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Am J Physiol Heart Circ Physiol 291: H1138-H1146, 2006. First published April 28, 2006; doi:10.1152/ajpheart.00187.2006
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Cardioprotective role of endogenous hydrogen peroxide during ischemia-reperfusion injury in canine coronary microcirculation in vivo

Toyotaka Yada,1 Hiroaki Shimokawa,3 Osamu Hiramatsu,1 Yoshisuke Haruna,2 Yoshitaka Morita,2 Naoki Kashihara,2 Yoshiro Shinozaki,4 Hidezo Mori,5 Masami Goto,1 Yasuo Ogasawara,1 and Fumihiko Kajiya1

1Department of Medical Engineering and Systems Cardiology and 2Division of Nephrology and Rheumatology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki; 3Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai; 4Department of Physiology, Tokai University School of Medicine, Isehara; and 5Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Suita, Japan

Submitted 22 February 2006 ; accepted in final form 18 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have recently demonstrated that endogenous H2O2 plays an important role in coronary autoregulation in vivo. However, the role of H2O2 during coronary ischemia-reperfusion (I/R) injury remains to be examined. In this study, we examined whether endogenous H2O2 also plays a protective role in coronary I/R injury in dogs in vivo. Canine subepicardial small coronary arteries (≥100 µm) and arterioles (<100 µm) were continuously observed by an intravital microscope during coronary I/R (90/60 min) under cyclooxygenase blockade (n = 50). Coronary vascular responses to endothelium-dependent vasodilators (ACh) were examined before and after I/R under the following seven conditions: control, nitric oxide (NO) synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA), catalase (a decomposer of H2O2), 8-sulfophenyltheophylline (8-SPT, an adenosine receptor blocker), L-NMMA + catalase, L-NMMA + tetraethylammonium (TEA, an inhibitor of large-conductance Ca2+-sensitive potassium channels), and L-NMMA + catalase + 8-SPT. Coronary I/R significantly impaired the coronary vasodilatation to ACh in both sized arteries (both P < 0.01); L-NMMA reduced the small arterial vasodilatation (both P < 0.01), whereas it increased (P < 0.05) the ACh-induced coronary arteriolar vasodilatation associated with fluorescent H2O2 production after I/R. Catalase increased the small arterial vasodilatation (P < 0.01) associated with fluorescent NO production and increased endothelial NOS expression, whereas it decreased the arteriolar response after I/R (P < 0.01). L-NMMA + catalase, L-NMMA + TEA, or L-NMMA + catalase + 8-SPT further decreased the coronary vasodilatation in both sized arteries (both, P < 0.01). L-NMMA + catalase, L-NMMA + TEA, and L-NMMA + catalase + 8-SPT significantly increased myocardial infarct area compared with the other four groups (control, L-NMMA, catalase, and 8-SPT; all, P < 0.01). These results indicate that endogenous H2O2, in cooperation with NO, plays an important cardioprotective role in coronary I/R injury in vivo.

endothelium-derived relaxing factor; myocardial infarction; vascular endothelial function

VASCULAR ENDOTHELIAL CELLS play an important role in maintaining vascular homeostasis by synthesizing and releasing endothelium-derived relaxing factors (EDRFs), including prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF) (6, 9, 26). Endothelial dysfunction is thus characterized by a reduction in the activity of PGI2, NO, and EDHF, thereby enhancing vasoconstrictor responses mediated by endothelin, serotonin, and thrombin (26). Endothelial injury secondary to myocardial ischemia-reperfusion (I/R) decreases the production and activity of EDRFs in acute myocardial infarction (18).

Among the three different EDRFs, the roles of PGI2 and NO have been extensively investigated (6, 9, 26). Regarding EDHF, since the first reports on its existence (6, 9), several candidates for EDHF have been proposed, including cytochrome P-450 metabolites (2, 4), endothelium-derived K+ (7), and electrical communications through gap junctions between endothelial cells and vascular smooth muscle cells (29). Matoba et al. (16, 17) have previously identified that endothelium-derived H2O2 is a primary EDHF in mesenteric arteries of mice and humans. Morikawa et al. (21) have recently confirmed that endothelial Cu,Zn-SOD plays an important role as an EDHF synthase in mice. We have subsequently confirmed the importance of H2O2 in canine coronary microcirculation during coronary autoregulation with reduced coronary perfusion pressure in vivo (35).

However, it remains to be examined whether H2O2 also exerts cardioprotective effects during I/R in the coronary microcirculation in vivo, and if so, whether such effects of H2O2 compensate the impaired NO-mediated responses due to I/R injury in vivo. In this study, we tested our hypothesis that H2O2 plays an important cardioprotective and compensatory role during coronary I/R injury in dogs in vivo.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study conformed to the Guideline on Animal Experiments of Kawasaki Medical School, and approved by an independent review committee from the same institution, and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Animal preparation. Anesthetized mongrel dogs (15–25 kg in body wt, n = 50) of either sex were ventilated with a ventilator (model VS600, IDC, Pittsburgh, PA). Aortic pressure and left ventricular (LV) pressure were continuously monitored with a catheter (SPC-784A, Millar, TX). The blood flow of the left anterior descending coronary artery (LAD) was continuously measured by a transonic flow probe (T206, Transonic Systems, Ithaca, NY).

Measurements of coronary diameter by intravital microscope. We continuously monitored coronary vascular responses by an intravital microscope (VMS 1210, Nihon-Kohden, Tokyo) with a needle probe in vivo as previously described (32). We gently placed the needle probe on subepicardial microvessels. When a clear vascular image was obtained, end-diastolic vascular images were taken with 30 pictures/s (32).

Measurements of regional myocardial blood flow. Regional myocardial blood flow was measured by the nonradioactive microsphere (Sekisui Plastic, Tokyo) technique, as previously described (20). Briefly, the microsphere suspension was injected into the left atrium 85 min after the onset of coronary occlusion. Myocardial collateral flow in the apex during suturing of the collateral vessels from the left circumflex artery (LCX) was calculated according to the formula: time flow = tissue counts x (reference flow/reference counts) and was expressed in milliliters per gram per minute (20).

Detection of H2O2 and NO production. 2',7'-Dichlorodihydrofluorescein diacetate (DCF, Molecular Probes, Eugene, OR) and diaminorhodamine-4M AM (DAR, Daiichi Pure Chemicals, Tokyo) were used to detect H2O2 and NO production in coronary microvessels without a different NO scavenger (e.g., methylene blue), respectively, as previously described (21). Briefly, fresh and unfixed heart tissue was cut into several blocks and frozen in optimal cutting temperature compound (Tissue-Tek, Sakura Fine Chemical, Tokyo) within a few hours. Fluorescent images of the microvessels were obtained 3 min after application of ACh by using a fluorescence microscope (Olympus BX51, Tokyo) (21). We used different animals for the fluorescent treatment (DCF and DAR) and the 2,3,5-triphenyltetrazolium chloride (TTC) treatment.

Western blotting. Portions of myocardial samples were homogenized in lysis buffer. After centrifugation, the supernatants were used for Western immunoblotting. The proteins were transferred by semidry electroblotting to polyvinylidene difluoride membranes. The blots were then blocked and incubated with horseradish peroxidase-conjugated rabbit anti-endothelial NO synthase (eNOS, dimer form) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-actin antibody (Santa Cruz Biotechnology). The antibody was visualized by using an enhanced chemiluminescence method (ECL; Amersham Biosciences, Tokyo). The integrated density of the bands was quantified by using NIH Image analysis, and the protein expression level of eNOS was normalized to that of actin (24).

Experimental protocols. After the surgical procedure and instrumentation, at least 30 min was allowed for stabilization while hemodynamic variables were monitored. The following protocols were examined.

Coronary vascular responses to endothelium-dependent [ACh, 0.5 and 1.0 µg/kg intracoronary (ic)] and -independent [sodium nitroprusside (SNP), 40 and 80 µg/min ic] vasodilators were examined before ischemia (90 min)-reperfusion (60 min) (I/R). ACh and SNP were continuously and retrogradely infused into the diagonal branch of the LAD by using a syringe pump (STC 525, Terumo, Tokyo). The coronary vascular responses to ACh and SNP were examined for 2 min, and the image of maximal vasodilatation was taken at 2 min of infusion of ACh or SNP.

Coronary vasodilator responses to ACh and SNP were examined before and after coronary ischemia (90 min)-reperfusion (60 min) by proximal LAD occlusion under the following seven conditions with cyclooxygenase blockade (ibuprofen, 12.5 mg/kg iv) to evaluate the role of H2O2 and NO without PGI2 in a different set of animals (Fig. 1); 1) control conditions, 2) NG-monomethyl-L-arginine (L-NMMA) alone (2 µmol/min ic for 20 min), 3) catalase alone (40,000 U/kg iv and 240,000 U·kg–1·min–1 ic for 10 min, an enzyme that dismutates H2O2 into water and oxygen), 4) adenosine receptor blockade alone [8-sulfophenyltheophylline (8-SPT), 25 µg·kg–1·min–1 ic for 5 min], 5) catalase plus L-NMMA, 6) catalase plus tetraethylammonium [TEA, 10 µg·kg–1·min–1 ic for 10 min, an inhibitor of large-conductance Ca2+-sensitive potassium (KCa) channels], and 7) catalase plus L-NMMA with 8-SPT (35). These inhibitors were given at 30 min before I/R. An interval between each treatment was 5 min. The basal coronary diameter was defined as that before administration of ACh or SNP either before or after I/R. L-NMMA, catalase, TEA, and 8-SPT were administered alone at 5 min after administration of ACh or SNP. Microspheres were administered at 85 min after the initiation of coronary occlusion. In the combined infusion (L-NMMA + catalase + 8-SPT), catalase solution was infused into the LAD at a rate of 0.5 ml/min at 5 min after infusion of L-NMMA, and then 8-SPT was added into the LAD at 15 min after the initiation of L-NMMA.


Figure 1
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  		Fig. 1. Experimental protocols. TEA, tetraethylammonium; 8-SPT, 8-sulfophenyltheophylline; ACh, acetylcholine; SNP, sodium nitroprusside; Occl, coronary occlusion; Cat, catalase; L-NMMA (L), NG-monomethyl-L-arginine; MI, myocardial infarction.

 
After 1 h of reperfusion, coronary vasodilator responses to ACh and SNP were examined.

After 5 h of reperfusion, we reoccluded the LAD and injected Evans blue dye into a systemic vein. Then, myocardial slices (5 µm thick) were incubated in 1% TTC (Sigma) solution to detect the infarct area (36). Different animals were used for fluorescent treatment (DCF and DAR) and TTC treatment.

Drugs. All drugs were obtained from Sigma Chemical and were diluted in a physiological saline immediately before use.

Statistical analysis. Results are expressed as means ± SE. Vascular responses (see Figs. 3C, 5F, 6F, 7, and 9A) were analyzed by one-way ANOVA followed by Scheffé's post hoc test for multiple comparisons. Difference in the effects of ACh and SNP on subepicardial coronary microvessels before and after I/R (see Figs. 3, A and B, 4, and 8, A and B), and difference between infarct size/risk area and transmural collateral flow in control and other inhibitors (see Fig. 9B) were examined by a multiple regression analysis by using a model in which the change in coronary diameter was set as a dependent variable (y) and vascular size as an explanatory variable (x), while the statuses of control and other inhibitors were set as dummy variables (D1, D2) in the following equation: y = a0 + a1x + a2D1 + a3D2, where a0 through a3 are partial regression coefficients (36). The criterion for statistical significance was at P < 0.05.


Figure 3
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  		Fig. 3. Endothelium-dependent coronary vasodilatation to ACh before and after coronary I/R injury in dogs in vivo. A: small artery (≥100 µm). B: arteriole (<100 µm). C: coronary blood flow (CBF). No. of small arteries or arterioles per animals (n/n) used was 7/5 for control, 8/5 for L-NMMA, 10/5 for catalase, 6/5 for 8-SPT, 8/5 for L-NMMA plus catalase, 5/5 for L-NMMA plus TEA, and 5/5 for L-NMMA plus catalase plus 8-SPT in small arteries; and 12/5 for control, 16/5 for L-NMMA, 12/5 for catalase, 5/5 for 8-SPT, 10/5 for L-NMMA plus catalase, 8/5 for L-NMMA plus TEA, and 7/5 for L-NMMA plus catalase plus 8-SPT in arterioles. No. of animals during the measuring CBF used was 5 for each group. B, before ACh; A, after ACh. *P < 0.05, **P < 0.01.

 

Figure 5
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  		Fig. 5. Detection of H2O2 production. A: left circumflex artery (LCX; baseline without ACh). B: LCX (control). C: left anterior descending coronary artery (LAD; control). D: LAD (L-NMMA). E: LAD (catalase). F: fluorescent intensity (B, baseline without ACh; C, control, L, L-NMMA; Cat, catalase). No. of arterioles per animals used was 5/5 for each group. Dashed line, outline of vessels. Bar, 100 µm.

 

Figure 6
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  		Fig. 6. Detection of nitric oxide (NO) production. A: LCX (baseline without ACh). B: LCX (control). C: LAD (control). D: LAD (L-NMMA). E: LAD (catalase). F: fluorescent intensity (B, baseline without ACh; C, control, L, L-NMMA; Cat, catalase). No. of small arteries per animals used was 5/5 for each group. Dashed line, outline of vessels.

 

Figure 7
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  		Fig. 7. Western blotting showing the effects of catalase on endothelial nitric oxide synthase (eNOS) protein expression in the myocardium of LAD and LCX. No. of animals used was 3 for each group.

 

Figure 9
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  		Fig. 9. Effects of H2O2, NO, and adenosine on I/R-induced MI in dogs in vivo. A: I/R-induced left ventricular infarct size in dogs in vivo. C, control. B: plot of infarct size expressed as a percentage of the risk area and regional collateral flow during I/R. **P < 0.01, #P < 0.05 vs. L-NMMA (L) or Cat or 8-SPT; ##P < 0.01 vs. L + TEA or L + Cat.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemodynamics and blood gases during I/R injury. Immediately after reperfusion, coronary blood flow was increased and some arrhythmias occurred; however, those changes returned to the control levels 1 h after reperfusion when we repeated the measurements. Thus, throughout the experiments, mean aortic pressure and heart rate at baseline were constant and comparable, and PO2, PCO2, and pH were maintained within the physiological ranges (pH 7.35–7.45, PO2 > 70 mmHg, and PCO2 25–40 mmHg,). Hemodynamic variables at baseline did not significantly change after I/R compared with those before I/R (Tables 1 and 2).


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  		Table 1. Hemodynamics during coronary ischemia-reperfusion injury in dogs

 

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  		Table 2. Baseline vascular diameter before I/R in response to ACh

 
Dose responses to ACh and SNP. ACh (0.5 and 1.0 µg/kg ic) and SNP (40 and 80 µg/min ic) caused coronary vasodilatation in a dose-dependent manner at both small arteries and arterioles (Fig. 2). Then we chose the maximal dose of the vasodilators (ACh, 1.0 µg/kg ic, and SNP, 80 µg/min ic) in the following experiments.


Figure 2
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  		Fig. 2. Dose responses to ACh (A and B) and SNP (C and D) before ischemia-reperfusion (I/R). Number of small arteries (A and C) and arterioles (B and D) per animals used was 5/5 for each group. *P < 0.05, **P < 0.01 vs. ACh (0.5 µg/kg) and SNP (40 µg).

 
Endothelium-dependent coronary vasodilatation before and after I/R. There was no significant difference in baseline diameter after ACh before I/R among the groups. All inhibitors did not affect resting coronary artery diameter or coronary blood flow. Under control conditions (before I/R), intracoronary administration of ACh caused a significantly greater coronary vasodilatation in arterioles than in small arteries (Fig. 3, A and B). Coronary I/R significantly impaired the coronary vasodilatation to ACh in both sized arteries (Figs. 3A and 4A), and L-NMMA reduced the vasodilatation in small arteries (Figs. 3A and 4B) but rather increased the response in arterioles compared with control (Figs. 3B and 4A) after I/R. Catalase and 8-SPT increased the ACh-induced vasodilatation in small arteries (Figs. 3A and 4, C and D) but decreased the response in arterioles (Fig. 3B) after I/R. There was no significant difference in coronary blood flow before and after I/R among the control, the L-NMMA, and the catalase group (Fig. 3C). L-NMMA + catalase (Figs. 3, A and B, and 4E) or L-NMMA + TEA (Figs. 3, A and B, and 4F) decreased the vasodilatation in both sized arteries (Fig. 3, A and B) with decrement of coronary blood flow (Fig. 3C), and L-NMMA plus catalase with 8-SPT further decreased the vasodilatation in both sized arteries (Figs. 3, A and B, and 4G) compared with other groups (Fig. 3, A–C).


Figure 4
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  		Fig. 4. Percent change in diameter after ACh before and after coronary I/R injury in dogs in vivo. No. of small arteries and arterioles per animals used was 7/5 for control (A), 8/5 for L-NMMA (B), 10/5 for catalase (C), 6/5 for 8-SPT (D), 8/5 for L-NMMA plus catalase (E), 5/5 for L-NMMA plus TEA (F), and 5/5 for L-NMMA plus catalase plus 8-SPT in small arteries (G); and 12/5 for control (A), 16/5 for L-NMMA (B), 12/5 for catalase (C), 5/5 for 8-SPT (D), 10/5 for L-NMMA plus catalase (E), 8/5 for L-NMMA plus TEA (F), and 7/5 for L-NMMA plus catalase plus 8-SPT in arterioles (G). *P < 0.05, **P < 0.01.

 
Detection of H2O2 and NO production. Fluorescent microscopy with DCF showed that I/R increased the vascular H2O2 production in control LCX (Fig. 5, B and F) compared with baseline conditions (Fig. 5, A and F) and decreased the H2O2 production in control LAD (Figs. 5, C and F), which was enhanced by L-NMMA (Fig. 5, D and F) and was abolished by catalase (Fig. 5, E and F) in arterioles. By contrast, the production of NO as assessed with DAR fluorescence was increased in control LCX (Fig. 6, B and F) compared with baseline LCX (Fig. 6, A and F) after I/R, decreased in control LAD (Fig. 6, C and F), inhibited by L-NMMA (Fig. 6, D and F), and was enhanced by catalase (Fig. 6, E and F) in small arteries.

Western blotting of eNOS protein expression in myocardium. In the control group, expression of eNOS protein in the ischemic LAD area was significantly decreased compared with the nonischemic LCX area (Fig. 7). In the catalase group, this decrease in the eNOS protein expression was inhibited by catalase (Fig. 7).

Endothelium-independent coronary vasodilatation. Coronary vasodilator responses to SNP were comparable under all conditions in both sized arteries (Fig. 8). Those coronary vasodilator responses were resistant to the blockade of NO synthesis with L-NMMA (Fig. 8).


Figure 8
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  		Fig. 8. Endothelium-independent coronary vasodilatation before and after coronary I/R injury in dogs in vivo. A: small artery (≥100 µm). B: arteriole (<100 µm). No. of small arteries and arterioles per animals used (n/n) was 7/5 for control, 8/5 for L-NMMA, 10/5 for catalase, 6/5 for 8-SPT, 8/5 for L-NMMA plus catalase, 5/5 for L-NMMA plus TEA, and 5/5 for L-NMMA plus catalase plus 8-SPT in small arteries; and 12/5 for control, 16/5 for L-NMMA, 12/5 for catalase, 5/5 for 8-SPT, 10/5 for L-NMMA plus catalase, 8/5 for L-NMMA plus TEA, and 7/5 for L-NMMA plus catalase plus 8-SPT in arterioles.

 
Effect of H2O2 on I/R-induced myocardial infarct size. I/R injury caused myocardial infarction, the size of which was ~40% of the LV risk area (Fig. 9A). Intracoronary L-NMMA, catalase, or 8-SPT alone did not further increase the I/R-induced infarct size (Fig. 9A). By contrast, intracoronary L-NMMA plus catalase or TEA markedly increased the infarct size, and L-NMMA plus catalase with 8-SPT further increased the infarct size (Fig. 9A). In the control group, there was an inverse relation between the infarct size and transmural collateral blood flow measured by microsphere technique (r = 0.90, P < 0.01). There was no significant difference in the relationship among the control, L-NMMA, and catalase treatment (Fig. 9B). L-NMMA plus catalase or TEA significantly shifted the regression line upward compared with the control group (both P < 0.01), and L-NMMA plus catalase with 8-SPT further shifted the regression line upward compared with L-NMMA plus catalase or TEA (Fig. 9B, both P < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of the present study is that endogenous H2O2, in cooperation with NO, plays an important cardioprotective role during coronary I/R injury as a compensatory mechanism for NO in vivo. To the best of our knowledge, this is the first report that demonstrates the important protective role of endogenous H2O2, in cooperation with NO, against coronary I/R injury in vivo.

Validations of experimental model and methodology. On the basis of the previous reports (22, 31), we chose the adequate dose of ACh, SNP, L-NMMA, catalase, TEA, and 8-SPT to examine the effects of endothelium-dependent and -independent coronary vasodilator responses and inhibition of NO synthesis, H2O2, KCa channels, and adenosine receptor, respectively. In addition, on the basis of previous studies and our own (31, 35), we choose the doses of ACh and SNP that cause maximal coronary vasodilatation in dogs in vivo. TEA at low doses is fairly specific for KCa channel, but at higher doses it may block a number of other K channels. Because several KCa channels are involved in H2O2-mediated responses (26), we selected the nonselective KCa inhibitor TEA to inhibit all KCa channels (15). We have previously confirmed the validity of the methods that we used in the present study (32). After 60–90 min of ischemia, ultrastructural damage of coronary endothelium was observed particularly in the subendocardium in the present study, a finding consistent with the previous study (8).

H2O2 during coronary I/R in vivo. It was previously reported that relaxations of isolated large canine coronary arteries to exogenous H2O2 were partially endothelium dependent (23). Recently, Matoba et al. (16, 17) identified that endothelium-derived H2O2 is an EDHF in mouse and human mesenteric microvessels. Subsequently, we (35) and others (19) have confirmed that endogenous H2O2 exerts important vasodilator effects in canine coronary microcirculation in vivo and in isolated human coronary microvessels, respectively. It is conceivable that H2O2 is produced from superoxide anions derived from several sources in endothelial cells, including eNOS, cyclooxygenase, lipoxygenase, cytochrome P-450 enzymes, and NAD(P)H oxidases (16). In the present study, L-NMMA or catalase alone did not completely abolish the ACh-induced vasodilatation in both sized arteries, whereas L-NMMA plus catalase markedly attenuated the residual vasodilatation in vivo as did TEA, indicating that H2O2 exerts important vasodilator effects during I/R injury in canine coronary microcirculation in vivo (Figs. 3 and 4). Furthermore, in the present study, endogenous H2O2-mediated coronary vasodilatation was noted to a greater extent in arterioles than in small arteries (Figs. 3 and 4), confirming the predominant role of H2O2 in microvessels and that of NO in relatively large arteries in vivo (25).

Compensatory vasodilator mechanism among H2O2, NO, and adenosine. It is well known that coronary vascular tone is regulated by the interactions among several endogenous vasodilators, including NO, H2O2, and adenosine (33). These vasodilators play an important role in compensatory vasodilatation of coronary microvessels during myocardial ischemia (35). In the present study (Figs. 3 and 4), endothelium-dependent arteriolar vasodilatation to ACh during coronary I/R was significantly increased by L-NMMA while small arterial vasodilatation to ACh was increased by catalase and 8-SPT, and the residual arteriolar dilation was further inhibited by both of them (L-NMMA plus catalase or TEA). Furthermore, fluorescent microscopy with DCF and DAR, respectively, showed that H2O2 and NO production after I/R were enhanced in small coronary arteries and arterioles by L-NMMA [fluorescent intensity (FI) 1.8] and catalase (FI 1.9) compared with those in the LAD of control group (Figs. 5 and 6, FI: DAR 1.2 and DCF 1.1). The residual small arteriolar dilatation after combined administration of L-NMMA + catalase was completely blocked by 8-SPT, an adenosine receptor blocker, indicating that adenosine also compensated for the loss of action of NO and H2O2. Taken together, these results indicate the compensatory vasodilator effects among NO, H2O2, and adenosine to maintain coronary blood flow during coronary I/R injury in vivo. H2O2 and NO were mutually compensatory in both small arteries and arterioles, and in the presence of their inhibitors (catalase and L-NMMA), adenosine also caused arteriolar vasodilatation, as we reported previously (35). This finding is consistent with our finding that NO, H2O2, and adenosine play an important compensatory role in coronary autoregulation in canine coronary microcirculation in vivo (35). It was reported that TEA inhibited adenosine-induced vasodilatation of canine subepicardial coronary arteries in vitro (3). Furthermore, H2O2 stimulates protein kinase C, phospholipase A2, and arachidonic acid release and increases intracellular cAMP levels (10). These findings suggest that cAMP-mediated pathway is involved, at least in part, during coronary vasodilatation through KCa channels after I/R injury.

Role of H2O2 during coronary I/R. It is known that KCa channels substantially contribute to coronary vasodilatation in myocardial ischemia (22) and that H2O2 also activates KCa channels (11). However, it remains to be examined whether H2O2 contributes to coronary vasodilatation during I/R in vivo. The present results demonstrate that H2O2 substantially contributes to coronary vasodilatation during I/R in vivo as a compensatory mechanism for the loss of NO. Several mechanisms have been proposed for KCa channel opening during coronary I/R, including cellular acidosis (27), increase in intracellular Ca2+ concentration after ischemia (28), and H2O2 production by inflammatory cells (5). Furthermore, an inhibitor of NO synthesis [NG-nitro-L-arginine methyl ester (L-NAME)] or that of KCa channels (charybdotoxin) partly inhibits the protective effect on myocardial infarct size (22). Liu et al. (14) demonstrated that peroxynitrite inhibits KCa channel activity in human coronary arterioles during I/R. This mechanism might contribute to impaired H2O2-mediated dilation in I/R where NO synthase activity is increased in the presence of excess of O2–. In the present study, inhibition of H2O2 or NO alone did not significantly increase myocardial infarct size compared with control conditions (Fig. 9). These results suggest that H2O2 and NO exert cardioprotective effects against the development of myocardial infarction in a compensatory manner.

Recently, we have demonstrated that the expression of eNOS protein is decreased in the ischemic myocardium, which is improved by a selective Rho-kinase inhibitor, hydroxyfasudil, during coronary I/R injury in dogs in vivo (36). Furthermore, a physiological concentration (2 µmol/l) of H2O2 improved the recovery of both cardiac contractile function and energy metabolism after I/R in perfused rat heart (37). In the present study, the expression of eNOS protein was decreased in the ischemic myocardium, which was increased by catalase during I/R injury (Fig. 7). All these mechanisms may be involved in the beneficial effects of H2O2 on the I/R-induced myocardial injury. It also is conceivable that I/R reduces endothelial tetrahydrobiopterin levels in coronary vessels and impairs eNOS function (30).

Limitations of the study. Several limitations should be mentioned for the present study. First, we did not examine coronary vasodilatation in response to SOD/SOD mimetic (e.g., Tempol) or peroxynitrite inhibitor (e.g., ebselen) after I/R. However, because of the complex interactions among the oxygen species, we consider that both Tempol and ebselen also affect H2O2 metabolism by scavenging superoxide anions and peroxynitrite, respectively. Second, in addition to catalase, endogenous glutathione peroxidase (GSH) also plays an important role in removing H2O2, and NO also could be a substrate for endogenous catalase (1). However, in the present study, we used exogenous catalase to remove H2O2 to examine the role of the reactive oxygen species. Third, the exact source of vascular H2O2 production remains to be elucidated (e.g., the endothelium, smooth muscle, or cardiomyocytes). Fourth, while we were able to demonstrate the production of H2O2 using fluorescent microscopy with DCF, we were unable to quantitatively measure the H2O2 production because DCF detects H2O2, ONOO–, and HOCl as well. Fifth, we were unable to find smaller arterioles because of the limited spatial resolution of our CCD intravital microscope. If we had an intravital camera with higher resolution, we would be able to observe coronary vasodilator responses of smaller arterioles.

Clinical implications and conclusions. During coronary I/R, microemboli of atherosclerosis debris and platelet plugs are released into the coronary microcirculation, particularly at revascularization with thrombolysis and/or percutaneous coronary intervention. Thus preexisting coronary endothelial dysfunction with various risk factors may be an important determinant for I/R injury in acute myocardial infarction. The synthesis and/or action of endothelium-derived NO are impaired under various pathological conditions, such as hypertension, hyperlipidemia, and diabetes mellitus (26, 34). In hypertension, K channel activities are increased in a compensatory manner with reduced NO activity (13). The present results suggest that NO and H2O2 compensate each other to cause coronary vasodilatation during I/R injury in vivo.

In conclusion, we were able to demonstrate that endogenous H2O2, in cooperation with NO, plays an important cardioprotective role in coronary I/R injury in vivo. The present findings may have important clinical implications because H2O2-mediated mechanisms substantially contribute to endothelium-dependent vasodilatation in coronary I/R in vivo.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology, Tokyo, Japan (Nos. 13307024, 13557068, 14657178, 15256003, 16209027, 16300164), and the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Yada, Dept. of Medical Engineering and Systems Cardiology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan (e-mail: yada{at}me.kawasaki-m.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Antunes F, Han D, and Cadenas E. Relative contributions of heart mitochondria glutathione peroxidase and catalase to H2O2 detoxification in in vivo conditions. Free Radic Biol Med 33: 1260–1267, 2002.[CrossRef][ISI][Medline]
  2. Bauersachs J, Hecker M, and Busse R. Display of the characteristics of endothelium-derived hyperpolarizing factor by a cytochrome P450-derived arachidonic acid metabolite in the coronary microcirculation. Br J Pharmacol 113: 1548–1553, 1994.[ISI][Medline]
  3. Cabell F, Weiss DS, and Price JM. Inhibition of adenosine-induced coronary vasodilation by block of large-conductance Ca2+ activated K+ channels. Am J Physiol Heart Circ Physiol 267: H1455–H1460, 1994.[Abstract/Free Full Text]
  4. Campbell WB, Gebremedhin D, Pratt PF, and Harder DR. Identification of epoxyeicosatrienoic acids as an endothelium-derived hyperpolarizing factor. Circ Res 78: 415–423, 1996.[Abstract/Free Full Text]
  5. Chandrasekar B, Colston JT, de la Rosa SD, Rao PP, and Freeman GL. TNF-{alpha} and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-{kappa}B activation. Biochem Biophys Res Commun 303:1152–1158, 2003.[CrossRef][ISI][Medline]
  6. Chen G, Suzuki H, and Weston AH. Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from blood vessels. Br J Pharmacol 95: 1165–1174, 1988.[ISI][Medline]
  7. Edwards G, Dora KA, Gardener MJ, Garland CJ, and Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 396: 269–272, 1998.[CrossRef][Medline]
  8. Ehring T, Krajcar M, Baumgart D, Kompa S, Hummelgen M, and Heusch G. Cholinergic and {alpha}-adrenergic coronary constriction with increasing ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 268: H886–H894, 1995.[Abstract/Free Full Text]
  9. Feletou M and Vanhoutte PM. Endothelium-dependent hyperpolarization of canine smooth muscle. Br J Pharmacol 93: 515–524, 1988.[ISI][Medline]
  10. Gao Y and Vanhoutte PM. Effects of hydrogen peroxide on the responsiveness of isolated canine bronchi: role of prostaglandin E2 and I2. Am J Physiol Lung Cell Mol Physiol 263: L402–L408, 1992.[Abstract/Free Full Text]
  11. Hayabuchi Y, Nakaya Y, Matsuoka S, and Kuroda Y. Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels. Heart Vessels. 13: 9–17, 1998.[ISI][Medline]
  12. Huang A, Sun D, Carroll MA, Jiang H, Smith CJ, Connetta JA, Falck JR, Shesely EG, Koller A, and Kaley G. EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice. Am J Physiol Heart Circ Physiol 280: H2462–H2469, 2001.[Abstract/Free Full Text]
  13. Liu Y, Hudetz AG, Knaus HG, and Rusch NJ. Increased expression of Ca2+-sensitive K+ channels in the cerebral microcirculation of genetically hypertensive rats: evidence for their protection against cerebral vasospasm. Circ Res 82: 729–737, 1998.[Abstract/Free Full Text]
  14. Liu Y, Terata K, Chai Q, Li H, Kleinman LH, and Gutterman DD. Peroxynitrite inhibits Ca2+-activated K+ channel activity in smooth muscle of human coronary arterioles. Circ Res 91: 1070–1076, 2002.[Abstract/Free Full Text]
  15. Masumoto A, Hirooka Y, Shimokawa H, Hironaga K, Setoguchi S, and Takeshita A. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension 38: 1307–1310, 2001.[Abstract/Free Full Text]
  16. Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H, and Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest 106: 1521–1530, 2000.[ISI][Medline]
  17. Matoba T, Shimokawa H, Kubota H, Morikawa K, Fujiki T, Kunihiro I, Mukai Y, Hirakawa Y, and Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries. Biochem Biophys Res Commun 290: 909–913, 2002.[CrossRef][ISI][Medline]
  18. Mehta JL, Nichols WW, Donnelly WH, Lawson DL, and Saldeen TG. Impaired canine coronary vasodilator response to acetylcholine and bradykinin after occlusion-reperfusion. Circ Res 64: 43–54, 1989.[Abstract/Free Full Text]
  19. Miura H, Bosnjak JJ, Ning G, Saito T, Miura M, and Gutterman DD. Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ Res 92: e31–e40, 2003.[Abstract/Free Full Text]
  20. Mori H, Haruyama Y, Shinozaki H, Okino H, Iida A, Takanashi R, Sakuma I, Husseini WK, Payne BD, and Hoffman JI. New nonradioactive microspheres and more sensitive X-ray fluorescence to measure regional blood flow. Am J Physiol Heart Circ Physiol 263: H1946–H1957, 1992.[Abstract/Free Full Text]
  21. Morikawa K, Shimokawa H, Matoba T, Kubota H, Akaike T, Talukder MA, Hatanaka M, Fujiki T, Maeda H, Takahashi S, and Takeshita A. Pivotal role of Cu,Zn-superoxide dismutase in endothelium-dependent hyperpolarization. J Clin Invest 112: 1871–1879, 2003.[CrossRef][ISI][Medline]
  22. Ogita H, Node K, Asanuma H, Sanada S, Liao Y, Takashima S, Asakura M, Mori H, Shinozaki Y, Hori M, and Kitakaze M. Amelioration of ischemia- and reperfusion-induced myocardial injury by the selective estrogen receptor modulator, raloxifene, in the canine heart. J Am Coll Cardiol 40: 998–1005, 2002.[Abstract/Free Full Text]
  23. Rubanyi GM and Vanhoutte PM. Oxygen-derived radicals, endothelium, and responsiveness of vascular smooth muscle. Am J Physiol Heart Circ Physiol 250: H815–H821, 1986.[Abstract/Free Full Text]
  24. Satoh M, Fujimoto S, Haruna Y, Arakawa S, Horike H, Komai N, Sasaki T, Tsujioka K, Makino H, and Kashihara N. NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy. Am J Physiol Renal Physiol 288: F1144–F1152, 2005.[Abstract/Free Full Text]
  25. Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike R, Fukumoto Y, Takayanagi T, Nagao T, Egashira K, Fujishima M, and Takeshita A. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulations. J Cardiovasc Pharmacol 28: 703–711, 1996.[CrossRef][ISI][Medline]
  26. Shimokawa H. Primary endothelial dysfunction: atherosclerosis. J Mol Cell Cardiol 31: 23–37, 1999.[CrossRef][ISI][Medline]
  27. Siegel G, Emden J, Wenzel K, Mironneau J, and Stock G. Potassium channel activation in vascular smooth muscle. Adv Exp Med Biol 311: 53–72, 1992.[Medline]
  28. Steenbergen C, Murphy E, Levy L, and London RE. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 60: 700–707, 1987.[Abstract/Free Full Text]
  29. Taylor HJ, Chaytor AT, Evance WH, and Griffith TM. Inhibition of the gap junctional component of endothelium-dependent relaxations in rabbit iliac artery by 18-alpha glycyrrhetinic acid. Br J Pharmacol 125: 1–3, 1998.[CrossRef][ISI][Medline]
  30. Tiefenbacher CP, Chilian WM, Mitchell M, and Defily DV. Restoration of endothelium-dependent vasodilation after reperfusion injury by tetrahydrobiopterin. Circulation 94: 1423–1429, 1996.[Abstract/Free Full Text]
  31. Traverse JH, Chen Y, Crampton M, Voss S, and Bache RJ. Increased extravascular forces limit endothelium-dependent and -independent coronary vasodilation in congestive heart failure. Cardiovasc Res 52: 454–461, 2001.[Abstract/Free Full Text]
  32. Yada T, Hiramatsu O, Kimura A, Goto M, Ogasawara Y, Tsujioka K, Yamamori S, Ohno K, Hosaka H, and Kajiya F. In vivo observation of subendocardial microvessels of the beating porcine heart using a needle-probe videomicroscope with a CCD camera. Circ Res 72: 939–946, 1993.[Abstract/Free Full Text]
  33. Yada T, Hiramatsu O, Kimura A, Tachibana H, Chiba Y, Lu S, Goto M, Ogasawara Y, Tsujioka K, and Kajiya F. Direct in vivo observation of subendocardial arteriolar responses during reactive hyperemia. Circ Res 77: 622–631, 1995.[Abstract/Free Full Text]
  34. Yada T, Goto M, Hiramatsu O, Tachibana H, Toyota E, Nakamoto H, Ogasawara Y, Matsuda H, Arakawa K, Hayashi K, Suzuki H, and Kajiya F. In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts. Am J Physiol Heart Circ Physiol 284: H1785–H1792, 2003.[Abstract/Free Full Text]
  35. Yada T, Shimokawa H, Hiramatsu O, Kajita T, Shigeto F, Goto M, Ogasawara Y, and Kajiya F. Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo. Circulation 107: 1040–1045, 2003.[Abstract/Free Full Text]
  36. Yada T, Shimokawa H, Hiramatsu O, Kajita T, Shigeto F, Tanaka E, Shinozaki Y, Mori H, Kiyooka T, Katsura M, Ohkuma S, Goto M, Ogasawara Y, and Kajiya F. Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia-reperfusion injury in canine coronary microcirculation in vivo. J Am Coll Cardiol 45: 599–607, 2005.[Abstract/Free Full Text]
  37. Yaguchi Y, Satoh H, Wakahara N, Katoh H, Uehara A, Terada H, Fujise Y, and Hayashi H. Protective effects of hydrogen peroxide against ischemia/reperfusion injury in perfused rat hearts. Circ J 67: 253–258, 2003.[CrossRef][ISI][Medline]



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