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Am J Physiol Heart Circ Physiol 286: H2296-H2304, 2004; doi:10.1152/ajpheart.00671.2003
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In vivo role of heme oxygenase in ischemic coronary vasodilation

Yasuhiro Nishikawa,1 David W. Stepp,2 Daphne Merkus,1 Deron Jones,3 and William M. Chilian4

1Physiology and 3Surgery, Medical College of Wisconsin, Watertown, Wisconsin 53226; 2Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912; and 4Health Sciences Center, Louisiana State University, New Orleans, Louisiana 70112

Submitted 15 July 2003 ; accepted in final form 10 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The heart constitutively expresses heme oxygenase (HO)-2, which catabolizes heme-containing proteins to produce biliverdin and carbon monoxide (CO). The heart also contains many possible substrates for HO-2 such as heme groups of myoglobin and cytochrome P-450s, which potentially could be metabolized into CO. As a result of observations that CO activates guanylyl cyclase and induces vascular relaxation and that HO appears to confer protection from ischemic injury, we hypothesized that the HO-CO pathway is involved in ischemic vasodilation in the coronary microcirculation. Responses of epicardial coronary arterioles to ischemia (perfusion pressure ~40 mmHg; flow velocity decreased by ~50%; dL/dt reduced by ~60%) were measured using stroboscopic fluorescence microangiography in 34 open-chest anesthetized dogs. Ischemia caused vasodilation of coronary arterioles by 36 ± 6%. Administration of NG-monomethyl-L-arginine (L-NMMA, 3 µmol·kg–1·min–1 intracoronary), indomethacin (10 mg/kg iv), and K+ (60 mM, epicardial suffusion) to prevent the actions of nitric oxide, prostaglandins, and hyperpolarizing factors, respectively, partially inhibited dilation during ischemia (36 ± 6 vs. 15 ± 4%; P < 0.05). The residual vasodilation during ischemia after antagonist administration was inhibited by tin mesoporphyrin IX (SnMP, 10 mg/kg iv), which is an inhibitor of HO (15 ± 4 vs. 7 ± 2%; P < 0.05 vs. before SnMP). The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (10–5 M, epicardial suffusion) also inhibited vasodilation during ischemia in the presence of L-NMMA with indomethacin and KCl. Moreover, administration of heme-L-arginate, which is a substrate for HO, produced dilation after ischemia but not after control conditions. We conclude that during myocardial ischemia, HO-2 activation can produce cGMP-mediated vasodilation presumably via the production of CO. This vasodilatory pathway appears to play a backup role and is activated only when other mechanisms of vasodilation during ischemia are exhausted.

microcirculation; carbon monoxide; hypoperfusion; ischemia

THE HEART REQUIRES CONTINUOUS oxygen delivery and blood flow to support the high levels of aerobic metabolism of working cardiac muscle. Interruption in coronary blood flow causes an imbalance between oxygen delivery and consumption. This imbalance results in the release of vasodilators that increase perfusion (15, 32) but to date, the identity of these vasodilators remains unclear.

Carbon monoxide (CO) is an endogenously generated gas that is produced from catabolism of heme by heme oxygenase (HO). CO stimulates soluble guanylyl cyclase (GC), increases cGMP in vascular tissue, and elicits vasodilation (9, 13). HO enzyme activity has been described in numerous tissues including heart (1, 33) and vascular endothelium (35, 36). Moreover, a growing consensus is that HO activity may be cardioprotective (10, 17). Recent studies suggest that CO participates in vascular regulation during normal (23, 49) and abnormal physiological conditions including endotoxin shock (48), chronic hypoxia (6), portal hypertension (16), and subarachnoid hemorrhage (44).

We hypothesized that CO may also play a significant role in the increase in vascular diameter that is elicited by reductions in coronary perfusion pressure. To test this hypothesis, we measured coronary microvascular diameter after 7 min of ischemia. Ischemia was verified by reductions in microvascular flow velocity and epicardial muscle shortening velocity (dL/dt). Vasodilator reactivity was examined in the presence and absence of the HO antagonist tin mesoporphyrin IX (SnMP; Ref. 25). The role of CO was also examined with the production or activity of endogenous dilators impaired via a combination of NG-monomethyl-L-arginine (L-NMMA), indomethacin (Indo), and elevated K+ suffusion. The involvement of cGMP was tested in the presence of the GC antagonist 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ). To determine whether HO enzyme activity was increased during ischemia, the HO substrate heme-L-arginate was applied to the microvasculature during reductions in perfusion pressure and in the presence of SnMP. For this pathway to be active, it is imperative that HO be expressed in the heart. Accordingly, HO expression in the canine heart was validated via Western analysis. The results show that HO-2 is expressed in the canine myocardium, is active during episodes of low coronary perfusion pressure, and results in vasodilation of the coronary microcirculation. We conclude that HO activity and its product CO may play significant roles in microvascular dilation during myocardial ischemia especially when other vasodilatory mechanisms are exhausted or compromised.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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All procedures were approved by the Institutional Animal Care and Use Committee and conformed to the guidelines for ethical treatment of animals as described by the American Physiological Society.

Coronary Microvascular Preparation

Adult mongrel dogs (body wt, 5–15 kg) were anesthetized with pentobarbital sodium (Nembutal, 30 mg/kg iv). A core body temperature of 37°C was maintained by placing the animals on an electric blanket. The right femoral artery and vein were cannulated for measurements of aortic pressure and arterial blood gases and for administration of drugs, respectively. A 5-Fr fluid-filled catheter was advanced into the left ventricle (LV) from the left carotid artery to measure LV pressure. The first derivative of the LV pressure trace (LV dP/dt) was obtained from an online differentiator. Hemodynamic data were acquired continuously using ACODAS data-acquisition software from DATAQ (Akron, OH). A tracheotomy was performed and ventilation was achieved via high-frequency jet ventilation. Using the maximum LV dP/dt value as a timing reference, a solenoid connected to a pressure source (100% O2, 6–12 lb/in2) was triggered to open for 20–35 ms at the same time in each cardiac cycle. The small tidal volume minimizes respiratory movement, which occurs at the same frequency as the heart beat. Arterial pH and blood gas measurements were monitored frequently and values were maintained within the following ranges by adjustment of the tracheal catheter or administration of sodium bicarbonate: PCO2, 25–40 mmHg; PO2, 100–200 mmHg; and pH, 7.35–7.45. All animals routinely received propranolol (1 mg/kg) and the H1-histamine receptor antagonist diphenhydramine (1 mg/kg) to limit tissue motion and prevent anaphylactic reactions to the high-molecular-weight dextrans, respectively.

To allow visualization of the epicardial surface, the heart was exposed by a left thoracotomy at the fifth intercostal space and stabilized in a partial pericardial cradle. A large [left anterior descending (LAD) or circumflex] coronary artery was exposed, and a 24-gauge Teflon cannula was inserted to allow measurement of coronary artery pressure and intracoronary administration of fluorescent microspheres, drugs, and fluorochromes. After an area of visible epicardial microvessels was identified, four 22-gauge pins were passed horizontally through the left ventricle to minimize vertical cardiac motion. Neither maneuver appears to compromise coronary tone, because resting blood flow or vasodilator reserve values are unaffected.

Measurement of Coronary Microvascular Diameter

To measure coronary microvascular diameter, the cardiac surface was illuminated by a stroboscope (100-W xenon arc; Chadwick-Helmuth; El Monte, CA) that was triggered by the maximum LV dP/dt signal to flash once for 20–30 µs at the same point during each cardiac cycle. The strobe trigger signal was monitored in relation with LV pressure for a precise determination of the strobe flash in the cardiac cycle. The combined use of low-tidal-volume jet ventilation and brief epicardial illumination (both of which were synchronized to the cardiac cycle) caused the surface coronary microvessels to appear virtually motionless when viewed through an intravital microscope (Leitz Ploemopak; Wild Leitz). The microscope objectives used were the Leitz EF4 (x4; numerical aperture, 0.22) and the Leitz L10 (x10; numerical aperture, 0.22).

To illuminate the inner diameter of the microvessels, 50- to 100-µl aliquots of fluorescein isothiocyanate-dextran (25 mg/ml) in 0.9% saline (mol wt, 500,000) were injected into the coronary artery through the coronary cannula. A Leitz H2 excitation-barrier filter was used to activate the fluorescein and receive the emitted light. Each injection causes arterial and venous vessels to fluoresce sequentially for 5–10 s. The anatomic landmarks of a particular vessel were identified, and 5–8 images were obtained over a period of <1 min using a Cohu silicon-intensified tube video camera (Cohu intensified charge-couple device camera). The images were digitized directly from the camera by a frame digitizer (Scion Image) and transferred to a Macintosh computer (Apple Computer) for offline diameter measurements using appropriate software (Image 2.18; National Institutes of Health Research Services Branch). Diameters were measured by aligning cursors at the vessel edges on the computer screen. Measurements in pixels were converted to micrometers using a conversion factor determined from a micrometer grid. Typically, microvascular measurements over each image-acquisition period vary by less than ±3% from the average value. A vessel was excluded from analysis if the control microvascular diameter after interventions varied from the prior baseline by >10%.

Measurement of Coronary Microvascular Velocity

A procedure similar to that designed by Nellis and Whitesell (38) was used to measure flow velocity in the epicardial coronary microcirculation in vivo as detailed elsewhere (45). The strobe was triple flashed during a single video frame (33 ms for 1 complete video image); thus three images separated in time (8 ms between flashes) were captured on the same video frame. The fluorescent particles appeared three times in each image, and the velocity was calculated as the quotient of the distance moved by the microspheres and the time interval between strobe flashes. Fluorescent microspheres (10-µm Fluoresbrite YG plain microspheres; Polyscience) suspended in a mixture of dilute fluorescein solution and 0.9% saline were injected into the coronary artery. Images were obtained during a fixed interval of the cardiac cycle.

To observe changes in systolic function on the epicardial surfaces, images of triple-flashed microspheres trapped in the tissue close to the vessels of interest were recorded. The LV dP/dt triggered the strobe at a point of 25% of the R-R interval per cardiac cycle. Thus we obtained triple-flashed images of microspheres in the tissue during the ejection phase of systole (Fig. 1), and these measurements reflect ventricular shortening.



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  		Fig. 1. Typical video-capture images of a small arteriole during diastole in the beating heart before and during reduced perfusion. Strobe was flashed three times per video scan during injection of microspheres. Microvascular velocity was determined as the distance the sphere traveled (between 2 microspheres) divided by the strobe-flash interval (8 ms). During reduced perfusion pressure, the microvascular flow velocity of the microspheres decreased by ~50%.

 
Preliminary Studies

Studying the coronary circulation during reductions in perfusion pressure is particularly challenging. Observation of coronary microvessels in situ requires a stable focal plane, and the ischemic myocardium becomes unstable in terms of rhythm and contractility. Thus we used a segmental approach whereby only a small portion of the myocardium was subject to underperfusion. To accomplish this, a ligature was placed around the distal LAD, and a catheter was inserted just distal to the ligature. The area supplied by the downstream branches of the LAD were underperfused. Because in this situation perfusion pressure is severely reduced to only a small subendocardial region, traditional measures such as reduced flow, changes in endocardial systolic shortening, and release of ischemic markers are not appropriate for evaluation of ischemic status in the epicardium where the microvascular diameters are to be measured. Thus we adapted the known effects of ischemia to verify ischemia based on two criteria: flow velocity and the rate of systolic wall shortening during systole dL/dt. Reduction in perfusion pressure to ~40 mmHg reduced microvascular flow velocity during diastole from 14 ± 3 to 7 ± 2 mm/s (88 ± 3 µm ID; 5 vessels each from 2 dogs), which indicates that a marked reduction in flow had occurred to the affected region (Fig. 1). Results of a second index suggested that ischemia was an observed reduction in systolic wall motion. As shown in Fig. 2, reduction in perfusion pressure caused parallel decreases in the systolic shortening velocity of fluorescent microspheres trapped in the subepicardium during systole (4.0 ± 1 to 1.7 ± 1 mm/s; control vs. occlusion, 5 vessels each from 2 dogs). Together with flow velocity, these data strongly suggest that during ischemia, the decreases in myocardial blood flow and systolic shortening function are consistent with the occurrence of ischemia in the epicardium. On the basis of these initial studies, we used these procedures to study the mechanisms of vasodilation during myocardial ischemia.



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  		Fig. 2. Typical video-capture images of tissue close to a vessel of interest during systole before and during reduced perfusion. When microspheres are lodged in tissue, the distance the spheres move represents the length of shortening of the epicardial surface during ejection phase (8 ms); dividing this value by the interval between flashes yields the shortening velocity. During ischemia, the distance was reduced compared with during baseline. This suggests that systolic shortening is decreased in the epicardial surface during ischemia.

 
An additional set of preliminary experiments was performed to assess the reproducibility of dilation during ischemia. The three periods of myocardial ischemia (7 min in duration; 1 occlusion/h) caused similar vasodilation (27 ± 3, 23 ± 3, and 21 ± 3%; P = not significant; n = 3) in coronary microvessels (11 vessels, 72 ± 5 µm), which suggests that our protocol, 7 min of reduced perfusion pressure with 60 min of recovery, did not significantly alter the ability of the coronary circulation to dilate during successive myocardial ischemia. Although the baseline diameter before each myocardial ischemia tended to increase (73 ± 5, 76 ± 5, and 79 ± 6 µm), these changes were minor.

Experimental Protocols

Protocol 1: role of nitric oxide, prostaglandins, and K+ channels in vasodilation during myocardial ischemia. In five animals, diameter measurements were performed during the following conditions: 1) baseline; 2) flow reduction; 3) Indo (10 mg/kg iv) and L-NMMA (3 µmol/min intracoronary for 5 min) administration; 4) flow reduction; 5) KCl suffusion (15 min); 6) baseline; and 7) flow reduction. KCl suffusion solution (in mM: 102 NaCl, 45.4 KCl, 2.0 CaCl2, 1.2 MgCl2, 11.0 dextrose, and 18 bicarbonate) was administered by suffusion onto the microvascular field of interest. The diameter measurements were performed during the last 5 min of KCl suffusion. Doses were verified to inhibit endothelium-dependent vasodilation identified in previous studies (39, 40).

Protocol 2: role of CO in vasodilation during myocardial ischemia without blockade of endothelium-dependent dilation. In four animals, diameter measurements were performed during the following conditions: 1) baseline; 2) flow reduction; 3) SnMP (HO inhibitor, 10 mg/kg iv) administration; 4) baseline; 5) flow reduction; 6) Indo (10 mg/kg iv), L-NMMA (3 µmol/min intracoronary for 5 min), and KCl (15 min) suffusion; 7) baseline; and 8) flow reduction.

Protocol 3: role of CO in vasodilation during myocardial ischemia with blockade of endothelium-dependent dilation. In five animals, diameter measurements were performed during the following conditions: 1) baseline; 2) flow reduction; 3) Indo (10 mg/kg iv), L-NMMA (3 µmol/min intracoronary for 5 min), and KCl suffusion (for 10–20 min); 4) baseline; 5) flow reduction; 6) SnMP (10 mg/kg iv) administration; and 7) flow reduction.

Protocol 4: role of cGMP production in vasodilation during myocardial ischemia with blockade of endothelium-dependent dilation. In five animals, diameter measurements were performed during the following conditions: 1) baseline; 2) flow reduction; 3) Indo (10 mg/kg iv), L-NMMA (3 µmol/min intracoronary for 5 min), and KCl (15 min) suffusion; 4) baseline; 5) flow reduction; 6) GC antagonist ODQ (10–5 M) with KCl suffusion; and 7) flow reduction.

Protocol 5: HO activation during ischemia. In five animals, diameter measurements were performed during the following conditions: 1) baseline; 2) flow reduction; 3) baseline; 4) heme-L-arginate application; and 5) flow reduction. In a parallel set of experiments (n = 5 dogs), the following sequence was used to assay the specificity of heme-L-arginate: 1) baseline; 2) heme-L-arginate (4 mM) administration; 3) flow reduction; 4) SnMP (10 mg/kg) and heme-L-arginate application; and 5) flow reduction.

At the end of each protocol, microvascular responses to papaverine (3 mg intracoronary) were determined to assess the intrinsic tone and thus the dilator ability of the preparation.

Western Analysis

To examine changes in HO-2 expression, Western blotting was used to assess protein levels in samples of cardiac muscle. Cardiac muscle was excised from the left ventricle and flash-frozen in liquid nitrogen. Individual samples (100 mg) were homogenized in TRIzol (GIBCO-BRL) with a Polytron homogenizer. RNA and DNA were removed by phenol-chloroform extraction and ethanol precipitation, respectively. Proteins were precipitated from the phenol-ethanol supernatant with isopropanol. Residual TRIzol components were removed via serial washes with 1 M guanidine HCl in ethanol, and residual guanidine was removed with washes in 100% ethanol. Pellets were dissolved in 1% SDS and heated overnight. Soluble protein was quantified by Bio-Rad microassay. Protein extracts were analyzed using Coomassie blue staining for integrity and relative loading efficiency before Western blotting was performed. For each sample, protein (30–50 µg) was separated using a 4–20% gradient polyacrylamide gel (Bio-Rad). After electrophoretic transfer to a polyvinylidene difluoride membrane (Hybond), the blot was blocked with Tris-buffered saline that contained Tween 20 [5% nonfat milk in 13 mM Tris·HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20]. Membranes were incubated with specific antibodies for HO-2 (Stressgen Biotechnologies) for 2 h in 1% buffered nonfat milk, washed, and then incubated with secondary antisera. Detection of bands was achieved using an enhanced chemiluminescence detection system (ECL; Amersham) and high-sensitivity film (Hyperfilm ECL; Amersham). In each gel, a molecular mass ladder of nine different-sized standards was included to ensure precise verification of the molecular mass of the analyzed protein. Positive controls were used to verify the identities of the detected bands. Signals from the film were digitized using a charge-couple device camera-frame digitizer and were analyzed with NIH Image software.

Drugs

Indo was dissolved in 95% ethanol to make a 5–10 mg/ml solution in 0.9% saline. L-NMMA was prepared as a 0.27 mg/ml solution in 0.9% saline brought to a physiological pH (between 7.3 and 7.5) by addition of small aliquots of 1 N NaOH immediately before use. The Krebs and high-KCl solutions were bubbled with 20% O2-5% CO-75% N2. ODQ was prepared as a suffusion in saline. SnMP (10 mg/kg; Poryphrin Products; Logan, UT) was made up in 1 N NaOH (~50 mg/ml) and then diluted into 20 ml (total volume) of saline. Heme-L-arginate was synthesized from hemin and L-arginate as described by Johnson et al. (24). All other drugs were obtained from Sigma Chemical. Although SnMP is light sensitive (46), our experiments were conducted in the dark (low-light fluorescence intravital microscopy), and the drug was stored in an opaque plastic bottle. We did not envision significant photooxidation of the drug during our procedures, because illumination of the heart averages 25 µs/beat, and therefore for an experiment with ~2 h of total stroboscopic illumination, there would be a total exposure of 0.76 s of light (25 µs flash x 120 min–1 x 120 min) during the entire experiment. Thus we were not concerned that photooxidation of the drug would occur during the experiments, owing to our precautions and experimental preparation.

Statistical Analysis

Microvascular diameter measurements in response to flow reduction were calculated as a percent change from the data before flow reduction at each state. Thus positive and negative percent changes indicate dilation and constriction, respectively. Two-way repeated-measures ANOVA was used to assess the effects of flow reduction on the diameters and hemodynamics in each condition. The data are presented as means ± SE. P < 0.05 was accepted as statistical significance.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Hemodynamics

Table 1 shows the hemodynamic variables that were measured during the various experimental protocols. Neither SnMP nor ODQ affected pressures from the baseline conditions; therefore, these data were combined with their respective basal data. Importantly, hemodynamics did not vary during each of the protocols. This suggests that alterations in diameters are not due to changes in myocardial oxygen demands but are rather due to the vasoactive mediators produced by ischemia.


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  		Table 1. Mean arterial pressure under control and ischemic conditions during various protocols

 
Protocol 1: role of endothelium-dependent dilators and K+ channels in vasodilation during myocardial ischemia. Figure 3 is a scatter plot of the percent changes in diameter vs. baseline diameter during reduced coronary perfusion pressure. Small arterioles (<100 µm) dilated to a greater extent during reduced perfusion pressure than large arterioles (>100 µm). In small arterioles (7 vessels, 73 ± 8 µm), the combined administration of L-NMMA and Indo reduced vasodilation during ischemia (from 36 ± 6 to 25 ± 4%; P < 0.05). Additional KCl suffusion further attenuated ischemia-induced vasodilation (15 ± 4%; P < 0.05 vs. before KCl). However, significant dilation remained after administration of L-NNMA with Indo and KCl. This indicates that small coronary arterioles can still dilate after inhibition of nitric oxide (NO) and prostaglandin dilator pathways in combination with blockade of hyperpolarization-induced vasodilation.



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  		Fig. 3. Scatter plot showing the percent changes in diameter and baseline diameters with reduced perfusion pressure for control vessels and vessels treated with either NG-monomethyL-L-arginine (L-NMMA) and indomethacin (Indo) or L-NMMA with Indo and KCl. In small arterioles (<100 µm diameter), the percent increase in diameter during ischemia was partially inhibited by L-NMMA and Indo treatment. Additional administration of KCl further inhibited vasodilation during ischemia, although the vessels still dilated in response to ischemia. In contrast, large arterioles (>100 µm diameter) exhibited less vasodilation during ischemia than small arterioles, and L-NMMA and Indo almost completely blocked vasodilation during ischemia.

 
Protocol 2: role of CO in vasodilation during myocardial ischemia in the absence of L-NMMA, Indo, and KCl. Coronary arterioles (13 vessels, 69 ± 8 µm) showed 27 ± 5% dilation during ischemia (Fig. 4). SnMP did not change the baseline diameter significantly (4 ± 2%; not significant vs. zero). In the presence of SnMP, the percent increase in diameter during ischemia did not change significantly (21 ± 5%), whereas the additional administration of L-NMMA with Indo and KCl inhibited ischemia-induced vasodilation (3 ± 2%). Papaverine dilated the microvessels by 24 ± 5% at the end of this protocol.



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  		Fig. 4. Changes in diameter during ischemia in control vessels and vessels treated with tin mesoporphyrin IX (SnMP); SnMP, L-NMMA, Indo, and KCl; and papaverine. SnMP alone did not attenuate dilation during ischemia, but administration of the combination of SnMP, L-NMMA, Indo, and KCl reduced vasodilation (P < 0.05 vs. SnMP and control). Vessels were still capable of vasodilation during administration of the combination of inhibitors, because papaverine produced robust vasodilation (P < 0.05 vs. SnMP with L-NMMA, Indo, and KCl). NS, not significant.

 
Protocol 3: role of CO in vasodilation during myocardial ischemia in presence of L-NMMA, Indo, and KCl. The effects of SnMP after combined blockade of endothelial dilator pathways are shown in Fig. 5. Combined administration of L-NMMA, Indo, and KCl reduced the baseline diameter by 7 ± 2% (P < 0.05), and SnMP did not affect the baseline diameter in the presence of L-NMMA with Indo and KCl (1 ± 2%; not significant). Ischemia-induced vasodilation was partially inhibited with L-NMMA with Indo and KCl (39 ± 6 vs. 18 ± 3%; P < 0.05), and additional administration of SnMP caused almost complete inhibition of vasodilation during ischemia (6 ± 2%; P < 0.05 vs. control and with L-NMMA with Indo and KCl). Papaverine dilated the vessels by 28 ± 5%, which demonstrates that the vessels retained vasodilator capability. These data show that CO contributes to coronary arteriolar vasodilation in vivo during reduced perfusion pressure particularly when the other vasodilatory mechanisms are impaired.



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  		Fig. 5. Changes in diameter in response to ischemia in control vessels and vessels treated with L-NMMA with Indo and KCl; SnMP with L-NMMA, Indo, and KCl; and papaverine. L-NMMA with Indo and KCl reduced vasodilation during ischemia (P < 0.05 vs. control). Administration of SnMP with L-NMMA, Indo, and KCl further reduced the dilation during ischemia (P < 0.05 vs. L-NMMA with Indo and KCl). Vessels were still capable of vasodilation during administration of the combination of inhibitors, because papaverine produced robust vasodilation (P < 0.05 vs. SnMP with L-NMMA, Indo, and KCl).

 
Protocol 4: role of cGMP production in vasodilation during myocardial ischemia with blockade of endothelium-dependent dilation. Figure 6 shows the effects of inhibition of GC production on dilation after reduced perfusion pressure. Combined administration of L-NMMA with Indo and KCl partially inhibited arteriolar vasodilation during myocardial ischemia from 25 ± 3 to 14 ± 4% (P < 0.05). ODQ had no significant effect on baseline diameter. In the presence of all of these antagonists, coronary arteriolar vasodilation during ischemia was severely depressed from 25 ± 3% at control to 6 ± 4% (P < 0.05). The response to papaverine was not impaired at the end of the protocol (22 ± 4%).



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  		Fig. 6. Changes in diameter in response to ischemia in control vessels and vessels treated with L-NMMA with Indo and KCl; L-NMMA with Indo, KCl, and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ); and papaverine. Administration of ODQ with L-NMMA, Indo, and KCl reduced dilation during ischemia (P < 0.05 vs. L-NMMA with Indo and KCl). Vessels were still capable of vasodilation during administration of the combination of inhibitors because papaverine produced striking vasodilation (P < 0.05 vs. L-NMMA with Indo, KCl, and ODQ). *P < 0.05 vs. control; #P < 0.05 vs. L-NMMA with Indo and KCl.

 
Protocol 5: activation of HO during ischemia. To evaluate activation of the HO enzyme during ischemia, we examined microvascular vasodilation to reduced perfusion pressure in the presence of heme-L-arginate, a substrate of HO. The data are presented in Figure 7. In five vessels from four animals, dilation was augmented by 39% in the presence of the HO substrate (13 ± 5 vs. 18 ± 5%; P < 0.05) compared with dilation during ischemia alone (Fig. 7A). When HO was inhibited with SnMP (Fig. 7B), the ischemic dilation was reduced by 33% after heme-L-arginate administration (14 ± 5 vs. 10 ± 5%; P < 0.05). These data support the hypothesis that HO activity is augmented in hypoperfused myocardium.



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  		Fig. 7. Responses of individual coronary arterioles to ischemic dilation before and after heme-L-arginate administration (A) and heme-L-arginate administration in the presence of SnMP (B). Heme-L-arginate increased dilation during ischemia (A), and this effect was abolished by SnMP (B).

 
Expression of HO Enzymes in Myocardium

Figure 8 shows representative Western blots of HO-2 expression in cardiac tissue from three hearts and the signal from recombinant protein. We consistently observed HO-2 expression in all preparations that we studied, but we did not usually observe expression of HO-1 (data not shown).



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  		Fig. 8. Western analysis of heme oxygenase-2 (HOX-2) expression in cardiac muscle. Constitutive form of the heme oxygenase enzyme was observed in all three samples (lanes 1–3); the heme oxygenase-2 standard is also shown (left lane).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major new findings of the present study are that activation of HO is involved in ischemia-induced vasodilation in canine heart particularly after other vasodilator pathways are blocked in the coronary microcirculation. Production of cGMP mediates this vasodilation in coronary microcirculation, and suffusion of the HO substrate heme-L-arginate augments it. Taken together, these observations suggest that HO activation during ischemia results in the production of CO, which produces vasodilation via activation of GC. Relevant to these conclusions are several issues concerning the limitations of the methodology, roles of endothelium-dependent dilators in ischemia, roles of CO in ischemia, and physiological implications.

Limitations of Methodology

The reduction of myocardial flow with critical stenosis occurs more severely in endocardium than in epicardium (14, 19). Thus it could be argued that ischemia has limited effects on epicardium. However, we previously reported that epicardial myocardial blood flow decreased 24% with coronary perfusion pressure of 40 mmHg in similar experimental preparations (8). Furthermore, in this study, both epicardial coronary flow velocity in small arterioles and epicardial systolic shortening values decreased during reduced perfusion pressure. Taken together, these data strongly suggest that the perfusion was inadequate to maintain normal function, i.e., some degree of ischemia was induced in epicardium as well. Because the extent of ischemia was undoubtedly greater in subendocardium than in subepicardium, the HO-CO pathway may be even more pronounced in endocardium.

Brief periods of coronary artery occlusion for 15 min induce endothelial dysfunction, i.e., "vascular stunning" (4, 5, 26). Thus the effects of repeated episodes of ischemia on vasodilation during subsequent ischemia should be discussed, because vascular stunning may be associated with reduced vasodilation after repeated ischemia. To determine the extent of this effect, we performed three 7-min periods of ischemia, each of which was followed by 1 h of recovery. These data showed that coronary microvessels dilate to a similar extent during repeated episodes of ischemia in the absence of antagonists. Thus we conclude that the possible confounding effects of vascular stunning on the coronary microcirculation were minimal in our experiments.

Comparisons with Literature

Roles of endothelium-dependent pathways in ischemia-induced vasodilation. Endothelium-dependent dilators have long been implicated in the coronary vasomotor response to ischemia (3, 28). The coronary arteriovenous difference in the endproducts of NO metabolism, nitrate and nitrite, are increased in the ischemic canine heart (41). Prostanoids have been largely discounted as a primary metabolic vasodilator (20, 21), but their role in ischemic vasodilation is largely unknown. Our data showing that Indo and L-NMMA partially inhibit coronary microvascular dilation during ischemia support the idea that NO plays a significant role in coronary adaptations during severe reductions in flow, which is consistent with the observations of Smith and Canty (43).

Because adenosine-induced dilation is mediated largely by ATP-sensitive K+ (KATP) channel activation (11, 12), KCl suffusion in our protocol would block this aspect of adenosine-induced dilation as well as the effects of endothelium-derived hyperpolarizing factor (39). Previous studies have suggested a significant contribution of K+ channels to coronary autoregulation. Within this context, glibenclamide, a KATP channel antagonist, abolished ischemia-induced vasodilation (27) or abrogated autoregulatory adjustments during decreasing coronary perfusion pressure (37). These data suggest that KATP channels play a major role in determining the coronary microvascular response to myocardial ischemia. Activation of Ca2+-activated K+ channels (KCa) may also be involved in coronary vasodilation during ischemia (7, 42), and KCl suffusion would also block this particular vasodilatory mechanism. Nevertheless, we should emphasize that in our studies, the microvascular dilation observed during reduced perfusion pressure could not be completely inhibited after blockade of NO production, prostanoid synthesis, and elimination of the effects of hyperpolarization (mediated by K+ channels) by high KCl suffusion. The residual dilation (discussed next) was mediated by CO, because it was inhibited by inhibitors of HO.

Role of CO in ischemia-induced vasodilation. CO is derived from the enzymatic activity of HO on free heme. HO activity has been demonstrated in several tissues relevant to this study. Early studies from Abraham et al. (1) documented HO activity in ventricular microsomes. Studies of normotensive rats showed that HO inhibition caused an increase in vasoconstriction (30), which suggests a physiological role of CO as one determinant of vascular tone. However, we observed that SnMP did not cause any change in baseline diameter in coronary microvessels either in the control state or in the presence of L-NMMA with Indo and KCl. Thus our data support the concept that CO does not regulate resting vascular tone in coronary microvessels.

Our study suggests that tissue ischemia could activate HO in the heart. There are two types of HO in tissue: one is constitutively expressed (HO-2), and the other is inducible (HO-1). In the present study, we documented by Western analysis the existence of HO-2 in cardiac tissue. Thus these previous data support the hypothesis that production of CO is increased in the setting of myocardial ischemia. Our observation is the first evidence that HO activity is involved in ischemic coronary dilation. Because CO is the active vasodilator produced by HO, we assume that this gaseous mediator is involved in dilation, although we did not measure CO production directly. A role for HO-2 in the regulation of coronary vasomotor reactions has been established in isolated coronary arteries (18). These investigators also found that CO-mediated dilation was mediated via cGMP and cGMP-dependent protein kinase modification of the KCa channel. This finding is also supported by evidence that CO modulates the KCa channel (47). Thus CO is an established dilator that signals through cGMP and certain ion channels.

Another aspect of our results as they relate to the literature also bear upon our conclusions. Johnson et al. (22) reported that SnMP has no basal effects on tone but has a significant effect after NO synthase (NOS) inhibition. Although the mechanisms that underlie this effect are obscure, our observations of the effects of SnMP share some similarities. The most striking effect of SnMP in our study was observed after inhibition of various dilator pathways including NOS during ischemia. Again, these observations suggest that the HO vasodilatory pathway assumes a more prominent role only after other pathways are corrupted.

Interestingly, even when no other dilator pathways were blocked, administration of heme-L-arginate caused significant augmentation of ischemia-induced vasodilation, and this dilation was antagonized with SnMP. This argues for activation of HO during ischemia. Although this substrate can be potentially catabolized by NOS to produce NO, our data argue against this possibility. First, there were no basal vasodilatory effects. Moreover, the effects of heme-L-arginate were blocked by SnMP. Both of these observations support our contention that heme-L-arginate was used as a substrate by HO. Moreover, this observation is similar to that of Kozma et al. (29), who reported that the dilation produced by heme-L-lysinate, another substrate of HO, was blocked by inhibitors of the oxygenase. These authors, like us, reached the conclusion that the blockade of dilation by the HO substrate was consistent with HO producing a vasodilatory metabolite that likely was CO. Our finding in the set of experiments using heme-L-arginate as a substrate for HO reinforces our conclusion that ischemia results in HO activation. We admit that this mechanism seems counterintuitive, because HO activity is dependent on PO2 (2). In fact, a grave reduction in PO2 values during ischemia could potentially limit HO activity and CO production. However, if adequate amounts of heme and sufficient oxygen are available, there may be enough HO activity to produce vasoactive amounts of CO. Our data support this contention.

Mechanisms of CO-induced vasodilation. It is known that CO stimulates soluble GC and thereby increases cGMP concentrations in vascular tissues and relaxes smooth muscle (34). Tissue cGMP content has been shown to increase in myocardium during 5 min of ischemia (33). Furthermore, in our study, ODQ inhibited dilation during ischemia in the presence of L-NMMA with Indo and KCl. Thus CO dilates microvessels during myocardial ischemia via cGMP production. CO can also relax smooth muscle cells via activation of KCa channels (18). Contribution of these two mechanisms, cGMP or K+ channel dependence, seems different between species and vascular preparations. In arterioles from newborn pig cerebellum, iberiotoxin, a KCa channel inhibitor, abolished CO-mediated vasodilation (31). CO is reported to modulate the activity of KCa channels (47). In isolated coronary arterioles, CO produced dilation via activation of a pathway involving GC, cGMP-dependent protein kinase, and KCa channels (18). Our protocols did not evaluate the contribution of K+ channels to CO-induced vasodilation in this setting, but such a contribution would have been eliminated by the KCl suffusion. However, we observed that SnMP almost abolished ischemia-induced vasodilation in the presence of L-NMMA with Indo and KCl, a dilation also abolished by ODQ. Thus our data suggest that cGMP plays a major role in CO-mediated vasodilation in coronary microcirculation.

Clinical Significance

We have shown that the HO antagonist SnMP inhibited ischemia-induced vasodilation in the coronary microcirculation in vivo. Our data indicate a role of CO as an ischemic vasodilator that becomes more prominent after inhibition of other vasodilatory mechanisms. Pathophysiological conditions such as hypertension, heart failure, and diabetes mellitus are associated with impaired endothelial function. Whether the CO-mediated response is augmented to compensate for the decreased NO-mediated response in these conditions remains unknown.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-32788 and HL-6520 (to W. M. Chilian) and HL-63880 and an American Heart Association (Northland Affiliate) Scientist Development Grant (to D. W. Stepp).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Alberto Nasjletti of the New York Medical College for guidance in the use of heme-L-arginate and porphyrin compounds and James Mintz for helpful reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. M. Chilian, Dept. of Physiology, LSU Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112 (E-mail: Chilian{at}LSUHSC.edu).

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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