A Long-Term and Slow-Releasing Hydrogen Sulfide Donor Protects against Myocardial Ischemia/Reperfusion Injury

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.


Preparation and Characterization of DATS-MSN
The synthesis of DATS-MSN was carried out by sol-gel method [1,2]. Briefly, 10 mg mesoporous silica nanoparticles (MSN) and 8 mg diallyl trisulfide (DATS) were dispersed in 2 mL distilled water to form the mixture with a ratio of drug/carrier 0.8/1, which was stirred at room temperature for 12 h, followed by centrifugation (14000 rpm, 10 min). The nanoparticles obtained were washed with distilled water to remove DATS on the surface of MSN.
The exact diameters of synthesized nanoparticles were acquired by averaging size of 50 nanoparticles in transmission electron microscopy (TEM) images, which were also applied to confirm the frameworks and structures of MSN. Unloaded DATS was subtracted from initial total DATS to calculate the mass of DATS loaded in MSN. The drug loading content was calculated as mass of drug loaded in nanoparticles /mass of drug loaded nanoparticles*100 %.
Cells were cultured in a humidified incubator at 37 °C with 95% air and 5% CO 2 for 72 h.

Most Protective Concentrations of NaHS and DATS in Hypoxia/reoxygenation Procedure
The hypoxia/reoxygenation procedure was described in the main text. According to the different concentrations of NaHS or DATS added into the culture medium, each drug was divided into 11 subgroups: NaHS groups (0 to 100 μM, each 10 μM as gradient) and DATS groups (0 to 10 μg/mL, each 1 μg/mL as gradient). After the 4 h hypoxia and 6 h reoxygenation process, the cell viabilities in different groups were measured by CCK-8 method. Most protective concentrations of NaHS and DATS were selected. The results were expressed as the percentages relative to 0 control (n = 3).
As shown in Fig. S2, the most protective concentration of NaHS emerged to be at 80 μM, and DATS appeared to most effectively protect cardiomyocytes at the concentration of 5 μg/mL.

Most Protective Dosages of NaHS, DATS and GYY4137 in Ischemia and Reperfusion Protocol
According to the different dosages of NaHS, DATS or GYY4137 injected into the tail vein of rats, each drug was divided into 11 subgroups: NaHS groups (0 to 100 μmol/kg, each 10 μmol/kg as gradient), DATS groups (0 to 10 mg/kg, each 1 mg/kg as gradient) and GYY4137 groups (0 to 250 mg/kg, each 25 mg/kg as gradient). After 30 min ischemia and 24 h reperfusion, the rat blood samples were obtained, and creatine kinase MB (CK-MB) was measured to indicate myocardial injury by Anilytics (Gaithersburg, MD, USA). The most protective dosages of each H 2 S donor were selected (n = 3).
As shown in Fig. S3, the most myocardial protective dose emerged to be of 30 μmol/kg for NaHS; 2 mg/kg for DATS and 100 mg/kg for GYY4137.

Measurement of H 2 S Concentration in Cell Culture Medium, Plasma and Heart Tissues
H 2 S concentrations in cell culture medium and in rat plasma were both measured to describe the release patterns of different H 2 S donors. NaHS (80 μM) and DATS-MSN (50 μg/mL) were separately added into the culture medium, then the culture medium was collected at every hour from 1 to 6 h after administration. Rat plasma was collected from rat blood in Vehicle, NaHS, DATS and DATS-MSN, as well as GYY4137 groups at every hour from 0 h to 12 h after reperfusion. Rat plasma and heart tissue in ischemia area were also collected at 24 h and 72 h after reperfusion before death. H 2 S concentration was measured by high performance liquid chromatography (HPLC) method as described in the previous study [3]. H 2 S concentration was determined using a standard curve of Na 2 S (0-100 μM). The H 2 S content in the heart tissues were quantified by protein content and expressed in proportion relative to Vehicle group. The protein content was measured by the BCA method mentioned above.

Arrhythmia Analysis and Scores
Ventricular tachycardia (VT) was defined as a run of four or more consecutive ventricular premature contractions. Ventricular fibrillation (VF) was defined as a ventricular rhythm in which one is unable to distinguish the individual QRS complexes of the ECG or to measure the rate. According to the cumulative duration of VT and VF, an arrhythmia score was graded as follows: 0: without arrhythmias; 1: less than three premature ventricular contractions per min; 2: greater than or equal to three premature ventricular contractions per min; 3: less than three episodes of VT per min; 4: greater than or equal to three episodes of VT per min or transient VF; and 5: frequent or sustained VF or death of the rat. The number corresponded to the most severe type of arrhythmia observed in each heart during the periods of ischemia and early reperfusion, and the scores were used for intergroup analysis of their severity.

Echocardiography
At 72 h after reperfusion, rats were anesthetized again by previous protocol and placed on a heating pad before sacrifice. Transthoracic echocardiography was performed using the Philips IE 33 system and a 12-4 MHz linear transducer (S12-4, Philips, AMS, NED). Left ventricular internal dimension in systole (LVIDs) and left ventricular internal dimension in diastole (LVIDd) were all obtained from the M-mode tracings. Ejection fraction (EF) and fractional shortening (FS) were also derived to evaluate cardiac function. All echocardiographic measurements were performed by skilled observer blindly.

DATS-MSN Exerts Superior Anti-apoptosis Ability than GYY4137
As shown in Fig. S4, DATS-MSN was associated with superior anti-apoptosis ability than GYY4137 at 24 h after reperfusion.

DATS-MSN Exerts Comparable Antioxidant and Anti-inflammation Ability to GYY4137
At 24 h after reperfusion, there was no obvious difference between GYY4137 and DATS-MSN groups in myocardial oxidative stress (Fig. S5) and inflammation (Fig. S6) evaluation.  Video.1: Schematic illustration of the mechanism of H 2 S slow-release from DATS-MSN. GSH molecules move into the mesopores to react with loaded DATS for H 2 S generation, after which H 2 S slowly releases into solution.