Hiroshi Kanatsuka

Mechanical interactions between four heart chambers with and without the pericardium in canine hearts.

By using excised postmortem hearts obtained from 15 mongrel dogs with the pericardium intact, we investigated mechanical interactions between the four heart chambers from the standpoint of ventricular pressure-volume relationships. The interactions investigated were those between (1) the atrium and the ventricle, (2) the right ventricle and left ventricles, (3) the atrium and one ventricle vs. the other ventricle, and finally (4) the left and right atrium and the right ventricle vs. the left ventricle. For these purposes, we inserted compliant balloons into the four heart chambers without injuring the pericardium, i.e., we incised the base of the atria which was not covered with the pericardium. We obtained the right and/or left ventricular pressure-volume relationships under a constant pressure in three other heart chambers by changing the height of the reservoir connected to each balloon. As a result, both ventricular pressure-volume relationships were hardly affected by an increase in the atrial pressure ranging from 5 to 30 cm H2O with the pericardium removed, although the ventricle became less compliant due to an increase of the same magnitude of the opposite ventricular pressure. On the other hand, the effect of an increase in atrial pressure was distinct with the pericardium intact. Also, all mechanical interactions were enhanced dramatically with the intact pericardium. Thus, the pericardium plays an important role in these mechanical interactions, especially when the filling pressures of all heart chambers increase simultaneously. Clinically, these findings may be important to understanding ventricular functions as related to various heart disease--especially acute heart failure.

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.

Phasic blood flow velocity pattern in epimyocardial microvessels in the beating canine left ventricle.

We quantitated phasic epimyocardial microcirculatory coronary blood flow velocity patterns in the beating left ventricle. Using a newly developed floating objective and high-speed cinematography, red cell velocities in small arterioles, capillaries, and small venules and microvascular diameters in the superficial layer of the epimyocardium of beating left ventricle were determined throughout the entire cardiac cycle in open-chest anesthetized dogs. Heart rate was maintained at 140 beats/min by means of left atrial pacing. Peak red cell velocity was observed in midsystole in small arterioles and capillaries, and in late systole in small venules. Abrupt decline in red cell velocity and, in many cases, a momentary cessation or reverse of flow, was observed in these microvessels during the pre-ejection period. The internal diameter of small venule was increased in late systole, while that of small arteriole remained almost constant during the cardiac cycle. Furthermore, in these epimyocardial microvessels, a higher percentage of the total area under the velocity curve occurred during the ejection phase; 51 % in small arterioles, 43% in capillaries, and 40% in small venules. These findings indicate that the phasic blood flow pattern is markedly different in the subepimyocardial microvessels from that in the large epicardial artery and the septal artery. During vasodilation following dilazep (50 fig/kg, i.v.), an adenosine potentiator, red cell velocity increased throughout the entire cardiac cycle in epimyocardial microvessels with significant increases in the total area under the velocity curves accompanied by significant dilation of the arterioles. The present data will provide information useful in predicting or simulating transmural differences in the phasic blood flow pattern.

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.

Upregulation of Nitric Oxide Production in Vascular Endothelial Cells by All-trans Retinoic Acid Through the Phosphoinositide 3-Kinase/Akt Pathway

Background—A natural retinoid all-trans retinoic acid (ATRA) contains various beneficial effects on vasculature, including suppression of neointima formation after balloon injury. However, little is known about the effects of ATRA on vascular endothelial function. We therefore studied its role in nitric oxide (NO) production of vascular endothelial cells (ECs). Methods and Results—Human dermal microvascular ECs, human umbilical vein ECs, and SV40-transformed rat lung vascular ECs were incubated with or without ATRA (1 &mgr;mol/L) for 48 hours. Their NO production was determined with the use of a fluorescent NO indicator, diaminofluorescein-2 diacetate. ATRA significantly increased their basal as well as acetylcholine-induced NO production. Treatment with N&ohgr;-nitro-l-arginine methyl ester or carboxy-PTIO suppressed their fluorescence. Increase of NO production was also observed by incubation with retinoic acid receptor (RAR) agonist Am580. ATRA-induced NO increase was abolished by coincubation with RAR antagonist LE540. Moreover, the NO increase was completely inhibited by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and LY294002. ATRA as well as Am580 enhanced endothelial NO synthase (eNOS) phosphorylation at Ser-1177 as well as Akt phosphorylation at Ser-473 without changing their protein expression. Overexpression of dominant-negative Akt inhibited the eNOS phosphorylation. Moreover, ATRA increased PI3K activity as well as PI3K catalytic subunit p110β protein expression, which was completely inhibited by LE540 treatment. Real-time polymerase chain reaction analyses demonstrated that ATRA increased PI3K catalytic subunit p110β mRNA expression without affecting its stability. Finally, ATRA-induced NO increase was observed in COS-1 cells transfected with wild-type eNOS and RARα, but not with mutated eNOS whose Ser-1177 was substituted. Conclusions—ATRA increases NO production by eNOS phosphorylation through RAR-mediated PI3K/Akt pathway activation in vascular ECs and possibly plays beneficial roles in vascular endothelium. Retinoids may therefore be candidates as novel therapeutic agents against vascular disorders with endothelial damage.

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.

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.

A new microscope system for the continuous observation of the coronary microcirculation in the beating canine left ventricle

A microscope system was designed using a new type of objective lens which makes possible the direct and continuous observation of the coronary microcirculation throughout the entire cardiac cycle in the beating canine heart. The microscope system consists of a standard microscope and a floating objective system which is composed of a pair of convex lenses and transmits a real image of the coronary microcirculatory bed to a standard microscope without any change in magnification. The convex lens facing the heart is supported by a weight-adjusting coil spring and low-resistance ball bearings which allow the lens to move perpendicularly in unison with cardiac motion. To reduce excessive cardiac movement, two 24-gauge needles connected to the animal table by a needle holder are horizontally inserted through the midmyocardium of the left ventricle beneath the area of interest. The epimyocardium of the left ventricle is transilluminated by means of a light pipe and a xenon-arc lamp. The distance between the floating lens and the cardiac surface is kept constant using a spacing device connected to the light pipe holder to prevent the compression of the tissue in the microscopic field of view. This improvement in the microscope system combined with high-speed cinematography greatly facilitates the continuous analysis of the coronary microcirculation in the beating left ventricle throughout the entire cardiac cycle, and may provide a useful approach to the understanding of the regulation mechanism of the coronary circulation.