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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2015 Jul 1;8(7):7740–7751.

Hydrogen sulfide preconditioning protects against myocardial ischemia/reperfusion injury in rats through inhibition of endo/sarcoplasmic reticulum stress

Changyong Li 1, Min Hu 1, Yuan Wang 1, Huan Lu 1, Jing Deng 1, Xiaohong Yan 1
PMCID: PMC4555667  PMID: 26339339

Abstract

Ischemia reperfusion (I/R) injury is a major cause of myocardial damage. Hydrogen sulfide (H2S), a gaseous signal molecule, has drawn considerable attention for its role in various pathophysiological processes. Multiple lines of evidence reveal the protective effects of H2S in various models of cardiac injury, however, the exact mechanism underlying this protective effect of H2S against myocardial I/R injury is not fully understood. The present study was designed to investigate whether H2S preconditioning attenuates myocardial I/R injury in rats and whether the observed protection is associated with reduced endo/sarcoplasmic reticulum (ER/SR) stress. We found that H2S preconditioning significantly reduced myocardial infarct size, preserved left ventricular function, and inhibited I/R-induced cardiomyocyte apoptosis in vivo. Furthermore, H2S preconditioning significantly attenuated I/R-induced ER/SR stress responses, including the increased expression of glucose-regulated protein 78, C/EBP homologous protein, and activate transcription factor in myocardium. Additionally, we demonstrate that H2S preconditioning attenuates ER/SR stress and inhibits cardiomyocyte apoptosis in an in vitro model of hypoxia/reoxygenation in rat H9c2 cardiac myocytes. In conclusion, these results suggest that H2S-attenuated ER/SR stress plays an important role in its protective effects against I/R-induced myocardial injury.

Keywords: Ischemia/reperfusion, hydrogen sulfide, endo/sarcoplasmic reticulum stress, myocardial protection

Introduction

Myocardial ischemia reperfusion (I/R) injury is the most important cause of cardiac damage. This process is mainly mediated by oxidative stress, calcium dysregulation, and inflammatory cell infiltration in infarcted myocardium [1]. The endo/sarcoplasmic reticulum (ER/SR) is responsible for synthesizing, modifying and folding of proteins, and senses oxidative stress. During ER/SR stress, unfolded proteins accumulate and aggregate during the pathological imbalance in ER/SR homeostasis, which is induced by perturbation of calcium homeostasis, glucose deprivation, and ischemia [2]. When ER/SR stress is intense or persistent, C/EBP homologous protein (CHOP), caspase-12, and JNK are activated, and ER/SR stress-induced apoptosis can be initiated [3]. ER/SR stress has been shown to play an important role in a broad spectrum of pathological conditions [3,4]. Recently, an increasing body of evidence has demonstrated that ER/SR stress was involved in myocardial I/R injury [5-8].

In order to reduce myocardial I/R injury, therapeutic strategies such as pre- and postconditioning, as well as pharmacological interventions have been intensively investigated [9-13]. Hydrogen sulfide (H2S) has become a molecule of great interest in recent years, and it is now recognized as the third endogenously produced gaseous messenger along with nitric oxide and carbon monoxide. Either endogenous or exogenous H2S plays a prominent role in modulating many physiological processes [14]. Several studies have explored the beneficial effects of H2S donors such as NaHS and Na2S in myocardial I/R injury and other models of cardiac injury [14-19]. In addition, a more recent report has demonstrated that gaseous administration of H2S also appears to be an effective way to attenuate the outcome of myocardial I/R injury [20]. However, the mechanisms underlying the protective effect of H2S against myocardial I/R injury are incompletely understood.

Recently, H2S was shown to protect rat H9c2 cardiac myocytes against doxorubicin-induced cardiotoxicity [21] and attenuates hyperhomocysteinemia-induced cardiomyocyte injury [22] through inhibition of ER/SR stress. In addition, Li et al reported that H2S exerts its protection against the neurotoxicity of formaldehyde by overcoming ER/SR stress in PC12 cells [23]. These studies suggest an emerging picture of the importance of H2S in regulating ER/SR stress. In this light, we hypothesized that H2S may protect against myocardial I/R injury by attenuating excessive ER/SR stress. In the present study, we establish that H2S preconditioning decreases infarct size, preserves left ventricular function, and reduces I/R-induced cardiomyocyte apoptosis in an in vivo model of myocardial I/R injury in rats. The observed protection is associated with reduced I/R-induced ER/SR stress responses, including the increased expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and activate transcription factor (ATF6). Additionally, we further demonstrate the cytoprotective effects of H2S in vitro cell culture experiments in rat H9c2 cardiac myocytes exposed to hypoxia and reoxygenation (H/R). Our results suggest that H2S plays an important role in myocardial cytoprotection during the evolution of myocardial infarction, and that H2S administration may be of clinical importance in ischemic disorders.

Materials and methods

Materials

Sodium hydrosulfide (NaHS) was from Sigma-Aldrich (St Louis, MO, USA). GRP78, ATF6, CHOP and PDI antibodies were from Santa Cruz Biotechnology (CA, USA). Enhanced chemiluminescence (ECL) kit was from Amersham Biosciences (Arlington Heights, IL). DMEM and fetal bovine serum (FBS) were from Gibico BRL (Calsbad, CA, USA). TRIzol reagent was from Invitrogen (Carlsbad, CA).

Cell culture and H/R injury

Rat H9c2 cardiac myocytes (Wuhan University Center for Animal Experiment, Wuhan, China) were cultured in DMEM supplemented with 10% FBS. Cells on culture plates were placed into the hypoxia chamber for 3 h to induce hypoxia, and then re-oxygenated with maintenance medium for 24 h to induce reoxygenation.

Myocardial ischemia-reperfusion and H2S treatment

Male adult Sprague-Dawley rats weighing 200-250 g were from Wuhan University Center for Animal Experiment. Surgical procedures used in the I/R were similar to methods described previously [20]. In brief, myocardial I/R injury was performed by temporary ligation of the left anterior descending coronary artery for 30 minutes through an incision in the fourth intercostal space under anesthesia. The heart was inspected for restoration of blood flow after removing the ligature. Sham operated rats underwent the same procedure, except the placement of the ligature. The rats received one time of NaHS (exogenous H2S donor, Sigma, St.Louis, MO) treatment within five seconds, at 1.4, 2.8, and 14 μmol/kg body weight (i.v.), respectively, starting 10 minutes prior to ischemia (n = 10 per group). All animal work were in agreement with institutional and legislator regulations and approved by the Committee on the Ethics of Animal Experiments of Wuhan University.

Myocardial infarct size determination

The infarct size was determined by 1% 2, 3, 5-triphenyltetrazolium chloride (TTC) staining as described previously [24]. In brief, at the end of reperfusion, the hearts were rapidly excised from the thorax and washed by 4°C physiological saline. The left ventricle (LV) was separated from the heart and was weighed, and then frozen for 3 h at -20°C. Then the LV was cut into 5 transverse slices (1-2 mm) and the slices were incubated in 1% TTC (pH 7.4) at 37°C for 10 min. The viable myocardium tissue was stained red while the infracted myocardium remained pale. The pale necrotic myocardial tissue was separated from the stained portions and weighed. The size of the myocardial infarction was determined by the following equation: (Wt-Inf1 + Wt-Inf2 + Wt-Inf3 + Wt-Inf4 + Wt-Inf5)/(Wt-LV1 + Wt-LV2 + Wt-LV3 + Wt-LV4 + Wt-LV5) × 100%, where Wt-Inf is the weight of the infracted myocardium from subscripted numbers 1-5 representing sections and Wt-LV is the weight of LV from the same numbered sections.

Cardiac functional parameters

One catheter was inserted into the left cardiac ventricle via right carotid artery. Left ventricular systolic pressure (LVSP) and maximal rate of increase and decline in left ventricular pressure (LV ± dp/dt max) were monitored continuously during the protocol of IRI with BL-420 multi-channels physiologic signal analysis system (Taimeng Technology Company, Chengdu, China).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for assessment of apoptosis

LV tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Apoptotic cells were identified by TUNEL using an apoptosis detection kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. TUNEL-positive myocytes and total myocytes were counted using Leica Qwin plus V3 software.

Measurement of H2S concentration in plasma

H2S concentration in the plasma was determined by the method described previously [25]. In brief, 0.5 mL of 1% zinc acetate and 2.5 mL of distilled water were mixed with 0.1 mL of plasma. Then 0.5 mL of 20 mmol/L N, N-dimethyl-pphenylenediamine dihydrochloride in 7.2 mol/L HCl and 0.4 mL of 30 mmol/L FeCl3 in 1.2 mol/L HCl were applied for 20 min at room temperature. After adding 1 mL of 10% trichloroacetic acid, the protein in the plasma was removed by centrifugation. The optical absorbance at 670 nm was measured with a spectrophotometer.

Western blot analysis

Western blot analysis of GRP78, CHOP and ATF6 were performed with 10 μg of protein extract, obtained as described previously [26], using rat monoclonal antibodies (1:1000 dilution; Santa Cruz Biotechnology, CA) and peroxidase-conjugated rabbit-anti-rat IgG antibody (1:2000 dilution; Santa Cruz Biotechnology) as a secondary antibody. The signals were normalized to the glyceraldehyde-3-phosphate dehydrogenase signals (rabbit monoclonal antibodies, 1:1,000; Sigma, St. Louis, MO).

Flow cytometry analysis for identification and quantification of cell death

Identification and quantification of cell death in H9c2 cells were determined as described previously [26]. In brief, treated H9c2 cells were digested with trypsin, and washed twice with PBS, and then stained for 30 min with 0.5 mL staining solution consisting of PI (50 mg/mL, Molecular Probes, Eugene, OR), RNase A (10 mg/mL), and 0.1% Triton X-100 for a total count of cell death. The stained cells were subjected to flow cytometric analysis with a FACSCalibur (Becton Dickinson, San Jose, CA). Cell death was quantified as percentage of the sub-G1 peak, an indicator of cell death, in a total of 104 collected counts.

Statistical analysis

The results are expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA. Dunns post-hoc analysis was applied where multiple comparisons were made. P < 0.05 was considered statistically significant.

Results

H2S preconditioning decreased myocardial infarct size and preserves lv function

Rats were subjected to 30 min of LV ischemia and reperfusion for 30, 60, 90 and 120 min, respectively. We found that there were no electrocardiogram (ECG) changes in the sham group, while I/R injury caused marked elevation in ST-segment and T wave (data not shown). In addition, at the end of reperfusion for the indicated periods, the myocardial infarct size was 34%, 30%, 22% and 10%, respectively. These results showed that our in vivo model of myocardial I/R in rats were successful. To observe whether endogenous H2S is involved in the myocardial I/R injury, H2S concentration in the plasma was measured at the end of reperfusion. As shown in Figure 1, H2S concentration was significantly decreased after myocardial I/R injury compared with that in the sham group.

Figure 1.

Figure 1

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H2S concentration in the plasma in rats. Rats were subjected to 30 min of left ventricle ischemia and reperfusion for 120 min, with or without different concentrations of NaHS preconditioning (1.4, 2.8 and 14 μmol/kg). The concentration of H2S was measured from rat plasma as described in Materials and Methods. Data are presented as the mean ± SEM. *P < 0.05, compared with sham-operated mice (n = 10). #P < 0.05, compared with I/R group (n = 8 per group).

To analyze the effect of H2S on protection against myocardial I/R injury, the rats were pretreated with NaHS (an H2S donor) at 1.4, 2.8, and 14 μmol/kg body weight, respectively, starting 10 minutes prior to ischemia. As expected, circulation plasma levels of H2S were significantly elevated following NaHS administration (Figure 1). Evaluation of myocardial infarct size revealed a cardioprotection with NaHS pretreatment, as assessed by TTC staining (Figure 2).

Figure 2.

Figure 2

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Effect of H2S on myocardial infarct size in rats. A. The representative photographs of TTC stained sections. TTC-stained sections were obtained from rat hearts subjected to 30 min of ischemia followed by 120 min of reperfusion, with or without different concentrations of NaHS preconditioning (1.4, 2.8 and 14 μmol/kg). B. Myocardial infarct size was expressed as a percentage of total left ventricle volume. Data are presented as the mean ± SEM. *P < 0.05, compared with I/R-operated rats (n = 8).

Additionally, the effect of H2S on LV function after myocardial I/R injury was determined. Myocardial functional parameters, such as left ventricular systolic pressure (LVSP) and the rate of pressure development (± dp/dt max) were measured at 30 min during ischemia, and 30, 60, 90 and 120 min during reperfusion, respectively. As shown in Figure 3, myocardial I/R caused marked decreases in LVSP, +dp/dt max and -dp/dt max, compared with those in the sham group. However, NaHS pretreatment significantly improved the cardiac contractile function. In addition, there were no significant differences in the above-mentioned parameters between NaHS-treated and vehicle-treated mice in the absence of I/R (data not shown).

Figure 3.

Figure 3

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Effect of H2S on left ventricle function after myocardial I/R injury in rats. Rats were subjected to 30 min of ischemia and reperfusion for 30, 60, 90 and 120 min, respectively, with or without different concentrations of NaHS preconditioning (1.4, 2.8 and 14 μmol/kg). A. LVSP. B. +dp/dtmax. C. -dp/dtmax. Data are presented as the mean ± SEM. *P < 0.05, compared with sham-operated rats (n = 10). #P < 0.05, compared with I/R group (n = 8).

Taken together, these results indicated that H2S preconditioning exerts a protective effect against myocardial I/R injury in rats.

H2S preconditioning reduces I/R-induced cardiomyocyte apoptosis in vivo

To further explore whether H2S has any effect on myocardial I/R-induced apoptosis in rats, we performed TUNEL assay. The number of TUNEL-positive cells in six random fields on the border of the infarcted area per left ventricle was counted and the apoptotic index expressed as a percentage of total cells counted (Figure 4). TUNEL staining showed that I/R increased cardiomyocyte apoptosis; however, NaHS pretreatment significantly reduced I/R-induced apoptosis. In addition, we used an ER/SR stress inhibitor, tauroursodeoxycholic acid (TUDCA) to test whether ER/SR stress is involved in myocardial I/R-induced apoptosis. As shown in Figure 4, TUDCA pretreatment also reduced cardiomyocyte apoptosis induced by I/R. Importantly, the combination of NaHS with TUDCA enhanced the protective effect against myocardial I/R-induced apoptosis.

Figure 4.

Figure 4

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Effect of H2S on I/R-induced myocardial apoptosis in rats. A-E. The representative images of TUNEL staining (original magnification, ×400). Tissue sections were obtained from rat hearts subjected to 30 min of ischemia followed by 120 min of reperfusion, with or without NaHS (14 μmol/kg) and/or TUDCA (an ER stress inhibitor, 25 mg/kg) pretreatment. The nuclei of TUNEL-positive cardiomyocytes were stained brown-yellow (dashed line arrow) and those of normal cells were stained blue (solid arrow). F. TUNEL-positive cells were counted in six random fields in the border of infarcted area of myocardium and expressed as percentage of the total number of nuclei. *P < 0.05, compared with sham-operated rats (n = 10). #P < 0.05, compared with I/R group (n = 8).

H2S attenuates myocardial I/R-induced ER/SR stress in rats

Next, we investigated the mechanism underlying the protective effect of H2S against myocardial I/R injury. We first sought to explore whether myocardial I/R induced ER/SR stress in our model by measuring the expression of protein markers of ER/SR stress such as GRP78, CHOP and ATF6 in myocardium using Western blot analysis. As illustrated in Figure 5, after myocardial I/R, the expression of GRP78, CHOP, and ATF6 were significantly increased. In keeping with the previous studies [5,6], our data indicate that ER/SR stress is involved in myocardial I/R injury in rats. Next, we tested the effect of H2S on myocardial I/R-induced ER/SR stress. As shown in Figure 5, pretreatment with NaHS significantly attenuated the increases in the expression levels of GRP78, CHOP and ATF6 in rats induced by myocardial I/R.

Figure 5.

Figure 5

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Effect of H2S on myocardial I/R-induced ER/SR stress in vivo. Rats were subjected to 30 min of ischemia followed by 120 min of reperfusion, with or without NaHS (14 μmol/kg) and/or TUDCA (25 mg/kg) pretreatment. The protein levels of GRP78 A. ATF6 B, and CHOP C. in myocardium were measured by Western blot analysis. In all blots, staining for GAPDH was used as a loading control. *P < 0.05, compared with sham-operated rats. #P < 0.05, compared with I/R group.

Furthermore, we used TUDCA to confirm that the observed protection is associated with reduced I/R-induced ER/SR stress responses. As shown in Figure 5, pretreatment with TUDCA mimicked the above protective effect of H2S through attenuating ER/SR stress. Taken together, these results suggest that H2S preconditioning protects against myocardial I/R injury in rats by attenuating excessive ER/SR stress.

H2S preconditioning inhibits H/R-induced apoptosis and attenuates ER/SR stress in h9c2 cells

Since ER/SR stress-induced myocardial apoptosis is known to play a significant role in the pathogenesis of myocardial I/R injury [7,8], we analyzed the cytoprotective effect of H2S on H/R-induced apoptosis in rat H9c2 cardiac myocytes in vitro. As shown in Figure 6, NaHS pretreatment markedly reduced H/R-induced H9c2 cell apoptosis, as determined by flow cytometric analysis.

Figure 6.

Figure 6

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Effect of H2S on H/R-induced cell death in rat H9c2 cardiac myocytes. H9c2 cells were exposed to hypoxia/reoxygenation (H/R) with or without different concentrations of NaHS pretreatment (100, 200, 300 and 400 μM). Representative histograms of flow cytometric analysis by propidium iodide (PI) staining were from 3 independent experiments. The peak of sub-G1 fraction (arrow) is an indicator of the total number of dead cells.

To further demonstrate that ER/SR stress was involved in the cytoprotective effect of H2S on H/R-induced apoptosis, we examined the expression of ER/SR stress-associated protein markers, including GRP78, ATF6 and CHOP in vitro. As expected, H/R induced a significant increase of GRP78, ATF6 and CHOP expression in H9c2 cells, and NaHS pretreatment inhibited their expression (Figure 7). CHOP is a critical pro-apoptotic factor in ER/SR stress-associated apoptosis [27,28]. Therefore, these results suggest that suppression of ER/SR stress may contribute to the cytoprotection of H2S against H/R-induced apoptosis in H9c2 Cells.

Figure 7.

Figure 7

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Effect of H2S on H/R-induced ER/SR stress in H9c2 cells. H9c2 cells were exposed to H/R with or without NaHS pretreatment. The levels of GRP78 A. ATF6 B. and CHOP C. were measured by Western blot analysis. In all blots, staining for GAPDH was used as a loading control.

Discussion

Growing evidence suggests that endogenous H2S, as a gas signal molecule, might be an important cardiovascular modulator and thus plays an important role in pathophysiological regulation of cardiovascular diseases [14-19]. However, the exact mechanism underlying this protective effect of H2S is not fully understood. The present study was designed to investigate whether H2S preconditioning attenuates myocardial I/R injury in rats and whether the observed protection is associated with reduced ER/SR stress. The main findings of the present work are the following: i) H2S preconditioning significantly reduces myocardial infarct size, preserves LV function, and reduces I/R-induced cardiomyocyte apoptosis in vivo; ii) H2S attenuates myocardial I/R-induced ER/SR stress; iii) H2S attenuates ER/SR stress and inhibits H/R-induced apoptosis in rat H9c2 cardiac myocytes. These results implicate excessive ER/SR stress inhibition in the protective effect of H2S against myocardial I/R injury.

It has been shown that excessive ER/SR stress is involved in myocardial I/R injury [5-8]. Therefore, regulation of ER/SR stress plays a crucial role in the protective effects of H2S against myocardial I/R injury. H2S is increasingly being recognized as an important gaseous messenger in the cardiovascular and nervous systems. Evidence is accumulating that therapeutic H2S donor compounds exert protective effects in various animal models of inflammation, I/R injury and circulatory shock [29]. Indeed, in our in vivo model of myocardial I/R injury in rats, we found that H2S concentration in plasma was significantly decreased after I/R injury, suggesting that endogenous H2S is associated with the myocardial I/R injury. In agreement with the previous studies [17], our findings further support the notion that H2S is able to exert an effective protection against the injury of hearts subjected to I/R. Elrod and his colleagues reported that H2S limited the extent of myocardial infarction in mice and that the protection was associated with a preservation of mitochondrial function [17]. However, in the present study, we demonstrated that pretreatment of NaHS attenuates the increased expressions of GRP78, CHOP and ATF6 in rats induced by myocardial I/R. It is well documented that ER/SR chaperon GRP78, a critical regulator of ER/SR homeostasis, is generally used as a biomarker of the presence of ER/SR stress [5,7,23]. CHOP, ubiquitously expressed at very low levels in normal cells, is an important mediator for ER/SR stress-induced apoptosis. Induction of CHOP is responsive to ER/SR stress [27]. ATF6, a 670 amino acid ER/SR-transmenbrane protein, is shown to activated in cardiac myocytes by ER/SR stress and is recognized as an important mediator of ER/SR stress [30]. Therefore, our results demonstrate that H2S is able to downregulate the elevated ER/SR stress induced by myocardial I/R. This is in agreement with recent studies that have demonstrated that H2S-suppressed excessive ER/SR stress contributes to its protective effect on doxorubicin-induced cardiotoxicity [21], hyperhomocysteinemia-induced cardiomyocytic injury [22], 6-hydroxydopamine-induced neurotoxicity [31], and formaldehyde-induced neurotoxicity [23]. These previous reports and our findings have shown that either exogenous or endogenous increases in H2S exert protective effect in various models of cardiac injury or neurotoxicity through inhibition of ER/SR stress.

It should be noted that, in the heart, ER/SR is an important organelle specializing in the regulation of Ca2+ fluxes in addition to protein synthesis functions. Multiple lines of evidence have demonstrated that disruption of the Ca2+ homeostasis in the ER/SR is a potent trigger of ER/SR stress, and that ER/SR stress sensor, BiP/GRP78, plays an important role as a Ca2+ buffer in the lumen of ER/SR [32]. In addition, recent studies have indicated that H2S and nitric oxide (NO), these two gaseous molecules may have overlapping pathophysiological functions [33]. Moreover, numerous reports suggest that H2S-NO cross-talk mediates their effects on several organ systems, including cardiovascular, immune and neurological tissues [33-36]. These findings led us to put forward the possibility that NO was also involved in H2S-mediated cardioprotection in myocardial I/R injury in rats. Thus, further studies will be needed to investigate the association between ER/SR stress and intracellular Ca2+ after H2S treatment, and to explore H2S-NO chemical interactions in I/R models.

Apoptosis of cardiomyocytes has been shown to play an important role in the development of myocardial I/R injury [37]. Furthermore, several reports have demonstrated that ER/SR stress-induced myocardial apoptosis plays a significant role in the pathogenesis of myocardial I/R injury [7,38]. One of the components of the ER/SR stress-mediated apoptosis pathway is CHOP, also known as growth arrest-and DNA damage-inducible gene 153 (GADD153). CHOP-mediated apoptosis is implicated in the pathophysiology of cardiovascular diseases [27,28]. In the present study, our results suggest that administration of H2S inhibits ER/SR stress-induced cardiomyocyte apoptosis both in vivo and in vitro. Additionally, we found that H2S attenuates H/R-induced ER/SR stress in rat H9c2 cardiac myocytes, as demonstrated by down-regulation of GRP78, ATF6 and CHOP expression. Several lines of evidence indicate that there are a number of potential mechanisms through which H2S may exert cardioprotective effects, including KATP channels [39], regulation of mitochondrial function [17], cytoprotective gene Nrf-2 signaling [40], reduction of myocardial ROS production and inhibition of inflammation, necrosis and fibrogenesis [20,41], mTORC2 phosphorylation of Akt1 [42], and upregulation of endothelial nitric oxide synthase [43,44]. Here, our results suggest that the cardioprotective effects of H2S against myocardial I/R injury in rats can be attributed, at least in part, to inhibition of ER/SR stress-induced apoptosis. Thus, studies conducted by our group and by other investigators have indicated that the pathways implicated in the cardioprotective action of H2S are multiple. Further insights into the mechanistic details of the protective effects of H2S in various models of cardiac injury are necessary to understand the pathophysiological actions of this intriguing gaseous mediator.

In conclusion, our findings indicate that H2S preconditioning could ameliorate myocardial I/R injury by attenuating excessive ER/SR stress and inhibiting myocardial apoptosis. Our study suggests a promising future of H2S based preventions and therapies for myocardial I/R injury.

Acknowledgements

This work was supported by grant from the National Natural and Science Foundation of China (No. 31071005).

Disclosure of conflict of interest

None.

References

  • 1.Yellon DM, Hausenloy DJ. Mechanisms of disease: Myocardial reperfusion injury. New Engl J Med. 2007;357:1121–1135. doi: 10.1056/NEJMra071667. [DOI] [PubMed] [Google Scholar]
  • 2.Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest. 2002;110:1389–1398. doi: 10.1172/JCI16886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ. 2006;13:363–373. doi: 10.1038/sj.cdd.4401817. [DOI] [PubMed] [Google Scholar]
  • 4.Kim I, Xu WJ, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 2008;7:1013–1030. doi: 10.1038/nrd2755. [DOI] [PubMed] [Google Scholar]
  • 5.Wang XB, Huang XM, Ochs T, Li XY, Jin HF, Tang CS, Du JB. Effect of sulfur dioxide preconditioning on rat myocardial ischemia/reperfusion injury by inducing endoplasmic reticulum stress. Basic Res Cardiol. 2011;106:865–878. doi: 10.1007/s00395-011-0176-x. [DOI] [PubMed] [Google Scholar]
  • 6.Liu XH, Zhang ZY, Sun S, Wu XD. Ischemic postconditioning protects myocardium from ischemia/reperfusion injury through attenuating endoplasmic reticulum stress. Shock. 2008;30:422–427. doi: 10.1097/SHK.0b013e318164ca29. [DOI] [PubMed] [Google Scholar]
  • 7.Martindale JJ, Fernandez R, Thuerauf D, Whittaker R, Gude N, Sussman MA, Glembotski CC. Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6. Circulation Res. 2006;98:1186–1193. doi: 10.1161/01.RES.0000220643.65941.8d. [DOI] [PubMed] [Google Scholar]
  • 8.Natarajan R, Salloum FN, Fisher BJ, Smithson L, Almenara J, Fowler AA. Prolyl hydroxylase inhibition attenuates post-ischemic cardiac injury via induction of endoplasmic reticulum stress genes. Vascul Pharmacol. 2009;51:110–118. doi: 10.1016/j.vph.2009.05.007. [DOI] [PubMed] [Google Scholar]
  • 9.Wojtovich AP, Brookes PS. The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial K-ATP channels. Basic Res Cardiol. 2009;104:121–129. doi: 10.1007/s00395-009-0001-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Zhao HX, Wang XL, Wang YH, Wu Y, Li XY, Lv XP, Zhao ZQ, Zhao RR, Liu HR. Attenuation of myocardial injury by postconditioning: role of hypoxia inducible factor-1 alpha. Basic Res Cardiol. 2010;105:109–118. doi: 10.1007/s00395-009-0044-0. [DOI] [PubMed] [Google Scholar]
  • 11.Ovize M, Baxter GF, Di Lisa F, Ferdinandy P, Garcia-Dorado D, Hausenloy DJ, Heusch G, Vinten-Johansen J, Yellon DM, Schulz R. Postconditioning and protection from reperfusion injury: where do we stand? Position Paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology. Cardiovasc Res. 2010;87:406–423. doi: 10.1093/cvr/cvq129. [DOI] [PubMed] [Google Scholar]
  • 12.Penna C, Tullio F, Perrelli MG, Moro F, Abbadessa G, Piccione F, Carriero V, Racca S, Pagliaro P. Ischemia/reperfusion injury is increased and cardioprotection by a postconditioning protocol is lost as cardiac hypertrophy develops in nandrolone treated rats. Basic Res Cardiol. 2011;106:409–420. doi: 10.1007/s00395-010-0143-y. [DOI] [PubMed] [Google Scholar]
  • 13.Dongo E, Hornyak I, Benko Z, Kiss L. The cardioprotective potential of hydrogen sulfide in myocardial ischemia/reperfusion injury (Review) Acta Physiol Hung. 2011;98:369–381. doi: 10.1556/APhysiol.98.2011.4.1. [DOI] [PubMed] [Google Scholar]
  • 14.Bos EM, van Goor H, Joles JA, Whiteman M, Leuvenink HG. Hydrogen sulfide-physiological properties and therapeutic potential in ischaemia. Br J Pharmacol. 2015;172:1479–1493. doi: 10.1111/bph.12869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang QQ, Fu HL, Zhang H, Xu FY, Zou Z, Liu M, Wang QX, Miao MY, Shi XY. Hydrogen Sulfide Preconditioning Protects Rat Liver against Ischemia/Reperfusion Injury by Activating Akt-GSK-3 beta Signaling and Inhibiting Mitochondrial Permeability Transition. PLos One. 2013;8:e74422. doi: 10.1371/journal.pone.0074422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Calvert JW, Elston M, Nicholson CK, Gundewar S, Jha S, Elrod JW, Ramachandran A, Lefer DJ. Genetic and Pharmacologic Hydrogen Sulfide Therapy Attenuates Ischemia-Induced Heart Failure in Mice. Circulation. 2010;122:11–19. doi: 10.1161/CIRCULATIONAHA.109.920991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, Jiao XY, Scalia R, Kiss L, Szabo C, Kimura H, Chow CW, Lefer DJ. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function. Proc Natl Acad Sci U S A. 2007;104:15560–15565. doi: 10.1073/pnas.0705891104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Minamishima S, Bougaki M, Sips PY, De Yu J, Minamishima YA, Elrod JW, Lefer DJ, Bloch KD, Ichinose F. Hydrogen Sulfide Improves Survival After Cardiac Arrest and Cardiopulmonary Resuscitation via a Nitric Oxide Synthase 3-Dependent Mechanism in Mice. Circulation. 2009;120:888–896. doi: 10.1161/CIRCULATIONAHA.108.833491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Szabo G, Veres G, Radovits T, Gero D, Modis K, Miesel-Groschel C, Horkay F, Karck M, Szabo C. Cardioprotective effects of hydrogen sulfide. Nitric Oxide. 2011;25:201–210. doi: 10.1016/j.niox.2010.11.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Snijder PM, de Boer RA, Bos EM, van den Born JC, Ruifrok WP, Vreeswijk-Baudoin I, van Dijk MC, Hillebrands JL, Leuvenink HG, van Goor H. Gaseous hydrogen sulfide protects against myocardial ischemia-reperfusion injury in mice partially independent from hypometabolism. PLoS One. 2013;8:e63291. doi: 10.1371/journal.pone.0063291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Wang XY, Yang CT, Zheng DD, Mo LQ, Lan AP, Yang ZL, Hu F, Chen PX, Liao XX, Feng JQ. Hydrogen sulfide protects H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of endoplasmic reticulum stress. Mol Cell Biochem. 2012;363:419–426. doi: 10.1007/s11010-011-1194-6. [DOI] [PubMed] [Google Scholar]
  • 22.Wei HL, Zhang RY, Jin HF, Liu DE, Tang XY, Tang CS, Du JB. Hydrogen Sulfide Attenuates Hyperhomocysteinemia-Induced Cardiomyocytic Endoplasmic Reticulum Stress in Rats. Antioxid Redox Signal. 2010;12:1079–1091. doi: 10.1089/ars.2009.2898. [DOI] [PubMed] [Google Scholar]
  • 23.Li X, Zhang KY, Zhang P, Chen X, Wang L, Xie M, Wang CY, Tang XQ. Hydrogen Sulfide Inhibits Formaldehyde-Induced Endoplasmic Reticulum Stress in PC12 Cells by Upregulation of SIRT-1. PLos One. 2014;9:e89856. doi: 10.1371/journal.pone.0089856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Rao PR, Kumar VK, Viswanath RK, Subbaraju GV. Cardioprotective activity of alcoholic extract of Tinospora cordifolia in ischemia-reperfusion induced myocardial infarction in rats. Biol Pharm Bull. 2005;28:2319–2322. doi: 10.1248/bpb.28.2319. [DOI] [PubMed] [Google Scholar]
  • 25.Chunyu Z, Junbao D, Dingfang B, Hui Y, Xiuying T, Chaoshu T. The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochem Biophys Res Commun. 2003;302:810–816. doi: 10.1016/s0006-291x(03)00256-0. [DOI] [PubMed] [Google Scholar]
  • 26.Yan XH, Lin L, Pan YX, Lu H, Rong WF, Lu Y, Ren AJ, Tang CS, Yuan WJ. Salusins protect neonatal rat cardiomyocytes from serum deprivation-induced cell death through upregulation of GRP78. J Cardiovasc Pharmacol. 2006;48:41–46. doi: 10.1097/01.fjc.0000242059.89430.ac. [DOI] [PubMed] [Google Scholar]
  • 27.Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ. 2004;11:381–389. doi: 10.1038/sj.cdd.4401373. [DOI] [PubMed] [Google Scholar]
  • 28.Fu HY, Okada K, Liao YL, Tsukamoto O, Isomura T, Asai M, Sawada T, Okuda K, Asano Y, Sanada S, Asanuma H, Asakura M, Takashima S, Komuro I, Kitakaze M, Minamino T. Ablation of C/EBP Homologous Protein Attenuates Endoplasmic Reticulum-Mediated Apoptosis and Cardiac Dysfunction Induced by Pressure Overload. Circulation. 2010;122:361–369. doi: 10.1161/CIRCULATIONAHA.109.917914. [DOI] [PubMed] [Google Scholar]
  • 29.Szabo C. Hydrogen sulphide and its therapeutic potential. Nat Rev Drug Discov. 2007;6:917–935. doi: 10.1038/nrd2425. [DOI] [PubMed] [Google Scholar]
  • 30.Thuerauf DJ, Hoover H, Meller J, Hernandez J, Su L, Andrews C, Dillmann WH, McDonough PM, Glembotski CC. Sarco/endoplasmic reticulum calcium ATPase-2 expression is regulated by ATF6 during the endoplasmic reticulum stress response: intracellular signaling of calcium stress in a cardiac myocyte model system. J Biol Chem. 2001;276:48309–48317. doi: 10.1074/jbc.M107146200. [DOI] [PubMed] [Google Scholar]
  • 31.Xie L, Tiong CX, Bian JS. Hydrogen sulfide protects SH-SY5Y cells against 6-hydroxydopamine-induced endoplasmic reticulum stress. Am J Physiol Cell Physiol. 2012;303:C81–91. doi: 10.1152/ajpcell.00281.2011. [DOI] [PubMed] [Google Scholar]
  • 32.Groenendyk J, Sreenivasaiah PK, Kim DH, Agellon LB, Michalak M. Biology of Endoplasmic Reticulum Stress in the Heart. Circulation Res. 2010;107:1185–1197. doi: 10.1161/CIRCRESAHA.110.227033. [DOI] [PubMed] [Google Scholar]
  • 33.Kolluru GK, Shen X, Kevil CG. A tale of two gases: NO and H2S, foes or friends for life? Redox Biol. 2013;1:313–8. doi: 10.1016/j.redox.2013.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Predmore BL, Kondo K, Bhushan S, Zlatopolsky MA, King AL, Aragon JP, Grinsfelder DB, Condit ME, Lefer DJ. The polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability. Am J Physiol Heart Circ Physiol. 2012;302:H2410–H2418. doi: 10.1152/ajpheart.00044.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Bir SC, Kolluru GK, McCarthy P, Shen XG, Pardue S, Pattillo CB, Kevil CG. Hydrogen Sulfide Stimulates Ischemic Vascular Remodeling Through Nitric Oxide Synthase and Nitrite Reduction Activity Regulating Hypoxia-Inducible Factor-1 alpha and Vascular Endothelial Growth Factor-Dependent Angiogenesis. J Am Heart Assoc. 2012;1:e004093. doi: 10.1161/JAHA.112.004093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Coletta C, Papapetropoulos A, Erdelyi K, Olah G, Modis K, Panopoulos P, Asimakopoulou A, Gero D, Sharina I, Martin E, Szabo C. Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation. Proc Natl Acad Sci U S A. 2012;109:9161–9166. doi: 10.1073/pnas.1202916109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Freude B, Masters TN, Robicsek F, Fokin A, Kostin S, Zimmermann R, Ullmann C, Lorenz-Meyer S, Schaper J. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J Mol Cell Cardiol. 2000;32:197–208. doi: 10.1006/jmcc.1999.1066. [DOI] [PubMed] [Google Scholar]
  • 38.Yu L, Lu MM, Wang P, Chen X. Trichostatin A Ameliorates Myocardial Ischemia/Reperfusion Injury Through Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis. Arch Med Res. 2012;43:190–196. doi: 10.1016/j.arcmed.2012.04.007. [DOI] [PubMed] [Google Scholar]
  • 39.Zhang Z, Huang H, Liu P, Tang C, Wang J. Hydrogen sulfide contributes to cardioprotection during ischemia-reperfusion injury by opening K-ATP channels. Can J Physiol Pharmacol. 2007;85:1248–1253. doi: 10.1139/Y07-120. [DOI] [PubMed] [Google Scholar]
  • 40.Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, Kevil CG, Lefer DJ. Hydrogen Sulfide Mediates Cardioprotection Through Nrf2 Signaling. Circulation Res. 2009;105:365–374. doi: 10.1161/CIRCRESAHA.109.199919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Sun WH, Liu F, Chen Y, Zhu YC. Hydrogen sulfide decreases the levels of ROS by inhibiting mitochondrial complex IV and increasing SOD activities in cardiomyocytes under ischemia/reperfusion. Biochem Biophys Res Commun. 2012;421:164–169. doi: 10.1016/j.bbrc.2012.03.121. [DOI] [PubMed] [Google Scholar]
  • 42.Zhou Y, Wang DY, Gao XF, Lew K, Richards AM, Wang PP. mTORC2 Phosphorylation of Akt1: A Possible Mechanism for Hydrogen Sulfide-Induced Cardioprotection. PLos One. 2014;9:e99665. doi: 10.1371/journal.pone.0099665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Kondo K, Bhushan S, King AL, Prabhu SD, Hamid T, Koenig S, Murohara T, Predmore BL, Gojon G, Gojon G, Wang R, Karusula N, Nicholson CK, Calvert JW, Lefer DJ. H2S Protects Against Pressure Overload-Induced Heart Failure via Upregulation of Endothelial Nitric Oxide Synthase. Circulation. 2013;127:1116–1127. doi: 10.1161/CIRCULATIONAHA.112.000855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.King AL, Polhemus DJ, Bhushan S, Otsuka H, Kondo K, Nicholson CK, Bradley JM, Islam KN, Calvert JW, Tao YX, Dugas TR, Kelley EE, Elrod JW, Huang PL, Wang R, Lefer DJ. Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent. Proc Natl Acad Sci U S A. 2014;111:3182–3187. doi: 10.1073/pnas.1321871111. [DOI] [PMC free article] [PubMed] [Google Scholar]

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