Cystathionine γ-lyase S-sulfhydrates SIRT1 to attenuate myocardial death in isoprenaline-induced heart failure

ABSTRACT Objective: Hydrogen sulfide (H2S), the third gasotransmitter, plays a critical role in protecting against heart failure. Sirtuin-1 (SIRT1) is a highly conserved histone deacetylase that has a protective role in the treatment of heart failure by regulating the deacetylation of some functional proteins. This study investigates the interaction between SIRT1 and H2S in heart failure and the underlying mechanisms. Methods and Results: Using endogenous H2S-generating enzyme cystathionine γ-lyase (CSE) knockout mice, we found that CSE deficiency aggravated isoprenaline-induced cardiac injury. Treatment with H2S attenuated atrial natriuretic peptide level, brain natriuretic peptide level, improved cardiac function. Moreover, H2S treatment potentiated myocardial SIRT1 expression. Silencing CSE abolished intracellular SIRT1 expression. Furthermore, CSE/ H2S S-sulfhydrated SIRT1 at its zinc finger domains and augmented its zinc ion binding activity to stabilize the alpha-helix structure. Discussion: In conclusion, these results uncover that a novel mechanism that CSE/H2S S-sulfhydrated SIRT1 to prevent heart dysfunction through modulating its activity.


Introduction
Heart failure is a major global health issue, affecting over 26 million people, but the pathologies of this complex syndrome remain unclear [1,2]. Heart failure may be caused by different etiologies; however, patients with heart failure or heart failurerelated disease have high mortality rates and poor quality of life. Statistics showed that the average duration of hospitalization for heart failure patients was 9-10 days, which brings a huge economic burden to society [3]. Mele et al. reported that cardiomyocyte apoptosis was one of the leading causes of heart failure [4]. Therefore, inhibition of cardiomyocyte apoptosis is important to protect against heart failure. Hydrogen sulfide (H 2 S) is the third gaseous mediator like carbon monoxide and nitric oxide, which can regulate cardiovascular functions [5,6]. The endogenous H 2 S is produced by three specific enzymes in mammalian cells: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) [5]. Some studies have shown that H 2 S has antioxidant properties [7], anti-atherosclerotic activity [8], pro-angiogenic effects [9], and vascular remodeling effects [10]. The decrease of H 2 S production in the cardiovascular system was observed in CSE knockout (CSE KO) mice, which caused more severe injury in different cardiovascular diseases, including heart failure [11] and myocardial infarction [12]. In contrast, CSE overexpression enhanced the endothelium-dependent vasorelaxation response in the thoracic aorta and abolished the myocardial reperfusion injury [13]. These findings indicate that the endogenous CSE/ H 2 S system has a cardiovascular protective effect.
Sirtuin-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide (NAD + )-dependent class III histone deacetylase that may play a crucial role in heart failure [14,15]. In mammals, SIRT1 is widely distributed in many tissues and organs and protects against apoptosis, oxidative stress, and ischemia/reperfusion injury [16]. Sirt1 mRNA and protein expression were significantly decreased, and the acetylation levels were increased in heart failure [17]. SIRT1 has a modulating effect on many target proteins, such as manganese superoxide dismutase (Mn-SOD), peroxisome proliferator-activated receptor-γ coactivator (PGC-1α), and Forkhead box O (FOXOs) through deacetylation [18]. Previous results suggested that SIRT1 protected against apoptosis through p53 and FoxO1 signaling pathways in cardiomyocytes [19]. H 2 S improved the survival of cardiomyocytes after H 2 O 2 -induced cell death through the SIRT1 signaling pathway [20].
The aim of this study was to determine whether H 2 S could increase SIRT1 activity to protected against heart failure and to unravel its potential role. Moreover, because H 2 S regulated the structure and biological function of the target protein [21] through S-sulfhydration the specific cysteine residue. Therefore, we set out to identify which cysteine residue in SIRT1 that may make a contribution in protective effect of H 2 S.

Cell culture and animals
H9c2 cardiomyocytes were purchased from American Type Culture Collection and were cultured in high glucose Dulbecco's minimum essential medium (DMEM), containing 100 U/ ml penicillin-streptomycin, 10% fetal bovine serum (FBS) in 5% CO 2 at 37°C. H 2 O 2 (150 μmol/L) was used to induce oxidative stress as described before [20].
Healthy male C57BL/6 (six to eight weeks old) mice were housed in plastic cages and maintained at 23°C and 12 h light/dark cycle (lights on from 7:00 to 19:00). Regular chow and water were available ad libitum. All experimental procedures followed the Shanghai Tongji Hospital Guideline for Animal Use (ethical code: SYXK(Hu) 2019-0005). The CSE knockout (CSE KO) mice (male, six to eight weeks) were a gift from Shanghai Research Center for Model Organisms, Shanghai, China and has been published before [22]. The model of heart failure was induced by subcutaneous injection of isoprenaline (ISO, 7.5 mg/kg) for four weeks once a day as described before [11].
In study-1, wild type (WT) and CSE KO mice were allotted into four groups (n = 10 in each group) as below: Group I -WT mice injected subcutaneously with normal saline; Group II -WT mice injected subcutaneously with ISO; Group III -CSE KO mice injected subcutaneously with normal saline; Group IV -CSE KO mice injected subcutaneously with ISO.
In study-2, WT mice were allotted into three groups (n = 10 in each group) as below: Group Iinjected subcutaneously with normal saline; Group IIinjected subcutaneously with ISO; Group IIIinjected subcutaneously with ISO and injected intraperitoneally with H 2 S donor sodium hydrosulfide (NaHS) (56 μmol/kg/day) as described previously [11].
After four weeks, animals were euthanized with an intraperitoneal injection of pentobarbital (0.1 g/kg), and then hearts as well as serum were harvested for further studies.
All drugs were obtained from Sigma except for the above drugs.

Western blot
The protein was quantified by the bicinchoninic acid reagent and then mixed with 4× loading buffer at 95°C for 10 min to denature. The target proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Ponceau S was used to observe the target band to facilitate subsequent operations, and then use 1× tris-buffered saline with tween-20 to wash the ponceau staining buffer. The membranes were blocked with 5% non-fat milk containing 0.1% tween-20 at room temperature for 1 h and then incubated with primary antibody at 4°C overnight. After washing, chemiluminescence was used to detect the membranes which were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature.

S-sulfhydration assay
The cells homogenized in HEN buffer (containing 1 mmol/L ethylenediaminetetraacetic acid, 250 mmol/L HEPES (4-(2hydroxyethyl) piperazine-1-ethane sulfonic acid) (pH 7.7) and 0.1 mmol/L neocuproine) were centrifuged at 12,000 r/min for 30 min at 4°C. The proteins were precipitated for 20 min at −20°C, centrifuged, and the supernatant were discarded. The pellet was resuspended in HENS buffer, and then the biotin-HPDP was added to the suspension for 3 h at room temperature. The obtained biotinylated proteins were separated and precipitated by protein A/G magnetic beads, and then the complex was washed with HENS buffer. The proteins were eluted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and analyzed by Western blot.

Echocardiography and cardiac parameters
The echocardiography of mice was performed by Vevo 770 with an RMV 707B high-frequency probe after the last isoproterenol administration. The mice were anesthetized by isoflurane and immobilized on a wooden board in a supine position. The ejection fraction (EF), fraction shortening (FS), left ventricular internal diameter-diastole (LVIDd) and left ventricular end-diastolic volume (LV Vold) were calculated from LV M-mode ultrasonography.

H 2 s concentration analysis
Plasma H 2 S levels were analyzed by reverse high-performance liquid chromatography system. Ten milliliters Tris-HCl (200 mmol/L, pH 8.5) and 70 μl methyl bromide (3.5 mmol/ L) were added into the 30 μl lysed myocardial tissue, and then the mixed solution was shaken on a shaker for 1 h at room temperature.

Picrosirius Red staining
The hearts were isolated and fixed in 4% paraformaldehyde solution and then embedded in paraffin. The paraffinembedded heart sections were cut into 5 µm thickness slices and stained with Picrosirius Red staining solution according to the operation manual. Quantitative histological analysis of myocardial fibrosis was performed using the Image J software.

Plasmid construction
The cDNA of human SIRT1 gene contains 747 amino acids (NCBI number NM_012238.5). The positions of the cysteine residues including C371, C374, C395 and C398 are according to the 747 as human SIRT1 variant (NCBI number NM_012238.5). The primers for site-directed mutagenesis of SIRT1 are as follows:

Statistical analysis
All experimental results are expressed as mean ± SEM. Statistical analyses used GraphPad Prism 7.02 software. Shapiro-Wilk normality test was performed to determine the data distribution. Non-normally distributed data were analyzed by nonparametric tests: Kruskal-Wallis test followed by Dunn's post hoc analysis for >2 groups or Mann-Whitney test for two groups. Normally distributed data were analyzed by using parametric tests: one-way analysis of variance (ANOVA) followed by Tukey test for >2 groups or unpaired Student's t-test for two groups. Multiple testing adjustment was included in the Dunn's test and Tukey test through controlling the Type I error for the family of comparisons. Experiments consisting of two categorically independent variables and one dependent variable were analyzed by two-way ANOVA. A P-value <.05 was considered statistically significant.

CSE/H 2 S played a critical role in cardiac function
To investigate whether CSE proteins can regulate cardiac function, we used CSE KO mice to perform experiments. We found that CSE protein expression, as well as H 2 S concentration, were significantly decreased in CSE KO mice compared with WT mice (Figure 1(A,B)). Compared with the control group, ISO significantly increased the levels of ANP mRNA (Accession: AK147180.1) and BNP mRNA (Accession: D82049.1) in the heart, GAPDH mRNA (Accession: AY618568.1) was used as loading control. The treatment of ISO led to higher ratios of left ventricular weight/body weight (LVW/BW) and heart weight/body weight (HW/BW), and increased mortality. Meanwhile, in the ISO-treated group, ANP mRNA levels, BNP mRNA levels, LVW/BW, and HW/BW were increased in CSE KO mice (Figue 1(C-F)). There was no death in WT and CSE KO group. In WT + ISO group, there were one mouse and two mice died in first and second days, respectively. In CSE KO + ISO group, two mice died on the second and third days respectively ( Figure  1(G)). Picrosirius Red staining showed an increase of myocardial fibrosis in ISO-treated group, which were severe in CSE KO mice (Figure 1(H)). The above results indicated that CSE protein has a role in regulating cardiac function.

H 2 S protected against the ISO-induced heart failure
NaHS acts as an exogenous H 2 S donor and could increase H 2 S concentration significantly in mouse hearts (Figure 2(A)). As shown by the Kaplan-Meier survival plot (Figure 2(B)), NaHS improved the survival rate of mice in ISO-induced heart failure. The HW/BW and LVW/BW were decreased in the NaHS + ISO group compared with the ISO group (Figure 2(C,D)). Furthermore, treatment of NaHS could inhibit ISO-induced elevation of hypertrophic markers (ANP and BNP) (Figure 2(E,  F)). The results of the ultrasonic electrocardiogram indicated that ISO treatment decreased ejection fraction and fractional shortening (Figure 2(G,H)). In contrast, ISO treatment increased left ventricular internal dimension diastole and left ventricular end-diastolic volume (Figure 2(I,J)). ISO treatment also increased myocardial fibrosis (Figure 2(K)). However, NaHS could decrease mortality rate, decrease ANP and BNP mRNA level, improve cardiac function, decrease myocardial fibrosis against ISOinduced abolished myocardial damage caused by ISO. These data suggested that H 2 S was protective against heart failure.

Endogenous CSE/H 2 S activated SIRT1/PGC-1α pathway
ISO could induce oxidative stress and apoptosis in heart. Indeed, a decrease activity of the antioxidant enzyme superoxide dismutase (SOD), an increase of malondialdehyde (MDA) content, as well as an increase of caspase 9 activity and cleaved caspase 9 level were noted in ISO-treated heart (Figure 3(A-D)), which could be attenuated by NaHS treatment. The treatment with NaHS significantly upregulated the levels of SIRT1 and PGC-1α in ISO-induced heart failure mice (Figure 3(D)). The protective function of SIRT1 is due to deacetylate its substrates and transcription factors [24]. We found that treatment with ISO increased acetylated protein levels, but NaHS could inhibit the effect of ISO (Figure 3(E)). PGC-1α plays an important role in mitochondrial biogenesis and function [25]. It was reported that PGC-1α was one of the target proteins of SIRT1 [26]. Compared with the ISO group, NaHS significantly decreased the level of acetylated PGC-1α (Figure 3(F)).
Upon ISO treatment, SIRT1 and PGC-1α expression levels in CSE KO mice were significantly lower than that in WT mice ( Figure 4). Moreover, the cleaved caspase 9 levels in ISO + CSE KO group decreased significantly different compared with the ISO + WT group (Figure 4). These results suggested that the endogenous CSE/H 2 S regulated the deacetylation activity of SIRT1.

H 2 S-sulfhydrated SIRT1 at two CXXC zinc finger domains
Cell viability was reduced in the treatment of H 2 O 2 , which could be increased H 2 S treatment ( Figure 5(A)). After H 2 S treatment, increased SOD activity, decreased MDA content and caspase 9 activity were observed in H 2 O 2 -treated H9c2 cardiomyocytes ( Figure 5(B-D)). These findings indicated that H 2 S had a marked effect on oxidative stress and apoptosis. To investigate the S-sulfhydration by H 2 S on SIRT1 activity, we determined the expressions of S-sulfhydration of SIRT1 (SHY-SIRT1) by immunoprecipitation-linked biotin switch assay. NaHS increased the expression of SHY-SIRT1 in heart failure ( Figure 5(E)). Furthermore, the SHY-SIRT1 was upregulated by NaHS treatment. To further demonstrate SIRT1 was Ssulfhydrated by H 2 S, we used de-sulfhydration reagent dithiothreitol (DTT, 1 mmol/L for 1 h) and found that DTT attenuated SHY-SIRT1 level in the H 2 O 2 -induced H9c2 cardiomyocytes ( Figure 5(F)). Cycloheximide (CHX, 25 µg/mL for 24 h) was used to inhibit protein synthesis. NaHS treatment inhibited the effect of CHX on the SIRT1 protein level ( Figure 5(G)). Therefore, S-sulfhydration could stabilize SIRT1 and prevent its degradation.
To explore the S-sulfhydrated cysteine residue sites, the mutated plasmids of domain a (Ma: C371A and C374A), domain b (Mb: C395A and C398A), or both domains (Ma + b: C371A, C374A, C395A, and C398A) were transfected into HEK-293 cells. We found that the SHY-SIRT1 levels were significantly decreased in the SIRT1 mutation proteins ( Figure  6(A)). Furthermore, SIRT1 mutations significantly increased the levels of acetylated protein (Figure 6(B)) and ac-PGC-1α ( Figure 6(C)). SIRT1 mutations increased the level of cleaved caspase 9 (Figure 6(D)). SIRT1 has a conserved zinc finger motif, which binds to Zn 2+ to enhance the structural integrity and deacetylation activity of SIRT1. These data suggested that H 2 S S-sulfhydrated SIRT1 at the two CXXC domains to increase Zn 2+ binding to SIRT1, augmented the stability of SIRT1, and abrogated oxidative stress-induced degradation of SIRT1.

Discussion
The growing evidence indicates that H 2 S protected against heart failure, but the mechanism remains still unclear [19]. In this study, we found that the severity of heart failure was much higher in CSE KO mice than in wild-type mice, suggesting that CSE-mediated H 2 S production was important in the cardiac system. Moreover, H 2 S donor could increase SIRT1 expression and its deacetylation activity through S-sulfhydrating at zinc finger domains.
CSE/H 2 S pathway has been proved to play an important role in physiology and pathophysiology [27,28]. CSE KO mice showed a significant decrease in H 2 S bioavailability [29], and worsened cardiac dysfunction and dilatation in heart failure [30]. In our study, the concentration of H 2 S was decreased, and the hypertrophic markers (BNP and ANP) were elevated in CSE KO mice. We also found that H 2 S improved cardiac function and prevented cardiac hypertrophy in ISO-induced heart failure. Endogenous H 2 S production via CSE had a cardioprotective effect on heart failure.
It was reported that H 2 S mediated the expression of SIRT1 and its activity [31]. We previously showed that H 2 S increased SIRT1 protein levels to protect against oxidative stress. Moreover, SIRT1 could be regulated by H 2 S to protect against ischemia/reperfusion injury in rats [32]. In this study, the expression of SIRT1 was downregulated by ISO, and NaHS could upregulate its level. However, the SIRT1 level was decreased more in CSE KO mice compared with wild-type mice in ISO-induced heart failure. PGC-1α is an important regulator of mitochondrial biogenesis, which could maintain cellular energy homeostasis [33]. SIRT1 can deacetylation PGC-1α to regulate its activity. The present study showed that H 2 S significantly decreased ac-PGC-1α levels and cleaved caspase-9 in heart failure. However, the levels of ac-PGC-1α and cleaved caspase-9 were abrogated in CSE KO mice as compared to wild-type animals. These results indicated that the CSE/H 2 S upregulated SIRT1 protein level and increased the deacetylation activity of SIRT1. Therefore, H 2 S protected against heart failure via activating SIRT1.
Evidence showed that SIRT1 deacetylated its downstream targets (PGC-1α, p53, FOXOs, etc.) to regulate the oxidative stress, inflammation, and apoptosis process in ischemia/ hypoxia injury [34]. Here, we showed that treatment with H 2 S increased the deacetylation activity of SIRT1, but this effect was attenuated by DTT. Therefore, H 2 S improved cardiomyocyte function by S-sulfhydrating SIRT1. SIRT1 protein level and its deacetylase activity were regulated by posttranslational modifications such as S-nitrosylation, SUMOylation, phosphorylation, etc. [35] SUMOylation of SIRT1 at Lys 734 increased its deacetylase activity and stabilized its protein [36]. Unlike SUMOylation, SIRT1 was S-nitrosated at Cys 387 and Cys 390 to reduce its deacetylase activity [37]. Du et al. found S-sulfhydration, a novel post-translational modification, at two zinc finger domains of SIRT1 increased SIRT1 deacetylation activity and attenuated atherosclerotic Figure 4. Lack of H 2 S caused severe heart failure. Western blotting images showing the protein levels of SIRT1, PGC-1α and cleaved-caspase 9 protein levels in indicated mice heart tissues. Data represent means ± SEM, n = 6. *p < .05, **p < .01.   plaque formation [8]. At the same zinc finger domains, SIRT1 activity could be inhibited through S-glutathionylation by GSNO [38]. Zn 2+ binding to the two conserved CXXC domains of SIRT1 stabilized its alpha-helix structure [39]. In our study, we found H 2 S S-sulfhydrated SIRT1 at Cys371, Cys374, Cys395 and Cys398 sites to increase SIRT1 deacetylation activity in response to oxidative stress. PGC-1α was the master regulator of mitochondrial biogenesis [40]. Acetylation could alter the localization of PGC-1α in the nucleus, and then inhibit PGC-1α transcriptional activity [41]. Multiple studies have shown that the deacetylation of PGC-1α was dependent on SIRT1 activity [42][43][44]. In this study, the mutation of cysteine residues in the zinc finger domains inhibited S-sulfhydration of SIRT1 and increased ac-PGC-1α and cleaved caspase-9 expression. These data suggested that S-sulfhydration in the zinc finger motif of SIRT1 by H 2 S, elevated the deacetylation of SIRT1 target genes, and then attenuated H 2 O 2 -induced cell apoptosis and oxidative stress.
In summary, we demonstrated that H 2 S directly S-sulfhydrated SIRT1 at zinc figure domains to activate SIRT1 to protective against heart failure treatment with H 2 S may serve as a therapeutic approach to attenuate heart failure development in humans. It should be noted that we cannot preclude that whether additional cysteine residues at SIRT1 could be S-sulfhydrated by H 2 S to regulate cardiac function. And further experiment will hopefully answer these questions.