Hydrogen Sulfide Mediates Cardioprotection Through Nrf2 Signaling John W. Calvert, Saurabh Jha, Susheel Gundewar, John W. Elrod, Arun Ramachandran, Christopher B. Pattillo, Christopher G. Kevil, David J. Lefer Rationale: The recent emergence of hydrogen sulfide (H2S) as a potent cardioprotective signaling molecule necessitates the elucidation of its cytoprotective mechanisms. Objective: The present study evaluated potential mechanisms of H2S-mediated cardioprotection using an in vivo model of pharmacological preconditioning. Methods and Results: H2S (100 ␮g/kg) or vehicle was administered to mice via an intravenous injection 24 hours before myocardial ischemia. Treated and untreated mice were then subjected to 45 minutes of myocardial ischemia followed by reperfusion for up to 24 hours, 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 minutes and 2 hours) and late (24 hours) preconditioning 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 preconditioning 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 protein kinase C␧ and STAT-3. During the late preconditioning period, H2S increased the expression of antioxidants (heme oxygenase-1 and thioredoxin 1), increased the expression of heat shock protein 90, heat shock protein 70, Bcl-2, Bcl-xL, and cyclooxygenase-2 and also inactivated the proapoptogen Bad. Conclusions: These results reveal that the cardioprotective effects of H2S are mediated in large part by a combination of antioxidant and antiapoptotic signaling. (Circ Res. 2009;105:365-374.) Key Words: hydrogen sulfide 䡲 cardioprotection 䡲 antioxidant signaling 䡲 myocardial infarction 䡲 Nrf2 H ydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with a diverse physiological profile. Its production in mammalian systems has been attributed to 2 key enzymes in the cysteine biosynthesis pathway, cystathionine ␤-synthase (CBS) and cystathionine ␥-lyase (CGL). The rate of H2S production in tissue homogenates is in the range of 1 to 10 pmol per second per milligram of 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-deoxyglucose.4 Recently, Elrod et al7 reported that the administration of H2S at the time of reperfusion decreased infarct (INF) size and preserved left ventricular (LV) 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 mech- Original received April 25, 2009; revision received July 5, 2009; accepted July 6, 2009. From the Department of Surgery (J.W.C., D.J.L.), Division of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, Ga; Department of Medicine (S.J., S.G., A.R.), Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY; Department of Molecular Cardiovascular Biology (J.W.E.), Cincinnati Children’s Hospital Medical Center, Ohio; and Department of Pathology (C.B.P., C.G.K.), Louisiana Statue University Health Sciences Center, Shreveport. Correspondence to David J. Lefer, PhD, Department of Surgery, Division of Cardiothoracic Surgery, Emory University School of Medicine, 550 Peachtree St NE, Atlanta, GA 30308. E-mail dlefer@emory.edu © 2009 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.109.199919 365 Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 366 Circulation Research August 14, 2009 Non-standard Abbreviations and Acronyms AAR CBS CGL Eh GSH GSSG H2 S HO HSP INF I/R Keap1 KO LCA LV Na2S Non-Tg Nrf2 PC PKC ROS STAT-3 Tg Trx Veh area at risk cystathionine ␤-synthase cystathionine ␥-lyase redox potential glutathione glutathione disulfide hydrogen sulfide heme oxygenase heat shock protein infarct ischemia/reperfusion Kelch ECH associating protein 1 knockout left coronary artery left ventricular sodium sulfide nontransgenic nuclear factor-E2–related factor preconditioning protein kinase C reactive oxygen species signal transducers and activators of transcription 3 transgenic thioredoxin vehicle anisms 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 present 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 cardiacspecific transgenic mouse that overexpresses CGL was used to evaluate signaling mechanisms mediated by endogenous H2S. Methods An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Animals Male C57BL6/J mice, 8 to 10 weeks of age, were used (The Jackson Laboratory, Bar Harbor, Me). The generation of cardiac-specific transgenic mice overexpressing CGL (␣MHC-CGL-Tg; FVB background),7 as well as the generation of Nrf2-deficient mice (Nrf2 knockout [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. Materials H2S was administered in the form of sodium sulfide (Na2S). Na2S was produced by Ikaria Holdings Inc (Seattle, Wash) using H2S gas (Matheson, Newark, Calif) as a starting material and was formulated to pH neutrality, and isoosmolarity. Na2S (stock solution at 0.55 mg/mL and 7.1 mmol/L) was diluted in normal (0.9%) saline to the desired concentration in a rapid fashion, immediately before administration. For all experiments, normal saline (100 ␮L) or Na2S (100 ␮g/kg) in a final volume of 100 ␮L was injected intravenously into the femoral vein using a 32-gauge needle. For this study, H2S denotes Na2S. Myocardial I/R Protocol and Myocardial Injury Assessment Surgical ligation of the left coronary artery (LCA), myocardial INF size determination, troponin I measurements, and LV echocardiography were performed similarly to methods described previously.9 Glutathione and Lipid Hydroperoxide Assays Cardiac glutathione and lipid hydroperoxides were measured in heart tissue collected from mice subjected to 45 minutes of myocardial ischemia and 1, 4, or 24 hours of reperfusion using commercially available kits (Cayman Chemicals) as previously described.3 Glutathione (GSH) and GSH disulfide (GSSG) values were used to calculate the steady-state redox potential using the Nernst equation, as described previously.10 Tissue Collection for Western Blot Analysis For the evaluation of cellular targets during early and late phase preconditioning, mice were administered H2S as described above and then euthanized 30 minutes, 2 hours, and 24 hours 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 minutes of myocardial ischemia and 4 hours of reperfusion. Western Blot Analysis Whole cell, cytosolic, membranous, nuclear, and mitochondrial fractions were prepared as described previously.11 Equal amounts of protein were loaded into lanes of polyacrylamide–sodium dodecyl sulfate gels and Western blot analysis was performed as previously described.9 TUNEL Staining Following 45 minutes of ischemia and 4 hours of reperfusion hearts from sham-, vehicle (Veh)-, 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 instructions of the manufacturer (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 (AAR) portion of the myocardium for each section (a total of 30 fields per heart). Statistical Analysis All data in this study are expressed as means⫾SEM. Differences in data between the groups were compared using Prism 4 (GraphPad Software Inc), with Student’s paired 2-tailed t test, 1-way ANOVA with post hoc Tukey test, or 2-way ANOVA with post hoc Bonferroni analysis where appropriate. A probability value of ⬍0.05 was considered significant. Results H2S PC Limited the Extent of Myocardial Injury Following I/R Initial experiments were conducted to investigate if H2S PC could limit myocardial I/R injury, For these experiments, Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Calvert et al H2S Protects via Nrf2 367 Figure 1. H2S PC reduced the extent of injury and improved LV function in mice following myocardial ischemia and reperfusion. A, Representative midventricular photomicrographs of hearts treated with Veh and H2S. B, AAR with respect to the left ventricle (LV) was similar between all groups. H2S PC significantly attenuated myocardial INF size with respect to the AAR (INF/AAR) and with respective to the left ventricle (INF/LV). C, Circulating levels of troponin I (ng/mL) were measured 24 hours after reperfusion. D, Ejection fraction (%) was calculated in separate groups of mice using high-resolution, 2D B-mode echocardiography images at baseline (BASE) and 7 days following myocardial ischemia (POST). Values are means⫾SEM. Numbers inside bars indicate the number of animals that were investigated in each group. ***P⬍0.001 vs sham or baseline. H2S denotes Na2S. mice were subjected to 45 minutes of LCA ischemia followed by 24 hours of reperfusion. H2S (Na2S; 100 ␮g/kg) or Veh was administered 24 hours before ischemia via an intravenous injection. The extent of myocardial infarction was then evaluated at 24 hours of reperfusion. Representative photomicrographs of midventricular cross sections of 2,3,5-triphenyltetrazolium chloride–stained hearts taken from Veh- and H2S PC–treated mice are shown in Figure 1A. The AAR per LV was similar (P⫽NS) in all of the groups (Figure 1B). H2S PC decreased the INF relative to the AAR (INF/AAR) by 46% (48⫾3 versus 26⫾3%, P⬍0.001) and the INF relative to the entire LV (INF/LV) by 48% (29⫾2.5 versus 15⫾2%, P⬍0.001) when compared to Veh-treated mice. Circulating levels of troponin I were evaluated as an additional marker of myocardial injury at 24 hours 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 Veh-treated group (P⬍0.001 versus sham). H2S PC significantly (P⫽0.026) attenuated the rise in circulating troponin I by 69% (75⫾9 versus 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 minutes of myocardial ischemia and 7 days of reperfusion. Myocardial I/R increased LV end-diastolic diameter (P⫽NS) and LV end-systolic diameter (LVESD, P⬍0.05) in both groups compared to their respective baseline readings (Online Figure I). However, the increase in LV end-diastolic diameter (P⫽0.12 versus I/R⫹Veh) and LVESD (P⫽0.03 versus 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 versus baseline). H2S PC did, however, significantly improve ejection fraction by 87% (P⫽0.006 versus I/R⫹Veh). Together, these results suggest that H2S PC limits the extent of damage to the myocardium following I/R injury. H2S PC Reduced Oxidative Stress and Apoptosis Following I/R 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 Veh and H2S PC treated mice. In response to myocardial I/R injury, the redox potential (Eh) (Figure 2A) significantly increased in the hearts of Veh-treated mice. This change in redox potential was accom- Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 368 Circulation Research August 14, 2009 Figure 2. H2S PC reduced oxidative stress and apoptotic cell death following myocardial ischemia and reperfusion. Cardiac redox state (Eh) for GSH and GSSG (A) and lipid hydroperoxide levels (␮mol/L) (B) from sham controls and Veh- and H2S PC–treated mice at 1 to 24 hours of reperfusion following myocardial ischemia. C, Representative immunoblots of uncleaved caspase-3, cleaved caspase-3, cytosolic cytochrome c, and mitochondrial cytochrome c from the hearts of sham controls and Veh- and H2S PC–treated mice at 4 hours of reperfusion following myocardial ischemia. COX indicates cytochrome c oxidase. D, Ratio of cleaved caspase-3 to uncleaved caspase-3. E, Ratio of cytosolic cytochrome c to mitochondrial cytochrome c. F, Number of TUNEL-positive cells (percentage of total nuclei) from the hearts of sham controls and Veh- and H2S PC–treated mice at 4 hours of reperfusion following myocardial ischemia. Values are means⫾SEM. Numbers inside bars indicate the number of animals that were investigated in each group. *P⬍0.05, **P⬍0.01, ***P⬍0.001 vs sham. panied by a significant rise in lipid hydroperoxide levels (Figure 2B, P⬍0.001 versus sham). In contrast, the redox potential was preserved in the H2S PC treated hearts (P⬍0.05 versus I/R⫹Veh) and a significant less rise in lipid hydroperoxide levels was also evident (P⬍0.05 versus 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 minutes of LCA and 4 hours of reperfusion in heart samples from sham-, Veh-, and H2S PC–treated mice (Figure 2C through 2F). Myocardial I/R induced a significant decrease in the expression of uncleaved caspase-3 and in- duced 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 Veh-treated mice (P⬍0.01 versus sham). This was accompanied by a significant increase in the number of TUNELpositive nuclei (P⬍0.001 versus sham). In contrast, the hearts of H2S PC-treated mice when compared to Veh-treated hearts exhibited a significant preservation of uncleaved caspase-3, a significant reduction in cleaved caspase-3 (P⬍0.01 versus I/R⫹Veh), a significant reduction in the translocation of cytochrome c (P⬍0.05 versus I/R⫹Veh), and fewer TUNELpositive nuclei (P⬍0.001 versus I/R⫹Veh). Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Calvert et al H2S Protects via Nrf2 369 Figure 3. H2S upregulated cellular antioxidant defenses. A and B, Representative immunoblots (A) and densitometric analysis (B) of cardiac Nrf2 in the cytosolic and nuclear fractions 30 minutes and 2 hours following the administration of H2S. C and D, Representative immunoblots (C) and densitometric analysis (D) of cardiac Trx1, HO-1, copper zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) 24 hours following the administration of H2S. Values are mean⫾SEM for n⫽4 to 5 animals for each group. *P⬍0.05, **P⬍0.01 vs sham. H2S Increased the Nuclear Accumulation of Nrf2 and Increased the Protein Expression of Thioredoxin and Heme Oxygenase-1 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 whether H2S induced Nrf2 signaling, H2S was administered to mice via an intravenous injection and heart tissue was excised at different times following this administration. As early as 30 minutes following the administration of H2S, Nrf2 accumulated in the nucleus of cardiac tissue and remained at an elevated level for at least 2 hours (Figure 3A and 3B). Subsequently, the protein expression (Figure 3C and 3D) of 2 downstream targets of Nrf2, thioredoxin (Trx)1 and heme oxygenase (HO)-1, were elevated 24 hours following the administration H2S. H2S did not increase the protein expression of either copper zinc superoxide dismutase or manganese superoxide dismutase (Figure 3C and 3D) at this latter time point. To determine whether 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 hours later (Figure 4A and 4B). H2S slightly increased the expression of Trx1 (P⬍0.05 versus sham) but failed to increase the expression of HO-1 in the hearts of Nrf2 KO mice. Subsequent experiments were then conducted to determine whether Nrf2 was critical for the cardioprotective actions of H2S PC. For these experiments, H2S (Na2S; 100 ␮g/kg) or Veh was administered 24 hours before 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 and 4D), as evidenced by an increase in INF/AAR (44⫾5 versus 25⫾2, P⬍0.01) and circulating troponin I levels (52.4⫾9.0 versus 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 INF 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), because past studies have reported the different susceptibility of various strains of mice to myocardial injury.9,13 H2S Activated a Protein Kinase C␧-p44/42-STAT-3 Prosurvival Signaling Pathway H2S has been reported to induce protein kinase (PK)C activation in isolated rat cardiomyocytes.14 To investigate whether H2S induced PKC␧ activation in vivo, the translocation of PKC␧ from the cytosolic fraction to the membranous fraction was evaluated after a single administration of H2S. A marked increase in the phosphorylated form of PKC␧ at serine residue 729 (PKC␧-PSer729) was evident in the membranous fraction (P⬍0.05 versus sham, Figure 5A) 30 minutes and 2 hours following the administration of H2S. The total membrane levels of PKC␧ were also increased during this time period (P⬍0.05 versus sham,), indicating translocation of PKC␧ to the membrane. In addition, H2S increased the phosphorylation of p44/42, a downstream effector of PKC␧ (P⬍0.05 versus sham, Figure 5B). H2S did not change the total levels of PKC␧ or alter the phosphorylation state PKC␧ in the cytosolic fraction and did not change the total levels of p44/42. The activation of prosurvival kinases, such as PKC␧ and p44/42, has been demonstrated to confer cardioprotection Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 370 Circulation Research August 14, 2009 Figure 4. Nrf2 mediates the cardioprotective effects of H2S. A and B, Representative immunoblots (A) and densitometric analysis (B) of cardiac Trx1 and HO-1 from the hearts of Nrf2 deficient (Nrf2 KO) mice with or without H2S. C and D, Myocardial INF size (C) and circulating levels of troponin I (D) were measured 24 hours after LCA ischemia in wild-type (WT) and Nrf2 KO mice receiving either Veh or H2S PC (100 ␮g/kg) treatment. Nrf2 KO mice experienced exacerbated myocardial injury when compared to wild-type mice. However, no differences in myocardial INF size or circulating troponin I levels were observed in Nrf2 KO following H2S PC treatment. Numbers inside bars indicate the number of animals that were investigated in each group. *P⬍0.05, **P⬍0.01 vs sham or WT. through an upregulation of antiapoptotic 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 versus sham) and an increase in the total nuclear levels of STAT-3 (P⬍0.05 versus sham) from 30 minutes to 2 hours after its administration. A marked increase in the phosphorylated form of STAT-3 (P⬍0.01 and P⬍0.05 versus sham) at serine residue 727 (STAT-3PSer727) was also evident in both Figure 5. H2S activated PKC␧-p/44/42-STAT-3 signaling. Representative immunoblots and densitometric analysis of phosphorylated PKC␧ at serine residue 729 (PKC␧Ser729) and total PKC␧ (cytosolic and membranous fractions) (A), phosphorylated p44/42 and total p44/42 (cytosolic fraction) (B), and phosphorylated STAT-3 at serine residue 727 (STAT-3Ser727) and total STAT-3 (cytosolic and nuclear fractions) (C) 30 minutes and 2 hours following the administration of H2S. Values are means⫾SEM for n⫽4 to 5 animals for each group. *P⬍0.05, **P⬍0.01 vs sham. Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Calvert et al H2S Protects via Nrf2 371 Figure 6. H2S increased the expression of antiapoptogens. A and B, Representative immunoblots and densitometric analysis of phosphorylated Bad at serine residue 112 (Bad-PSer112) and total Bad (cytosolic and mitochondrial fractions). C and D, HSP90, HSP70, Bcl-2, Bcl-xL, and cyclooxygenase-2 (COX-2) 24 hours following the administration of H2S. Values are means⫾SEM for n⫽4 to 5 animals for each group. *P⬍0.05, **P⬍0.01 vs sham. the cytosolic fraction and nuclear fraction of H2S-treated hearts during this time period. H2S also increased the phosphorylation of Bad (Figure 6A and 6B) at serine residue 112 (Bad-PSer112) in both the mitochondrial (P⫽NS) and the cytosolic fraction (P⬍0.05 versus sham) 24 hours 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 versus sham). Further analysis revealed that H2S increased the protein expression of heat shock protein (HSP)90, HSP70, Bcl-2, Bcl-xL, and cyclooxygenase-2 (Figure 6C and 6D, P⬍0.05 and P⬍0.01 versus sham) 24 hours after its administration. These data suggest that in addition to promoting antioxidants, H2S also increased antiapoptogens. The Overexpression of CGL Increased Nrf2 Nuclear Accumulation and Activated PKC␧, p44/42, and STAT-3 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 versus nontransgenic [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 PKC␧PSer729 was evident in both the cytosolic (P⬍0.05 versus Non-Tg, Figure 7B) and membranous fractions (P⬍0.01 versus Non-Tg) of hearts isolated from CGL-Tg⫹ mice. The total membrane levels of PKC␧ were also increased in the hearts of CGL-Tg⫹ mice (P⬍0.05 versus Non-Tg), indicating translocation of PKC␧ 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 versus Non-Tg, Figure 7C). A marked increase in STAT-3PSer727 was also evident in the cytosolic (P⬍0.01 versus Non-Tg) and nuclear fractions (P⬍0.05 versus 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 versus Non-Tg, Figure 7D). Discussion 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 present study highlights 2 signaling cascades which lead to the upregulation of antioxidants and antiapoptogens and provides evidence that pharmacological preconditioning with H2S results in profound protection against myocardial I/R injury, as evidenced by a significant Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 372 Circulation Research August 14, 2009 Figure 7. Cardiac-specific overexpression of CGL increased Nrf2 nuclear accumulation and PKC␧-STAT-3 signaling. Representative immunoblots and densitometric analysis of Nrf2 (cytosolic and nuclear fractions) (A), phosphorylated PKC␧Ser729 and total PKC␧ (cytosolic and membranous fractions) (B), phosphorylated STAT-3Ser727 and total STAT-3 (cytosolic and nuclear fractions) (C), Trx1, HO-1, HSP90, and Bcl-2 (D) from the hearts of CGL-Tg⫹ (n⫽4) and Non-Tg (n⫽4) mice. Values are means⫾SEM *P⬍0.05, **P⬍0.01 vs Non-Tg. decrease in INF 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 Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Calvert et al activate or induce protective enzymes.17 However, during I/R the activity of many of the endogenous antioxidant enzyme systems are compromised or even abolished,18 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 myocytes,20 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 upregulates endogenous antioxidant defenses. Kimura and Kimura21 demonstrated that H2S protects neurons from cell death by increasing GSH levels through an enhancement of ␥-glutamylcysteine synthetase activity and an upregulation of cystine transport. A major finding of the present study supports the latter notion of H2S inducing a signaling mechanism to combat oxidative stress, as evidenced by the ability of H2S to upregulate 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 prooxidative stressors.22 This regulation is mediated by Nrf2 binding to the antioxidant responsive element, a cis-acting regulatory element or enhancer sequence found in the promoter region of certain genes, including HO-1 and Trx1. In the present 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 present 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 versus 3.3⫾0.7), suggesting that H2S upregulates HO-1 in an Nrf2dependent manner but only partially upregulates Trx1 in an Nrf2-dependent manner. This indicates 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, because 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 present study relates to H2S-medaited activation of a PKC␧-STAT-3 signaling cascade. Although, there is in vitro evidence in the literature suggesting that H2S activates PKC,14 the present study is the H2S Protects via Nrf2 373 first to report that H2S activates PKC␧ in vivo. The activation of this prosurvival signaling cascade has previously been shown to confer cardioprotection against myocardial I/R through inhibition of apoptotic cell death25 and activation of cyclooxygenase-2 and has been shown to play a prominent role in the cardioprotective signaling of ischemic PC.26 The antiapoptotic actions of this pathway are mediated in part by the phosphorylation and inhibition of the proapoptotic factor Bad,27 upregulation of the prosurvival factors Bcl-2 and Bcl-xL,28 and 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 cardioprotection,31 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 present 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 antiapoptotic signaling. The activation of multiple pathways by H2S in the present 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, Keap1 (Kelch ECH associating protein 1) 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 antioxidant responsive elements. It is thought that several critical cysteine residues in Keap1 serve as the primary sensor for stress signals (ie, 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 (ie, PKC) remains an important unanswered question that requires further study. Together, the findings of the present study indicate that the cardioprotective effects of H2S are mediated in large part by a combination of antioxidant and antiapoptotic 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. Acknowledgments We thank D. B. Grinsfelder and M. Elston for invaluable technical expertise in conducting these experiments. Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 374 Circulation Research August 14, 2009 Sources of Funding Supported by grants from the NIH (2 R01 HL-060849-09 and 5R01 HL– 092141-01) and the American Diabetes Association (7-04-RA59) 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. References 1. Doeller JE, Isbell TS, Benavides G, Koenitzer J, Patel H, Patel RP, Lancaster JR Jr, Darley-Usmar VM, Kraus DW. Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues. Anal Biochem. 2005;341:40 –51. 2. Szabo C. Hydrogen sulphide and its therapeutic potential. Nat Rev Drug Discov. 2007;6:917–935. 3. Jha S, Calvert JW, Duranski MR, Ramachandran A, Lefer DJ. Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling. Am J Physiol Heart Circ Physiol. 2008;295: H801–H806. 4. Pan TT, Feng ZN, Lee SW, Moore PK, Bian JS. Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes. J Mol Cell Cardiol. 2006; 40:119 –130. 5. Lefer DJ. A new gaseous signaling molecule emerges: cardioprotective role of hydrogen sulfide. Proc Natl Acad Sci U S A. 2007;104: 17907–17908. 6. Bian JS, Yong QC, Pan TT, Feng ZN, Ali MY, Zhou S, Moore PK. Role of hydrogen sulfide in the cardioprotection caused by ischemic preconditioning in the rat heart and cardiac myocytes. J Pharmacol Exp Ther. 2006;316:670 – 678. 7. Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, Jiao X, 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. 8. Prince M, Li Y, Childers A, Itoh K, Yamamoto M, Kleiner HE. Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice. Toxicol Lett. 2009;185:180 –186. 9. Calvert JW, Gundewar S, Jha S, Greer JJ, Bestermann WH, Tian R, Lefer DJ. Acute metformin therapy confers cardioprotection against myocardial infarction via AMPK-eNOS-mediated signaling. Diabetes. 2008;57: 696 –705. 10. Go YM, Ziegler TR, Johnson JM, Gu L, Hansen JM, Jones DP. Selective protection of nuclear thioredoxin-1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells. Free Radic Biol Med. 2007;42:363–370. 11. Hunter JC, Kostyak JC, Novotny JL, Simpson AM, Korzick DH. Estrogen deficiency decreases ischemic tolerance in the aged rat heart: roles of PKCdelta, PKCepsilon, Akt, and GSK3beta. Am J Physiol Regul Integr Comp Physiol. 2007;292:R800 –R809. 12. Venardos KM, Perkins A, Headrick J, Kaye DM. Myocardial ischemiareperfusion injury, antioxidant enzyme systems, and selenium: a review. Curr Med Chem. 2007;14:1539 –1549. 13. Yamamoto M, Yang G, Hong C, Liu J, Holle E, Yu X, Wagner T, Vatner SF, Sadoshima J. Inhibition of endogenous thioredoxin in the heart increases oxidative stress and cardiac hypertrophy. J Clin Invest. 2003; 112:1395–1406. 14. Pan TT, Neo KL, Hu LF, Yong QC, Bian JS. H2S preconditioninginduced PKC activation regulates intracellular calcium handling in rat cardiomyocytes. Am J Physiol Cell Physiol. 2008;294:C169 –C177. 15. Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, Bolli R. Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway. Circulation. 2007;116:535–544. 16. Kaminski KA, Bonda TA, Korecki J, Musial WJ. Oxidative stress and neutrophil activation–the two keystones of ischemia/reperfusion injury. Int J Cardiol. 2002;86:41–59. 17. Kang KW, Lee SJ, Kim SG. Molecular mechanism of nrf2 activation by oxidative stress. Antioxid Redox Signal. 2005;7:1664 –1673. 18. Zweier JL, Talukder MA. The role of oxidants and free radicals in reperfusion injury. Cardiovasc Res. 2006;70:181–190. 19. Chen Z, Siu B, Ho YS, Vincent R, Chua CC, Hamdy RC, Chua BH. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J Mol Cell Cardiol. 1998;30:2281–2289. 20. Geng B, Chang L, Pan C, Qi Y, Zhao J, Pang Y, Du J, Tang C. Endogenous hydrogen sulfide regulation of myocardial injury induced by isoproterenol. Biochem Biophys Res Commun. 2004;318:756 –763. 21. Kimura Y, Kimura H. Hydrogen sulfide protects neurons from oxidative stress. FASEB J. 2004;18:1165–1167. 22. Fisher CD, Augustine LM, Maher JM, Nelson DM, Slitt AL, Klaassen CD, Lehman-McKeeman LD, Cherrington NJ. Induction of drugmetabolizing enzymes by garlic and allyl sulfide compounds via activation of constitutive androstane receptor and nuclear factor E2-related factor 2. Drug Metab Dispos. 2007;35:995–1000. 23. He X, Kan H, Cai L, Ma Q. Nrf2 is critical in defense against high glucose-induced oxidative damage in cardiomyocytes. J Mol Cell Cardiol. 2009;46:47–58. 24. Yoh K, Hirayama A, Ishizaki K, Yamada A, Takeuchi M, Yamagishi S, Morito N, Nakano T, Ojima M, Shimohata H, Itoh K, Takahashi S, Yamamoto M. Hyperglycemia induces oxidative and nitrosative stress and increases renal functional impairment in Nrf2-deficient mice. Genes Cells. 2008;13:1159 –1170. 25. Yellon DM, Baxter GF. Reperfusion injury revisited: is there a role for growth factor signaling in limiting lethal reperfusion injury? Trends Cardiovasc Med. 1999;9:245–249. 26. Xuan YT, Guo Y, Zhu Y, Wang OL, Rokosh G, Messing RO, Bolli R. Role of the protein kinase C-epsilon-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning. Circulation. 2005;112:1971–1978. 27. Bertolotto C, Maulon L, Filippa N, Baier G, Auberger P. Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD. J Biol Chem. 2000;275: 37246 –37250. 28. Griner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. Nat Rev Cancer. 2007;7:281–294. 29. Madamanchi NR, Li S, Patterson C, Runge MS. Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway. J Biol Chem. 2001;276:18915–18924. 30. Barry SP, Townsend PA, Latchman DS, Stephanou A. Role of the JAK-STAT pathway in myocardial injury. Trends Mol Med. 2007;13:82– 89. 31. Kunisada K, Negoro S, Tone E, Funamoto M, Osugi T, Yamada S, Okabe M, Kishimoto T, Yamauchi-Takihara K. Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy. Proc Natl Acad Sci U S A. 2000;97:315–319. 32. Hilfiker-Kleiner D, Hilfiker A, Fuchs M, Kaminski K, Schaefer A, Schieffer B, Hillmer A, Schmiedl A, Ding Z, Podewski E, Poli V, Schneider MD, Schulz R, Park JK, Wollert KC, Drexler H. Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury. Circ Res. 2004;95:187–195. 33. Chen H, Wu XJ, Lu XY, Zhu L, Wang LP, Yang HT, Chen HZ, Yuan WJ. Phosphorylated heat shock protein 27 is involved in enhanced heart tolerance to ischemia in short-term type 1 diabetic rats. Acta Pharmacol Sin. 2005;26:806 – 812. 34. Saleh A, Srinivasula SM, Balkir L, Robbins PD, Alnemri ES. Negative regulation of the Apaf-1 apoptosome by Hsp70. Nat Cell Biol. 2000;2: 476 – 483. 35. Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, Zamzami N, Mak T, Jaattela M, Penninger JM, Garrido C, Kroemer G. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol. 2001;3:839 – 843. 36. Wakabayashi N, Dinkova-Kostova AT, Holtzclaw WD, Kang MI, Kobayashi A, Yamamoto M, Kensler TW, Talalay P. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers. Proc Natl Acad Sci U S A. 2004;101:2040 –2045. 37. Huang HC, Nguyen T, Pickett CB. Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J Biol Chem. 2002;277:42769 – 42774. Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Hydrogen Sulfide Mediates Cardioprotection Through Nrf2 Signaling John W. Calvert, Saurabh Jha, Susheel Gundewar, John W. Elrod, Arun Ramachandran, Christopher B. Pattillo, Christopher G. Kevil and David J. Lefer Circ Res. 2009;105:365-374; originally published online July 16, 2009; doi: 10.1161/CIRCRESAHA.109.199919 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2009 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/105/4/365 Data Supplement (unedited) at: http://circres.ahajournals.org/content/suppl/2009/07/16/CIRCRESAHA.109.199919.DC1.html Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Online Data Supplement Circulation Research 2009 199919.R1 Supplemental Data Expanded Materials and Methods Animals. Three different stains of mice were utilized: (1) Male C57BL6/J mice, 810 weeks of age (Jackson Labs, Bar Harbor, ME), (2) Male mice (8-10 weeks of age) with a cardiac-specific overexpression of CGL (αMHC-CGL-Tg) and nontransgenic littermates. (3) Male mice deficient in Nrf2 (Nrf2 KO) and wild-type littermates. The αMHC-CGL-Tg mice were generated on an FVB background.1 Nrf2 knockout mice were originally generated on an ICR/129SV background and then backcrossed onto an ICR background.3 2 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. Myocardial Ischemia-Reperfusion (I/R) Protocol. Prior to any surgical procedure, mice were anesthetized with intraperitoneal injections of ketamine (60 mg/kg) and sodium pentobarbital (20 mg/kg). Mice also received 200 Units/kg of sodium heparin via intraperitoneal injection before surgery to prevent clot formation and allow for consistent and complete reperfusion postligation. The mice were then attached to a surgical board with their ventral side up and orally intubated with polyethylene-60 (PE-60) tubing connected via loose junction to a rodent ventilator (MiniVent Type 845, Hugo-Sachs Elektronik) set at a tidal volume 240 µL of and a rate of 110 breaths per minute and supplemented with 100% oxygen (0.1-0.2 liters/minute flow rate) via a side port on the ventilator. Effective ventilation was visually confirmed by vapor condensation in the endotracheal tube and rhythmic rising of the chest. Mice were maintained at a constant temperature of 37°C with a water-filled heating pad connected to a circulating water pump (Gaymar). Temperature was monitored via a rectal probe 1 Online Data Supplement Circulation Research 2009 199919.R1 connected to a Digisense K-Type digital thermometer. Hair remover (i.e., Nair®) was placed on the chest with a cotton swab and then removed along with the chest hair. The exposed regions were wiped with alcohol and betadine solution. A midline incision was then made along the sternum exposing the ribcage. Next, a median sternotomy was performed and the wound edges were cauterized with an electrocautery device. The proximal left coronary artery (LCA) was visually identified with the aid of an Olympus stereomicroscope with a fiber optic light source. The LCA was ligated with a 7-0 silk suture passed with a tapered BV-1 needle in close approximation just under the coronary artery. A short segment of PE-10 tubing was placed between the LCA and the 7-0 silk suture to minimized damage to the coronary artery and allow for complete reperfusion following the ischemic period. Ischemia was visually confirmed by cyanosis of the affected left ventricle. During the ischemic period the incision was covered with parafilm creating an effective barrier against desiccation and dehydration. Following 45 minutes of LCA occlusion, the ligature was removed, and reperfusion was visually confirmed. The chest wall and skin incision was carefully closed in layers with a 4-0 BIOSYN suture (CV-23 tapered needle). Animal recovery was supplemented by 100% oxygen and butorphanol (0.15 mg/kg) analgesia as well as a single dose of the antibiotic Cefazolin (80 mg/kg) to prevent infection. In the surgical recovery area, a heat lamp was utilized to maintain the appropriate body temperature of the mice. In addition, food and water were made available immediately and for the remainder of the first 24 hours of recovery. Myocardial Infarct Size Determination. At 24 hours of reperfusion, the mice were anesthetized, intubated, and connected to a rodent ventilator. A catheter (PE-10 tubing) was placed in the common carotid artery to allow for Evans Blue dye injection. A median sternotomy was performed and the LCA was re-ligated in the same location as before. Evans Blue dye (1.2 mL of a 7.0% solution, Sigma) was injected into the carotid artery catheter into the heart to delineate the ischemic zone from the non-ischemic zone. The heart was rapidly excised and serially sectioned along the long axis in five, 1 mm thick sections that were then 2 Online Data Supplement Circulation Research 2009 199919.R1 incubated in 1.0% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) for 5 minutes at 37°C to demarcate the viable and nonviable myocardium within the risk zone. Each of the five, 1 mm thick myocardial slices were weighed and the areas of infarction, risk, and non-ischemic left ventricle were assessed by a blinded observer using computer-assisted planimetry (NIH Image 1.57). All of the procedures for the left ventricular area-at-risk and infarct size determination have been previously described 4. Troponin I. A blood sample (500 µL) was collected from mice prior to the Evans blue dye injection. Serum was obtained and the levels of the cardiac-specific isoform of Troponin-I (ng/mL) were assessed using an ELISA kit from Life Diagnostics (West Chester, PA). Echocardiograhic Assessment of Left Ventricular Structure and Function. Baseline two-dimensional echocardiography images were obtained one week prior to LCA ischemia as previously described4. The mice were lightly anesthetized with isoflurane (3% for induction and 2% for maintenance) in 100% O2 and in vivo transthoracic echocardiography of the left ventricle (LV) using a 30-MHz RMV scanhead interfaced with a Vevo 770 (Visualsonics) was used to obtain high-resolution, two-dimensional ECG-based kilohertz visualization, B mode images acquired at the rate of 1,000 frames/sec over 7 min. These images were used to measure LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) and to calculate LV ejection fraction (EF). One week after the baseline images were acquired, the mice were subjected to 45 min of LCA occlusion followed by reperfusion as described above. At 1 week of reperfusion, post I/R echocardiographic images were obtained and analyzed. TUNEL Staining. After 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 3 Online Data Supplement Circulation Research 2009 199919.R1 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 highpowered field were counted in a minimum of 10 fields from the area-at-risk for each section (a total of 30 fields per heart). Subcellular Fractionation. Whole cell, cytosolic, membranous, nuclear, and mitochondrial fractions were prepared as described previously4,5. Briefly, frozen LV samples were powdered under liquid nitrogen with mortar and pestle prior to homogenization in 10 volumes of buffer A (containing in mM: 250 sucrose; 20 Tris, pH 7.4; 2 EDTA, pH 7.15; phosphatase inhibitors; 5 µg/ml each of leupeptin and aprotinin; 0.5 µg/ml pepstatin A; 0.3 PMSF) and subjected to serial centrifugations of 1,000 g, 10,000 g, and 100,000 g. The 1,000 g pellet (nuclear fraction) and the 10,000 g pellet (mitochondrial fraction) were washed in buffer A and recentrifuged. The final pellets were resuspended in buffer B (containing in mM: 150 NaCl; 20 Tris·HCl, pH 7.4; 10 EDTA; phosphatase inhibitors; 5 µg/ml each of leupeptin and aprotinin; 0.5 µg/ml pepstatin A; 0.1 PMSF) and subjected to a 21,000 g centrifugation for 10 min. The resultant supernatants were defined as the nuclear and mitochondrial fractions. The 100,000 g supernatant was defined as the cytosolic fraction and the 100,000 g pellet was resuspended in buffer B and defined as the membranous fraction. For whole cell preparations, samples were homogenized in 1 ml of ice-cold RIPA lysis buffer (Cell Signaling). Homogenates were then centrifuged at 1,300 g to remove any cellular debris. The pellet was discarded, and the supernatant was again centrifuged at 16,000 g for 30 min at 4°C. The resultant supernatant was collected. Protein concentrations of all cellular fractions were measured with the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Western blot analysis. Western blot analysis was performed as described previously (16). Equal amounts of protein were loaded into lanes of polyacrylamide-SDS gels. The gels were electrophoresed, followed by transfer of the protein to a PVDF membrane. The membrane was then blocked and probed with primary antibodies overnight at 4°C. The following primary antibodies were 4 Online Data Supplement Circulation Research 2009 199919.R1 used: anti-rabbit Trx1 (1:3000; Cell Signaling Technology, Danvers, MA); antirabbit Nrf2 (1:3000; abcam, Cambridge, MA); anti-mouse heme oxygenase-1 (HO-1) (1:3000; abcam); anti-rabbit phospho-PKCε Ser729 (1:3000; Upstate, Lake Placid, NY); anti-rabbit PKCε (1:5000; Upstate, Lake Placid, NY); anti-rabbit phospho-STAT-3 Ser727 (1:1500; Cell Signaling); anti-rabbit STAT-3 (1:3000; Cell Signaling); anti-rabbit p44/42 (1:3000; Cell Signaling); anti-rabbit phosphop44/42 (1:1500; Cell Signaling); anti-rabbit HSP70 (1:3000; Cell Signaling); antirabbit HSP90 (1:5000; Cell Signaling); anti-rabbit Caspase-3 (1:3000; Cell Signaling); anti-rabbit cleaved Caspase-3 Asp 175 (1:1500; Cell Signaling); antirabbit cytochrome C (1:5000; Cell Signaling); anti-rabbit fibrillarin (1:5000; Cell Signaling); anti-rabbit Na/K ATPase (1:5000; Cell Signaling); anti-rat α-tubulin (1:40,000; Santa Cruz Biotechnology Inc, Santa Cruz, CA); anti-rabbit phosphoBad Ser112 (1:1000; Cell Signaling); anti-rabbit Bad (1:1000; Cell Signaling); anti-rabbit Bcl-2 (1:1500; Cell Signaling); anti-rabbit Bcl-xL (1:3000; Cell Signaling); anti-rabbit cyclooxygenase-2 (COX-2) (1:2000; Cell Signaling); antirabbit CuZnSOD (1:3000; Cell Signaling); anti-rabbit MnSOD (1:3000; Santa Cruz); anti-rabbit cytochrome c oxidase IV (1:20,000; Cell Signaling). Immunoblots were next processed with secondary antibodies (anti-rabbit, antimouse, or anti-rat; Cell Signaling or Santa Cruz) for 1 hr at room temperature. Immunoblots were then probed with an ECL+Plus chemiluminescence reagent kit (GE Healthcare) to visualize signal, followed by exposure to X-ray film (Denville Scientific). The film was scanned to make a digital copy and densitometric analysis was performed to calculate relative intensity with ImageJ software from the National Institutes of Health (version 1.40g) using the Rodbard function. In all cases, the membranes were incubated with the phospho-specific antibody first. Membranes were then stripped and incubated with the total-specific antibody. Results were presented as the ratio of the expression of phosphorylated protein to total protein. The following antibodies were used as loading controls: anti-fibrillarin served as the subcellular marker for the nuclear fraction; anti-α-tubulin served as the 5 Online Data Supplement Circulation Research 2009 199919.R1 subcellular marker for the cytosolic fraction; anti-cytochrome c oxidase served as the subcellular marker for the mitochondrial fraction; anti-Na/K-ATPase served as the subcellular marker for the membraneous fraction. All experiments were performed in triplicate. For each membrane the relative intensity of each band was normalized to the value of the weakest band (smallest intensity). The values for each individual sample were averaged to obtain one value for each sample. The values for each group were then averaged and subsequently normalized to the mean of the control group (Sham or Non-Tg group). Gluthathione and Lipid hydroperoxide assays. 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.6 Reduced glutathione (GSH) and oxidized glutathione (GSSG) values were used to calculate the steady-state redox potential using the Nernst equation, Eh = E0 + 2.3 x RT/nF x log([GSSG]/[GSH]2 where E0= -264 at pH 7.4 and n=2 as described previously.7,8 6 Online Data Supplement Circulation Research 2009 199919.R1 Online Figure. Hydrogen sulfide preconditioning (H2S PC) preserved left ventricular dimensions in mice following myocardial ischemia and reperfusion. (A) Left ventricular end-diastolic diameter (LVEDD) and (B) left ventricular endsystolic diameter (LVESD) were calculated using high-resolution, two- dimensional B-mode echocardiography images at baseline and 7 days following myocardial ischemia. Values are means ± S.E.M. Numbers inside bars indicate the number of animals that were investigated in each group. *p<0.05, ***p<0.001 vs. BASE. H2S denotes Na2S. 7 Online Data Supplement Circulation Research 2009 199919.R1 References 1. Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, Jiao X, 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;104:1556015565. 2. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997;236:313-322. 3. Prince M, Li Y, Childers A, Itoh K, Yamamoto M, Kleiner HE. Comparison of citrus coumarins on carcinogen-detoxifying enzymes in Nrf2 knockout mice. Toxicol Lett. 2009;185:180-186. 4. Calvert JW, Gundewar S, Jha S, Greer JJ, Bestermann WH, Tian R, Lefer DJ. Acute metformin therapy confers cardioprotection against myocardial infarction via AMPK-eNOS-mediated signaling. Diabetes. 2008;57:696705. 5. Hunter JC, Kostyak JC, Novotny JL, Simpson AM, Korzick DH. Estrogen deficiency decreases ischemic tolerance in the aged rat heart: Roles of PKCdelta, PKCepsilon, Akt, and GSK3beta. Am J Physiol Regul Integr Comp Physiol. 2007;292:R800-809. 6. Jha S, Calvert JW, Duranski MR, Ramachandran A, Lefer DJ. Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling. Am J Physiol Heart Circ Physiol. 2008;295:H801-806. 7. Jones DP. Redox potential of GSH/GSSG couple: assay and biological significance. Methods Enzymol. 2002;348:93-112. 8. Go YM, Ziegler TR, Johnson JM, Gu L, Hansen JM, Jones DP. Selective protection of nuclear thioredoxin-1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells. Free Radic Biol Med. 2007;42:363-370. 8