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a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States
b Department of Surgery, University of Medicine and Dentistry of New Jersey, Newark, NJ, United States
c Ikaria, Inc., Seattle, WA, United States
Received 3 September 2007; received in revised form 23 January 2008; accepted 24 January 2008.
* Corresponding author. Address: 110 Francis Street, LMOB 2A, Boston, MA 02215, United States. Tel.: +1 617 632 8385; fax: +1 617 632 8387. (Email: fsellke{at}caregroup.harvard.edu).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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Key Words: Ischemia • Reperfusion • Apoptosis • Cardiac • Sulfide • Protection
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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Investigation into the pathogenesis of myocardial I/R injury has identified an endogenous inflammatory response and the release of damaging free radicals and oxidants associated with this response to be key in inducing myocardial damage [4,5]. Recently, research into the gaseous signaling molecule hydrogen sulfide (H2S) has demonstrated it may in fact serve as an endogenous mediator to limit inflammation and free radical damage [6]. Specifically, H2S has been shown to limit neutrophil adhesion and activation in response to inflammatory stimuli, as well as suppress the release of the pro-inflammatory mediator tumor necrosis factor-alpha (TNF-
) [7,8]. In addition to its effects on neutrophils, H2S, which is a strong reducing agent, is able to react with multiple oxidant stressors including superoxide radical anion [9], hydrogen peroxide [10], peroxynitrite [6], and hypochlorite [7]. H2S, which is synthesized endogenously from L-cysteine via cystathionine-
-lyase (CSE) in the heart and vasculature, has also become the subject of recent investigation in the context of myocardial protection. These studies, which have all utilized rodent models of myocardial injury, have demonstrated sulfide is able to limit myocardial infarction size and oxidative stress in response to I/R injury [10–12]. Given the ability of H2S to mitigate the production of stimuli known to induce apoptosis, free radical stressors [13] and inflammatory cytokines such as TNF [14], we hypothesized administration of exogenous therapeutic H2S prior to the onset of reperfusion after acute myocardial ischemia would reduce the apoptotic response to I/R injury. Utilizing a preclinical large animal model, this study investigates the effects of H2S on apoptotic signaling in response to acute myocardial ischemia followed by reperfusion.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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2.2 Experimental design
Yorkshire pigs of either sex (35–40 kg) were divided randomly into control (n
= 6) and sulfide treatment (n
= 6) groups. Animals were subjected to regional left ventricular (LV) ischemia by left anterior descending (LAD) arterial occlusion distal to the second diagonal branch for 60 min. The treatment group received sodium sulfide (100 µg/kg bolus + 1 mg/kg/h infusion) 10 min prior to the onset of reperfusion, whereas the control group received a placebo carrier solution of equal volume. Sodium sulfide was produced and formulated to pH neutrality and iso-osmolarity by Ikaria Inc. (Seattle, WA) using H2S gas (Mattheson, Newark, CA) as the starting material. The myocardium was reperfused for 120 min following ischemia. Arterial blood gas (ABG), arterial blood pressure, hematocrit (Hct), LV pressure, heart rate (HR), EKG, O2 saturation, core temperature, and intravenous fluid requirements were measured and recorded. Myocardial segmental shortening in the long-axis (parallel to the LAD) and short-axis (perpendicular to the LAD) were recorded at baseline prior to the onset of ischemia, and prior to harvest after 120 min of reperfusion. At the completion of the protocol, the heart was excised, and tissue samples from the ischemic-reperfused, distal LAD territory were collected for molecular analyses as described below.
2.3 Surgical protocol
Swine were sedated with ketamine hydrochloride (20 mg/kg, intramuscularly, Abbott Laboratories, North Chicago, IL), and anesthetized with a bolus infusion of thiopental sodium (Baxter Healthcare Corporation, Inc., Deerfield, IL; 5.0–7.0 mg/kg intravenously), followed by endotracheal intubation. Ventilation was begun with a volume-cycled ventilator (model Narkomed II-A; North American Drager, Telford, PA; oxygen, 40%; tidal volume, 600 cc; ventilation rate, 12 breaths/min; positive end-expiratory pressure, 3 cm H2O; inspiratory to expiratory time, 1:2). General endotracheal anesthesia was established with 3.0% sevoflurane (Ultane; Abbott Laboratories) at the beginning of the surgical preparation, and then maintained with 1.0% throughout the experiment. One liter of Lactated Ringer's intravenous (IV) fluid was administered after induction of anesthesia and continued thereafter throughout the surgical protocol at 150 cc/h. A right groin dissection was performed and the femoral vein and common femoral artery were isolated and cannulated utilizing 8 F sheaths (Cordis Corporation, Miami, FL). The femoral vein was cannulated for intravenous access, drug/placebo delivery, and the right common femoral artery was cannulated for arterial blood sampling and continuous intra-arterial blood pressure monitoring (Millar Instruments, Houston, TX). A median sternotomy was performed exposing the pericardial sac, which was then opened to form a pericardial cradle. A catheter-tipped manometer (Millar Instruments, Houston, TX) was introduced through the apex of the left ventricle to record LV pressure. Segmental shortening in the area-at-risk was assessed utilizing a sonometric digital ultrasonic crystal measurement system (Sonometrics Corp., London, ON, Canada) using four 2 mm digital ultrasonic probes implanted in the subepicardial layer approximately 10 mm apart within the ischemic LV area. Cardiosoft software (Sonometrics Corp., London, ON, Canada) was used for data recording (LV dP/dt, segmental shortening, arterial blood pressure, heart rate) and subsequent data analysis to determine myocardial function. Baseline hemodynamic, functional measurement (global: + LV dP/dt, regional: segmental shortening), arterial blood gas analysis, and hematocrit were obtained. ABG analysis was continued every 15 min throughout the protocol and hematocrit was measured every 20 min. All animals received 75 mg of lidocaine and 20 mEq of potassium chloride as prophylaxis against ventricular dysrhythmia, as well as 60 units/kg of intravenous heparin bolus prior to occlusion of the LAD. The LAD coronary artery was occluded 3 mm distal to the origin of the second diagonal branch utilizing a Rommel tourniquet. Myocardial ischemia was confirmed visually by regional cyanosis of the myocardial surface. Fifty minutes after the initiation of regional ischemia (10 min prior to the onset of reperfusion), control pigs received a placebo carrier solution infusion intravenously, and treatment animals received exogenous sulfide, generated as sodium sulfide (NaH2S), (100 µg/kg bolus + 1 mg/kg/h infusion) until the end of the experimental protocol. The Rommel tourniquet was released 60 min after the onset of acute ischemia and the myocardium was reperfused for 120 min. At the end of the reperfusion period, hemodynamic and functional measurements were recorded as described above, followed by religation of the LAD and injection of monastral blue pigment (Engelhard Corp., Louisville, KY) at a 1:150 dilution in PBS into the aortic root after placement of an aortic cross-clamp distal to the coronary arterial ostia to demarcate the area-at-risk. The heart was rapidly excised and the entire left ventricle, including the septum, was dissected free. The LV was cut into 1 cm thick slices perpendicular to the axis of the LAD. The area-at-risk was clearly identified by lack of blue pigment staining. Tissue from the area-at-risk of the slice 1 cm proximal to the LV apex was isolated and divided for use in molecular and microvascular studies. The remaining slices were weighed utilized for infarct size calculation as described below. Ventricular dysrhythmia (ventricular fibrillation or pulseless ventricular tachycardia) events were recorded and treated with immediate electrical cardioversion (50 J, internal paddles).
2.4 Measurement of global and regional myocardial function
Global myocardial function was assessed by calculating the maximum positive first derivative of LV pressure over time (+dP/dt). Regional myocardial function was determined by using subepicardial 2 mm ultrasonic probes to calculate the percentage segment shortening (%SS), which was normalized to the baseline. Measurements were taken at baseline prior to the onset of ischemia and at the end of reperfusion. The ventilator was stopped during data acquisition to eliminate the effects of respiration. Measurements were made during at least three cardiac cycles in normal sinus rhythm and then averaged. Digital data were inspected for the correct identification of end-diastole and end-systole. End-diastolic segment length (EDL) was measured at the onset of the positive dP/dt, and the end-systolic segment length (ESL) at the peak negative dP/dt.
2.5 Quantification of myocardial infarct size
The left ventricle was isolated and cut into 1 cm slices perpendicular to the axis of the LAD. Briefly, slices were immediately immersed in 1% triphenyl tetrazolium chloride (TTC, Sigma Chemical Co., St Louis, MO) in phosphate buffer (pH 7.4) at 38 °C for 30 min. The infarct area (characterized by absence of staining), the non-infarcted area-at-risk (characterized by red tissue staining), and the non-ischemic portion of the LV (characterized by purple tissue staining) were sharply dissected from one another and weighed. The percentage area-at-risk was defined as: (infarct mass + non-infarct area-at-risk mass)/total LV mass x 100. Infarct size was calculated as a percentage of area-at-risk to normalize for any variation in AAR size using the following equation: (infarct mass/total mass AAR) x 100.
2.6 Western blotting
Whole-cell lysates were isolated from the homogenized myocardial samples with a RIPA buffer (Boston Bioproducts, Worcester, MA) and centrifuged at 12,000 x
g for 10 min at 4 °C to separate soluble from insoluble fractions. Protein concentration was measured spectrophotometrically at a 595-nm wavelength with a DC protein assay kit (BioRAD, Hercules, CA). Forty to eighty milligrams of total protein were fractionated by 4–20% gradient, SDS polyacrylamide gel electrophoresis (Invitrogen, San Diego, CA) and transferred to PVDF membranes (Millipore, Bedford, MA). Each membrane was incubated with specific antibodies (Cell Signaling Technology, Beverly, MA) as follows: anti-apoptosis inducing factor (AIF) (1:1000 dilution), anti-Bcl-2 (1:1000 dilution), anti-Bad (1:1000 dilution), anti-phospho Bad (Serine 136) (1:500 dilution), anti-phospho Bad (Serine 112) (1:2000 dilution), anti-caspase-3 (1:1000 dilution), anti-cleaved caspase-3 (1:1000 dilution), anti-poly (ADP) ribose polymerase (PARP) (1:1000 dilution), anti-cleaved PARP (1:1000 dilution). The membranes were subsequently incubated for 1 h in diluted appropriate secondary antibody (Jackson Immunolab, West Grove, PA). Immune complexes were visualized with the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ). Bands were quantified by densitometry of radioautograph films. Ponceau S staining was performed to confirm equivalent protein loading.
2.7 Serum creatine kinase-MB, troponin I, and fatty acid binding protein quantification
Serum collected prior to sacrifice was utilized for quantification of creatine kinase-MB (CK-MB), troponin I, and fatty acid binding protein (FABP) utilizing a protein microarray (Allied Biotech Inc., Ijamsville, MD) in triplicate as described previously [15]. Serum levels of markers were calculated based on standards provided by the manufacturer.
2.8 Immunohistochemical staining
Myocardial tissue from the ischemic territory was placed in 10% buffered formalin for 24 h, followed by paraffin mounting and sectioning into 4 µm slices.
2.8.1 Poly (ADP) ribosylation staining
For the immunohistochemical detection of poly (ADP-ribose) polymerase activity, mouse monoclonal anti-poly (ADP-ribose) (PAR) antibody (Calbiochem, San Diego, CA) (1:1000, overnight, 4 °C) was used. Secondary labeling was achieved by using biotinylated horse anti-mouse antibody (Vector Laboratories, Burlingame, CA) (30 min room temperature). Horseradish peroxidase-conjugated avidin (30 min, room temperature) and brown colored diaminobenzidine (6 min, room temperature) was used to visualize the labeling (Vector Laboratories, Burlingame, CA). The sections were counterstained with hematoxylin (blue color). The intensity of specific staining of individual sections was determined by a blinded experimenter. The semiquantitative PAR-positivity score is the following: 1: no specific staining, 2: light cytoplasmic staining, 3: few positive nuclei, 4: light nuclear staining in approximately 10% of cells, 5: light nuclear staining in approximately 25% of cells, 6: light nuclear staining in approximately 50% of cells, 7: strong nuclear staining in approximately 50% of cells, 8: approximately 75% of the nuclei are positive, 9: approximately 90% of the nuclei are positive, 10: few negative cells).
2.8.2 TUNEL staining
The apoptotic cells were identified by dUTP nick-end labeling (TUNEL) using an apoptosis detection kit according to the manufacturer's protocol (Chemicon Inc., Temecula, CA). Five photographs (magnification 20x) of each tissue section were taken. The nuclei were viewed and manually counted by an observer blinded to the experimental conditions. The number of TUNEL-positive cardiomyocytes, indicating apoptosis, was expressed in mean number per/100 cells/microscopic field.
2.9 Data analysis
Data are reported as mean ± SD. Immunoblots are expressed as a ratio of protein to loading band density and were analyzed after digitization and quantification of X-ray films with ImageJ 1.33 (National Institutes of Health, USA). Blots were analyzed using an unpaired t-test. Bonferroni corrections were applied to multiple tests and probability values of less than 0.05 were considered statistically significant.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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3.2 Hemodynamic parameters
Heart rate (HR) and mean arterial blood pressure (MAP) were similar between groups at baseline (HR placebo 74.0 ± 7.2 vs sulfide 68.2 ± 20.4 beats/min, p
= 0.53), MAP placebo 62.5 ± 7.2 vs sulfide 56.4 ± 9.6 mmHg, p
= 0.24) (Fig. 1
). The HR remained similar between groups at the end of reperfusion (placebo 82.0 ± 9.0 vs sulfide 78.00 ± 10.0 beats/min, p
= 0.49), but MAP was significantly lower in the placebo treated group (placebo 32.3 ± 7.7 vs sulfide 49.6 ± 11.6 mmHg, p
= 0.01) (Fig. 1).
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3.5 Myocardial infarct size
The ischemic area-at-risk, as a percentage of total LV mass was similar between groups (placebo 31.1 ± 5.1 vs sulfide 33.5 ± 4.2% of LV mass, p
= 0.48). Myocardial infarct size was significantly reduced by 2.36-fold in sulfide treated groups relative to placebo (placebo 47.4 ± 15.2 vs sulfide 20.1 ± 7.9% of area-at-risk, p
= 0.003) (Fig. 4
).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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Previous work in murine models has demonstrated that exogenous administration of sulfide can reduce myocardial infarct size in response to acute I/R injury [10–12]. This study, which utilizes a more clinically relevant porcine model found results consistent with these prior reports. Additionally, we evaluated clinically utilized serum biomarkers of myocardial injury; CK-MB, troponin I, and FABP. Our study found a trend towards reduction in CK-MB levels (not reaching significance), and a significant reduction in serum levels of FABP, whereas troponin I levels were not detectable. Our time point for serum collection (3 h after the onset of ischemia) likely failed to capture a rise in troponin I, which generally occurs several hours after this timepoint. While a trend towards differences in serum CK-MB was observed, the single timepoint collection also may have limited discerning the true differences in this marker of myocardial injury. Serum FABP, a protein released rapidly from cardiomyocytes in response to ischemic injury [16] and detectable as early as 90–120 min after injury [16], was significantly lower in sulfide treated animals. This finding may add clinical relevance to the therapeutic benefits of sulfide administration as increased levels of FABP after acute coronary syndrome are prognostic for an increased risk of death, recurrent MI, and congestive heart failure in patients [17].
While myocardial necrosis likely produces the greatest degree of injury after acute I/R injury, myocardial apoptosis in response to I/R injury continues to be a relatively unavoidable consequence during restoration of perfusion. While essential to maximize salvage of viable tissue, the resulting initiation of the apoptotic cascade may have important clinical consequences relating to stunning [2] and survival [3]. The apoptotic signaling cascade can be initiated by free radicals and TNF [1,18], both of which are produced in myocardial I/R injury [13,14]. H2S can act as both an effective anti-oxidant [6], and limit the generation of TNF [8], and in this study has been shown to reduce expression of two key downstream effectors of cell death, cleaved caspase-3 and cleaved PARP, with a resulting decrease in the number of apoptotic cardiomyocytes.
Early in apoptotic signaling the anti-apoptotic Bcl-2, and pro-apoptotic Bad are in opposition. A shift in favor of the pro-apoptotic proteins subsequently results in increased mitochondrial pore permeability, releasing cytochrome C and AIF, which can activate caspase-3, and facilitate DNA fragmentation, respectively [1]. Upon cleavage of terminal caspases, such as caspase-3, cell death is generally thought to be inevitable. In this study we found no significant differences in early mediators of apoptotic signaling, such as Bcl-2, and Bad, but did observe significant reduction in expression of downstream effectors including caspase-3 and PARP. The absence of significant differences in expression of the phosphorylated forms of Bad may be due to tissue collection at a single time point, 3 h after the onset of ischemia, which may not capture transient changes in the activation status of these proteins. This time point was able to capture a significant reduction in the expression of cleaved caspase-3, a terminal effector of apoptosis that is responsible for intracellular degradation of structural proteins, regulatory proteins, and DNA repair enzymes. The reduction in I/R injury seen in our study is consistent with prior investigations which have demonstrated caspase inhibition reduces myocardial apoptosis and improves function in response to I/R injury [19]. Poly (ADP) ribose polymerase (PARP) is a nuclear enzyme activated by DNA damage that catalyzes the synthesis of poly (ADP) ribose (PAR) from nicotine adenine dinucleotide (NAD). Over-activation of PARP results in the depletion of NAD and subsequent ATP formation leading to cell death [20]. PARP inhibition has been shown to ameliorate myocardial damage in response to I/R injury [21]. The findings of this study indicate sulfide treated animals display reduced expression of the active form of PARP, cleaved PARP, and reduced poly ADP ribosylation of proteins, providing mechanistic insight into sulfide's ability to provide myocardial protection. Taken in sum, the ability of sulfide to limit production of key stimuli for initiation of apoptosis may allow for it to limit the generation of multiple pro-apoptotic signaling molecules which have previously been individual targets for myocardial protection agents.
4.1 Limitations
While providing functional and molecular data into the effects of sulfide therapy in myocardial I/R injury, this study has several limitations. Our time course for tissue harvest (3 h after the onset of ischemia) is not able to account for long-term effects of sulfide on myocardial function and infarct extension, and conversely may miss rapid changes in the activation/phosphorylation status of certain apoptotic signaling proteins such as Bad. Additionally, the use of TUNEL staining may not capture cells in the initial stages of the apoptotic process and thus may underestimate the total number of apoptotic cells.
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5. Conclusions |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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Appendix A |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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Dr D. Chambers (London, United Kingdom): It's a very interesting study which uses a clinically relevant model of myocardial ischemia and reperfusion and I think the results are very clear. However, I have a problem with the interpretation of your study. My understanding of ischemia and reperfusion is that injury is primarily caused by necrosis. I/R can initiate apoptosis, but the main injury is necrosis, and your interpretation seems to me to be that there is an apoptotic effect. You’ve suggested that apoptosis is occurring with increases in caspase-3 expression and TUNEL-stained cells, but there is increasing evidence that these initial stages of apoptosis can actually cause an oncosis which goes on to a secondary necrosis with actually little apoptotic activity, and to a certain extent you’ve shown that in your TUNEL staining where you only see that 6% and 3% of the cells of your infarct are apoptotic. So to a certain extent my criticism relates to the title of your study in that you are implicating the sulfide effect as being an apoptotic effect as the main effect. You also suggest that sulfide has an anti-inflammatory effect, and, again, these effects are fairly long-term effects, whereas you’re suggesting that these could be short-term in the 1 to 2 h of your study.
Firstly, I wonder how the sulfide concentrations used in this study relate to the H2S levels that you would see endogenously and have been shown to be protective. Are these higher?
The other question is, I wonder what would happen if you would have stopped your sulfide treatment after, say, an hour. Would you still see these beneficial effects, or does it have to be a long-term treatment?
Dr Clements: I would have to agree with you on the effects of necrosis being the primary injury in an infarction model. With that said, I think there are effects associated with apoptosis in that we can clearly see some induction of apoptotic signals. How important that is when we’re talking about 5% or less of the cells that actually see TUNEL-positive staining, I think that's a very good question. Nevertheless, if you have the initiation of apoptotic signals, what could that mean for long-term, or potentially longer-term events than the 2 h of reperfusion. Apoptosis could potentially play more of a role in long-term effects. As far as the apoptotic affects of hydrogen sulfide and relating those to the functional effects, I don’t think we could necessarily do that at this time, and I’d be much more comfortable, as you said, equating the immediate beneficial effects of hydrogen sulfide to a limiting of necrosis as shown by infarct staining, but it seems to have effects on both apoptosis and necrosis.
In relation to your question regarding inflammation and inflammatory signals, I didn’t present it here for the sake of time, but we also have studies from this experiment that looked at cytokine levels and neutrophil migration to the tissue as assessed by MPO activity, and those were greatly reduced with the hydrogen sulfide as well.
I think there could be a couple of different mechanisms where there is the chance for sulfide to provide a therapeutic benefit in the setting of ischemia–reperfusion injury. Also I think where you could have the apoptosis signaling being more important is in necrotic-free cell damage, such as transient hypoxia, where that could play more of a role for functional outcomes.
Dr Chambers: Your endogenous levels and the treatment levels?
Dr Clements: These exogenous levels would be higher than those usually seen endogenously. And right now there is a very big question as to what those endogenous levels are, because of questions concerning the methodology for measuring it. There is a question as to whether or not you are truly measuring free hydrogen sulfide or you are actually liberating sulfhydryl groups that wouldn’t normally exist endogenously. Those estimates are anywhere from zero-detectable amounts at baseline up to 100 µM, which I think, in my opinion, seems relatively high. But overall, in terms of dosing compared to other animal experiments, this would be on the middle to low end of the scale for when you look at effects of low-dose versus high-dose hydrogen sulfide, and high-dose has been shown to have numerous toxic effects as well.
Dr Chambers: Could you speculate on whether you need to have a long-term treatment?
Dr Clements: Well, that is one question that these experiments didn’t address because we did have continuous infusion throughout which was intentional to preempt the potentially short half-life of the hydrogen sulfide. So if you were able to give just the bolus injection without infusion, would we see the same results, I really couldn’t comment. My guess is as long as it's prior to reperfusion and at least in the early reperfusion phase, I think that would be the most important time to target, but as far as long-term, I really couldn’t speculate.
Dr J. Vaage (Oslo, Norway): You probably know about the study in Science a couple years ago showing that hydrogen sulfide injected into mice actually caused a serious reduction in body temperature and reduced metabolic rate. This made me a little bit sceptical about having it in a clinical setting. My first question is, did you notice anything like this in your model? Second, do you have any estimate, measurements, or calculations of the concentration of hydrogen sulfide in the blood of your animals?
Dr Clements: For the first question, we did expect that as well, that potentially there could be effects, and we actually did not record any change in body temperature. I think you probably need a higher dose for those effects. The second question, we could go back and calculate it, but right now I just know the dosing, the 100 µg/kg.
Dr Vaage: So how did you choose the dose?
Dr Clements: That was based on previous experimentation in rats and mice that were associated with Ikaria.
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Acknowledgments |
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Footnotes |
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Presented at the 21st Annual Meeting of the European Association for Cardio-thoracic Surgery, Geneva, Switzerland, September 16–19, 2007.
This work was supported by NHLBI grant HL46716. Drs Sodha and Clements are supported in part by NIH grant T-32HL076130-02 and the Irving Bard Memorial Fellowship. Dr Liu is supported by NIH grant T-32HL076130-02. Partial funding for this project was provided by Ikaria, Inc., Seattle, Washington.
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. ConclusionsAppendix AReferences |
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