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a Institute of Experimental Medical Research, Faculty Division, Ullevål University Hospital, University of Oslo, Oslo, Norway
b Department of Cardiothoracic Surgery, Ullevål University Hospital, Oslo, Norway
c Department of Surgery, Ullevål University Hospital, Oslo, Norway
Received 24 November 2007; received in revised form 11 March 2008; accepted 13 March 2008.
* Corresponding author. Address: Institute of Experimental Medical Research, Ullevål University Hospital, N-0407 Oslo, Norway. Tel.: +47 97 69 07 30; fax: +47 23 01 67 99. (Email: k.o.stenslokken{at}medisin.uio.no).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Hydrogen sulphide • Ischaemia • Reperfusion • Heat shock protein 72 • Mitogen activated protein kinases • Adenine nucleotides • Energy charge
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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H2S is produced in many mammalian tissues including the brain [8], vascular smooth muscles and the heart [9]. The enzymes responsible for H2S production appear to be tissue-specific, cystathionine β synthase is found in the brain [8], while cystathionine 
synthase (CSE) is in the cardiovascular system and smooth muscle cells [9,10]. Both enzymes use L-cysteine as substrate, which is believed to be the rate-limiting step in H2S production [11]. H2S concentration in rat brain has been found as high as 160 µM [8], while concentration in rat serum is approximately 45 µM [10]. H2S induces relaxation of vascular smooth muscle cells, probably by activation of K+
ATP channels [10].
Mitogen activated protein kinases (MAPK, including extracellular signal-regulated kinases (ERK 1/2), the stress-activated/c-Jun NH2 terminal kinases (JNK), P38 in addition to protein kinase B/AKT (AKT) and adenosine monophosphate dependent protein kinase (AMPK) may be important in the signal transduction pathway of ischaemic preconditioning [12,13]. AMPK regulates K+ ATP channels during preconditioning [14] and is also regarded as a key regulator of metabolism [15]. Recently it has been shown that brief cyclic perfusion with NaHS reduces infarct size in isolated rat hearts and involves ERK 1/2 and AKT because blockade of these kinases abolishes protection [16]. Another possible protective mechanism is heat shock proteins (HSP) such as the inducible HSP 72. Nothing is known about HSP72 and AMPK in H2S-induced cardioprotection.
Recently, it was reported that mice breathing H2S-enriched gas had a reduced metabolism of 90% and entered a ‘suspended animation’ [17]. Reduction of metabolic rate might be another possible mechanism of myocardial protection in cardiac surgery.
Studies investigating H2S and heart protection have used either low flow ischaemia [4] or regional ischaemia [3,5]. We hypothesised that: (1) H2S protects the heart against ischaemia–reperfusion injury also after global ischaemia, (2) H2S plays a mechanistic role in ischaemic preconditioning, and (3) phosphorylation of survival protein kinase AKT, ERK 1/2, JNK, P38, AMPK and expression of inducible heat shock protein 72 are induced by H2S.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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2.1 Langendorff perfusion
All rats were anaesthetised with 5% sodium pentobarbital (60–80 mg kg–1 intraperitoneally (i.p)) and heparinised (500 IU i.p). After anaesthesia, rat hearts were rapidly excised and placed in ice-cold Krebs–Henseleit buffer (KHB) (mmol l–1: NaCl 118.5; NaHCO3 25; KCl 4.7; KH2PO4 1.2; MgSO4·7H2O 1.2; glucose/1H2O 11.1; CaCl2 1.8) for further dissection. After aortic cannulation the hearts were mounted on a Langendorff system (AD Instruments Pty Ltd, Castle Hill, NSW 2154, Australia) and retrogradely perfused with warm (37 °C), oxygenated (95% O2, 5% CO2) KHB at constant pressure of 70 mmHg. The heart temperature was kept constant during the experiment by the surrounding glass tube (inner diameter 40 mm, height 80 mm) perfused with water from the heating chamber. A fluid-filled latex balloon (Hugo Sachs Elektronik-Harvard Apparatus GmbH, Hugstetten, Germany) was inserted into the left ventricle to measure ventricular pressures by a Powerlab system (AD Instruments Pty Ltd, Castle Hill, NSW 2154, Australia). Left ventricular end-diastolic pressure (LVEDP) was set to 5–10 mmHg and changes in LVEDP were measured. Left ventricular developed pressure (LVDevP = left ventricular systolic pressure (LVSP) – LVEDP) and maximum and minimum of left ventricular pressure development (LVdp/dt
max and LVdp/dt
min) were calculated. Coronary flow (CF) was measured by timed collections of the coronary effluent. Arrhythmias were counted as an all or nothing response (asystolia or ventricular fibrillation) during the first 30 min of reperfusion and were evaluated from pressure curves as was heart rate (HR). Myocardial temperature was measured by inserting a temperature probe in the right ventricle. The hearts with LVSP 
100 mmHg, CF 8 or 
20 ml min–1, HR 220 beats per min before ischaemia or irreversible arrhythmias for more than 30 min during reperfusion were excluded from the study.
After 120 min of reperfusion the hearts were cut in four slices of 1 mm and three slices of 2 mm (hearts fixed in acrylic rat brain matrix by AgnThor's AB, Lidingö, Sweden). The 2 mm slices were freeze-clamped in liquid nitrogen and stored for later analyses (Western blot and energy status). The four ventricular 1 mm slices (5–8 mm from apex) were incubated at 37 °C in 1% triphenyltetrazolium chloride for 15 min. After incubation the slices were gently pressed between two glass plates and photographed (Nikon, Colorfix5400). The infarct area was measured as percentage of total area and calculated with Adobe Photoshop and ScionImage (Infarct Area Calculation Macro file, Copyright ©1998 Rob Bell, Hatter Institute, UCL, UK).
2.2 Experimental groups
The study was divided into two series with three groups in each series.
2.2.1 Series 1
Hearts were stabilised for 30 min and randomised into the following groups:
2.2.2 Series 2
This series was performed some months after series 1. Due to variations in tolerance to ischaemia, this series was performed with 30 min of ischaemia. This variation of ischaemia tolerance over time is repeatedly observed in isolated, perfused rodent hearts. The explanation is unknown. The hearts were randomised to the following groups:
2.3 Drugs and chemicals
Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich. A stock solution of NaHS was freshly prepared by dissolving NaHS into the perfusate (KHB) immediately before use. 40 µM of NaHS is likely to give a H2S concentration of approximately 8 µM [9]. This was also the highest concentration that did not alter cardiac function during pilot dose response studies. NaHS dissociates to Na+ and HS– in the solution, then the HS– associates with H+ and forms H2S. NaHS is preferred instead of bubbling with H2S gas, because it is easier to monitor the H2S concentration in the solution. Dombkowski et al. [18] report that under physiological conditions (pH 7.4 at 37 °C), 18.5% of the NaHS will exist as H2S gas. The biochemical properties of NaHS have previously been discussed [3,9].
PAG has been frequently used as an inhibitor of CSE. PAG reduces H2S production by 80% in isolated myocytes [6] and 50 mg/kg significantly reduce H2S production in rats in vivo [19]. Serum concentrations of PAG after both intraperitoneal and intravenous injections have been investigated and found to peak between 1 and 2 h after intraperitoneal injections and more rapidly after intravenous injections [20,21].
2.4 Western blots
Frozen heart samples collected at the end of reperfusion were transferred to an ice cold lysis buffer containing 210 mM sucrose, 40 mM NaCl, 30 mM Hepes, 5 mM EDTA, 100 µM sodium orthovanadate, 1% Tween-20. In addition, one tablet complete EDTA-free protease inhibitor (Roche) and 250 µl phosphatase inhibitor cocktail 1 were added to 50 ml of extraction buffer (20 mg tissue ml–1 extraction buffer). The tissue was homogenised using a Polytron PT 1200 homogeniser. Lysates were subsequently centrifuged at 12000 x
g and 4 °C for 10 min to remove insoluble material. One percent SDS was added to the supernatant and the samples were vortexed for 15 min at room temperature. The samples were then frozen in liquid nitrogen and stored for later analysis. Protein content was determined by Micro BCA protein assay kit (Pierce, Rockford, USA). Protein (20 µg lane–1 except for total AKT antibody when 10 µg lane–1 was used to avoid saturation of the membranes) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the fractionated products were electrophoretically transferred onto a hybond-P membrane (Amersham Biosciences Europe, Freiburg, Germany). The membrane was incubated for 1 h with 5% skimmed milk in Tris-buffered saline (TBS) (20 mM Trizma base and 140 mM NaCl) containing 0.1% Tween-20 (TBST) in order to block non-specific reactions. The membrane was then incubated over night with primary antibodies (1:1000) both phosphospecific and total protein of AKT, ERK, JNK, P38 and AMPK, (Cell Signaling Technology). After washing with TBST (three times for 15 min), the membrane was incubated for 1 h with secondary antibody (Goat anti rabbit, Southern Biotech, 1:2500) conjugated to horseradish peroxidase. After washing (three times for 15 min), the immunoreactions were visualised by chemiluminescence (ECL+, Amersham Biosciences Europe, Freiburg, Germany) and pictures taken with ImageReader LAS-1000 (Fujifilm, Europe). The densitometry of each band was investigated using ImageQuant (Amersham Biosciences Europe, Freiburg, Germany). The membranes were stained with coomassie blue (Bio-Rad laboratories), scanned (CanonScan Lide 35) and equal loading was investigated using ImageQuant on the scanned membranes. Membranes with uneven blotting were removed from the analysis.
2.5 High performance liquid chromatography (HPLC)
Frozen heart tissue collected at the end of reperfusion in series 1 was weighed (180 ± 4 mg) and homogenised in 2.5 ml 6% perchloric acid. After centrifugation at 15000 x
g for 10 min, the supernatant was neutralised with 0.85 M K2CO3 in the ratio 1:0.75 and centrifuged again as described above. Contents of ATP, ADP and AMP were analysed with a HPLC apparatus consisting of a reversed-phase column (4 mm x 120 mm, nucleosil 120, C18 3 µm; Machery-Nagel, Düren, Germany), a SpectroMonitor 4100 detector and a ConstaMetric III pump (both from LDC Analytical, Riviera Beach, FL, USA). The mobile phase buffer contained 215 mM NaH2PO4, 2.5 mM tetrabutylammonium bromide (pH 6.25) and 1.5% acetonitrile. The energy charge was calculated as ([ATP + 0.5[ADP])/([ATP] + [ADP] + [AMP]).
2.6 Statistics
Data are shown as mean ± SD except Fig. 1
(median value and individual data) and Fig. 3 (box plots with median value and 5, 25, 75 and 95% percentiles). Data were analysed with Graph Pad Prism, Graph Pad Instat or Sigma Stat. Infarct size after reperfusion was analysed using one-way ANOVA with Bonferroni post-test. Differences in nucleotide content and energy charge were compared with Kruskal–Wallis and a Dunn post-test. Functional heart data was compared using repeated measures two-way ANOVA with Bonferroni post-tests.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.1.2 Series 2
Ischaemic preconditioning reduced infarct size from 37 ± 10% to 20 ± 4%. A further reduction to 16 ± 6% (Fig. 1b) was seen when endogenous H2S production was blocked with PAG, but this reduction was not significant compared to ischaemic preconditioning.
3.2 Heart function
There were small differences in cardiac function between H2S and PAG treated groups compared to controls in series 1 and we observed no change in LVEDP (Fig. 2a). H2S had a small negative pre-ischaemic effect on LVdevP (Fig. 2b) and increased pre- and post-ischaemic coronary flow (Fig. 2c). Ischaemic preconditioning improved post-ischaemic heart function (Fig. 2d–f). PAG tended to improve LVdevP further (Fig. 2e).
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When adenine nucleotides were investigated in hearts in series 1, H2S increased the concentration of ADP. No difference in energy charge was found between groups (Fig. 4 ).
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Our results are in agreement with recent findings of the cardio protective effects of H2S [3–6]. Johansen and co-workers [3] showed that perfusing rat hearts with NaHS, commencing 10 min prior to the onset of coronary occlusion (with regional ischaemia), and maintained during the first 10 min of reperfusion, caused a concentration-dependent reduction of infarct size. Maximal effect was observed with 1 µM NaHS whereas no infarct reduction was observed with a concentration of 10 µM NaHS. NaHS had no effect on heart function. In another study NaHS was given as an intravenous bolus injection of 3 mg kg–1, which would give an average plasma concentration of about 50 µM and would be expected to rapidly decrease. This treatment reduced infarct size in rats in vivo [5]. In isolated rat ventricular myocytes 100 µM NaHS was chosen as the dosage with the optimal protection against metabolic inhibition but 10 µM also improved protection [6]. The same group recently reported that cyclic intervention of 100 µM NaHS before an ischaemic episode protects the heart possibly through PI3K/AKT and ERK signal transduction pathways [16]. It is difficult to interpret these differences between these studies and ours due to different models, but it appears that either a low dose or cyclic intervention with NaHS induces better protection against ischaemia than a constant dose of 40 µM NaHS.
Although infarct size was reduced by H2S, function was not improved. A small pre-ischaemic reduction in LVdevP, due to decreased systolic pressure was observed with 40 µM NaHS (Fig. 2b). H2S activates K+ ATP channels [10] causing hyperpolarisation, which may reduce the excitability of the myocyte. This negative dromotropic and inotropic effect of H2S, might explain why changes of infarct size did not correlate with change in heart function. H2S caused a vasodilatation (Fig. 2c), which may be beneficial in situations with hypoxia or ischaemia.
Cardiac ischaemia–reperfusion injury in rat hearts increases plasma levels of H2S and concentration of CSE enzyme in the ischaemic area [19]. PAG is an irreversible inhibitor of CSE, penetrates cell membranes and decreases H2S production in many rat tissues including the heart and is frequently used for this purpose [5,6,19]. It is possible that the increase in infarct size seen in the PAG treated group is due to a reduced H2S production. In support of this is the increased infarct size found in rats in vivo after a week with daily injection of 50 mg/kg PAG [19]. Plasma concentration of H2S decreased significantly in these rats. However, CSE inhibition may also decrease glutathione [22], and a reduction in this endogenous antioxidant might explain the increase in infarct size [23].
Recently, it was found that mice breathing H2S gas decreased their metabolic rate by 90% together with a fall in body temperature [17]. This dramatic reduction in metabolic rate occurred within 10 min, and when H2S was withdrawn after 6 h, metabolic rate normalised within 1 h. It is tempting to speculate that H2S reduced metabolism of the hearts in the present investigation. Any substance reducing metabolic rate will protect against ischaemic injury.
If metabolism was unaltered we would expect to find differences in nucleotide content between the groups because of the different infarct size. However, there were no significant differences in energy charge although the level of ADP was higher in the group given H2S. The nucleotide concentration was higher in the PAG treated groups compared to controls, although they had larger infarcts. We have no good explanation for this unexpected finding. As expected the nucleotide contents in the ischaemic hearts were low [24].
H2S might activate protective cellular mechanisms, as suggested by increased inducible HSP 72 level in the H2S-treated group. Increased HSP 72 protects the heart against ischaemia–reperfusion injury [25]. Recently the so-called reperfusion injury salvage kinases have been suggested to play an important mechanistic role in preconditioning [12]. These kinases include ERK 1/2, AKT, P38 and JNK. Cyclic intervention with NaHS before sustained ischaemia induces protection and phosphorylates AKT and ERK [16]. Blocking the kinase ERK 1/2 or AKT abolished the protection. We could not find any increase in phosphorylation of these kinases in the H2S group. However, PAG decreased phosphorylation of AKT corroborating the increase in infarct size (Fig. 3). Activation of AKT at the time of reperfusion is believed to be important in endogenous heart protection [12] and a reduced activation of this kinase might explain the increased infarct size in the PAG group. The discrepancy for kinase activity might possibly be related to the different protocols as we used continuous supplementation of H2S (both pre- and post-ischaemia), while Hu et al. [16] used it as a preconditioning stimuli.
4.2 H2S and preconditioning
When endogenous H2S production was blocked by PAG, there was no effect on preconditioning-induced cardioprotection. PAG had no effect on phosphorylation of AKT, ERK, JNK, P38 or AMPK in preconditioned hearts. These kinases have been linked to ischaemic preconditioning [12,13]. As no change was seen in these kinases with PAG treatment, it seems that endogenous H2S is less important in these signaling pathways in ischaemic preconditioning. This is in agreement with in vivo findings by Siverajah et al. in rat hearts [5]. These investigators showed a possible role of H2S in delayed preconditioning. Although we found no role for endogenous H2S in ischaemic preconditioning, another model with a brief bolus of H2S appears to trigger the preconditioning transduction system [16].
4.3 Implications of present study
In heart surgery reduced excitability and metabolism attenuating ischaemia–reperfusion injury would be beneficial in connection with cardioplegia. Further studies on the possible role of H2S in myocardial protection is warranted in more surgically relevant models. The presently used model is not directly relevant to cardiac surgery as it involves neither cardioplegia nor myocardial hypothermia. However, the present study is of H2S-induced cardioprotection in a model with global ischaemia. More studies are also needed to better understand the molecular mechanisms behind the protective effect of H2S before, during or after ischaemia. Understanding the molecular mechanisms of cardioprotection by H2S may assist in understanding the signaling and effector mechanisms of endogenous cardioprotection, and thus help to open up new therapeutic interventions against ischaemia–reperfusion injury.
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Footnotes |
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This study was supported by the University of Oslo (JV), Ullevål University Hospital (KOS, JV), Eastern Norway Regional Health Authority and Norwegian National Health Association (JV), and the Norwegian Council on Cardiovascular Diseases (JV). |
References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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