Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter exerting various physiological effects, especially in the cardiovascular system. Herein we investigated the cardioprotective effects of a novel long-term and slow-releasing H2S donor, DATS-MSN, using in vivo myocardial ischemia/reperfusion (I/R) models and in vitro hypoxia/reoxygenation cardiomyocyte models. Unlike the instant-releasing pattern of sodium hydrosulphide (NaHS), the release of H2S from DATS-MSN was quite slow and continuous both in the cell culture medium and in rat plasma (elevated H2S concentrations during 24 h and 72 h reperfusion). Correspondingly, DATS-MSN demonstrated superior cardioprotective effects over NaHS in I/R models, which were associated with greater survival rates, reduced CK-MB and troponin I levels, decreased cardiomyocyte apoptosis index, increased antioxidant enzyme activities, inhibited myocardial inflammation, greater reduction in the infarct area and preserved cardiac ejection fraction. Some of these effects of DATS-MSN were also found to be superior to classic slow-releasing H2S donor, GYY4137. In in vitro experiments, cardiomyocytes injury was also found to be relived with the use of DATS-MSN compared to NaHS after the hypoxia/reoxygenation processes. The present work provides a novel long-term and slow-releasing H2S donor and an insight into how the release patterns of H2S donors affect its physiological functionality.
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Introduction
Hydrogen sulfide (H2S) is a novel gasotransmitter that can exert various physiological and pathophysiological effects, particularly in the cardiovascular system. Increasing evidences suggest that the production of endogenous H2S or the administration of exogenous H2S can successfully attenuate myocardial infarction (MI) following ischemia and reperfusion (I/R) injury. Sodium hydrosulphide (NaHS), the most commonly used H2S donor, can reduce the myocardial infarct size and preserve left ventricular (LV) function following I/R injury in both preconditioning or postconditioning experiments1, 2. However, the instant release of H2S from NaHS cannot mimic the slow and continuous process of H2S generation in vivo 3. The rapid production and loss of H2S may also lead to imprecise experimental results and detrimental effects in the body (e.g., hypotension and neurotoxicity)4. Furthermore, the unstable features of NaHS in an aqueous solution make it difficult to be reformulated by drug carriers, such as nanoparticles. Therefore, many other long-term release compounds have emerged as H2S donors, including morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137)3, cysteine analogs5, dithiolethione and its chimeras6, and cysteine-activated H2S donors7. However, the release processes of these H2S donors are hardly regulated, and the H2S concentrations are relatively low both in vitro and in vivo. Recently, a natural garlic-derived polysulfide compound–diallyl trisulfide (DATS) has drawn increased attention as a potential H2S donor, regarding its ability to generate H2S in the presence of reduced glutathione (GSH) both in red blood cells and phosphate buffers (PBS)8. Furthermore, the beneficial effects of DATS for cardiovascular diseases are also shown to be derived from H2S9. However, the poor solubility of DATS in aqueous media limits its use as an H2S donor, and its H2S release still remains relatively rapid after the addition of GSH10.
Recently, by exploiting mesoporous silica nanoparticles (MSN) as the carrier of DATS, a novel GSH-activated, water-dispersible, slow and controllable releasing H2S system (DATS-MSN) was synthesized in our laboratory (Supporting Information, Fig. S1). Unlike NaHS, DATS is quite stable in an aqueous solution with minimal spontaneous H2S release, which makes it an excellent drug to be loaded and reformulated. Due to the unique mesoporous structures of MSN and its loading ability, the new H2S donor DATS-MSN can be activated by GSH with slow and controllable H2S release rates10. Therefore, DATS-MSN presents a much slower process of H2S generation than free DATS, both in vitro and in vivo with excellent biocompatibility, which makes it an ideal slow-releasing H2S donor10. The present study aimed to investigate the cardioprotective effects of DATS-MSN treatment in a rat I/R model compared with the classic H2S donors NaHS and DATS, as well as the slow-releasing H2S donor GYY4137, and to explore how the different release patterns of H2S affect its physiological functions.
Methods
Materials
Newborn (6 g, 24 h) and adult male Sprague-Dawley rats (250–280 g, 8 w) were used in this study. The adult rats were housed under a 12-h/12-h light/dark cycle with food and water provided ad libitum during the experimental protocol. All animal experiments were approved by Institutional Review Board and Institutional Animal Care and Use Committee Protocols of Fudan University, and confirmed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996). DATS-MSN was synthesized and characterized by the method described previously10 (Supporting Information). NaHS, GSH, DATS, GYY4137 and other chemical reagents were obtained from Sigma-Aldrich.
In vitro Cell Viability and Cytotoxicity Assay after Hypoxia/reoxygenation Procedure
The isolation and culture of primary neonatal cardiomyocytes were performed using the method described previously10 (Supporting Information). Cardiomyocytes were divided into seven groups (n = 3): Control group: same volume of PBS (normoxia); Vehicle group: same volume of PBS; GSH group: GSH (2 mM); MSN group: MSN (5 μg/mL); NaHS group: NaHS (80 μM); DATS group: DATS (5 μg/mL) + GSH (2 mM); DATS-MSN group: DATS-MSN (10 μg/mL) + GSH (2 mM). The concentrations of NaHS and DATS were selected based on their most effective concentrations described in the Supporting Information (Fig. S2). The concentration of DATS-MSN was determined to compare the amounts of total sulfur atoms with that in the NaHS and DATS groups (S atoms: 80 μM of NaHS vs. 84.3 μmol/kg of DATS vs. 74.2 μmol/kg of DATS-MSN).
The culture medium of each group (except for the Control group) was removed and replaced with DMEM/F-12 without glucose and serum, and the cells were exposed to hypoxic conditions (94% N2, 5% CO2, 1% O2) for 4 h in a CO2 incubator (Forma SERIES II WATER JACKET, Thermo Scientific, MA, USA), followed by reoxygenation (95% O2, 5% CO2) with fetal bovine serum for 6 h. Drugs were administrated at the time of reoxygenation, and cell viability, lactate dehydrogenase (LDH) activity and creatine kinase (CK) activity were evaluated within 6 h following reoxygenation. The H2S concentrations in the culture medium were also measured using a high-performance liquid chromatography (HPLC) method as described previously10 (Supporting Information).
After the hypoxia/reoxygenation procedure, the culture medium was removed; the cells were washed with PBS, and then resuspended in cell counting kit-8 (CCK-8) solution (Dojindo Laboratories, Kumamoto, JA) as the medium. The absorbance of the individual wells was measured at 450 nm via a microplate reader (Molecular Devices, FlexStation 3, CA, USA). The results were expressed as the mean percentage of cell viability relative to the Control group. The LDH and CK activities for each group were also measured using an assay kit (JianCheng, Nanjing, China) in accordance with the manufacturer’s instructions.
In Vivo Myocardial I/R Model
Male Sprague-Dawley rats undergoing the I/R protocol were randomly assigned into five groups (n = 6, counting alive animals): Vehicle group: 500 μL of PBS; MSN group: 2 mg/kg of MSN; NaHS group: 30 μmol/kg of NaHS; DATS group: 2 mg/kg of DATS; and DATS-MSN group: 4 mg/kg of DATS-MSN. There were six extra rats undergoing an open-close chest procedure that comprised the Sham group (500 μL of PBS). The dosages of NaHS and DATS were chosen based on their most effective dosages for the reduction of myocardial injury during the I/R process, which is described in the Supporting Information (Fig. S3). The dose of DATS-MSN was determined by comparing the amounts of total sulfur atoms with NaHS and DATS administrated (S: 30 μmol/kg of NaHS vs. 33.7 μmol/kg of DATS vs. 29.7 μmol/kg of DATS-MSN).
Ischemic/reperfusion protocol
The I/R model was induced by ligating the left anterior descending coronary artery (LAD) as previously described1. Briefly, the rats were anesthetized with medetomidine hydrochloride (Domitor, 250 μg/kg, ip.) and ketamine hydrochloride (Ketamine, 50 mg/kg, ip.), followed by an endotracheal intubation. The carotid arteries were cannulated for blood withdrawal and monitoring of the blood pressure and heart rates. The LAD was temporarily ligated using a 6–0 silk suture 3 mm from its origin between the artery conus and the left atrium. Successful ligation of the coronary artery was verified by the color change in the ischemic area (anterior ventricular wall and the apex) of the heart. The rats were subjected to 30 min of ischemia followed by 24 h reperfusion. All drugs were administrated via a tail vein injection at the time of reperfusion. The mean arterial pressure (MAP) and heart rates were recorded at baseline, 30 min after ischemia, as well as 30 min and 6 h after reperfusion; the arrhythmia scores were also calculated at the same time point as described in the previous study11 (Supporting Information). The survival rates were evaluated based on the animals surviving throughout the experimental protocol until 24 h after reperfusion. At 24 h after reperfusion, the hearts were excised, washed, and stored for subsequent experimental analysis.
Determination of Myocardial Injury and the H2S Concentration
The levels of creatine kinase-MB (CK-MB) and cardiac troponin I were measured to indicate and evaluate the extent of myocardial injury. Blood samples from the rats were obtained at 24 h after reperfusion just before death, and analyzed using Analytics (Gaithersburg, MD, USA). The H2S concentrations in plasma were evaluated within the first 12 h and at 24 h after reperfusion before death, and the H2S concentrations in the myocardium were also evaluated immediately after heart excision by HPLC as previous described10 (Supporting Information).
Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assay
A piece of ischemic heart tissue was excised, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (H&E), followed by a TUNEL assay: the cell nuclei were stained with 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) color development kits (Roche, Basil, CH) in accordance with the manufacturers’ instructions. The cell nuclei that stained green were defined as TUNEL-positive nuclei and were monitored using a fluorescence microscope (Olympus IX-71, Japan). The proportion of TUNEL positive nuclei per 500 nuclei was quantified at 200x magnification.
Western Blot Assay
A piece of ischemic heart tissue was homogenized by a rotor-stator homogenizer in ice-cold RIPA buffer (Pierce, Pittsburgh, PA, USA), and incubated at 4 °C overnight. After boiling with loading buffer (Fermentas, Glen Burnie, MD, USA), denatured proteins were separated in SDS PAGE gel, and transferred onto PVDF membrane. The membrane was blocked with 5% nonfat milk, followed by incubation with primary antibody of Bcl-2 and Bax (Abcam, Cambridge, MA, USA) at 4 °C overnight. HRP-conjugated secondary antibody (Kangchen Bio-tech, Beijing, China) was used to incubate the membrane for another 2 h. SuperSignal West Pico Chemiluminescent Substrate (Pierce, Pittsburgh, PA, USA) was poured on the membrane to develop the band captured by FluorChem Image System (Alpha Innotech, Santa Clara, CA, USA).
Determination of Caspases-3 Activity
The activity of caspase 3 in the border zone of infarcted area was determined using the colorimetric assay via a microplate reader at 400 nm. The assay kits were purchased from Biovision (Milpitas, CA).
Antioxidant Enzyme Activities
A total of 50 mg ischemic heart tissue was homogenized in a 50 mM ice-cold potassium phosphate buffer (pH of 6.8). Superoxide dismutase (SOD) activity was measured as described by Dieterich12, which was determined by monitoring the inhibition of pyrogallol autooxidation at 420 nm. The catalase (CAT) activity was determined by the modified method of Alvarez13. The GSH levels were measured by using a commercially available kit according to the manufacturer’s instructions (Beytime Institute of Biotechnology, Nantong, China). The malonydialdehyde (MDA) levels were measured using the thiobarbituric acid (TBA) assay, with 1, 1, 3, 3-tetramethoxypropane as an external standard for the construction of standard curves. The activity of SOD and CAT, and the levels of MDA and GSH were all standardized by protein content, determined using a bicinchoninic acid (BCA) protein assay kit (Beytime Institute of Biotechnology, Nantong, China).
Quantitative Assessment of Neutrophil Accumulation
Ischemic heart tissues were assessed for the myeloperoxidase (MPO) activity as a marker of neutrophil accumulation. Tissues were homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mM K3PO4 buffer (pH of 7) and centrifuged for 30 min (20, 000 g, 4 °C). An aliquot of the supernatant was allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The change in absorbance was measured by spectrophotometry at 650 nm. The MPO activity was defined as the quantity of enzyme degrading 1 mmol of hydrogen peroxide per minute at 37 °C and expressed in milliunits per milligram protein.
Measurement of cytokines
Blood was collected at 24 h after reperfusion before the animals were sacrificed. The samples were centrifuged, and then the plasma was collected according to the manufacturer protocol. The expressions of TNF-a and IL-1β cytokines were measured with an ELISA Kit (R&D Systems).
Determination of the Infarct Size and Cardiac Function at 72 h Reperfusion
Repeated I/R protocol (n = 6) and then evaluated the infarct size and cardiac function at 72 h after reperfusion. The rats were anesthetized again after 72 h of reperfusion, and the LAD was ligated again in the same location. Evans blue (5.0%, 1.0 mL) was injected into the carotid artery to delineate between the ischemic and nonischemic zones. Then, the heart was rapidly excised and cross-sectioned into 1-mm-thick sections, and incubated in a 1% triphenyltetrazolium chloride (TTC) solution for 30 min. The stained slices were photographed with a digital camera, and infarct zones were quantified as a percentage of the total myocardium using Image J software (NIH, Boston, USA). The myocardial area at risk (AAR) and infarct per left ventricle (INF) were determined by a blinded observer.
Cardiac function was evaluated before the animals were sacrificed using an echocardiography method. The ejection fraction (EF), fractional shortening (FS), left ventricular internal dimension in diastole (LVIDd), and the left ventricular internal dimension in systole (LVIDs) were obtained and compared with the baseline values before the infarction (Supporting Information). The H2S concentrations in plasma and myocardium were also evaluated after 72 h reperfusion just before the sacrifice as previously described10.
Comparison of DATS-MSN to Classic Slow-releasing H2S Donor in Myocardial Protection
To compare myocardial protective effects of the novel slow-releasing H2S donor to traditional slow-releasing donors, the most popular and classic H2S slow-releasing donor GYY4137 was utilized. The in vivo experiments above were repeated, and the protective effects such as myocardial injury protection, anti-apoptosis, antioxidant, anti-inflammation (at 24 h after reperfusion), and reducing infarct size and preserving cardiac function (at 72 h after reperfusion) were evaluated in two H2S slow-releasing donors. The dose of GYY4137 (100 mg/kg) was chosen based on its most effective dose for the reduction of myocardial injury during the I/R process, which is described in the Supporting Information (Fig. S3). The in vivo releasing feature of H2S of GYY4137 was also determined using HPLC as previous described10.
Statistical Analysis
The data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used to examine statistical comparisons between groups. The significant difference between two groups was analyzed by Student’s t test. The Chi-square test (or Fisher’s exact test when appropriate) was used to compare discrete variables between different groups. A value of P < 0.05 was considered to be significant. All authors had full access to, and take full responsibility for the integrity of the data.
Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Results
DATS-MSN Protected Rat Cardiomyocytes from Hypoxia/Reoxygenation Induced Damage
As shown in Fig. 1A–C, all three H2S donors effectively protected cardiomyocytes from hypoxia/reoxygenation injury, with greater cell viability and decreased LDH and CK activity observed at 6 h after reoxygenation. However, the DATS-MSN group exhibited an obvious advantage with regards to cellular protection compared with the NaHS and DATS groups. This advantage became even more significant along with the reoxygenation time expanding. Moreover, Fig. 1D showed that the in vitro H2S release from NaHS was quite rapid, peaked at 30 min, and rapidly decreased to nearly baseline levels after 3 h without reaching a plateau. In contrast, the release of H2S from DATS-MSN was much slower and continuous. In addition, the curve continued to increase after the 6 h experiment. The DATS release curve was between NaHS and DATS-MSN, peaking at nearly 1 h. There was no obvious difference between the Vehicle, GSH, and MSN groups, suggesting that the cytoprotective effects were not be mediated by the administration GSH or MSN.
DATS-MSN Improved the Hemodynamics and Survival during the I/R Process
As shown in Fig. 2A, the H2S donor groups exhibited elevated MAP levels compared with the Vehicle group at 6 h after reperfusion, which was most obvious in the DATS-MSN group; however, there was no difference in the alteration in the heart rates between groups (Fig. 2B). The DATS-MSN was also associated with substantially lower arrhythmia scores compared with the NaHS and DATS groups during the 30 min ischemia and 6 h reperfusion period (Fig. 2C). At 24 h after reperfusion, the survival was 100% in the Sham and DATS-MSN groups, compared to 66.7% (6 of 9 rats) in the Vehicle and MSN groups, and to 80.0% (6 of 8 rats) in NaHS and DATS groups (Fig. 2D). The main cause of death was due to lethal ventricular arrhythmias occurring after reperfusion.
DATS-MSN Reduced Myocardial Injury after the I/R Process
As shown in Fig. 3A and B, the CK-MB and troponin I levels were reduced by three H2S donors at 24 h after reperfusion, among which the DATS-MSN group exhibited the greatest decrease in these biomarkers of myocardial injury. Following the intravenous injection of three H2S donors with a similar amount of total sulfur atoms (S: 30 μmol/kg of NaHS vs. 29.7 μmol/kg of DATS-MSN vs. 33.7 μmol/kg of DATS), the H2S concentration in plasma of the DATS-MSN group increased from the first time point (1 h), peaked at 4 h after administration, and remained elevated over the course of the 12 h experiment. In contrast, NaHS rapidly increased the plasma H2S concentration after administration (peaking at 1 h), which, however, quickly decreased to near baseline levels after 6 h (Fig. 3C). DATS elevated the plasma H2S concentration more rapidly than DATS-MSN, but slowly than NaHS, peaking at nearly 3 h after administration. Furthermore, the H2S levels in plasma and heart tissue remained elevated in the DATS-MSN group following the 24 h reperfusion process, which did not occur in NaHS, DATS, and Vehicle groups at this time point (Fig. 3D and E).
DATS-MSN Protected against Myocardial Apoptosis
Figure 4A presents the representative TUNEL results following 24 h of reperfusion. Although the apoptosis index was significantly decreased in all the H2S donor groups, the DATS-MSN was associated with the most potent anti-apoptosis ability among the three H2S donors (Fig. 4B).
As shown in Fig. 4C–E, the I/R injury induced the expression of Bax and reduced the expression of Bcl-2 in the Vehicle group. The NaHS, DATS, and DATS-MSN groups all exhibited increased levels of Bcl-2, but decreased levels of Bax, respectively, providing protection against myocardial apoptosis. These effects were also most remarkable in the DATS-MSN group. Accordingly, the Caspase-3 activity was associated with similar results, exhibiting the greatest activity decrease in the DATS-MSN group (Fig. 4F).
DATS-MSN Resisted Oxidative Stress following the I/R Protocol
As shown in Fig. 5, the NaHS, DATS and DATS-MSN groups all displayed an effective preservation of SOD activity, CAT activity, and GSH levels compared with the Vehicle group, and DATS-MSN exerted more significant antioxidant effects than the other two H2S donors (Fig. 5A–C). In addition, the tissue levels of MDA, an important biomarker of oxidative stress injury, were also found to be much lower in the DATS-MSN group compared to the NaHS and DATS groups (Fig. 5D).
DATS-MSN Reduced Myocardial Inflammation following the I/R Protocol
Heart sections stained with H&E were presented at 24 h after reperfusion (Fig. 6A). The rats receiving H2S donors displayed a reduced degree of myocardial neutrophilic infiltrate and necrosis in comparison with the Vehicle group, with a reduction in MPO activities (Fig. 6B). Furthermore, the histological analysis and MPO activity assessment both revealed that DATS-MSN exerted the most potent anti-inflammatory effects among the three H2S donors. The serum levels of TNF-α and IL-1β measured at 24 h after reperfusion also revealed that the most significant decrease was observed in the DATS-MSN group compared with the other two H2S donor groups and the Vehicle group.
DATS-MSN Reduced Infarct Size and Preserved Cardiac Function after 72 h of Reperfusion
As presented in Fig. 7A and B, although all groups displayed a similar AAR% to the total LV, the percent of INF to AAR was substantially reduced by the three H2S donors at 72 h after reperfusion, among which, the DATS-MSN decreased the INF/AAR percent to the greatest extent. The echocardiography results (Fig. 7C) revealed that DATS-MSN most effectively preserved the cardiac function at 72 h after the infarction and reperfusion, presenting an increased ejection fraction and fractional shortening (Fig. 7D and E), as well as reduced LVIDd and LVIDs (Fig. 7F and G) when compared with the NaHS, DATS, and Vehicle groups. Moreover, the levels of H2S in plasma and myocardium remained elevated in the DATS-MSN group after the 72 h reperfusion process, but not in the NaHS and DATS groups (Fig. 7H and I).
DATS-MSN Demonstrated Potential Myocardial Protective Advantages to GYY4137
The CK-MB (Fig. 8A) and troponin I (Fig. 8B) levels were more effectively reduced by DATS-MSN compared with GYY4137 at 24 h after reperfusion, while INF/AAR was also more obviously reduced by the DATS-MSN at 72 h after reperfusion (Fig. 8C and D), with more preserved cardiac function (Fig. 8E and F). Meanwhile, DATS-MSN was also associated with superior anti-apoptosis ability than GYY4137 (Fig. S4). However, there was no obvious difference between groups in myocardial oxidative stress (Fig. S5) and inflammation (Fig. S6) assessment. In H2S releasing measurement, although GYY4137 presented a quite slow releasing pattern of H2S, H2S levels of which remained lower than DATS-MSN in 12 h after reperfusion in plasma (Fig. 8G); and at 24 h and 72 h after reperfusion in plasma and myocardium (Fig. 8H and I).
Discussion
H2S has been shown to effectively reduce the infarct size, preserve cardiac function, and improve the survival rates in I/R injury models1, 2. To date, although several mechanisms of the cardioprotective effects of H2S have been elucidated including anti-apoptosis, anti-inflammatory, and antioxidant effects4, how the different H2S releasing patterns mediate its physiological effects remains unknown. Herein we present a novel slow-releasing H2S donor as an ideal platform for studying the slow release pattern of H2S both in vitro and in vivo. To clarify the advantage of the slow-releasing H2S donor with respect to myocardial protection, all of the drugs were administrated at the onset of reperfusion as post-conditioning. Ischemic post-conditioning has been previously shown to attenuate I/R injury; however, it has failed to easily translate into therapeutically useful cardioprotective strategies in clinical practice14, 15. In comparison, pharmacological post-conditioning may provide more clinical advantages, which would be fairly easy to be used in an intravenous formulation, and effectively cover the necessary time frame to mediate protection against lethal reperfusion injury16. H2S donors with the same possible H2S production administrated at the same time point (onset of reperfusion) can eliminate the majority of possible disturbances, and the effects of the different H2S releasing patterns are clearly presented.
H2S has been widely studied using in vitro models of cardiovascular diseases, demonstrating various significant cytoprotective effects against cell loss and cellular injury1, 17. In the present study, DATS-MSN manifested a superior cytoprotective effect compared with both NaHS and DATS. Along with the expansion of the reperfusion time, the protective advantage of DATS-MSN became increasingly more remarkable. Correspondingly, DATS-MSN also presented a more slowly releasing H2S pattern than NaHS and DATS, suggesting that the superior protective effects of DATS-MSN may be attributed to its slow-releasing pattern and long-term H2S effects in vitro.
In the in vivo I/R models, DATS-MSN also demonstrated superior cardioprotective effects over other H2S donors, associated with decreased CK-MB and troponin I levels and improved survival rates at 24 h after reperfusion, as well as elevated MAP levels and decreased arrhythmia scores in the I/R process. In contrast to the rapidly increased H2S concentrations in plasma associated with NaHS, DATS-MSN released H2S slowly and stably, and H2S remained elevated in plasma as well as in ischemic myocardium after 24 h of reperfusion. In addition, DATS increased the H2S concentration in plasma more rapidly than DATS-MSN, but slowly than NaHS, with plasma and myocardial H2S levels between the NaHS and DATS-MSN groups at 12 h following reperfusion. Therefore, with the same amount of S atoms, both the different releasing speeds of the H2S donors and the maintenance of the H2S levels are related to the extent of myocardial injury.
It was shown previously that H2S exerts anti-apoptotic effects in different organs, especially in I/R hearts18. In the present study, the DATS-MSN group exhibited a significantly decreased apoptosis index, suggesting that an anti-apoptotic mechanism may play an important role in the cardioprotective effects of the novel H2S donor. Multiple studies have reported that H2S exerts anti-apoptotic effects via the inactivation of caspases caused by I/R19. In this study, DATS-MSN administration was associated with H2S-mediated modulation through increasing the levels of Bcl-2, decreasing the levels of Bax, and reducing the activity of caspases-3. Although NaHS and DATS were associated with the same anti-apoptotic mechanisms, they were not as adequate as DATS-MSN due to their relatively short duration of efficacy.
It was found that oxidative stress plays an important role in the I/R injury, and H2S potently protects cells from oxidative stress through multiple mechanisms20. In the present study, the elevated levels of MDA were reduced to the greatest extent by DATS-MSN among the three H2S donors. In addition, DATS-MSN was also associated with the greatest preservation of antioxidant levels and activity (e.g., SOD, CAT, and GSH), greatly reinforcing the antioxidant defenses. DATS-MSN also effectively attenuated the level of myocardial inflammation following the I/R process. Ischemic myocardial neutrophils assessed by an MPO analysis and the level of inflammatory cytokines were all markedly reduced by the DATS-MSN administration compared to those observed in other H2S donor groups and the Vehicle control. The advantage of DATS-MSN over NaHS and DATS regarding the antioxidant and anti-inflammatory effects may also attribute to its slow release and long-term effects of H2S.
To further identify the advantage of the slow and stable release feature of DATS-MSN, an I/R experiment with a longer reperfusion time was performed. Due to the slow release ability of DATS-MSN, the H2S concentration in either plasma or myocardium remained elevated at 72 h after reperfusion, of which returned to the baseline levels in both the DATS and NaHS groups. Correspondingly, DATS-MSN also demonstrated superior cardioprotective effects over other H2S donors, with most reduced infarct size in the long-term reperfusion experiment. Furthermore, DATS-MSN was also superior regarding the preservation of cardiac function and preventing LV dilatation following an infarction; this could potentially reverse LV remodeling and ameliorate the development of heart failure after an infarction, similar to some other endogenous H2S donors21. The greater protective effects combined with expanded reperfusion time highlight the advantage of this slow-releasing H2S donor.
To date, the release pattern of H2S from relevant H2S donors has rarely been identified. NaHS is the most frequently applied donor in H2S studies; however, the extremely rapid release and loss pattern of this donor serves to limit its biofunctionality. H2S is easily transferred across respiratory membranes22, 23, thus, the residence time in tissues is relatively short for fast-releasing H2S donors, which limits their therapeutic potential and clinical application. In contrast, slow-releasing H2S donors may offer an accumulation of H2S over a long period, and they are more potent for providing therapeutic functions3. As previously reported, the slow-release mechanism of DATS-MSN could be due to GSH molecules moving into the mesopores and reacting with loaded DATS to generate H2S, after which the H2S is slowly released into solution and participates in the protective process (Fig. 9, Video 1). Moreover, the cardiovascular protective effects of DATS have also been found to be derived from H2S9. Therefore, the differences in the cardioprotective abilities between different H2S donors may be attributed to the different release patterns of H2S. In this study, DATS-MSN was associated with an overall superiority regarding its anti-apoptotic, antioxidant, and anti-inflammatory abilities over NaHS and DATS, contributing to the long-range effects of H2S, which can precisely mimic the physiological production and function of H2S.
Besides releasing pattern, the physiological effects of H2S also rely on its local effecting concentration. Sharing the same in vivo I/R model, time of administration and evaluating method, DATS-MSN presented some superior myocardial protective abilities than GYY4137, which may be attributed to its relatively high concentrations of H2S in plasma and myocardium. A satisfactory level of H2S over a long period conforms to an ideal H2S donor’s demand; DATS-MSN presented a more elevated H2S level than GYY4137 in the 72 h reperfusion process, which is a vital stage to myocardial preservation and LV remodeling. This may explain the potential advantages of DATS-MSN in myocardial injury protection. However, GYY4137 demonstrated comparable antioxidant and anti-inflammation effects to DATS-MSN, suggesting that it may involve additional protective mechanisms in GYY4137, which develops multiple cardioprotective effects by diverse mechanisms in a long time manner24, 25. Therefore, it is currently too early to conclude DATS-MSN exerting overall superiority to traditional slow-releasing H2S donors; actually it mainly provides us a novel slow-releasing alternative with new insight into control-releasing H2S based on nano-system, which has the potential to further regulate H2S releasing speed by adjusting structures of the nanoparticle10, 26.
In summary, we demonstrated that DATS-MSN as a novel slow-releasing H2S donor, can exert potent protective effects in rats and isolated cardiomyocytes following an I/R injury. Moreover, DATS-MSN exhibited superior cardioprotective effects in comparison to NaHS, DATS and GYY4137, which may be related to its specific show-releasing properties both in vitro and in vivo. Finally, DATS-MSN may primarily meditate the protective effects of H2S via various anti-apoptotic, antioxidant, and anti-inflammatory mechanisms. The present work provides a novel insight into how the H2S release patterns derived from different H2S donors affect their physiological functionality.
References
Yao, L., Huang, X., Wang, Y. et al. Hydrogen sulfide protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by preventing GSK-3beta-dependent opening of mPTP. Am J Physiol Heart Circ Physiol 298, 310–319 (2010).
Elrod, J., Calvert, J., Morrison, J. et al. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function. Proc Natl Acad Sci USA 104, 15560–15565 (2007).
Li, L., Whiteman, M., Guan, Y. et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 117, 2351–2360 (2008).
Caliendo, G., Cirino, G., Santagada, V. et al. Synthesis and biological effects of hydrogen sulfide (H2S): development of H2S-releasing drugs as pharmaceuticals. J Med Chem 53, 6275–6286 (2010).
Wang, Q., Wang, X., Liu, H. et al. Protective effects of cysteine analogues on acute myocardial ischemia: novel modulators of endogenous H(2)S production. Antioxid Redox Signal 12, 1155–1165 (2010).
Lee, M., Tazzari, V., Giustarini, D. et al. Effects of hydrogen sulfide-releasing L-DOPA derivatives on glial activation: potential for treating Parkinson disease. J Biol Chem 285, 17318–17328 (2010).
Zhao, Y., Wang, H. & Xian, M. Cysteine-activated hydrogen sulfide (H2S) donors. J Am Chem Soc 133, 15–17 (2011).
Benavides, G., Squadrito, G., Mills, R. et al. Hydrogen sulfide mediates the vasoactivity of garlic. Proc Natl Acad Sci USA 104, 17977–17982 (2007).
Tsai, C., Wen, S., Shibu, M. et al. Diallyl trisulfide protects against high glucose-induced cardiac apoptosis by stimulating the production of cystathionine gamma-lyase-derived hydrogen sulfide. Int J Cardiol 195, 300–310 (2015).
Sun, X., Kong, B., Wang, W. et al. Mesoporous silica nanoparticles for glutathione-triggered long-range and stable release of hydrogen sulfide. J Med Chem B 3, 4451–4417 (2015).
Wang, S., Cui, X., Xue, F. et al. Combined morphine and limb remote ischemic perconditioning provides an enhanced protection against myocardial ischemia/reperfusion injury by antiapoptosis. J Surg Res 202, 13–25 (2016).
Dieterich, S., Bieligk, U., Beulich, K. et al. Gene expression of antioxidative enzymes in the human heart: increased expression of catalase in the end-stage failing heart. Circulation 101, 33–39 (2000).
Alvarez, M., Caldiz, C., Fantinelli, J. et al. Is cardiac hypertrophy in spontaneously hypertensive rats the cause or the consequence of oxidative stress? Hypertens Res 31, 1465–1476 (2008).
Soriano, F., Liaudet, L., Szabó, E. et al. Resistance to acute septic peritonitis in poly (ADP-ribose) polymerase-1-deficient mice. Shock 17, 286–292 (2002).
Heusch, G. Cardioprotection: chances and challenges of its translation to the clinic. Lancet 381, 166–175 (2013).
Ovize, M., Thibault, H. & Przyklenk, K. Myocardial conditioning: opportunities for clinical translation. Circ Res 113, 439–450 (2013).
Pan, T., Feng, Z. N., Lee, S. et al. Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes. J Mol Cell Cardiol 40, 119–130 (2006).
Calvert, J., Jha, S., Gundewar, S. et al. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res 105, 365–374 (2009).
Sodha, N., Clements, R., Feng, J. et al. The effects of therapeutic sulfide on myocardial apoptosis in response to ischemia-reperfusion injury. Eur J Cardiothorac Surg 33, 906–913 (2008).
Jha, S., Calvert, J., Duranski, M. et al. Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling. Am J Physiol Heart Circ Physiol 295, H801–H806.
Huang, C., Kan, J., Liu, X. et al. Cardioprotective effects of a novel hydrogen sulfide agent-controlled release formulation of S-propargyl-cysteine on heart failure rats and molecular mechanisms. PLoS One 8, e69205 (2013).
Derwall, M., Francis, R., Kida, K. et al. Administration of hydrogen sulfide via extracorporeal membrane lung ventilation in sheep with partial cardiopulmonary bypass perfusion: a proof of concept study on metabolic and vasomotor effects. Crit Care 15, R51 (2011).
Insko, M., Deckwerth, T., Hill, P. et al. Detection of exhaled hydrogen sulphide gas in rats exposed to intravenous sodium sulphide. Br J Pharmacol 157, 944–951 (2009).
Meng, G., Wang, J., Xiao, Y. et al. GYY4137 protects against myocardial ischemia and reperfusion injury by attenuating oxidative stress and apoptosis in rats. J Biomed Res 29, 203–213 (2015).
Lilyanna, S., Peh, M. T., Liew, O. W. et al. GYY4137 attenuates remodeling, preserves cardiac function and modulates the natriuretic peptide response to ischemia. J Mol Cell Cardiol 87, 27–37 (2015).
Slowing, I.I., Vivero-Escoto, J. L., Wu, C. W. et al. Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv Drug Deliv Rev 60, 1278–88 (2008).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81601663, 81270326); Shanghai Municipal Health and Planning Commission Foundation (20164Y0177).
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X.S. and W.W. conceived and designed the experiments. J.D., S.J. and J.H. carried out the experiments. C.G. and C.W. collected and analyzed the data. X.S., L.P. and Y.W. wrote the paper. All authors reviewed and edited the manuscript.
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Sun, X., Wang, W., Dai, J. et al. A Long-Term and Slow-Releasing Hydrogen Sulfide Donor Protects against Myocardial Ischemia/Reperfusion Injury. Sci Rep 7, 3541 (2017). https://doi.org/10.1038/s41598-017-03941-0
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DOI: https://doi.org/10.1038/s41598-017-03941-0
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