C57Bl/6J mice were purchased from the Jakson Laboratory. The CTH knockout (Cth ^−/− ) and MPST knockout (Mpst ^−/− ) mice have been previously described (Yang et al., 2008; Nagahara et al., 2013). All animals used for experimentation were bred/housed in individual ventilated cages, under specific pathogen-free, temperature controlled (22°C) and 12 h light/dark cycle conditions in full compliance with the guidelines of the Federation of Laboratory Animal Science Association recommendations in the Laboratory Animal Unit of Biomedical Research Foundation of the Academy of Athens (BRFAA) and allowed free access to diets and water. All studies were performed on male 8–12 week old mice. The lung and kidney from the right side of the experimental animals were used to determine the tissue weight. The left lateral lobe was used to determine the weight of the liver. All experimental procedures reported here were approved by the veterinary authority of the Prefecture of Athens, in accordance with the National Registration (Presidential Decree 56/2013) in harmonization with the European Directive 63/2010.

Tissues were lyophilized with mortar and pestle and then homogenized in lysis Buffer 150 mM NaCl (Calbiochem, 7760), 1% NP-40 (Sigma-Aldrich, 74,385), 0.5% Na-deoxycholate (AppliChem, A1531,0025), 0.1% SDS (PanReac AppliChem, A2572), 50 mM Tris-HCL, pH = 7.4 (Sigma-Aldrich, T1503), 2 mM EDTA (Merck, 4005) supplemented with a cocktail of protease (PI, Roche, 5,892,970,001) and phosphatase inhibitors (PhoI, Roche, 4906837001). Lysates were centrifuged (13.000 rpm, 15min, 4°C) and the protein concentration in the supernatants was quantified using the DC protein assay (BIO-RAD, 5000116). Concentration was normalized before western blot analysis. Samples were separated on 10% or 12% SDS–PAGE and transferred to a nitrocellulose membrane (Macherey-Nagel; Düren, Germany), after Laemmli buffer containing 4% SDS, 10% β-mercaptoethanol (Sigma-Aldrich, M6250), 20% glycerol (Melford, GI345), 0,004% blue bromophenol (AppliChem, A2331,0025) and 0,125M Tris-HCL, was added. The membranes were blocked [5% milk (PanReac AppliChem, A0830)] and probed with the following antibodies: anti-β-Αctin (Abcam, ab8227), anti-β-Τubulin (Abcam, ab15568), anti-GAPDH (Proteintech, 10494-1-AP), anti-CBS (Proteintech, 14787-1-AP), anti-CTH (Proteintech, 12217-1-AP), anti-MPST (Atlas Antibodies, HPA001240), anti-ETHE1 (Invitrogen, PA5-56040), anti-TST (Proteintech, 16311-1-AP), anti-SQRDL (Proteintech, 17256-1-AP), anti-eNOS (Cell signaling, 32027s), anti-peNOS_s1177 (Cell signaling, 9571) anti-PKG-I (Cell signaling, 32485s), anti-sGCβ1 (Cayman chemical, 160,897) and, anti-sGCα (Cayman chemical, 160,895). Immunoblots were next processed with anti-rabbit secondary antibody (Merck, AP132P) and visualized using the Western HRP substrate (Merck). Quantification of western blots was performed using ImageJ software (NIH Image, National Institutes of Health, United States).

The dimedone switch method was performed as previously described (Zivanovic et al., 2019). In brief, aortas were homogenized in Hens Buffer [50 mM Hepes, 1 mM EDTA, 2% SDS, 0.1 mM neucoproine (Cayman Chemical, 208,745)] supplemented with 1% PI and 20 mM 4-chloro-7-nitrobenzofurazan (NBF-CL, Merck, 10,199-89-0). Lysates were centrifuged (13.000 rpm, 15 min, 4°C) and supernatants were incubated at 37°C for 1 h. Samples were then precipitated by methanol/chloroform precipitation, organic and aqueous layers were aspirated and H₂O/MeOH/CHCl₃ was added to the protein pellets and centrifuged. Supernatants were aspirated again, and the pellets were washed (MeOH) and resuspended in 50 mM Hepes containing 1% SDS and 1% PI. Samples were incubated with 50 μΜ cysteine sulfenic acid probe, (DCP-Bio1, Merck, NS1266) for 1 h at 37 °C, precipitated with methanol/chloroform and resuspended in 50 mM Hepes containing 1% SDS and 1% PI. Detection of persulfhydated proteins was achieved using western blood method and a HRP-conjugated anti-biotin specific antibody (Cell Signaling, 5571).

Blood was collected from the orbital venous sinus of mice. Samples were next centrifuged (8,000 rpm, 8 min, 4°C) and serum was isolated. Serum biochemical parameters (alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), α-amylase, creatinine, urea, uric acid, albumin, transferrin, ferritin, total-bilirubin, direct-bilirubin, glucose, cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol and triglycerides of WT and Cth/Mpst ^−/− mice were measured.

Blood pressure was measured with the non-invasive plethysmography tail-cuff method (Kent Scientific, Torrington, CT, United States). Baseline blood pressure was measured in WT and Cth/Mpst ^−/− mice for 3 days before actually beginning the formal measurements. This is the established training period that allows the mice to acclimatize with the technique and eliminate any stress response. Once, confirmed that all mice showed no signs of stress response, measurements for 2 consecutive days were performed and averaged for the calculation of mean, systolic (SBP) and diastolic blood pressure (DBP); mean arterial blood pressure (MABP) was computed using the equation MABP=(SBP+2DBP)/3. Inhibition of nitric oxide synthase was achieved using N _ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, N5751, Merck). L-NAME was added in drinking water at a concentration of 0.5 g/L for 10 days.

WT and double Cth/Mpst knockout mice were anaesthetized using ketamine at a dose of 100 mg/kg by intraperitoneal injection (i.p.) and echocardiographic assessment of left ventricular (LV) function was performed using an ultrasound system (Vivid 7; GE Healthcare) with a 13-MHz linear transducer. Parameters such as heart rate (HR), left ventricular (LV) end-diastolic and end-systolic diameter (LV EDD, LV ESD), LV posterior wall thickness at diastole and systole (PWd, PWs), fractional shortening [FS % = (EDD -ESD)/EDD * 100], ejection fraction [EF% = [(LVEDD³-LVESD³)/LVEDD³]*100] were calculated LV radius to LV posterior wall thickness ratio (r/h) were calculated.

Vascular reactivity was assessed by evaluation of phenylephrine- (PE), acetylcholine- (Ach), the NO donor, DEA-NONOate- and the H₂S donor, NaHS- induced responses in isolated aortic rings. Mice were anaesthetized with enflurane (5%) and then killed in CO₂ chamber (70%). The thoracic aorta was rapidly harvested and adherent connective and fat tissue were removed. Aorta was cut in rings of 1–1.5 mm in length and placed in organ baths (3.0 mL) filled with oxygenated (95% O₂–5% CO₂) Krebs’ solution (NaCl 118 mM, KCl 4.7 mM, MgCl₂ 1.2 mM, KH₂PO₄ 1.2 mM, CaCl₂ 2.5 mM, NaHCO₃ 25 mM and glucose 10.1 mM) and kept at 37°C. The rings were connected to an isometric transducer (Fort 25, World Precision Instruments, 2Biological Instruments, Varese, Italy) associated to PowerLab 8/35 (World Precision Instruments, Biological Instruments, Varese, Italy). The optimal resting tension applied has been previously determined for each mouse strain. The rings were initially stretched until a resting tension of 1.0 g and then were allowed to equilibrate for at least 30 min. During this period, when necessary, the tension was adjusted to 1.0 g, and the bath solution was periodically changed (Mitidieri et al., 2018; Mitidieri et al., 2021). In each set of experiments, rings were firstly challenged with PE (1 μM; Sigma-Aldrich, P16126) until the responses were reproducible. Then PE cumulative concentration-response curve was performed (1 nM–3 µM). In a separate set of experiments, the rings were contracted with PE (1 μM) and, once a plateau was reached, a cumulative concentration-response curve of the following drugs was performed: Acetylcholine (10 nM–30 µM, Sigma-Aldrich, A9101), DEANONOate (10 nM–30 μM, Sigma-Aldrich, D184), N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO; Sigma-Aldrich I134) and sodium hydrosulfide NaHS (10 nM–3 mM, Sigma-Aldrich, 161,527).

Data are presented as means ± S.E.M. Differences were analyzed using two-tailed unpaired Student’s t-test for comparisons between two-groups. For vascular relaxation studies, differences were analyzed using two-way ANOVA, followed by Bonferroni post hoc test. All statistical calculations were made using Graphpad Prism statistical software. Sample sizes are reported in all figure captions. p was considered significant when it was less than 0.05.

Mice lacking both Cth and Mpst were generated by crossing Cth ^−/− and Mpst ^−/− mice to homozygosity. Lack of CTH and MPST was confirmed in the aorta of Cth/Mpst ^−/− animals at the protein level (Figure 1A). To determine if lack of the two H₂S-producing enzymes leads to a compensatory increase in the remaining H₂S-producing enzyme, we measured CBS levels. Lack of Cth and Mpst did not affect CBS expression. Similarly, no changes in the levels of the H₂S degrading enzymes ethylmalonic encephalopathy 1 protein (ETHE1), thiosulfate sulfurtransferase (TST) and sulfide quinone reductase (SQRLD) were evident in aortic lysates of double knockout mice (Figure 1B). In line with the attenuated CTH and MPST levels, a reduction in the persulfidation of proteins (a footprint of H₂S concentration) was detected in aorta of Cth/Mpst ^−/− (Figure 1C). Experiments to measure the levels of CBS and H₂S-degrading enzymes in the heart revealed that no major changes were noted in this tissue either (Figures 2A, B). As has been reported before (Fu et al., 2012), CTH was not detectable in the hearts of wild-type mice at the protein level. Body weight, as well as heart and lung weight did not differ between the two strains of mice, while we observed an increase in the kidney and liver mass of double knockout animals (Table 1).

We next assessed basic biochemical parameters in the serum of the new mouse strain. Alkaline phosphatase (ALT) and aspartate aminotransferase (AST) were increased in double knockout mice, in line with their grater liver weight observed (Figure 3A). Similarly, double knockout mice had higher serum creatine kinase activity (Figure 3B) and marginally lower creatinine, urea and uric acid levels (Figure 3C). Although these reductions were statistically significant, they were deemed to be of limited or no biological significance. Transferrin (Figure 3D), glucose and triglycerides (Figure 3F) were reduced. Levels of the remaining biochemical parameters tested including lipid levels, bilirubin, ferritin and albumin were not different between the two strains of mice (Figures 3C–F).

To evaluate the effect of simultaneous deletion of the two most prominent H₂S-producing enzymes in the cardiovascular system, blood pressure and cardiac structure and function were measured. Surprisingly, both systolic and diastolic (and therefore mean) arterial blood pressure were lower in double knockout mice (Figures 4A–C). Echocardiography measurements revealed marginal changes in cardiac parameters. Double knockout mice exhibited reduced heart rate (HR, Figure 5A), posterior wall thickness at diastole (PWTd) (Figure 5D), fractional shortening (FS, Figure 5F) and ejection fraction (EF, Figure 5G). The reductions in FS and in EF are too small to be of biological interest. All other parameters measured were similar between the two strains of mice (Figures 5B, C, E, H).

We next determined the vascular reactivity of aortic rings to vasodilators and vasoconstrictors. In contrast to what would be expected from the literature, but in line with a reduced blood pressure of double knockout mice, relaxation responses to the endothelium-dependent dilator acetylcholine where enhanced in the new mouse strain (Figure 6A). Relaxation to the endothelium-independent NO donor DEANONOate was also slightly enhanced in the double knockout mice (Figure 6B), while responses to the H₂S donor sodium hydrosulfide were not different between the two strains (Figure 6C). Moreover, phenylephrine caused smaller contractions in the aortic rings of Cth/Mpst ^−/− mice (Figure 6D). In another set of experiments, the selective eNOS inhibitor L-NIO (10 μM) was added on PE-precontracted aortic rings (300 nM) of both WT and Mpst/Cth^−/− mice. After a 20 min incubation, such treatment resulted in a greater increase in tension in Mpst/Cth^−/− indicating an enhanced NO production (Figure 6E). To study the mechanism responsible for the enhanced relaxation seen in the double knockout mice, we determined the expression of endothelial nitric oxide synthase (eNOS), soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). Both the α1 and the β1 sGC subunit, as well as eNOS, peNOS_s1177 and PKG-Ι were more abundant in the aorta of Cth/Mpst ^−/− mice at the protein level (Figure 7A). In contrast, only sGCα1 was increased in the hearts of double knockout mice (Figure 7B).

To evaluate the contribution of NO to the reduced blood pressure in vivo, we administered the NOS inhibitor L-NAME to mice for 10 days (Figure 8). This treatment lead to elevated blood pressure in both strains of mice; systolic, diastolic and mean arterial blood pressures were similar in wild-type and Cth/Mpst ^−/− mice after L-NAME treatment. These findings suggests that the NO/cGMP pathway is responsible for the lower blood pressure observed in mice lacking both H₂S-producing enzymes under baseline conditions.