPDH and anti-β-actin (Abcam, Cambridge, UK); rabbit polyclonal anti-inducible NOS (Bio-Rad, Inc., Hercules, CA, USA); rabbit polyclonal anti-Ang1-7, Mas receptor (Alomone Labs, Jerusalem, Israel); rabbit polyclonal anti-CBS and mouse monoclonal anti-CSE antibodies (Proteintech, Manchester, UK); and rabbit monoclonal anti-ACE2 (Invitrogen, Waltham, MA, USA) overnight at 4 °C. Primary antibody binding was determined using a secondary horseradish peroxidase-conjugated antirabbit antibody (Abcam, Cambridge, UK) or horseradish peroxidase-conjugated anti-mouse antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 2 h. An enhanced chemiluminescence system (ECL, Amersham, UK) was used for band intensity visualization. The ChemiDocTM Touch Imagine System (Image LabTM Touch software, Bio-Rad, Inc., Hercules, CA, USA) was used for quantification of the bands, with β-actin or GAPDH used as an internal loading control in aorta and LV, respectively.

Fluorescent staining of proteins

The distribution of the enzymes (CSE, CBS) was stained using 10 μm thick cryosections of TA (Tissue-Tek O.C.T. compound, Leica Biosystems, Deer Park, IL, USA). The sections were mounted onto SUPERFROST PLUS adhesion microscope slides (Epredia™, Fisher Scientific, Waltham, MA, USA) and fixed in 4% paraformaldehyde. Then, the slides were immersed in 0.1% Triton to permeabilize the membrane and blocked in 1% bovine serum albumin (BSA), both diluted with phosphate-buffered saline (PBS, pH = 7.4). The samples were incubated at 4 °C (overnight) with primary antibodies: mouse monoclonal CSE antibody and rabbit CBS polyclonal antibody (both diluted 1:100 in 1% BSA, Proteintech, Manchester, UK). The next day, after washing in PBS, the slides were incubated for 90 min in the dark with secondary antibodies: green FITC-fluorescein isothiocyanate for CBS (diluted: 1:2000, Abcam, Cambridge, UK) and red Alexa Fluor 532 for CSE (diluted 1:2000, Thermo Fisher Scientific, Waltham, MA, USA). To reduce the nonspecific signals (autofluorescence of the laminae in the media), staining with 0.25% sudan black B (in 70% isopropyl alcohol) was used. After 90 min of incubation in the dark, the slides were washed first with 70% isopropyl alcohol and 3x in PBS, and tissues were mounted in Vectashield antifade mounting medium containing 4´,6´- diamidino-2-phenylindole (DAPI) for staining of the nuclei (Vector Laboratories Inc., Burlingame, CA, USA). The samples were captured by confocal microscopy (NikonC4) with a Nikon APO-TIRF 60/1.49 oil objective and visualized by NIS-Elements software.

Statistical analysis

The group size was calculated by a priori analysis using G*Power software v3.1 [29] based on the expected effect of ACE inhibition on blood pressure, using the following values: effect size 0.55, α-error 0.05, power 0.85. Total sample size was calculated to 32 (n = 8/group). This number was increased to 40 because endothelial damage during in vitro experiment or death of the rat during the in vivo experiment may occur. Thus, the final number of rats in individual parameters was 8–10/group. The normality of the data was tested by the Shapiro-Wilks test. Three-way analysis of variance (ANOVA) for repeated measurements with the Bonferroni post hoc test was used to evaluate vasoactive responses. To evaluate cardiovascular and biochemical data, NO synthase activity, superoxide production, H2S concentrations, gene, and protein expression, and cardiac (systolic, diastolic) function, 2-way ANOVA was used. Data were analyzed using OriginPro (OriginLab Corporation, Northampton, MA, USA), GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) and Statistica 13.5 (StatSoft, Hamburg, Germany). The data are expressed as the mean ± S.E.M.

Drugs

All the chemicals used in this study were purchased from Merck (Bratislava, Slovakia) unless stated otherwise.