Tatsuya Komaru

Microvascular sites and mechanisms responsible for reactive hyperemia in the coronary circulation of the beating canine heart.

Our aim was to elucidate the site and mechanism responsible for reactive hyperemia in coronary circulation. In in vivo beating canine hearts, microvessels of the left anterior descending coronary artery (LAD) were observed through a microscope equipped with a floating objective. Flow velocity of the LAD was measured with a suction-type Doppler probe. The LAD was occluded for 20 or 30 seconds and then released, and reactive hyperemia was observed before and after 8-phenyltheophylline (7.5 mg/kg i.v.) or glibenclamide (200 micrograms/kg into the LAD) infusion. During the occlusion, only arterial microvessels smaller than 100 microns in diameter dilated. Dilation of those vessels was partially attenuated by 8-phenyltheophylline and completely abolished with glibenclamide. In the early phase of reactive hyperemia, all arterial microvessels dilated, and the magnitude of peak dilation was greater in vessels smaller than 100 microns compared with those larger than 100 microns. Vasodilation during reactive hyperemia ceased within 60 seconds in vessels smaller than 100 microns but was sustained for more than 120 seconds in those larger than 100 microns. 8-Phenyltheophylline did not change peak dilation of arterial microvessels but reduced dilation after the peak. Glibenclamide remarkably attenuated dilation of all arterial microvessels in the whole phase of reactive hyperemia. These results indicate that all arterial microvessels are responsible for reactive hyperemia after coronary artery occlusions of 20-30 seconds, but there is greater participation of vessels smaller than 100 microns in the early phase of reactive hyperemia. Dilation of vessels larger than 100 microns assumes an important role in the later phase. ATP-sensitive K+ channels mediate dilation of arterial microvessels both in brief ischemia and reactive hyperemia.

Vasodilatory effect of nicorandil on coronary arterial microvessels : its dependency on vessel size and the involvement of the ATP-sensitive potassium channels

We aimed to clarify the size dependency of nicorandil-induced dilation in coronary microcirculation and the involvement of adenosine triphosphate (ATP)-sensitive potassium channels. Coronary arterial microvessels were observed through a microscope equipped with a floating objective in anesthetized open-chest dogs (n = 29). Heart rate and mean aortic pressure were maintained at control level. In 16 dogs, nicorandil was infused into the coronary in a cumulative fashion (0.1, 1.0, 10, and 100 micrograms/kg/min, for 5 min for each dose). In 13 dogs, glibenclamide (10 microM) was topically applied onto the observed area, and nicorandil was similarly infused. Nicorandil dilated vessels < 100 microns in diameter at all applied doses in a dose-dependent manner. Glibenclamide abolished the dilation of these vessels at the lower two doses. Vessels > 100 microns in diameter dilated only at the two higher doses and the dilation was not affected by glibenclamide. These data suggest that the vessels < 100 microns are more sensitive to this agent than other size vessels, and that ATP-sensitive potassium channels are involved in the nicorandil-induced dilation of vessels smaller than 100 microns, whereas the dilation of other size vessels occurs independently of this channel.

Coronary microcirculation: physiology and pharmacology.

Coronary microvessels play a pivotal role in determining the supply of oxygen and nutrients to the myocardium by regulating the coronary flow conductance and substance transport. Direct approaches analyzing the coronary microvessels have provided a large body of knowledge concerning the physiological and pharmacological characteristics of the coronary circulation, as has the rapid accumulation of biochemical findings about the substances that mediate vascular functions. Myogenic and flow-induced intrinsic vascular controls that determine basal tone have been observed in coronary microvessels in vitro. Coronary microvascular responses during metabolic stimulation, autoregulation, and reactive hyperemia have been analyzed in vivo, and are known to be largely mediated by metabolic factors, although the involvement of other factors should also be taken into account. The importance of ATP-sensitive K(+) channels in the metabolic control has been increasingly recognized. Furthermore, many neurohumoral mediators significantly affect coronary microvascular control in endothelium-dependent and -independent manners. The striking size-dependent heterogeneity of microvascular responses to all of these intrinsic, metabolic, and neurohumoral factors is orchestrated for optimal perfusion of the myocardium by synergistic and competitive interactions. The regulation of coronary microvascular permeability is another important factor for the nutrient supply and for edema formation. Analyses of collateral microvessels and subendocardial microvessels are important for understanding the pathophysiology of ischemic hearts and hypertrophied hearts. Studies of the microvascular responses to drugs and of the impairment of coronary microvessels in diseased conditions provide useful information for treating microvascular dysfunctions. In this article, the endogenous regulatory system and pharmacological responses of the coronary circulation are reviewed from the microvascular point of view.

Mechanisms of Coronary Microvascular Dilation Induced by the Activation of Pertussis Toxin–Sensitive G Proteins Are Vessel-Size Dependent: Heterogeneous Involvement of Nitric Oxide Pathway and ATP-Sensitive K+ Channels

G proteins are critically important mediators of many signal transduction systems. In the present study, we investigated the effect of direct activation of pertussis toxin (PTX)-sensitive G protein (GPTX) on coronary arterial microvascular tone in 37 open-chest anesthetized dogs in vivo. Coronary arterial microvessels on the surface of the beating left ventricle were visualized by performing fluorescence coronary microangiography using an intravital microscope with a floating objective system. Microvessels were divided into two groups, small microvessels (inner diameter, < or = 130 microns) and large microvessels (inner diameter, > 130 microns). Topically applied mastoparan (G protein activator, 10, 30, and 100 mumol/L) produced homogeneous microvascular dilation in a concentration-dependent manner (10 mumol/L, 7.9 +/- 2.0%; 30 mumol/L, 10.3 +/- 2.4%; and 100 mumol/L, 16.7 +/- 4.5% in small microvessels; 10 mumol/L, 5.3 +/- 1.2%; 30 mumol/L, 9.8 +/- 2.5%; and 100 mumol/L, 15.5 +/- 3.9% in large microvessels). These dilations were reversed to constriction by pretreatment with PTX (300 ng/mL, 2 hours) in both microvessel groups. Blockade of nitric oxide production by NG-nitro-L-arginine (LNNA, 300 mumol/L) offset the mastoparan-induced dilation in large microvessels but not in small microvessels. Cosuperfusion of glibenclamide (10 mumol/L) with LNNA produced constriction of all sizes of microvessels in response to mastoparan, whereas charybdotoxin (10 nmol/L) did not affect the mastoparan effect. Pretreatment with glibenclamide alone reversed mastoparan dilation to constriction in small microvessels, whereas it only offset the dilation without producing constriction in large microvessels. We conclude that the activation of GPTX produces homogeneous coronary arterial microvascular dilation and that the underlining mechanisms of the dilation are vessel size dependent. The L-arginine-nitric oxide pathway mediates the dilation only in large microvessels, whereas ATP-sensitive K+ channel activation plays a central role in the dilation of small microvessels when GPTX is directly activated. ATP-sensitive K+ channels are also involved in the dilation of large microvessels in a synergistic fashion with nitric oxide production.

Effect of calcitonin gene-related peptide on coronary microvessels and its role in acute myocardial ischemia.

BACKGROUND Calcitonin gene-related peptide (CGRP) is a potent dilator of epicardial conduit vessels and is released during myocardial ischemia in humans. However, the effect of CGRP on coronary arterial microvessels is still unclear, and it is unknown if CGRP modulates the tone of coronary arterial microvessels during acute myocardial ischemia. METHODS AND RESULTS Epimyocardial microvessels were observed through a microscope equipped with a floating objective system in anesthetized open-chest dogs. Heart rate and aortic pressure were maintained at control levels. Flow velocity of the left anterior descending coronary artery (LAD) was measured with a suction-cup Doppler probe. When CGRP was cumulatively infused into the LAD (0.05, 0.5, 5.0, and 50 pmol/kg per minute) or superfused (0.03, 0.3, 3.0, and 30 nmol/L) over the left ventricular surface, arterial control microvessels > 100 microns in diameter dilated dose dependently at dosages of 0.5 to 50 pmol/kg per minute (infused) or 0.3 to 30 nmol/L (superfused), but those < 100 microns dilated only at the highest dose, and those > 100 microns had greater dilation in both groups. Only the highest dose of CGRP (infused) significantly increased coronary flow. The superfusion of CGRP(8-37) (CGRP receptor antagonist, 300 nmol/L) did not affect the control diameters of coronary arterial microvessels but completely abolished CGRP-induced vasodilation at the same doses (infused and superfused). However, 300 nmol/L of CGRP(8-37) did not affect the response of coronary arterial microvessels to the LAD occlusion in any size. CONCLUSIONS CGRP preferentially dilates the coronary arterial microvessels > 100 microns in diameter but has only a small effect on those < 100 microns. Endogenous CGRP does not modulate the tone of coronary arterial microvessels during acute myocardial ischemia in beating canine hearts.

Clinical impacts of additive use of olmesartan in hypertensive patients with chronic heart failure: the supplemental benefit of an angiotensin receptor blocker in hypertensive patients with stable heart failure using olmesartan (SUPPORT) trial

We examined whether an additive treatment with an angiotensin receptor blocker, olmesartan, reduces the mortality and morbidity in hypertensive patients with chronic heart failure (CHF) treated with angiotensin-converting enzyme (ACE) inhibitors, β-blockers, or both. In this prospective, randomized, open-label, blinded endpoint study, a total of 1147 hypertensive patients with symptomatic CHF (mean age 66 years, 75% male) were randomized to the addition of olmesartan (n = 578) to baseline therapy vs. control (n = 569). The primary endpoint was a composite of all-cause death, non-fatal acute myocardial infarction, non-fatal stroke, and hospitalization for worsening heart failure. During a median follow-up of 4.4 years, the primary endpoint occurred in 192 patients (33.2%) in the olmesartan group and in 166 patients (29.2%) in the control group [hazard ratio (HR) 1.18; 95% confidence interval (CI), 0.96–1.46, P = 0.112], while renal dysfunction developed more frequently in the olmesartan group (16.8 vs. 10.7%, HR 1.64; 95% CI 1.19–2.26, P = 0.003). Subgroup analysis revealed that addition of olmesartan to combination of ACE inhibitors and β-blockers was associated with increased incidence of the primary endpoint (38.1 vs. 28.2%, HR 1.47; 95% CI 1.11–1.95, P = 0.006), all-cause death (19.4 vs. 13.5%, HR 1.50; 95% CI 1.01–2.23, P = 0.046), and renal dysfunction (21.1 vs. 12.5%, HR 1.85; 95% CI 1.24–2.76, P = 0.003). Additive use of olmesartan did not improve clinical outcomes but worsened renal function in hypertensive CHF patients treated with evidence-based medications. Particularly, the triple combination therapy with olmesartan, ACE inhibitors and β-blockers was associated with increased adverse cardiac events. This study is registered at clinicaltrials.gov-NCT00417222.

Neuropeptide Y modulates vasoconstriction in coronary microvessels in the beating canine heart.

The purpose of this study was to determine whether neuropeptide Y has a direct vasoconstrictor effect at low doses, mimicking the physiological plasma concentration on the specific site(s) of coronary arterial microvessels in in situ beating canine left ventricles. Coronary microvessels were directly observed by means of an intravital microscope and video system equipped with a floating objective. Epi-illuminated fluorescence coronary microangiography was performed in open-chest anesthetized dogs (n = 14) to examine the changes in internal diameter of epimyocardial arterial microvessels. Flow velocity of fluorescently labeled microshperes in capillaries was also measured (n = 6). To eliminate secondary effects of neuropeptide Y on coronary microvessels via autonomic nervous modulation, experiments were conducted under pharmacological blockade of the regional autonomic nervous system by intracoronary injection of propranolol, 50 micrograms/kg; phentolamine, 100 micrograms/kg; and atropine, 5 micrograms/kg. Aortic pressure and heart rate were kept constant during the experiments. Intracoronary infusion of three different doses of neuropeptide Y (1, 10, and 100 pmol/kg/min) for 5 minutes significantly constricted small microvessels (less than 100 microns in diameter) (-5.2 +/- 1.4%, -8.5 +/- 1.5%, and -14.0 +/- 1.7%; p less than 0.05 versus before neuropeptide Y at each dose), medium microvessels (100-200 microns in diameter) (-5.5 +/- 1.6%, -10.6 +/- 1.8%, and -16.8 +/- 2.1%, p less than 0.05 versus before neuropeptide Y at each dose), and large microvessels (greater than 200 microns in diameter) (-3.6 +/- 0.6%, -5.8 +/- 0.8%, and -10.0 +/- 1.1%; p less than 0.05 versus before neuropeptide Y at each dose) in a dose-dependent manner. Capillary flow velocity was reduced by 17.2 +/- 3.1% by an intracoronary dose of 100 pmol/kg/min of neuropeptide Y (p less than 0.05). The present study indicates that low doses of neuropeptide Y exert a homogeneous direct vasoconstrictor effect on various sizes of coronary arterial microvessels and reduce capillary flow velocity. These results suggest that neuropeptide Y may play a physiological role in modulating coronary microvascular tone.

Pertussis toxin-sensitive G protein mediates coronary microvascular control during autoregulation and ischemia in canine heart.

GTP-binding regulatory proteins (G proteins) regulate various biological functions, but their participation in controlling coronary microvascular tone has not been established yet. The goal of the present study was to elucidate the role of pertussis toxin (PTX)-sensitive G protein in regulating coronary microvascular tone during autoregulation and ischemia. In 42 open-chest dogs, coronary arterial microvessels on the surface of the left ventricle were directly observed by epi-illuminated fluorescence microangiography using a floating objective system. PTX (300 ng/mL) was superfused onto the surface of the left ventricle for 2 hours to block Gi and G(o) protein in epimyocardial coronary microvessels in vivo. PTX superfusion caused no change in the resting diameters of microvessels and significantly blocked the vasoconstriction induced by BHT 920 (a selective alpha 2-agonist). After pretreatment with PTX or its vehicle, the left anterior descending coronary artery (LAD) was occluded by a hydraulic occluder to reduce coronary perfusion pressure (CPP) in a stepwise fashion. A mild stenosis (CPP, 60 mm Hg), a severe stenosis (CPP, 40 mm Hg), and complete occlusion were sequentially produced. Coronary flow velocity in the LAD distal to the stenotic site was continuously monitored. In both PTX and vehicle groups, flow velocity did not significantly decrease during mild stenosis, proving that transmural coronary autoregulatory function was well preserved in the preparation. During severe stenosis and complete occlusion, the coronary flow velocity significantly decreased. In the vehicle group, microvessels < 100 microns in inner diameter significantly dilated in response to the reduction in perfusion pressure (mild stenosis, 6.2 +/- 1.9%; severe stenosis, 21.1 +/- 4.4%; and complete occlusion, 16.8 +/- 5.9%; P < .05 versus baseline diameters). In the PTX group, microvessels did not dilate during each occlusion level (mild stenosis, -2.0 +/- 0.9%; severe stenosis, -3.9 +/- 1.9%; and complete occlusion, -13.4 +/- 2.9%; P < .05 versus vehicle group). PTX did not affect the microvascular dilation caused by nitroprusside. The present data indicate that PTX-sensitive G protein is crucially involved in microvascular control during autoregulation and ischemia.

Role of adenosine in vasodilation of epimyocardial coronary microvessels during reduction in perfusion pressure.

Summary Previous studies in which an isolated heart or in situ constant pressure preparation was used suggested a minimal role for adenosine in autoregulatory control of coronary circulation. These results, however, are controversial, and the role of adenosine in autoregulation of flow in heart is uncertain. To test the hypothesis that adenosine mediates microvascular dilation in response to reduction in perfusion pressure (PP), we performed experiments in 41 open-chest chloralose-anesthetized dogs. Internal diameters (ID) of epicardial small arterioles <100 μm were measured with an intravital microscope and stroboscopic epiillumination synchronized to cardiac cycle. PP was reduced by graded stenoses of the left anterior descending coronary artery (LAD, mild stenosis PP = 60 mm Hg; critical stenosis PP = 40 mm Hg) and complete occlusion. 8-Phenyltheophylline (8-PT 10 μM) or adenosine deaminase (ADA 10 U/min) was topically superfused onto the heart. Arteriolar dilation induced by topically applied adenosine ≤10 μM was completely blocked by 8-PT. Without 8-PT (vehicle group), mild critical stenosis and complete occlusion caused arteriolar dilation (percentage of change in diameter 8.6 ± 2.6, 16.0 ± 2.7, and 13.6 ± 4.8%). 8-PT did not inhibit this dilation (8.5 ± 2.8, 16.1 ± 4.6, 15.1 ± 5.7%, NS vs. vehicle group). Topically applied ADA significantly inhibited intravenously (i.v.) administered adenosine-induced arteriolar dilation. Without ADA, arteriolar dilation occurred (16.6 ± 3.0, 28.2 ± 4.3, 15.4 ± 6.2%, at each PP). However, ADA did not inhibit dilation induced by gradual stenoses (10.6 ± 1.4, 24.2 ± 4.3,17.5 ± 6.9%, at each PP, NS vs. vehicle group). These data indicate that adenosine does not play a primary role in autoregulatory or ischemia-induced coronary microvascular dilation.

Hydrogen Peroxide Derived From Beating Heart Mediates Coronary Microvascular Dilation During Tachycardia

Objective—Coronary flow is closely correlated to the myocardial metabolic demand. We tested the hypothesis that hydrogen peroxide (H2O2) derived from beating hearts mediates metabolic coronary microvascular dilation. Methods and Results—We used a bioassay method in which an isolated microvessel is placed on a beating heart to detect myocardium-derived vasoactive mediators. A rabbit coronary arterial microvessel (detector vessel [DV], n=25) was pressurized and placed on a canine beating heart. After intrinsic tone of DV had developed, we observed DV at rest (heart rate, 120 bpm) and during tachypacing (heart rate, 240 bpm) using an intravital microscope equipped with a floating objective. The tachypacing produced DV dilation by 8.2% (P<0.01 versus baseline), and the dilation was abolished by cell-impermeable catalase (a H2O2 scavenger, 500 U/mL). We performed myocardial biopsy at rest and tachypacing. The biopsy specimens were loaded with 2′,7′-dichlorodihydrofluorescein diacetate (10 &mgr;mol/L) to visualize H2O2, and observed with confocal microscopy. Dichlorofluorescein fluorescence was diffusely identified in the myocardium and the tachypacing increased the fluorescence intensity (P<0.01). Exogenous H2O2 caused vasodilation of arterial microvessels in vitro in a concentration-dependent manner that was abolished by catalase. Conclusions—H2O2 derived from the beating heart mediates tachypacing-induced metabolic coronary vasodilation in vivo.