Corresponding author S. Shimizu: Department of Pathophysiology, Showa University School of Pharmacy, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Email: firstname.lastname@example.org
The goal of this study was to elucidate whether there is an increase in myocardial tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase, during the late phase of ischaemic preconditioning (IPC) leading to cardioprotection against myocardial infarction and, if so, to examine the induction mechanisms of BH4 synthesis. Rats were preconditioned with four cycles of 3 min left main coronary artery (LCA) occlusion followed by 10 min reperfusion. Twenty-four hours later, the rats were subjected to 20 min ischaemia by LCA ligation and 2 h reperfusion, and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. The IPC protocol reduced the infarct size, and increased the BH4 content and expression of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme for BH4 synthesis. Administration of a GTPCH inhibitor attenuated both the reduction in infarct size and the increase in BH4 levels. Moreover, the increase in BH4 content was reduced by administration of catalase or a Janus tyrosine kinase-2 (JAK2) inhibitor. These observations suggest that upregulation of BH4 synthesis in the heart contributes to an acquisition of ischaemic tolerance in late IPC, and the increase in myocardial BH4 content seems to be mediated by the induction of GTPCH via the H2O2–JAK2 pathway.
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Ischaemia–reperfusion (I–R) injury is a common clinical problem that occurs in various situations, including thrombolytic therapy, coronary angioplasty and vascular or heart surgery (Yellon & Hausenloy, 2007). A variety of mechanisms are implicated in I–R injury, such as Ca2+ overload, generation of reactive oxygen species (ROS), inflammation due to neutrophil infiltration, and endothelial dysfunction (Wang et al. 2002). The late phase of ischaemic preconditioning (IPC) is a phenomenon in which brief episodes of I–R increase the tolerance of the heart to subsequent I–R injury 12–24 h later, and lasts 3–4 days (Bolli, 2000; Das & Das, 2008). Although the underlying mechanisms of late IPC remain incompletely understood, accumulated evidence suggests that the induction of late IPC involves various factors, including ROS, nitric oxide synthase (NOS), cyclooxygenase and mitochondrial ATP-sensitive K+ channels (Takano et al. 1998; Fryer et al. 2000; Wang et al. 2004; Xiao-Qing et al. 2005).
Nitric oxide synthase is an enzyme which produces NO by the conversion of l-arginine to l-citrulline. The resulting NO is an important signalling molecule for vascular homeostasis by the regulation of blood vessel diameter, leukocyte adhesion and platelet aggregation. Nitric oxide also seems to have a protective role against I–R injury by the activation of mitochondrial ATP-sensitive K+ channels (Sasaki et al. 2000; Wajima et al. 2006); therefore, the reduced bioavailability of NO could enhance the development of reperfusion injury following myocardial ischaemia. There are three isoforms of NOS, including two constitutive synthases, neuronal NOS (nNOS or NOS I) and endothelial NOS (eNOS or NOS III), and inducible NOS (iNOS or NOS II). All three isoforms of NOS require tetrahydrobiopterin (BH4) as a critical cofactor (Kwon et al. 1989; Tayeh & Marletta, 1989; Werner-Felmayer et al. 1990; Schmidt et al. 1992). Importantly, a decrease of BH4 leads to the uncoupling of NOS, resulting in reduced production of NO and increased production of ROS (Wever et al. 1997; Vásquez-Vivar et al. 1998; Xia et al. 1998). Oxidative stress is a well-known mechanism underlying I–R injury. The BH4 content in the heart during oxidative stress appears to be decreased, because it is one of the most potent naturally occurring reducing agents. Dumitrescu et al. (2007) have shown that myocardial BH4 contents are decreased by I–R in the isolated rat heart. In fact, recent studies have shown that BH4 has a protective effect against I–R injury in various organs, such as heart and stomach (Ishii et al. 2000; Yamashiro et al. 2002; Tiefenbacher et al. 2003; Wajima et al. 2006), although whether BH4 changes are related to the late IPC effect was not elucidated. Tang et al. (2005) recently described that brief I–R for late IPC increases BH4 levels in the rabbit heart, and hypercholesterolaemia abrogates late IPC by preventing the upregulation of BH4 as a cofactor for iNOS. Thus, the increase in BH4 levels appears to be involved in late IPC; however, the underlying mechanism for stimulating BH4 synthesis during late IPC is unknown.
The biosynthesis of BH4 occurs from GTP via a de novo pathway in which the enzyme GTP-cyclohydrolase I (GTPCH) is the rate-limiting step. Synthesis of BH4 has been shown to be increased by various cytokines, including tumour necrosis factor α (TNFα) and interferon γ (IFNγ), through the induction of GTPCH (Blau & Niederwieser, 1985; Nichol et al. 1985; Huang et al. 2005). We recently found that hydrogen peroxide (H2O2) increases BH4 content in vascular endothelial cells through the induction of GTPCH (Shimizu et al. 2003). The induction of GTPCH by H2O2 is mediated by Janus tyrosine kinase-2 (JAK2)–signal transducer and activator of transcription 1 (STAT1) pathway, which controls the expression of stress-responsive genes (Shimizu et al. 2008). The acquisition of resistance to I–R injury has been shown to involve H2O2-mediated mechanisms (Tang et al. 2006). Moreover, recent studies have shown that activation of the JAK–STAT pathway has a central role in late IPC (Xuan et al. 2001; Bolli et al. 2003). These reports allow us to speculate that the induction of BH4 synthesis during late IPC is mediated by the ROS–JAK2–STAT1 signalling pathway and the ROS-mediated induction is modified by coproduced cytokines, such as TNFα and IFNγ.
In the present study, we first confirmed that the increase in myocardial BH4 levels in the late IPC confers cardioprotection against myocardial infarction in a rat model of I–R, and then examined whether the induction of BH4 synthesis during late IPC was mediated by the H2O2–JAK2 signalling pathway. Moreover, whether or not the H2O2-induced BH4 synthesis is enhanced by cytokines was investigated in cultured vascular endothelial cells.
The animals used in this study were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996), and the experimental protocol was approved by the Experimental Animal Committee of Showa University.
Ischaemic preconditioning model
Male Sprague–Dawley rats (300–450 g; Saitama Experimental Animal Supply, Inc., Saitama, Japan) were anaesthetized with sodium pentobarbitone (50 mg kg−1, i.p.). Additional anaesthetic was given during the experiment as needed. Aseptic surgical techniques were used throughout. Each rat was intubated and ventilated with room air (10 ml kg−1 per stroke and 54 strokes min−1) using a mechanical ventilator (model SN-480-7; Shinano, Tokyo, Japan). Left thoracotomy at the fourth to sixth ribs was performed, and the heart was exteriorized to pass a silk thread around the origin of the left coronary artery (LCA). Both ends of the silk thread were passed through a polyethylene tube, and the heart was placed back in the thorax. The LCA was occluded by pressing the polyethylene tube against the heart. The hearts were preconditioned with four cycles of 1 or 3 min ischaemia with a polyethylene tube, and with subsequent 10 min reperfusion. After brief I–R, the chest was closed, and the rats were then administered benzylpenicillin (30 000 U ml−1 kg−1i.m.) and allowed to recover from anaesthesia for 24 h.
Production of ischaemia and reperfusion
Twenty-four hours after brief I–R, rats were again anaesthetized with sodium pentobarbitone (50 mg kg−1, i.p.). Additional anaesthetic was given during the experiment as needed. Each rat was intubated and ventilated with room air (10 ml kg−1 per stroke and 54 strokes min−1). Blood pressure was measured with a carrier amplifier (N4438; Nihon Kohden, Tokyo, Japan) after connecting it to a pressure transducer via a catheter inserted into the left carotid artery. Left thoracotomy at the fourth to sixth ribs was performed, and the LCA was occluded by pressing the polyethylene tube against the heart again at the same site with brief I–R. After 20 min ischaemia, the polyethylene tube was removed, and reperfusion was performed for 2 h.
Administration of drugs
Aminoguanidine and catalase were dissolved in physiological saline, and 2,4-diamino-6-hydroxypyrimidine (DAHP) and tyrphostin AG490 (AG490) were dissolved in dimethyl sulphoxide (DMSO). The left jugular vein was cannulated for intravenous administration of catalase. The protocol of drug treatment is shown in Fig. 1. Catalase (95 800 U ml−1 kg−1) or physiological saline (vehicle) was administered i.v. 30 min before the brief I–R, and also continuously injected (31 900 U ml−1 h−1 kg−1) by infusion pump from 30 min before brief ischaemia to the end of the brief I–R. 2,4-Diamino-6-hydroxypyrimidine (100 mg ml−1 kg−1) or its vehicle (100% DMSO) was administered i.p. 120 min before the sustained ischaemia. AG490 (1 mg ml−1 kg−1) or its vehicle (100% DMSO) was injected i.p. 120 min before the brief I–R.
Determination of myocardial infarct size
Two hours after reperfusion, the LCA was occluded by pressing the polyethylene tube against in the heart at the same site as for the sustained ischaemia, and 3 ml of 5% Evans Blue solution was administered into the vena cava. The heart was then removed, and the left ventricular muscle immediately below the polyethylene tube was cut into four myocardial slices with a thickness of about 2 mm. All slices were stained in 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution for 10 min at 37°C. Each slice was weighed and then photographed. The area stained blue by Evans Blue (normal myocardium), the area stained red by TTC (ischaemic myocardium) and the area unstained by TTC (necrotic myocardium) were measured using ImageJ (Media Cybernetics Inc., Silver Spring, MD, USA). The area at risk (risk mass/left ventricular mass) and the infarct size (infarct mass/risk mass) were calculated as described by Linz et al. (1998).
Cell culture and treatment
Bovine coronary endothelial cells were purchased from Toyobo Co. (Osaka, Japan). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. Cells were used at four to six passages after purchase.
The cells were grown in six-well plates. The confluent cells were washed twice with FBS-free DMEM containing 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (FBS-free DMEM), and then treated with various reagents at 37°C in 2 ml FBS-free DMEM.
Determination of BH4 levels
Tetrahydrobiopterin levels in the ischaemic areas of the hearts were determined using an adaptation of the protocol described previously in our laboratory (Wajima et al. 2006). Briefly, the ischaemic areas of the cardiac tissues were homogenized in ice-cold 10 mm Tris–HCl buffer (pH 7.4) containing 1 mm dithiothreitol and 1 mm EDTA. The aliquots were separately oxidized by iodine in an acidic solution (0.04 m KI/I2 in 0.2 m HCl) and a base solution (0.04 m KI/I2 in 0.2 m NaOH). When BH4 contents in culture cells were determined, cells were treated with (0.02 m KI/I2 in 0.1 m HCl) and a base solution (0.02 m KI/I2 in 0.1 m NaOH) as previous described (Shimizu et al. 2008). Quantification of biopterin was performed by reverse-phase, high-performance liquid chromatography with fluorometric detection. The amount of BH4 was calculated from the difference in biopterin concentrations measured after oxidation in the acid (total) and the base (7,8-dihydrobiopterin plus biopterin).
Determination of nitrite plus nitrate (NOx) levels
The NOx levels in the ischaemic areas of the hearts were determined using NO2/NO3 assay kit-FX (Dojindo Laboratories, Kumamoto, Japan). Briefly, the ischaemic areas of the cardiac tissues were homogenized in ice-cold phosphate-buffered saline. The obtained supernatants were treated with Ultracel YM-10 (Millipore Co., Bedford, MA, USA) to remove haemoglobin and proteins, and assayed to determine NOx levels (Wajima et al. 2006).
Western blot analysis
Hearts were extracted 5 min after the IPC protocol for JAK2, 30 min after the IPC protocol for STAT1, 6 h after the IPC protocol for TNFα, and 24 h after the IPC protocol for iNOS. The ischaemic areas of the cardiac tissues were then homogenized in lysis buffer containing 50 mm Tris–HCl (pH 7.4), 1% polyoxyethylene (9) octylphenyl ether (NP-40), 0.25% deoxycholic acid, 15 mm NaCl, 0.1 mm EDTA, 1 mm phenylmethylsulphonyl fluoride, 1 mm sodium orthovanadate, 1 mm sodium fluoride and protease inhibitor cocktail (Roche, Mannheim, Germany), and then treated with ultrasonication for 5 s. The lysates were centrifuged at 15 000 g for 15 min at 4°C. After the supernatants were collected, the protein concentration was determined with a Dc protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples containing equal amounts of protein (80 μg) were separated on SDS-polyacrylamide gels under reducing conditions and transferred onto a Hybond ECL nitrocellulose membrane (Amersham, UK). Non-specific binding was blocked with 5% skim milk in TBS containing 0.1% Tween 20 (TBS-T) for 60 min. The membranes were incubated overnight at 4°C with a 1:1000 dilution of rabbit polyclonal anti-JAK2 antibody, rabbit polyclonal anti-phospho-JAK2 antibody (Y1007/1008), rabbit polyclonal anti-STAT1 antibody, rabbit polyclonal anti-phospho-STAT1 antibody (Y701), mouse monoclonal anti-TNFα antibody, rabbit polyclonal anti-iNOS antibody, rabbit polyclonal anti-β-actin antibody or goat polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody in TBS-T containing 5% BSA, and developed with an enhanced chemiluminescence Western blotting detection system (GE Healthcare, Tokyo, Japan). For the second antibody, horseradish peroxidase (P)-conjugated anti-rabbit IgG antibody, anti-mouse IgG antibody or anti-goat IgG antibody was used. The membranes were exposed to chemiluminescence-sensitive film (Hyperfilm, Amersham). Densities of signals on the blots were measured using NIH image software.
Hearts were extracted 24 h after the IPC protocol for the measurement of GTPCH mRNA. Total RNA was extracted from the ischaemic areas of the cardiac tissues by a modified guanidinum isothiocyanate method with TRIzol® reagent (Invitrogen, Tokyo, Japan). Twenty micrograms of total RNA from each sample was treated with a solution containing 2.2 m formaldehyde, 50% formamide, 5 mm sodium acetate, 1 mm EDTA and 20 mm Mops (pH 7.0) for 15 min at 65°C, and the RNA was separated by electrophoresis using 1% agarose gel containing 2.2 m formaldehyde in Mops buffer containing 5 mm sodium acetate, 1 mm EDTA and 20 mm Mops (pH 7.0). The RNA samples were transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech, UK), and hybridized to the indicated random prime-labelled cDNA probe (Takara, Otsu, Japan). The cDNA probe of GTPCH was prepared by RT-PCR using the pair of primers of 5′-GGATACCAGGAGACCATCTCA-3′ and 5′-TAGCATGGTGCTAGTGACAGT-3′, as previously described (Shimizu et al. 2003). Hybridization reactions were carried out for 1 h at 68°C in ExpressHyb® hybridization solution (Takara Bio Company, Tokyo, Japan). The membrane was washed in 20 mm Na2HPO4 (pH 7.2), 1 mm EDTA (pH 8.0) and 0.1% SDS. The washed membrane was exposed to Kodak Biomax® film at −80°C for 24–48 h. The membrane was rehybridized by GAPDH cDNA, which is a constitutive gene. The cDNA probe of GAPDH was prepared by RT-PCR as previously described (Shimizu et al. 2003).
2,3,5-Triphenyltetrazolium chloride (TTC), Evans Blue, aminoguanidine and anti-TNFα antibody were purchased from Sigma Chemical Co. (St Louis, MO, USA). Sodium pentobarbitone was purchased from Dainippon Pharmaceutical Co. (Osaka, Japan). 2,4-Diamino-6-hydroxypyrimidine (DAHP) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Tyrphostin AG490 (AG490), catalase, TNFα (2 × 107 U mg−1), IFNγ (2 × 107 U mg−1) and interleukin-1β (IL-1β, 1 × 107 U mg−1) were purchased from Wako Pure Chemicals (Osaka, Japan). Anti-STAT1 antibody, anti-phospho-STAT1 antibody (Y701), anti-phospho-JAK2 antibody (Y1007/1008) and anti-β-actin antibody were from Daiichi Pure Chemicals (Tokyo, Japan). Anti-JAK2 antibody was from Upstate Biotechnology (Charlottesville, VA, USA). Anti-iNOS antibody and anti-GAPDH antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
All data are expressed as the means ±s.e.m. The statistical significance of observed differences was determined by the Student's unpaired t test or by ANOVA followed by Bonferroni test, as appropriate. Differences between means were considered significant when P < 0.05.
Mortality and exclusions
A total of 247 rats were used for this study. One hundred and eighty-eight animals survived the preparation protocols and were analysed for this study. Eighteen animals were lost due to sustained ventricular fibrillation and/or systemic circulatory failure during the infarct protocol (one in sham for 1 min IPC, one in IPC (1 min), one in sham for 3 min IPC, one in IPC (3 min), one in sham + DMSO for DAHP, one in sham + DAHP, one in IPC + DMSO for DAHP, two in IPC + DAHP, one in sham + DMSO for AG490, two in sham + AG490, one in IPC + DMSO for AG490 and five in IPC + AG490). Twenty-five animals were lost due to technical failure.
Induction of late IPC
We first examined how the condition of brief I–R reduces the infarct size after 20 min ischaemia and 2 h reperfusion (Fig. 2). Rats were preconditioned with four cycles of 1 or 3 min LCA occlusion followed by 10 min reperfusion. The area at risk (risk mass/left ventricular mass) in the sham-operated group was 48.3 ± 3.7% for the 1 min ischaemia protocol, and no significant differences were observed among all the groups (Fig. 2B). The infarct size (infarct mass/risk mass) in the sham-operated group was 30.0 ± 2.2% for the 1 min ischaemia protocol and 30.0 ± 2.4% for the 3 min ischaemia protocol. Preconditioning with 1 min ischaemia and 10 min reperfusion did not reduce the infarct size (29.4 ± 2.3%), whereas when preconditioning with 3 min ischaemia and 10 min reperfusion was performed, the infarct size was significantly reduced to 17.5 ± 4.2% (Fig. 2C).
Increases in BH4 content, iNOS and NOx levels, and cardioprotection by IPC
Changes in BH4 contents and the expression of GTPCH mRNA in hearts were determined 24 h after the IPC protocol. Consistent with the infarct size-limiting effect, BH4 contents in hearts were increased by preconditioning with a 3 min ischaemia protocol, but not with a 1 min ischaemia protocol (Fig. 3A). Oxidized biopterin (BH2 + biopterin) levels in the sham group for the 3 min ischaemia protocol and in the IPC group for the 3 min ischaemia protocol were 0.019 ± 0.001 and 0.035 ± 0.004 pmol (mg wet weight)−1, respectively, and total biopterin levels were 0.225 ± 0.016 and 0.350 ± 0.025 pmol (mg wet weight)−1, respectively. Thus, the ratio of BH4 levels to total biopterin levels was slightly decreased by IPC (sham group, 0.957 ± 0.001; IPC group, 0.897 ± 0.014; P < 0.05). The GTPCH mRNA levels were also increased by preconditioning with a 3 min ischaemic protocol, but not with a 1 min ischaemic protocol (Fig. 3B). For subsequent experiments, we chose four cycles of 3 min ischaemia and 10 min reperfusion to induce late IPC effects.
Tetrahydrobiopterin is an essential cofactor for all three isoforms of NOS, and an increase in iNOS expression has been shown to be implicated in the acquisition of resistance to I–R injury by IPC (Guo et al. 1999; Xuan et al. 2000). We next examined the effects of brief I–R on iNOS expression and NOx levels. As shown in Fig. 3C, the brief I–R increased the expression of iNOS protein. Moreover, the brief I–R also increased NOx levels, and the increases in NOx levels were blocked by treatment with aminoguanidine, a specific inhibitor for iNOS (Fig. 3D).
To determine whether the elevation of BH4 contents in hearts is implicated in the acquisition of resistance to I–R injury, the effects of DAHP, an inhibitor of BH4 synthesis, were examined. Hamadate et al. (2008) reported that BH4 content in rat aorta was diminished to 65% of the control value by administration of DAHP (100 mg kg−1, i.p.) for 5 h. Although administration of DAHP (100 mg kg−1, i.p.) to sham-operated rats 2 h before determination of BH4 slightly decreased the cardiac BH4 levels (Fig. 4A), its administration eliminated both the elevation of BH4 content by IPC (Fig. 4A) and the infarct size-limiting effect by IPC (Fig. 4C). The area at risk (risk mass/left ventricular mass) among all the groups was not significantly different (Fig. 4B). These results strongly suggest that increased BH4 levels through the induction of GTPCH are crucial for the acquisition of ischaemic resistance by IPC.
Stimulation mechanism of BH4 synthesis by IPC
We previously showed that BH4 synthesis was stimulated by H2O2 treatment through the induction of GTPCH in vascular endothelial cells, and the mechanisms include the JAK2–STAT1 signalling pathway (Shimizu et al. 2008). To examine whether the JAK2 signalling pathway is associated with increased BH4 contents through the induction of GTPCH by IPC, the effect of administration of the JAK2-specific inhibitor, AG490, on BH4 contents and GTPCH mRNA expression was investigated. The administration of AG490 (1 mg kg−1) 2 h before IPC abrogated the elevation of BH4 contents and GTPCH expression (Fig. 5A and B). Moreover, administration of AG490 (1 mg kg−1) eliminated the infarct size-limiting effect of IPC (Fig. 5D). The area at risk (risk mass/left ventricular mass) among all the groups was not significantly different (Fig. 5C). Therefore, levels of phosphorylated JAK2 (phospho-JAK2) and phospho-STAT1, which are the active forms of JAK2 and STAT1, respectively, were measured. Both phospho-JAK2 levels and phospho-STAT1 levels were increased by IPC, and their increases were abrogated by the administration of AG490 (Fig. 6A and B).
It is well known that I–R generates ROS and the inflammatory cytokine TNFα (Pain et al. 2000; Yamashita et al. 2000; Samavati et al. 2002; Reil et al. 2007). We next examined the involvement of H2O2 in the IPC-induced increase in myocardial BH4 contents. Administration of the H2O2 scavenging enzyme, catalase, during the IPC protocol inhibited the IPC-induced increase in BH4 contents 24 h after the last brief I–R (Fig. 7A) and inhibited the induction of GTPCH mRNA levels (Fig. 7B). These results suggest that the IPC-induced increase in BH4 levels through GTPCH induction is mediated by the produced H2O2. Moreover, an increase in TNFα levels following IPC was also observed (Fig. 7C).
Enhancement of BH4 synthesis by cotreatment with H2O2 and cytokines
Although H2O2 and various cytokines, including TNFα, IFNγ and IL-1β, have been shown to be produced during I–R (Yamashita et al. 2000; Reil et al. 2007), whether H2O2 interacts with these cytokines in BH4 synthesis is unknown. As shown in Fig. 8, BH4 contents in coronary endothelial cells were increased by treatment with 50 μm H2O2 and TNFα (20 ng ml−1), but not with IFNγ (50 ng ml−1) and IL-1β (20 ng ml−1). Interestingly, although H2O2 (10 μm) only increased BH4 levels slightly, it markedly stimulated the TNFα (20 ng ml−1)-induced increase in BH4 contents.
At 5 min before 20 min ischaemia, mean arterial pressure and heart rate averaged 78 ± 13 mmHg (n= 5) and 420 ± 20 beats min−1 (n= 5), respectively, in sham-operated rats with 3 min IPC. In rats treated with IPC + DAHP, IPC + DMSO (the DAHP vehicle), IPC + AG490 and IPC + DMSO (the AG490 vehicle), mean arterial pressures (in mmHg) were 73 ± 8 (n= 5), 80 ± 9 (n= 5), 73 ± 13 (n= 5) and 76 ± 8 (n= 5), respectively, and heart rates (in beats min−1) were 417 ± 31 (n= 5), 420 ± 14 (n= 5), 400 ± 24 (n= 5) and 411 ± 8 (n= 5), respectively. Thus, administration of DAHP, AG490 and DMSO had no effect on these parameters.
Late IPC is a phenomenon in which brief episodes of I–R increase the tolerance of the heart to subsequent I–R injury 12–24 h later, and lasts 3–4 days. In the present study, preconditioning with four cycles of 3 min ischaemia and 10 min reperfusion reduced the infarct size induced by subsequent I–R. In these experimental conditions, BH4 contents and GTPCH mRNA levels were increased by IPC, and the infarct size-limiting effect was abrogated by treatment with a GTPCH inhibitor. These findings suggest that BH4 levels increased through the induction of GTPCH are implicated in the infarct size-limiting effect of IPC. Only one report shows changes in BH4 levels induced by IPC. Tang et al. (2005) showed that brief I–R for IPC increases BH4 levels in rabbit hearts, and hypercholesterolaemia abrogates IPC by preventing the upregulation of BH4. Thus, our observations are consistent with the report of Tang et al. (2005) showing the importance of increased BH4 levels in the effect of IPC; however, the mechanisms underlying the increase in BH4 levels induced by IPC are unknown.
The biosynthesis of BH4 is regulated by GTPCH, which is the rate-limiting enzyme. Several hypotheses can be proposed to describe the mechanisms underlying the increase in BH4 contents by IPC. Activity of GTPCH has been shown to be stimulated by various factors, including Ca2+ (De Saizieu et al. 1995), and intracellular Ca2+ is likely to be increased transiently by brief I–R. Therefore, it is possible that the increase in BH4 contents was mediated by the stimulation of BH4 synthesis by transient increase of intracellular Ca2+. However, this hypothesis seems unlikely, because elevation of BH4 contents through increase of intracellular Ca2+ by brief I–R may be transient, whereas BH4 contents were measured 24 h after brief I–R. A favoured hypothesis is that induction of GTPCH expression was caused by brief I–R. The stimulation of BH4 synthesis through GTPCH induction by various stimuli, including cytokines and lipopolysaccharide, has been well documented in cultured cells and isolated tissues (Nichol et al. 1985; Huang et al. 2005). In fact, the induction of GTPCH mRNA expression was observed following brief I–R. It is well known that I–R produces ROS (Vanden Hoek et al. 1998), and ROS have been shown to induce chemical preconditioning in various cells (Yu et al. 2006). Moreover, it has been shown that ROS mediate IPC in hearts (Sharma & Singh, 2001). We previously showed that treatment with H2O2 increased BH4 levels through the induction of GTPCH in vascular endothelial cells (Shimizu et al. 2003). In the present study, the increase of BH4 levels following brief I–R was strongly reduced by catalase. Moreover, we previously found that the induction of GTPCH expression by H2O2 is mediated by activation of the JAK2–STAT1 pathway (Shimizu et al. 2008). In fact, the increase in BH4 levels was inhibited by the JAK2 inhibitor AG490, and the level of phospho-STAT1, which is the activated form of STAT1, was also increased by brief I–R. These observations suggest that the increase of BH4 levels following brief I–R is mediated by GTPCH induction through activation of the JAK2–STAT1 pathway by produced H2O2.
As mentioned above, H2O2 is a critical factor in the increase of BH4 levels through GTPCH induction during IPC. Huang et al. (2005) have shown that co-administration of IFNγ and TNFα markedly induces GTPCH expression by the activation of STAT1 and nuclear factor-κB (NF-κB), respectively, compared with single treatment in vascular endothelial cells. Both IFNγ and TNFα have been shown to be produced during I–R (Vaddi et al. 1994; Herskowitz et al. 1995; Yamashita et al. 2000; Reil et al. 2007), and we also observed that TNFα levels were increased by brief I–R. However, whether H2O2 and inflammatory cytokines are associated with the induction of BH4 synthesis during IPC is unknown; therefore, we investigated the interaction between H2O2 and inflammatory cytokines in the stimulation of BH4 synthesis in cultured vascular endothelial cells. We found that H2O2 stimulated TNFα-induced BH4 synthesis. The phenomenon that H2O2 stimulates cytokine-induced BH4 synthesis is the first finding in the regulation of BH4 synthesis. Although future studies are needed to examine this phenomenon in cardiac cells, this interaction may be important in the increase of BH4 levels during IPC.
Tetrahydrobiopterin is an essential cofactor for NO production by all three isoforms of NOS. The inhibition of iNOS activity or disruption of the iNOS gene completely abrogated the infarct-sparing effect of late IPC (Guo et al. 1999; Xuan et al. 2000). Thus, the enhanced biosynthesis of NO is probably necessary to trigger the infarct size-limiting effect of late IPC. In the present study, we observed increases in both BH4 contents and iNOS expression following IPC. Moreover, cardiac NOx levels were increased by IPC, and the increases were blocked by a specific inhibitor for iNOS. Therefore, the increased BH4 seems to function as a cofactor for simultaneously increased iNOS following IPC, and increased production of NO is likely to be implicated in the effect of IPC. We previously described that administration of BH4 strongly reduced cardiac I–R injury without brief I–R, and this effect might be involved in opening ATP-sensitive mitochondrial potassium channels through the stimulation of NO production (Wajima et al. 2006). The acquisition of resistance to I–R injury by IPC may be implicated in the activation of ATP-sensitive mitochondrial potassium channels by NO produced through the induction of iNOS and the increase in BH4 content. However, alternative mechanisms can also be speculated. Importantly, although NOS mainly produces NO in normal conditions, the NOS enzymes also generate superoxide anion and H2O2 by uncoupling when BH4 levels are decreased (Wever et al. 1997; Vásquez-Vivar et al. 1998; Xia et al. 1998). Thus, a decrease in BH4 levels leads not only to impairment of NO production but also to the production of ROS by NOS, while the increased BH4 reduces the uncoupling of constitutive NOS. In fact, many reports, including our report, show that supplemented BH4 strongly reduces I–R injury in hearts (Yamashiro et al. 2002; Tiefenbacher et al. 2003; Wajima et al. 2006). Tetrahydrobiopterin appears to have a protective effect against I–R injury through the attenuation of NOS dysfunction, which is the decrease in NO production and increase in ROS formation; therefore, the induction of BH4 synthesis accompanied by brief I–R for IPC is likely to be due not only the to induced iNOS function but also to the improvement of constitutive NOS function. The role of constitutive NOS during the effect of IPC needs to be examined in the future.
In conclusion, we showed here the possibility that the induction of BH4 synthesis is critical for the formation of resistance to I–R injury following late IPC. The mechanism seems to be mediated by the induction of GTPCH through the JAK2–STAT1 signalling cascade. The interaction of H2O2 and TNFα may be important to increase BH4 through induction of GTPCH by late IPC.