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Departments of 1Pharmacology and 2Biochemistry, National University of Singapore, Singapore; 3Departments of Physiology and Pathophysiology, and 4Pharmacology, School of Pharmacy and Institute of Biological Sciences, Fudan University, Shanghai, People's Republic of China; and 5Defence Medical and Environmental Research Institute, Singapore
Submitted 25 January 2006 ; accepted in final form 25 September 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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cardioprotection; gasomediator; cardiac protection; ischemic animal model
-synthase and cystathionine 
-lyase (CSE), each of which utilize L-cysteine as substrate (17). Although cystathionine -synthase does not appear to play a major role in generating H2S in cardiovascular tissue under physiological conditions (21), the expression of CSE and the associated production of endogenous H2S was demonstrated in rat mesenteric artery and other vascular tissue several years ago. Hosoki and colleagues (7) reported that sodium hydrosulfide (NaHS), an H2S donor, relaxed vascular smooth muscle (including rat portal vein and aorta) in vitro. By analogy with other endogenous gaseous molecules such as nitric oxide and carbon monoxide, H2S has been implicated as a gaseous transmitter fulfilling a physiological role in regulating cardiovascular function. The mechanisms underlying the vascular relaxant effect of H2S are incompletely understood, although opening of ATP-sensitive K+ (KATP) channels in vascular smooth muscle cells may play an important role (21). Similarly, the effect of H2S on cardiac function was reported by Geng et al. (6) and their subsequent study on isoproterenol-induced myocardial injury (5). Additional studies suggesting a role for H2S in the pathogenesis of spontaneous hypertension (19) as well as hypoxic pulmonary hypotension (12) prompted us to study the potential role of H2S in myocardial infarction (MI). |
MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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In Vivo Ischemic Model (MI)
Male Wistar rats (250–300 g) were anesthetized with 7% wt/vol chloral hydrate (350 mg/kg ip) followed by endotracheal intubation. After thoracotomy, the heart was exteriorized, and the left anterior descending coronary artery was permanently ligated using 6-0 silk suture 2–3 mm from its origin between the pulmonary artery conus and the left atrium as previously reported (15, 21). Successful ligation of the coronary artery was verified by the color change immediately in the ischemic area (anterior ventricular wall and the apex) of the heart, and MI was confirmed by electrocardiography (ECG) (4, 21).
Homodynamic Parameter Measurement
Rat blood pressure was measured in anesthetized rats using a tail-cuff noninvasive blood pressure system (ML125/R, AD Instruments PowerLab System) and BioAmp Amplifier (ML 136, AD Instruments PowerLab), respectively, as described previously (22, 24, 25). ECG and blood pressure measurements were performed on day 1 (baseline), day 7 (the day of surgery), and day 9 (shortly before killing the animals).
Infarct Size Measurement
The entire ventricular tissue was dissected and cut into four horizontal slices and sections. After incubation in 1% wt/vol 2,3,5- triphenyl tetrazolium chloride solution for 15 min (37°C), the sections were immersed in formalin (4% wt/vol) for another 30 min. After fixing, slices were photographed and captured separately as digital images. Total area and infarct area were determined by computerized planimetry (Scion Image). Percent infarct size was calculated as the infarct area/total left ventricular area (22). The infarct area was corrected for tissue weight, summed for each ventricle, and calculated as the ratio of the weight of the necrotic zone to the whole ventricle.
Cell Viability and Cell Damage Measurement
Cell viability of vascular smooth muscle cells (American Type Culture Collection, Manassas, VA) was determined by measuring exclusion of trypan blue. Cells were detached with 1 ml of trypsin, pelleted by 2,000 g (5 min), and resuspended in DMEM (Sigma). After staining with trypan blue, viable cells in five random fields of view were counted and quantified using a hemocytometer. The percentage of trypan blue exclusive viable cells was determined as a percentage of the total number of cells.
The lactate dehydrogenase (LDH) in vitro toxicology assay kit (TOX-7, Sigma-Aldrich) was further used to measure membrane viability as a function of the amount of cytoplasmic LDH released into the medium. LDH assay mixture (30 µl) was added to sample aliquots (60 µl) taken from the receiver chamber at each sampling point time, followed by mixtures incubated at room temperature for 30 min, 1 M HCl (9 µl) added, and absorbance was measured at 490 nm using a spectrophotometer (TECAN Systems). LDH activity is shown as percentage of LDH release compared with normoxic control.
Measurement of H2S Concentration in Plasma and Culture Medium
H2S concentrations were measured in plasma as described previously (9, 10). Briefly, plasma was collected 48 h after surgery from rat blood before death followed by centrifugation. We modified the methods for measuring in cell culture medium recently. Culture medium was collected from vascular smooth muscle cell flask. Seventy-five microliters of plasma or medium were mixed with 250 µl of 1% (wt/vol) zinc acetate and 425 µl distilled water, depending on the volume of plasma used, in a glass test tube. Then 20 mM N-dimethyl-p-phenylenediamine sulfate in 7.2 mM HCl (133 µl) and 30 mM FeCl3 in 1.2 mM HCl (133 µl) were also added to the test tube for 10-min incubation at room temperature. The protein in the plasma was removed by adding 250 µl of 10% tricholoacetic acid to the reaction mixture and pelleted by centrifugation at 14,000 g (5 min). The absorbance of the resulting solution at 670 nm was measured with a spectrophotometer (TECAN Systems) in a 96-well plate. All samples were assayed in duplicate, and concentration in the solution was calculated against a calibration curve of NaHS (3.125–250 µM). Results show plasma or medium H2S concentration in micromolar.
RNA Extraction and RT-PCR Analysis of CSE mRNA in Ventricular Myocardium
Total RNA from each left ventricle (LV), right ventricle (RV) myocardium, or harvested cells were extracted using the TRIzol reagent as in the manufacturer's instructions (Invitrogen). The concentration of isolated nucleic acid was determined spectrophotometrically by measuring the absorbance at 260 nm.
RT-PCR was performed with QIAGEN OneStep RT-PCR kit (QIAGEN). Each reaction contained 0.5 µg of total RNA as template, 4 µl of 5x RT-PCR buffer, 0.8 µl of dNTP mix (400 µM), 1.2 µl of each primer (0.6 µM), 0.8 µl of enzyme mix (contains Omniscript RT, Sensiscript RT, and HotStarTaq DNA polymerase), and RNase-free water in a final volume of 20 µl. Rat -actin served as an internal control gene. The RT-PCR profile was one cycle of cDNA synthesis at 50°C for 30 min and one cycle of initial PCR activation at 95°C for 15 min, followed by 20 cycles (-actin) or 33 cycles (CSE) of penetration at 94°C for 30 s, annealing at 60°C (-actin) or 61°C (CSE) and extension at 72°C for 35 s (-actin) or 30 s (CSE), followed by one cycle of final extension at 72°C for 10 min. After RT-PCR, aliquots of the RT-PCR products were electrophoresed through 1.2% agarose gels (Bio-Rad, Hercules, CA) containing 0.5 g/ml of ethidium bromide (Bio-Rad) and visualized under ultraviolet light and photographed. Semiquantitative analysis was obtained using Gel analysis software (Syngene). The primer sequences of -actin and CSE were designed to be intron spanning, such that genomic DNA contamination was excluded, and were as follows: -actin (forward) 5'-GGG CTG TAT TCC CCT CCA TC-3', -actin (reverse) 5'-GTC ACG CAC GAT TTC CCT CTC-3'; CSE (forward) 5'-GAC CTC AAT AGT CGG CTT CGT TTC-3', CSE (reverse) 5'-CAG TTC TGC GTA TGC TCC GTA ATG-3'. The RT-PCR product size for -actin and CSE were 552 and 618 bp, respectively.
Morphological Examination and CSE Localization Identification
After dewaxing with serial concentrations of xyline and rehydration with serial concentrations of ethanol (from high to low concentrations gradually), the heart sections for vehicle or PAG pretreatment groups were stained in Gill's hematoxylin for 1 min followed by staining in alcoholic eosin for 30 s, dehydrated with serial concentrations of ethanol (from low to high concentrations gradually), and mounted in Permount (BDH Laboratory Supplies, Poole, UK).
In another experiment, a two-step immunostaining Vectastain ABC kit was employed for detecting the expression of CSE antigens in heart sections. Briefly, after dewaxing and rehydration as described above, sections were treated with 3% (wt/vol) H2O2 for 10 min to quench endogenous peroxidase activity and incubated with normal serum for a further 30 min at room temperature. Sections were then sequentially incubated with 1:1,000 diluted CSE antibody (immunized from an antagonistic piece of rat CSE sequence designed by ourselves), and serum was collected and purified by Biogenes (Berlin, Germany) for 1 h. Biotinylated secondary antibody (Vectastain) was then added for 30 min at room temperature. Slides were subjected to microwave antigen retrieval before each round of immunohistochemistry as specified individually. Colorimetric detection was performed with 0.02% (wt/vol) 3,3'-diaminobenzidine tetrahydrochloride in Tris·HCl buffer. After being washed, sections were mildly counterstained with methyl green and then dehydrated, cleared, and mounted. The specificity of labeling was confirmed by the absence of staining on substitution of sodium phosphorylate buffer or an equal concentration of an irrelevant nonimmune species-matched serum for the primary antibody (1:200), or on omission of secondary antibody (1:200).
Experimental Protocol
Protocol 1. Wistar rats (250–300 g) were randomly assigned into three groups (n = 20–21), namely MI with vehicle, MI with NaHS pretreatment, and MI with PAG (DL-propargylglycine). In brief, rats were injected intraperitoneally either with saline (1 ml·kg–1·day–1), NaHS (14 µmol·kg–1·day–1), or PAG (50 mg·kg–1·day–1) for 7 days before the occlusion of coronary artery to induce MI as we previously described (15, 22, 25). Blood pressure and ECG were measured before the day of experiment initiation, on the day of MI induction, and on the day of death. Surviving rats were killed 48 h after MI induction, and the ventricular myocardium was harvested and separated into LV and RV for total RNA extraction. In a separate experiment for the preparation of paraffin tissue sections, the hearts were perfused with sodium phosphorylate buffer for 20 min, then fixed in paraformaldehyde followed by tissue processing and paraffin embedding, and then sectioned at a thickness of 4 µm.
Protocol 2. Vascular smooth muscle cells were cultured at 37°C in a humidified incubator with 95% air and 5% CO2 in DMEM supplemented with 10% (wt/vol) heat-inactivated FBS (Invitrogen) and 0.4% (wt/vol) Genticin (Invitrogen). The experiments were performed when the cells reached 80–90% confluence. The cells were placed in serum-free DMEM for 48 h. They were then exposed to DMEM supplemented with either no addition (groups 1 and 2), 300 µM NaHS, or 1,000 µM PAG. The concentrations of NaHS and PAG were chosen based on their effective dosages in previous studies (17). After 1 h of treatment, hypoxia was induced by placing treated or nontreated culture into a BBL GasPak Pouch System (Becton Dickinson), which was then tightly sealed and placed back into the incubator for a further 8 h. Pouches contain an iron-based O2-consuming chemical, an O2 indicator strip, and a CO2 generator. The GasPak Pouch System generates a CO2-enriched hypoxic microenvironment with an O2 concentration of <2% and a CO2 concentration of >4% within a 2-h incubation. The cells were used thereafter for viability measurement, LDH assay, or RNA isolation. The concentration of H2S release was detected from culture medium.
Statistics
Data show means ± SE. Statistical analysis was by one-way ANOVA followed by post hoc Tukey's test. A P value of <0.05 was taken to indicate statistical significance. A 
2 test was employed for calculating the significance of mortality.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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To evaluate the effect of H2S on MI, we measured infarct size by triphenyltetrazolium chloride staining (Fig. 1). The infarct size/total area of myocardium was significantly less in rats subjected to NaHS treatment than in vehicle-injected rats (43.4 ± 2.8% vs. 52.9 ± 3.5%; P < 0.05). However, infarct size was greater (62.9 ± 7.6%; P < 0.05) in PAG-treated animals. This result was further confirmed by the ratio of weight of ischemic area vs. weight of whole LV (0.29 ± 0.05 in NaHS-treated animals vs. 0.42 ± 0.06 in vehicle-treated animals; P < 0.05; Fig. 1B).
Hemodynamic Parameters
Hemodynamic measurements of systolic blood pressure in the three groups are shown in Fig. 2. Systolic blood pressure tends to dwindle in all groups after MI induction. However, this change occurred in the NaHS-pretreated group before MI induction (114.8 ± 7.9 mmHg; P < 0.05 vs. vehicle) and reduced more obviously after MI, indicating that NaHS may have an ability to reduce the cardiac postload, especially after MI. Rat heart rate measured from ECG showed an average heart rate of between 440 ± 24.9 and 474 ± 20.0 beats/min in all rats. However, there was no significant difference in heart rate among groups.
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Cell viability measurement demonstrated that cell death was increased under the stimuli of hypoxia (71 ± 1.2% cell viability in hypoxic vehicle vs. 95 ± 2.3% in nonhypoxic control; P < 0.05). The increased cell death was attenuated by the pretreatment of NaHS (85 ± 1.8%), but this effect was abolished significantly by PAG pretreatment (62.2 ± 2.1%) (Fig. 3A).
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Plasma and Culture Medium H2S-Level Measurement
Plasma H2S concentration in the vehicle rats 48 h after MI induction was elevated to 59.2 ± 7.16 µM compared with the baseline value at 38.2 ± 2.07 µM (n = 6; P < 0.05). Plasma H2S concentration was significantly decreased in animals treated with PAG (39.2 ± 5.02 µM; n = 6; P < 0.05) but was significantly increased in animals administered NaHS (92.2 ± 12.40 µM; n = 6; P < 0.05). The plasma H2S concentration in NaHS-treated rats was 
2.4 times higher than PAG-pretreated rats (Fig. 4A). The measurement of H2S content from culture medium is shown in Fig. 4B. H2S content was 81.2 ± 3.2 µM in the culture medium from vehicle and 69.3 ± 1.2 µM in the PAG-treated cells. NaHS-treated cells had the highest H2S content in their culture medium (107.6 ± 5.3 µM).
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To further verify the role of endogenous H2S in myocardium and identify the alternations after ischemic injury, we accessed the expression of CSE mRNA by RT-PCR (Fig. 5) at 618 bp. An isoform (486 bp) of CSE gene was detected as we reported by GenBank (access no. AY641456). The relative amount of CSE mRNA in myocardium of control rats subjected to MI group decreased 2.70-fold compared with that in the sham control group [61.75 ± 3.45 vs. 166.84 ± 1.18 (arbitrary unit, gene expression density) in LV and 66.84 ± 2.36 vs. 147.56 ± 2.36 in RV, respectively; n = 4; P < 0.05], which indicated that CSE gene expression was downregulated in the development of myocardial ischemia.
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Morphological Change and the Localization of CSE in Myocardium
Histologically, at 48 h after MI, the infarcted cardiac muscle fibers exhibited patchy loss or blurring of striation, which were expressed intensely eosinophilic, and most had lost their nuclei (Fig. 6A, left). Nonetheless, there was marked infiltration by neutrophils into the edematous interstitium with the necrotic myocardium undergoing autolysis and fragmentation in the PAG-pretreated myocardium after MI induction (Fig. 6A, right).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Role and Protective Effects of H2S in MI
Beneficial effects were produced by H2S on MI in rats, as seen from a decreased mortality rate of MI rats that underwent NaHS treatment, with only 27.5% of the rats dying after induction of MI. This was approximately three-quarters that of the vehicle group, which had a mortality rate of 35% (Fig. 1). The size of the infarct area is an important gauge of the extent of ischemia as well as remodeling in MI, and thus it is critical for prognosis. Therefore, beneficial effects of H2S were seen again as the infarct size was also diminished in MI rats treated with NaHS, whereas decreased levels of H2S produced by inhibiting CSE with PAG resulted in a larger infarct area (Fig. 1). Administration of PAG caused a significantly higher mortality rate in animals.
Cardiac function was evaluated by looking at the hemodynamic parameters such as blood pressure, ECG, and heart rate. Heart rate was not affected, either before or after MI. Systolic blood pressure was observed to be reduced from the NaHS 7 days after treatment (Fig. 2), which indicated a hypotensive effect of H2S after chronic administration.
H2S has been found to be a potent vasodilator (21), and its inhibition or downregulation should produce the opposite effects. This was indeed the case, as shown by systolic blood pressure measurements in PAG treatment rats (Fig. 2). PAG successfully decreased the levels of H2S in the plasma (Fig. 4A) and in the medium of cultured cells (Fig. 4B), most likely via an irreversible inhibition of its producing enzyme (CSE), and this effectively increased the systolic blood pressure after 7 days of PAG administration in rats.
The possible role of H2S in the pathogenesis of MI has not been reported. This study demonstrates for the first time a role of H2S in a rat model of MI. NaHS significantly decreased the infarct size of the LV and mortality after acute MI in rats. It could be assumed that vessel dilating/relaxing effects of NaHS could dilate coronary arteries and increase coronary blood flow in ischemic diseases, thus reducing cellular damage from ischemia. This result is also consistent with our finding that the NaHS-treated group had a lower mortality rate.
Similar results have been noted in the hypoxic in vitro cell culture model. The cell viability of vehicle-treated hypoxic cells was the lowest, which was correlated with high levels of LDH being detected at the same time. This may offer a probable explanation in that hypoxic cells in vitro, similarly, produce a lower amount of H2S, and thus more cellular damage was inflicted. Expectedly, the addition of NaHS significantly increased the cell viability compared with the cells treated with vehicle in hypoxia. This result was consistent with the LDH assay, where the LDH level was significantly decreased in the NaHS-treated group. NaHS treatment elevated the plasma concentrations of H2S, whereas PAG caused a considerable drop in plasma H2S levels (Fig. 4A). A similar pattern of changes was observed in culture medium of H2S content (Fig. 4B).
Results for the assays for plasma H2S concentration and H2S biosynthesis activity in myocardial tissue, as well as in CSE gene expressions, opened an avenue for understanding the possible role of H2S in ischemic conditions. MI and PAG treatment resulted in a decreased CSE gene expression (Fig. 5). However, Geng et al. (5) reported that upregulated CSE gene expression in an isoproterenol-injected rat heart-induced myocardial ischemic injury model. A possible explanation could be that the animal models were different: surgically induced cardiac ischemia was used in the present study, whereas Geng and colleagues used the model of chemically caused cardiac damage. Another possible reason could be the different timing of tissue collection: our animal hearts were collected 48 h after MI, but Geng and colleagues collected their samples on the last day of isoproterenol treatment. It is well known that most genes (including CSE) are transiently expressed.
In another study, Geng et al. (6) showed that mRNA of CSE was expressed in myocardial tissues and that H2S could endogenously be produced in myocardial tissues. Other than detecting CSE gene expression in heart, we have also localized CSE protein expression by using our own raised CSE antibodies. In the infarct area, obvious increased CSE expression was observed (Fig. 7), which could be a "compensatory effect" to produce H2S to cope with the damage of LV. CSE expression was also found at the area-at-risk beside the necrotic tissue, whereas little staining was seen in cardiomyocytes. The results demonstrated further that production of H2S should be cardioprotective against ischemic insult. Our morphological study confirmed again this statement: more significant autolysis and fragmentation were seen under the microscope in the PAG-pretreated myocardium when compared with NaHS-treated myocardium (Fig. 6).
In conclusion, we demonstrate for the first time that decreases in infarct size and mortality are linked to elevated plasma H2S concentrations after MI, and the opposite, where decreased H2S levels in the plasma were associated with an increased infarct size and mortality. Similar observation were found in our in vitro study whereby higher level H2S content in the culture medium increased cell viability after exposure to hypoxic conditions. Our results suggested a significance of H2S in the reduction of cellular damage inflicted on cells exposed to hypoxia. Exogenously administered H2S via NaHS showed improved conditions in MI rats compared with the vehicle, whereas PAG administration showed worsened conditions. The same was seen in the in vitro study, where inhibition of endogenous H2S had a negative consequence on the cell viability as well as a subsequent increase in LDH production.
Last, this study strengthens the possibility of H2S to be a potent cardioprotecive agent; however, exact mechanisms remain to be further investigated.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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P < 0.05 mortality in NaHS group vs. vehicle and PAG-treated groups. B: comparison of the weight ratio of ischemic area (IA) against whole ventricular myocardium in vehicle-treated rat, in PAG-pretreated rats, and in [left ventricle (LV)] NaHS-pretreated rats. *P < 0.05 vs. PAG-treated group.
NaHS vs. PAG, P < 0.01; n = 12 each. B: the measurement of H2S content from culture medium 8 h after hypoxia. *PAG vs. vehicle, P < 0.05; NaHS vs. vehicle, P < 0.05; 
