CTH/MPST double ablation results in enhanced vasorelaxation and reduced blood pressure via upregulation of the eNOS/sGC pathway

Hydrogen sulfide (H2S), a gasotransmitter with protective effects in the cardiovascular system, is endogenously generated by three main enzymatic pathways: cystathionine gamma lyase (CTH), cystathionine beta synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) enzymes. CTH and MPST are the predominant sources of H2S in the heart and blood vessels, exhibiting distinct effects in the cardiovascular system. To better understand the impact of H2S in cardiovascular homeostasis, we generated a double Cth/Mpst knockout (Cth/Mpst −/− ) mouse and characterized its cardiovascular phenotype. CTH/MPST-deficient mice were viable, fertile and exhibited no gross abnormalities. Lack of both CTH and MPST did not affect the levels of CBS and H2S-degrading enzymes in the heart and the aorta. Cth/Mpst −/− mice also exhibited reduced systolic, diastolic and mean arterial blood pressure, and presented normal left ventricular structure and fraction. Aortic ring relaxation in response to exogenously applied H2S was similar between the two genotypes. Interestingly, an enhanced endothelium-dependent relaxation to acetylcholine was observed in mice in which both enzymes were deleted. This paradoxical change was associated with upregulated levels of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) α1 and β1 subunits and increased NO-donor-induced vasorelaxation. Administration of a NOS-inhibitor, increased mean arterial blood pressure to a similar extent in wild-type and Cth/Mpst −/− mice. We conclude that chronic elimination of the two major H2S sources in the cardiovascular system, leads to an adaptive upregulation of eNOS/sGC signaling, revealing novel ways through which H2S affects the NO/cGMP pathway.

Several sources contribute to H 2 S levels in mammalian tissues. H 2 S can be generated by enzymatic and non-enzymatic reactions; additional H 2 S is released from the consumption of sulfurcontaining compounds that are present in the diet and by the gut microbiome (Shen et al., 2013;Kabil and Banerjee, 2014;Filipovic et al., 2018;Yang et al., 2019;Cirino et al., 2023). The main mammalian enzymes that are responsible for H 2 S production are two enzymes of the transulfuration pathway, cystathionine-γ lyase (CTH) and cystathionine-β synthase (CBS), along with 3mercaptopyruvate sulfurtransferase (MPST), an enzyme of a minor cysteine breakdown pathway (Kabil and Banerjee, 2014;Kimura, 2014;Cirino et al., 2023). The three enzymes use different substrates to generate H 2 S, and have distinct expression profiles and different subcellular distribution. While MPST is equally distributed between the cytosol and the mitochondria, CTH and CBS are predominantly cytosolic under physiological conditions (Fräsdorf et al., 2014;Cirino et al., 2023). It is well known that CTH and MPST are the major sources of H 2 S in the cardiovascular system; CTH and MPST are more abundantly present in both the heart and blood vessels of mice and humans compared to CBS (Peleli et al., 2020).
Although CTH and MPST exhibit some overlapping biological actions, they also exhibit distinct physiological functions. For example, Cth −/− mice are hypertensive from a young age and exhibit reduced endothelium-dependent relaxations, while Mpst −/− knockout mice have normal responses to vasodilators (Yang et al., 2008;Peleli et al., 2020). In contrast, both CTH and MPST are important for angiogenesis (Papapetropoulos et al., 2009;Coletta et al., 2015). In the heart CTH is cardioprotective; CTH knockout mice exhibited greater infarct sizes after ischemia-reperfusion and a worse phenotype in animal models of heart failure (Kondo et al., 2013;King et al., 2014). On the other hand, MPST knockout mice are protected from cardiac ischemia-perfusion injury, while they exhibit greater deterioration of left ventricular function in heart failure with reduced injection (Peleli et al., 2020;Li et al., 2022).
Given the importance of H 2 S in cardiovascular homeostasis and the importance of CTH and MPST in cardiovascular physiology and disease, we set out to generate and characterize mice lacking both H 2 S-generating enzymes. Surprisingly, the double knockout mice had lower mean arterial blood pressure and exhibited enhanced vasorelaxation due to increased endothelial NO synthase/soluble guanylate cyclase expression. Our findings unravel a novel mechanism of crosstalk between H 2 S and NO.

Mice
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.

Blood pressure measurements
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.

Evaluation of vascular function
Vascular reactivity was assessed by evaluation of phenylephrine-(PE), acetylcholine-(Ach), the NO donor, DEA-NONOate-and the H 2 S donor, NaHS-induced responses in isolated aortic rings. Mice were anaesthetized with enflurane (5%) and then killed in CO 2 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% O 2 -5% CO 2 ) Krebs' solution (NaCl 118 mM, KCl 4.7 mM, MgCl 2 1.2 mM, KH 2 PO 4 1.2 mM, CaCl 2 2.5 mM, NaHCO 3 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).

Statistical analysis
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.  Figure 1A). To determine if lack of the two H 2 S-producing enzymes leads to a compensatory increase in the remaining H 2 S-producing enzyme, we measured CBS levels. Lack of Cth and Mpst did not affect CBS expression. Similarly, no changes in the levels of the H 2 S 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 H 2 S concentration) was detected in aorta of Cth/Mpst −/− ( Figure 1C). Experiments to measure the levels of CBS and H 2 S-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).
Blood biochemistry of Cth/Mpst −/− mice 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).

Characterization of basic cardiovascular parameters in Cth/Mpst −/− mice
To evaluate the effect of simultaneous deletion of the two most prominent H 2 S-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).

Vascular responses in Cth/Mpst −/− mice
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 endotheliumindependent NO donor DEANONOate was also slightly enhanced in the double knockout mice ( Figure 6B), while responses to the H 2 S 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  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).

Inhibition of NO production restores blood pressure in Cth/Mpst −/− mice
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 H 2 S-producing enzymes under baseline conditions.

Discussion
The major findings of our study are that simultaneous global deletion of Cth and Mpst 1) does not have a substantial impact on cardiac physiology and architecture, 2) results in reduced diastolic and systolic arterial blood pressure, 3) leads to enhanced endothelium-dependent and endothelium-independent vasorelaxation and 4) is linked to an increase in protein levels of eNOS, sGC and PKG-I in the vessel wall.

FIGURE 2
Cth/Mpst double deletion does not affect the expression of CBS and sulfide-metabolism enzymes in heart. WT and Cth/Mpst −/− mice were sacrificied, proteins were extracted from heart tissues and enzymes leves were determined by western blot. Representative western blots and quantification of (A) MPST, CTH, CBS and (B) ETHE1, TST, SQRDL levels in heart. Protein expression is presented as ratio over WT group. Data were normalized to GAPDH and presented as means ± S.E.M. N = 6 mice per group. It should be noted that mice lacking both Cth and Mpst have been previously generated independently using a CRISPR/Cas9 approach (Akahoshi et al., 2020); however, the cardiovascular phenotype of these mice was not evaluated. The only measurements performed in this strain were basic serum biochemical analytes and amino acid levels, as well as serum, urine and liver levels of compounds related to the general cellular redox state. Serum levels of histidine, cystathionine and citrulline were increased in Cth/Mpst −/− animals. The increase in citrulline is in line with the increased expression of eNOS since citrulline is produced during the conversion of arginine to NO that is catalyzed by eNOS. In addition, lack of Cth leads to accumulation of the CTH substrate cystathionine; serum cystathionine levels have been proposed as a biomarker to assess the reduction in CTH activity that is associated with endothelial dysfunction (Bibli et al., 2019). Cth/Mpst −/− mice were also found to have increased serum homocystathionine and reduced cysteine levels, both of which are expected based on the catalytic activity of CTH (Kabil and Banerjee, 2014;Cirino et al., 2023). In line with the antioxidant properties of CTH and MPST (Nagahara, 2018;Cirino et al., 2023), markers of oxidative stress (oxidized glutathione, total glutathione and thiobarbituric acid-reactive substances) were increased in the serum and liver of Cth/Mpst −/− mice compared to wild-type control animals. The above observations confirm that lack of the two H 2 S-generation enzymes leads to a pro-oxidant environment in vivo.
Additional biochemical measurements in the serum of double knockout mice generated during the course of our study, revealed increased transaminase levels which is in agreement with the  Frontiers in Pharmacology frontiersin.org 06 observed increase in liver mass. With the exception of creatine kinase which exhibited a two-fold increase and triglycerides which showed a 50% reduction, the remaining analytes measured showed either no difference or minor changes in the range of approximately 10% that although statistically significant in some cases, are of little biological significance.  Hydrogen sulfide levels are determined by both the rate of its production, as well as its degradation rate. Oxidation is the main enzymatic pathway for sulfide elimination and occurs in the mitochondria in two steps (Murphy et al., 2019;Cirino et al., 2023). Sulfide is first oxidized by sulfide quinone oxidoreductase (SQRLD) giving rise to a persulfide. The persulfide is further oxidized to sulfite by persulfide dioxygenase (ETHE1). Sulfite is, in turn, converted to sulfate or thiosulfate by sulfite oxidase (SUOX) and rhodanese (also called thiosulfate transferase, TST), respectively. To evaluate possible compensatory changes in the levels of H 2 S-degrading enzymes in the double knockout mice, we assessed the expression of SQRLD, ETHE1 and TST. None of these was found to be altered in the aorta or in the heart. In agreement with our findings, hepatic TST levels were unchanged in the double knockout mice of the CRISP/Cas9-generated mouse line (Akahoshi et al., 2020). It should be noted that CBS levels were also unchanged in our Cth/Mpst −/− mouse line.
Cardiac function in Cth −/− and Mpst −/− has been shown to remain unaffected under baseline conditions (Donnarumma et al., 2017;Peleli et al., 2020;Cirino et al., 2023). To determine if simultaneous deletion of both Cth and Mpst results in alterations in cardiac physiology, Cth/Mpst −/− mice were subjected to echocardiography. The most notable feature of these mice that might have physiological significance was a modest decrease in heart rate; the borderline reduction in ejection is likely of minor biological importance. Although no changes in baseline cardiac performance have been documented in Cth −/− and Mpst −/− , both types of mice exhibit a more severe disease phenotype in heart failure and other cardiac pathologies (Kondo et al., 2013;King et al., 2014;Li et al., 2022). Additional studies would be required to evaluate whether double knockout mice exhibit an exacerbated form of cardiac dysfunction in disease models.

FIGURE 8
No differences in blood pressure between WT and double Cth/Mpst knockout mice after eNOS inhibition. WT and Cth/Mpst −/− mice were exposed to eNOS-inhibitor, L-NAME (0.5 g/L in drinking water) for 10 days and blood pressure was measured. (A) Systolic, (B) diastolic and (C) mean arterial blood pressure of WT and Cth/Mpst −/− mice after L-NAME administration. Data are presented as means ± S.E.M, N = 5 mice per group.
Frontiers in Pharmacology frontiersin.org 08 To further characterize the cardiovascular phenotype of Cth/ Mpst −/− mice, we measured arterial blood pressure in awake mice. Although the parental Cth −/− mouse strain used to generate the double knockout mice is hypertensive and the Mpst −/− normotensive (Yang et al., 2008;Peleli et al., 2020), mice carrying a double Cth/Mpst gene deletion have reduced systolic and diastolic blood pressure. This observation is in line with the reduced heart rate in these mice. Notably, when mice were given a NOS inhibitor, blood pressure of both wt and Cth/Mpst −/− increased to the same level, suggesting an involvement of NO in the hypotensive response observed in Cth/Mpst −/− animals. To determine the vascular reactivity of mice lacking both Cth and Mpst, we tested the response of aortic rings to dilating and constricting agents. In agreement to the reduced arterial blood pressure of double knockout mice, contractile responses to the α1 agonist phenylephrine were reduced in rings from Cth/Mpst −/− animals. Moreover, we noted significantly greater endotheliumdependent vasorelaxation to Ach and enhanced relaxation to an endothelium-independent NO donor. In contrast to the current observations, acute pharmacological inhibition of H 2 S production reduces endothelium-dependent relaxation and ablation of Cth only attenuates endothelium-dependent responses (Yang et al., 2008;Bucci et al., 2010;Xia et al., 2020). It should be noted that our tension measurements were performed in conductance, rather than resistance arteries, which would are important for determining peripheral vascular resistance and blood pressure.
The enhanced dilatory responses to Ach and DEANONOate correlated with increased expression of all of the components of the eNOS/cGMP pathway, namely eNOS, the α1 and β1 subunits of sGC and PKG1. As these changes are tissue-selective occurring only in the aorta (only sGC α1 was increased in the heart), so they are most likely not related to genetic alterations of the double knockout mice. It should be kept in mind that very few stimuli have been shown to increase sGC subunit expression and that to the best of our knowledge there is no known stimulus that can increase the expression of eNOS, sGC and PKG at the same time (Andreopoulos and Papapetropoulos, 2000). Interestingly, H 2 S has been shown to affect mRNA stability and to alter the rate of translation of selected transcripts (Lee et al., 2012;Bibli et al., 2019;Wang et al., 2019). Further experiments would be required to test the mechanism(s) through which lack of CTH and MPST in the vessel wall increases expression of components of the eNOS/cGMP pathway.
In summary, we report that double ablation of Cth and Mpst results in mice with reduced arterial blood pressure and enhanced responses to vasodilators. Interestingly, the majority of the literature points towards synergistic and/or mutually dependent effects of NO and H 2 S. For example, H 2 S inhibits phosphodiesterase 5 and shifts the sGC redox balance towards ferrous heme to increase its responsiveness to NO (Bucci et al., 2010;Zhou et al., 2016). Also, the angiogenic and cardioprotective responses to H 2 S donors are reduced in eNOS knockout mice, while vasodilation to H 2 S donors is reduced in mice lacking eNOS (Coletta et al., 2012;King et al., 2014;Bibli et al., 2015). Given the interdependence and complementarity in the actions of the two gasotransmitters in the vascular wall, upregulation of the NO arm in the face of complete blockade of the H 2 S production would be homeostatically beneficial. Whether this is of relevance to human pathophysiology remains to be investigated.

Data availability statement
The original contributions presented in the study are included in the article material, further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by the 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.