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The recent emergence of hydrogen sulfide (H2S) as a potent cardioprotective signaling molecule necessitates the elucidation of its cytoprotective mechanisms.
The current study evaluated potential mechanisms of H2S-mediated cardioprotection using an in vivo model of pharmacological preconditioning (PC).
H2S (100 μg/kg), or vehicle was administered to mice via an intravenous injection 24 hr prior to myocardial ischemia. Treated and untreated mice were then subjected to 45 min of myocardial ischemia followed by reperfusion for up to 24 hr, during which time the extent of myocardial infarction was evaluated, circulating troponin-I levels were measured, and the degree of oxidative stress was evaluated. In separate studies myocardial tissue was collected from treated and untreated mice during the early (30 min and 2hr) and late (24 hr) PC periods to evaluate potential cellular targets of H2S. Initial studies revealed that H2S provided profound protection against ischemic injury as evidenced by significant decreases in infarct size, circulating troponin-I levels, and oxidative stress. During the early PC period H2S increased the nuclear localization of Nrf2, a transcription factor that regulates the gene expression of a number of antioxidants, and increased the phosphorylation of PKCε and STAT-3. During the late PC period, H2S increased the expression of antioxidants (HO-1 and Trx1), increased the expression of HSP90, HSP70, Bcl-2, Bcl-xL, and COX-2 and also inactivated the pro-apoptogen Bad.
These results reveal that the cardioprotective effects of H2S are mediated in large part by a combination of antioxidant and anti-apoptotic signaling.
Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with a diverse physiological profile. Its production in mammalian systems has been attributed to two key enzymes in the cysteine biosynthesis pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). The rate of H2S production in tissue homogenates is in the range of 1–10 pmoles per second per mg protein, resulting in low micromolar extracellular concentrations.1,2 It is at these physiological concentrations that H2S is cytoprotective in various models of cellular injury.3,4 The reported cytoprotective effects of H2S are partially related to its ability to neutralize reactive oxygen species (ROS), to inhibit leukocyte-endothelial cell interactions, to promote vascular smooth muscle relaxation, to reduce apoptotic signaling, and to reversibly modulate mitochondrial respiration.5 Pretreatment with NaHS has been reported to reduce the number and duration of arrhythmias in isolated hearts subjected to global ischemia-reperfusion (I/R)6 and to enhanced the viability of isolated rat ventricular myocytes exposed to glucose deprivation and 2-deoxy-glucose.4 Recently, Elrod et al7 reported that the administration of H2S at the time of reperfusion decreased infarct size and preserved left ventricular function in an in vivo model of myocardial I/R. Additional findings from this study also demonstrated that cardiac-specific overexpression of CGL likewise limited the extent of myocardial I/R injury. The findings of these studies and others suggest that H2S is cytoprotective during myocardial I/R injury and that either direct H2S administration or the modulation of endogenous H2S production may be of clinical importance.
Although the physiological and cardioprotective effects of H2S have previously been documented, the signaling mechanisms that mediate these effects have not been fully evaluated. Moreover, the signaling mechanisms that have been attributed to the cardioprotective effects of H2S have predominantly been studied in in vitro model systems with very few studies actually exploring the protective effects in in vivo systems. Therefore, the purpose of the current study was to evaluate signaling mechanisms triggered by H2S treatment using an in vivo model of pharmacological preconditioning (PC). This model was chosen as a model for which early and delayed cellular targets responsible for H2S-mediated cardioprotection could be identified. Additionally, a cardiac-specific transgenic mouse that overexpresses CGL was utilized to evaluate signaling mechanisms mediated by endogenous H2S.
Male C57BL6/J mice, 8–10 weeks of age, were utilized (Jackson Labs, Bar Harbor, ME). The generation of cardiac-specific transgenic mice overexpressing CGL (αMHC-CGL-Tg; FVB background)7, as well as the generation of Nrf2 deficient mice (Nrf2 KO; ICR background)8 has been described previously. All experimental mouse procedures were approved by the Institute for Animal Care and Use Committee at Albert Einstein College of Medicine and Emory University and conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 86-23, Revised 1996) and with federal and state regulations.
H2S was administered in the form of sodium sulfide (Na2S). Na2S was produced by Ikaria Holdings Inc. (Seattle, WA) using H2S gas (Matheson, Newark, CA) as a starting material and was formulated to pH neutrality, and iso-osmolarity. Na2S (stock solution at 0.55 mg/ml and 7.1 mM) was diluted in normal (0.9%) saline to the desired concentration in a rapid fashion, immediately before administration For all experiments, normal saline (100 μL) or Na2S (100 μg/kg) in a final volume of 100 μL was injected intravenously into the femoral vein using a 32-gauge needle. For this study, H2S denotes Na2S.
Surgical ligation of the left coronary artery (LCA), myocardial infarct size determination, Troponin-I measurements and LV echocardiography was performed similar to methods described previously.9
Cardiac glutathione and lipid hydroperoxides were measured in heart tissue collected from mice subjected to 45 min of myocardial ischemia and 1, 4, or 24 hr of reperfusion using commercially available kits (Cayman Chemicals) as previously described.3 GSH and GSSG values were used to calculate the steady-state redox potential using the Nernst equation, as described previously.10
For the evaluation of cellular targets during early and late phase preconditioning, mice were administered H2S as described above and then sacrificed 30 min, 2 hr, and 24 hr after the injection. The hearts were rapidly excised and the LV was isolated and snap frozen in liquid nitrogen. The samples were then stored at −80°C. In a separate group of mice, heart samples were obtained following 45 min of myocardial ischemia and 4 hr of reperfusion.
Whole cell, cytosolic, membranous, nuclear, and mitochondrial fractions were prepared as described previously.11 Equal amounts of protein were loaded into lanes of polyacrylamide-SDS gels and Western blot analysis was performed as previously described.9
Following 45 min of ischemia and 4 hr of reperfusion hearts from sham, vehicle, and H2S PC treated mice were rapidly excised, cross-sectioned into 3 sections and fixed in 10% buffered formalin. Fixed tissue was then paraffin embedded and sectioned in a standard fashion. TUNEL staining was conducted using a kit according to the manufacturer's instructions (ApopTag HRP kit, DBA). The number of TUNEL-positive nuclei and the total number of nuclei per high-powered field were counted in a minimum of 10 fields from the area-at-risk portion of the myocardium for each section (a total of 30 fields per heart).
All data in this study are expressed as mean ± standard error (SEM). Differences in data between the groups were compared using Prism 4 (GraphPad Software, Inc) with Student's paired 2-tailed t test, one-way analysis of variance (ANOVA) with post hoc Tukey test, or two-way ANOVA with post hoc Bonferroni analysis where appropriate. A p value less than 0.05 was considered significant.
Initial experiments were conducted to investigate if H2S PC could limit myocardial I/R injury, For these experiments, mice were subjected to 45 min of LCA ischemia followed by 24 hr of reperfusion. H2S (Na2S; 100 μg/kg) or vehicle was administered 24 hr prior to ischemia via an intravenous injection. The extent of myocardial infarction was then evaluated at 24 hr of reperfusion. Representative photomicrographs of mid-ventricular cross sections of TTC-stained hearts taken from vehicle and H2S PC treated mice are shown in Figure 1A. The area-at-risk (AAR) per LV was similar (p=not significant, NS) in all of the groups (Figure 1B). H2S PC decreased the infarct (Inf) relative to the AAR (Inf/AAR) by 46% (48±3 vs. 26±3%, p<0.001) and the Inf relative to the entire LV (Inf/LV) by 48% (29±2.5 vs. 15±2%, p<0.001) when compared to vehicle treated mice. Circulating levels of troponin-I were evaluated as an additional marker of myocardial injury at 24 hr of reperfusion (Figure 1C). Following myocardial I/R, circulating levels of troponin-I rose from undetectable levels in sham operated mice to 75±9 ng/mL in the vehicle treated group (p<0.001 vs. Sham). H2S PC significantly (p=0.026) attenuated the rise in circulating troponin-I by 69% (75±9 vs. 23±7 ng/mL), thus confirming the cardioprotective effects of H2S PC.
The effects of H2S PC on LV structure and function following myocardial I/R were evaluated in separate groups of mice using in vivo transthoracic echocardiography. For these experiments, mice were subjected to 45 min of myocardial ischemia and 7 days of reperfusion. Myocardial I/R increased left ventricular end-diastolic diameter (LVEDD, p=NS) and LV end-systolic diameter (LVESD, p<0.05) in both groups compared to their respective baseline (BASE) readings (Online Figure). However, the increase in LVEDD (p=0.12 vs. I/R+Veh) and LVESD (p=0.03 vs. I/R+Veh) was attenuated in mice pretreated with H2S. Following myocardial I/R, LV ejection fraction (Figure 1D) decreased in both groups (p<0.001 vs. BASE). H2S PC did, however, significantly improve ejection fraction by 87% (p=0.006 vs. I/R+Veh). Together, these results suggest that H2S PC limits the extent of damage to the myocardium following I/R injury.
There is considerable evidence that implicates the production of ROS as an initial cause of injury to the myocardium following I/R.12 To evaluate the effects of H2S PC on cellular oxidative stress, lipid hydroperoxide levels were measured in tissue isolated from the hearts of vehicle and H2S PC treated mice. In response to myocardial I/R injury, the redox potential (Figure 2A, Eh) significantly increased in the hearts of vehicle treated mice. This change in redox potential was accompanied by a significant rise in lipid hydroperoxide levels (Figure 2B, p<0.001 vs. Sham). In contrast, the redox potential was preserved in the H2S PC treated hearts (p<0.05 vs. I/R+Veh) and a significant less rise in lipid hydroperoxide levels was also evident (p<0.05 vs. I/R+Veh). To investigate the effects of H2S PC on apoptosis, the expression of uncleaved caspase-3, cleaved caspase-3 and cytochrome C, as well as the extent of TUNEL-positive staining was evaluated after 45 min of LCA and 4 hr of reperfusion in heart samples from sham, vehicle, and H2S PC-treated mice (Figure 2C–F). Myocardial I/R induced a significant decrease in the expression of uncleaved caspase-3 and induced a significant increase in the expression of cleaved caspase-3 as well as induced the translocation of cytochrome C from the mitochondria to the cytosol in the hearts of vehicle-treated mice (p<0.01 vs. Sham). This was accompanied by a significant increase in the number of TUNEL-positive nuclei (p<0.001 vs. Sham). In contrast, the hearts of H2S PC-treated mice when compared to vehicle treated hearts exhibited a significant preservation of uncleaved caspase-3, a significant reduction in cleaved caspase-3 (p<0.01 vs. I/R+Veh), a significant reduction in the translocation of cytochrome C (p<0.05 vs. I/R+Veh), and fewer TUNEL-positive nuclei (p<0.001 vs. I/R+Veh).
Nuclear-factor-E2-related factor (Nrf2) is a key transcription factor that regulates antioxidant genes as an adaptive response to oxidative stress or pharmacological stimuli. To investigate if H2S induced Nrf2 signaling, Na2S was administered to mice via an intravenous injection and heart tissue was excised at different times following this administration. As early as 30 min following the administration of H2S, Nrf2 accumulated in the nucleus of cardiac tissue and remained at an elevated level for at least 2 hr (Figure 3A–B). Subsequently, the protein expression (Figure 3C–D) of two downstream targets of Nrf2, Thioredoxin-1 (Trx1) and heme oxygenase-1 (HO-1), were elevated 24 hr following the administration H2S. H2S did not increase the protein expression of either CuZnSOD or MnSOD (Figure 3C–D) at this latter time point.
To determine if H2S upregulated HO-1 and Trx1 in an Nrf2-dependent manner, subsequent experiments were performed with mice deficient in Nrf2 (Nrf2 KO). For these experiments, H2S was administered as noted above and heart tissue was excised 24 hr later (Figure 4A–B). H2S slightly increased the expression of Trx1 (p<0.05 vs Sham), but failed to increase the expression of HO-1 in the hearts of Nrf2 KO mice. Subsequent experiments were then conducted to determine if Nrf2 was critical for the cardioprotective actions of H2S PC. For these experiments, H2S (Na2S; 100 μg/kg) or vehicle was administered 24 hr prior to ischemia via an intravenous injection to Nrf2 KO mice and Wild-Type littermates. H2S PC reduced Inf/AAR and reduced circulating troponin-I levels when it was administered to Wild-Type mice. Myocardial injury following I/R was found to be exacerbated in Nrf2 KO mice compared with Wild-Type mice (Figure 4C–D), as evidenced by an increase in Inf/AAR (44±5 vs. 25±2, p<0.01) and circulating troponin-I levels (52.4±9.0 vs. 26±5.2, p<0.01). H2S failed to provide protection in the Nrf2 KO mice, suggesting that Nrf2 plays a role in the cardioprotection actions mediated by H2S. The smaller infarct size reported here for the Wild-Type mice in these experiments as compared to the size reported in Figure 1 is indicative of the background strain of these mice (ICR), as past studies have reported the different susceptibility of various strains of mice to myocardial injury.9,13
H2S has been reported to induce PKC activation in isolated rat cardiomyocytes.14 To investigate if H2S induced PKCε activation in vivo, the translocation of PKCε from the cytosolic fraction to the membranous fraction was evaluated after a single administration of H2S. A marked increase in the phosphorylated form of PKCε at serine residue 729 (PKCε–PSer729) was evident in the membranous fraction (p<0.05 vs. Sham, Figure 5A) 30 min and 2 hr following the administration of H2S. The total membrane levels of PKCε were also increased during this time period (p<0.05 vs. Sham,), indicating translocation of PKCε to the membrane. In addition, H2S increased the phosphorylation of p44/42, a downstream effector of PKCε (p<0.05 vs. Sham, Figure 5B). H2S did not change the total levels of PKCε or alter the phosphorylation state PKCε in the cytosolic fraction and did not change the total levels of p44/42.
The activation of pro-survival kinases, such as PKCε and p44/42, has been demonstrated to confer cardioprotection through an upregulation of anti-apoptotic signaling mediated in part by signal transducers and activators of transcription-3 (STAT-3).15 In the present study, H2S increased the translocation of STAT-3 to the nucleus (Figure 5C), as evidenced by a decrease in the total cytosolic levels of STAT-3 (p<0.01 vs. Sham) and an increase in the total nuclear levels of STAT-3 (p<0.05 vs. Sham) from 30 min to 2 hr after its administration. A marked increase in the phosphorylated form of STAT-3 (p<0.01 and p<0.05 vs. Sham) at serine residue 727 (STAT-3PSer727) was also evident in both the cytosolic fraction and nuclear fraction of H2S treated hearts during this time period. H2S also increased the phosphorylation of Bad (Figure 6A–B) at serine residue 112 (Bad-PSer112) in both the mitochondrial (p=NS) and the cytosolic fraction (p<0.05 vs. Sham) 24 hr after its administration. As a result of this phosphorylation, H2S induced the translocation of Bad from the mitochondria to the cytosol, as evidenced by a significant increase in the total cytosolic levels of Bad (p<0.05 vs. Sham). Further analysis revealed that H2S increased the protein expression of HSP90, HSP70, Bcl-2, Bcl-xL, and cyclooxygenase-2 (COX-2) (Figure 6C–D, p<0.05 and p<0.01 vs. Sham) 24 hr after its administration. These data suggest that in addition to promoting antioxidants, H2S also increased anti-apoptogens.
Previously, the cardiac-specific overexpression of CGL was shown to increase H2S production in the heart and reduce the degree of injury following myocardial I/R.7 Therefore, the next series of experiments were conducted to evaluate if endogenous H2S activated the same cardioprotective signaling pathways as exogenous H2S. The overexpression of CGL resulted in the accumulation of Nrf2 in the nucleus of hearts isolated from transgenic mice (p<0.01 vs. Non-Tg, Figure 7A). Although there was a trend for a decline, cytosolic Nrf2 levels were not significantly different between the groups of mice. A marked increase in PKCε–PSer729 was evident in both the cytosolic (p<0.05 vs. Non-Tg, Figure 7B) and membranous fractions (p<0.01 vs. Non-Tg) of hearts isolated from CGL-Tg+ mice. The total membrane levels of PKCε were also increased in the hearts of CGL-Tg+ mice (p<0.05 vs. Non-Tg), indicating translocation of PKCε to the membrane. CGL overexpression also induced the translocation of STAT-3 to the nucleus, as evidenced by an increase in the total nuclear levels of STAT-3 (p<0.01 vs. Non-Tg, Figure 7C). A marked increase in STAT-3PSer727 was also evident in the cytosolic (p<0.01 vs. Non-Tg) and nuclear fractions (p<0.05 vs. Non-Tg) of CGL-Tg+ hearts. Further analysis revealed that hearts from CGL-Tg+ mice have increased protein expressions of HSP90, HO-1, Trx-1, and Bcl-2 (p<0.05 and p<0.01 vs. Non-Tg, Figure 7D).
As a gaseous signaling molecule, H2S is able to freely diffuse across cell membranes in a receptor-independent manner and activate various cellular targets. This distinct ability makes H2S an attractive pharmacological agent for the treatment of cardiovascular disease. The current study highlights two signaling cascades which lead to the upregulation of antioxidants and anti-apoptogens and provides evidence that pharmacological preconditioning with H2S results in profound protection against myocardial I/R injury, as evidenced by a significant decrease in infarct size and a preservation of LV geometry and cardiac function.
Under physiological conditions, small amounts of ROS produced in cells are quenched by cellular antioxidant defense systems. Antioxidants act by scavenging oxidative species and their precursors, inhibiting their formation and enhancing endogenous antioxidant defenses.16 There is considerable evidence that implicates the production of ROS and subsequent related cellular damage as an initial cause of injury to the myocardium following I/R injury.12 Therefore, the capacity of cardiac myocytes to maintain homeostasis during periods of oxidative stress resides in the ability to activate or induce protective enzymes.17 However, during I/R the activity of many of the endogenous antioxidant enzyme systems are compromised or even abolished18, suggesting that increasing the activity of cellular antioxidant enzymes may protect tissues against reperfusion damage.19 Previous studies have reported that H2S protects various cell types, including myocytes20 from oxidative stress. There is some debate, however, regarding the nature in which H2S reduces oxidative stress. H2S acts as a direct scavenger of ROS and up-regulates endogenous antioxidant defenses. Kimura and colleagues21 demonstrated that H2S protects neurons from cell death by increasing GSH levels through an enhancement of γ-glutamylcysteine synthetase activity and an up-regulation of cystine transport. A major finding of the current study supports the latter notion of H2S inducing a signaling mechanism to combat oxidative stress, as evidenced by the ability of H2S to up-regulate cellular antioxidants in the heart in a Nrf2-dependent manner. Nrf2, a member of the NF-E2 family of nuclear basic leucine zipper transcription factors, regulates the gene expression of a number of enzymes that serve to detoxify pro-oxidative stressors.22 This regulation is mediated by Nrf2 binding to the antioxidant responsive element (ARE), a cis-acting regulatory element or enhancer sequence, found in the promoter region of certain genes, including HO-1 and Trx1. In the current study, H2S was shown for the first time to induce the nuclear accumulation of Nrf2 very rapidly after its administration and to subsequently increase the protein expression of HO-1 and Trx1. Additionally, this is the first study to report that Nrf2 deficient mice experienced an exacerbated injury in response to myocardial I/R, suggesting that Nrf2 is an important endogenous cardioprotective signal that protects against oxidant-mediated injury. This is further supported by previous reports indicating that Nrf2 deficiency is associated with enhanced oxidative stress and cell death.23,24
In the current study, H2S modestly increased the expression of Trx1, but failed to increase the expression of HO-1 in the hearts of Nrf2 KO mice. This is an important finding because the increase in Trx1 was found to be only 58% of the increase observed in wild-type mice (1.4±0.1 vs. 3.3±0.7), suggesting that H2S upregulates HO-1 in an Nrf2-dependent manner, but only partially upregulates Trx1 in an Nrf2-dependent manner, suggesting that H2S regulates Trx1 expression through an additional unidentified mechanism. Interestingly, the modest increase in Trx1 levels in the Nrf2 KO hearts was not sufficient to induce protection, as H2S failed to reduce myocardial injury following I/R in these mice. Our data indicate that Nrf2 plays an important role in mediating the cardioprotective effects of H2S and provides important evidence linking Nrf2 and its downstream effectors to the antioxidant effects of H2S. Moreover, these results suggest that H2S therapy enhances the endogenous antioxidant defenses of myocytes and create an environment resistant to the oxidative stress associated with myocardial I/R injury as evidenced by the preservation of redox state and a reduction in lipid peroxidation.
Another major finding of the current study relates to H2S-medaited activation of a PKCε-STAT-3 signaling cascade. Although, there is in vitro evidence in the literature suggesting that H2S activates PKC14, the current study is the first to report that H2S activates PKCε in vivo. The activation of this pro-survival signaling cascade has previously been shown to confer cardioprotection against myocardial I/R through an inhibition of apoptotic cell death25, an activation of COX-2, and has been shown to play a prominent role in the cardioprotective signaling of ischemic PC.26 The anti-apoptotic actions of this pathway are mediated in part by the phosphorylation and inhibition of the pro-apoptotic factor Bad27, an upregulation of the pro-survival factors Bcl-2 and Bcl-xL28, and an upregulation of HSPs.29 p44/42 promotes the phosphorylation of Bad through the actions of p90RSK, whereas it upregulates Bcl-2, Bcl-xL, and HSPs through STAT-3. The STAT pathway has recently been shown to be an integral part of the response of the myocardium to various cardiac insults, including myocardial infarction.30 In particular, the overexpression of STAT-3 results in cardioprotection31, whereas cardiac-specific deficiency of STAT-3 exacerbates cardiac injury.32 HSPs have also been demonstrated to provide cardioprotection in the setting of I/R.33 In particular, HSP70 suppresses apoptosis in a caspase-dependent34 and caspase-independent manner.35 The findings of the current study suggest that H2S therapy does not simply reduce apoptotic cell death following myocardial I/R through a reduction in oxidative stress, but actually promotes direct anti-apoptotic signaling.
The activation of multiple pathways by H2S in the current study highlights the diversity of this gasotransmitter. Unlike other pharmacological agents that rely on receptor-mediated signaling, H2S activates multiple pathways simultaneously. In addition to the pathways studied here, H2S could promote cardioprotection via the activation of KATP channels and by inhibiting leukocyte-endothelial interactions and subsequent inflammation.2 An important question that remains unanswered relates to the mechanism by which H2S induces the nuclear accumulation of Nrf2. Under basal conditions, Kelch ECH associating protein 1 (Keap1) represses the ability of Nrf2 to induce endogenous antioxidants by binding very tightly to Nrf2, anchoring it in the cytoplasm, and targeting it for ubiquitination and proteasome degradation.36 Only when this association is disrupted can Nrf2 translocate to the nucleus and bind AREs. It is thought that several critical cysteine residues in Keap1 serve as the primary sensor for stress signals (i.e. ROS) and that their modification leads to conformational changes in Keap1, which results in the release of Nrf2.36 In addition to Keap1 being a target, Nrf2 can also be directly modified by kinases, such as PKC, to induce its release.37 Whether H2S alters Keap1 and/or Nrf2 directly or through upstream signaling (i.e. PKC) remains an important unanswered question that requires further study.
Together, the findings of the current study indicate that the cardioprotective effects of H2S are mediated in large part by a combination of antioxidant and anti-apoptotic signaling and highlight a novel signaling cascade involving Nrf2. Furthermore, this study suggests that either the administration of H2S donors or the modulation of the endogenous production of H2S may be of therapeutic benefit in the setting of myocardial I/R.
The authors would like to thank D.B. Grinsfelder and M. Elston for invaluable technical expertise in conducting these experiments.
Sources of funding Supported by grants from the NIH (2 RO1 HL-060849-09 and 5R01 HL-092141-01) and the American Diabetes Association (7-04-RA-59) to D.J.L and by a grant from the NIH (F32 DK 077380-01) to J.W.C. as well as funds from the Carlyle Fraser Heart Center of Emory University Hospital Midtown.
Disclosures Ikaria Holdings, Inc provided the Na2S.