Vasodilatory Effect of Pulsatile Pressure on Coronary Resistance Vessels
Intramyocardial pressure becomes high in systole and decreases in diastole. Therefore, the transmural pressure of the intramyocardial vessels is pulsatile, resulting in the cyclic distension of these vessels. However, the effect of pulsatility on the behavior of the coronary resistance vessels has not been evaluated. To assess the influence of pulsatile pressure on the behavior of the coronary arterioles, we measured the luminal cross-sectional area (CSA) of coronary arterioles under cyclically changing transmural pressure. Isolated porcine coronary arterioles (internal diameter, 100 to 150 μm) were cannulated with two micropipettes and pressurized with square waves (1 Hz) through both pipettes so as not to induce flow-dependent vasodilation. During the presence (active, induced by acetylcholine; n=7) or absence (passive, abolished by bradykinin; n=7) of vascular tone, the CSA was measured under the following conditions: (1) The amplitude of the pressure pulse was changed at a fixed mean pressure. (2) The mean pressure was changed at a fixed pressure pulse. With increasing pulse pressure, the mean CSA at steady state increased under active conditions, whereas it decreased under passive conditions (P<.0001). This vasodilatory effect of pulse pressure remained present after endothelial denudation (P<.0001; n=6 vessels with basal tone, n=9 vessels with U46619-induced tone). The mean steady state CSA under passive conditions increased with the mean pressure (P<.05), whereas under active conditions it remained constant in the range of mean pressures between 50 and 100 mm Hg, reflecting myogenic responsiveness. These results indicate that an increase in amplitude of the pressure pulse dilates coronary arterioles. The vasodilating effect of the pulsation may compensate partly for the extra compressing effect of cardiac contraction on the intramyocardial vessels.
Blood flow distribution in the heart muscle is determined, apart from anatomy, by smooth muscle tone in resistance arteries and compression by cardiac contraction.1 Recently, Kuo and colleagues2 3 reported that coronary arterioles show a myogenic response to a change in static transmural pressure. However, transmural pressure of in situ coronary arterioles is not static but varies strongly within the heart cycles,1 4 and vascular tone of the coronary resistance vessels may be modulated by this pulsation. Although cardiac contraction is known to squeeze the intramyocardial vessels and impede coronary arterial flow,4 5 6 7 8 particularly in the subendocardium,9 10 the effect of pulsatility on the behavior of the coronary resistance vessels has not been evaluated.
We postulated that the coronary resistance vessels have an intrinsic mechanism causing dilation in response to pressure pulsation, which allows them to compensate for the impeding effect of heart contraction. As a first step in establishing evidence for such a mechanism, we studied the effect of pressure pulsation on vascular tone in isolated cannulated coronary resistance arteries. The level of vasoconstriction was measured at various luminal pulse pressure amplitudes with a physiological frequency (1 Hz) and a fixed mean pressure. We also determined whether the endothelial cells are involved in the response to pulsatile pressure. Furthermore, to understand the possible role of myogenic mechanisms in autoregulation of the beating heart, we determined the level of vasoconstriction at various mean pressures in the presence of a pressure pulse of constant amplitude.
Materials and Methods
Seventeen pigs weighing 6 to 10 kg were anesthetized by metomidate (Hypnodil, 13.3 mg/kg IP, Janssen) or 4% halothane, followed by 4 to 5 μg/kg sufentanil. All animals received pancuronium bromide (Pavulon, 0.27 to 0.80 mg/kg IV, Organon). After the animal was intubated and artificially ventilated (O2/N2O, 1:2), a midsternal thoracotomy was performed. The pericardium was opened, and the heart was exposed. After heparinization with Thromboliquine (250 to 670 IU/kg IV, Organon), the heart was fibrillated electrically, excised, and immediately placed in cold (4°C) MOPS-buffered Ringer's solution (mmol/L: NaCl 145.0, KCl 4.7, MgSO4·7H2O 1.17, CaCl2·2H2O 2.0, NaH2PO4·H2O 1.2, glucose 5.0, and pyruvate 2.0; the solution was equilibrated with air [pH 7.35±0.05]). The coronary microvessels were visualized by injecting an india ink/gelatin/physiological saline solution into the left coronary arteries.2 Dissection was performed in MOPS-buffered Ringer's solution containing 1% dialyzed albumin (4°C) with the aid of a dissection microscope with epi-illumination (Wild M7, ×9 to ×46.3). Dissection was started from the diagonal branch of the left anterior descending coronary artery or the obtuse marginal branch of the left circumflex coronary artery. Usually, we followed the arteries to a peripheral segment, which was located in the myocardium after three or four branching points from the starting branch. After the dissection of the arteriole (one to two vessels from one heart), we excised the vascular segment, and the surrounding tissue was removed carefully. The vessels had internal diameters of ≈100 to 150 μm at 60 mm Hg and full dilation.
In the vessel chamber filled with MOPS-buffered Ringer's solution containing 1% dialyzed albumin (4°C), the isolated arteriole was cannulated with two micropipettes. The vessel was set to its in situ length. The cannulated arteriole was filled with MOPS-buffered Ringer's solution containing FITC-dextran (40 mg/L, Sigma Chemical Co) and 1% dialyzed albumin. The vessel chamber was placed on an inverted microscope (Olympus IM2), and the temperature was raised gradually to 37°C. The vessels were allowed to equilibrate for 30 to 60 minutes before any measurements were performed.
For experimental protocol I (see below), the luminal cross-sectional area (CSA) of the cannulated vessels was measured by a fluorescence technique.11 The system used for the CSA measurement and luminal pressure control has been described in detail and validated elsewhere.11 12 In brief, the FITC-dextran present in the lumen of the vessel was excited by a weak light source (wavelength, 400 to 480 nm). The total amount of fluorescence light (wavelength, >515 nm) coming from the lumen was measured using a photomultiplier tube. A constricting vessel would push the dye back into the cannulas and out of the field of illumination, causing a drop of the fluorescence signal. Since the length of the vessel and the concentration of the dye were kept constant, the fluorescence signal was proportional to the CSA. A two-point calibration was performed by comparing the amount of the fluorescent light with the microscopically measured luminal diameter of the vessel at two different luminal diameters, namely those at 20 and 80 mm Hg perfusion pressure during maximal dilatation.
The above CSA measuring technique is not applicable to vessels with removed endothelium, since the fluorescing luminal marker would readily diffuse into the wall. For that reason, for experimental protocol II, we used a video technique instead: the internal diameter of the vessel was automatically determined from the distances between the dark walls and lighter lumen in the video image. This was done at a video frequency of 25 Hz, and on average, a vessel length of ≈100 μm was used. In validation experiments in which we combined the video technique with the CSA technique on vessels with intact endothelium, we found results from these two techniques to be fully consistent (data not shown).
Luminal pressure was varied by using an electropneumatic valve (Fairchild T5200-9) controlled electrically by either a function generator or computer and measured at the end of the micropipettes (see Fig 1⇓ of Reference 12).
Experimental Protocol I: Effect of Pulsatile Pressure on Coronary Arteriolar CSA
We studied the vessels in the presence and absence of tone. Tone was induced by 10−5 mol/L acetylcholine (n=7), which is an endothelium-independent constrictor in the porcine coronary arterial bed.13 Only those vessels were included that had acetylcholine-induced reduction of the CSA to <60% of the dilated value at 60 mm Hg. To study vessels in the passive state, tone was abolished by 10−8 mol/L bradykinin (n=7), which was reported to cause maximal dilation of porcine coronary arterioles.14 Full dilation was confirmed from the absence of myogenic responses to changes in pressure.
The vessels were pressurized with square waves at a frequency of 1 Hz. In order to prevent flow and subsequent flow-dependent vasodilation, this procedure was performed through both the pipettes. During active and passive conditions, the CSA of each vessel was measured under the following conditions: (1) The amplitude of the pressure pulse was changed stepwise (four to six steps; range, 0 to 100 mm Hg) at a fixed mean pressure of 60 mm Hg. (2) The mean pressure was changed stepwise (10 mm Hg step) at a fixed pressure pulse of 60 mm Hg. Each pressure was kept until the CSA reached a steady level.
Experimental Protocol II: Role of the Endothelial Cells in the Pulsation-Induced Vasodilation
Although net flow during pulsation was prevented by applying the same pressure to both ends of the isolated vessel, a capacitive flow component that was due to the changes in volume of the vessel was present. Therefore, we also examined a possible contribution of transient flow-induced endothelium-dependent vascular responses by comparing the effect of pulsatile pressure in vessels before and after removal of the endothelial cells. Endothelial cells were removed from the cannulated vessels by gentle rubbing with a thin platinum wire (diameter, 100 μm). In order to do this, the vessel was removed from one of the cannulas. After the rubbing procedure, the vessel was flushed in order to remove the debris from the lumen and recannulated. The success of removing the endothelial cells was determined from the effect of 10−8 mol/L bradykinin, which is an endothelium-dependent vasodilator in the porcine coronary bed. The effects of pulse pressure and bradykinin were tested on vessels with either basal tone (n=6) or with tone induced by the stable thromboxane analogue U46619 (3 μmol/L, n=9). We chose not to use acetylcholine as a preconstrictor in these experiments, since this substance may be expected to have an endothelium-dependent vasodilator effect in addition to its vasoconstrictor action in the porcine coronary circulation, which would complicate the comparison of the pulse pressure effects in vessels with and without endothelium. The internal diameter was measured before and after an application of pulse pressure (100 mm Hg) at a fixed mean pressure of 60 mm Hg.
For protocol I, the CSA and left and right cannula pressure signals were sampled at 10 Hz. Mean steady state values of the CSA were determined by calculating the time average over at least 20 seconds. For the comparisons of the mean CSAs, the CSAs were normalized by the CSA at constant transmural pressure of 60 mm Hg under passive conditions. The percent changes of mean CSA in response to the changes in pressure pulse amplitude were grouped into two groups, ie, low (range, 0 to 50 mm Hg) and high (range, 50 to 100 mm Hg) pressure pulse ranges. The mean CSAs at various mean pressures (with fixed pressure pulse amplitude) in the pressure range of coronary autoregulation (range, 50 to 100 mm Hg) were divided into two mean pressure groups, ie, low mean pressure (range, 50 to 75 mm Hg) and high mean pressure (range, 75 to 100 mm Hg). Comparisons of mean CSAs between various pressures and between passive and active conditions were made by two-way ANOVA, followed by a post hoc Scheffe´'s multiple comparison test. For protocol II, the internal diameter was sampled at 25 Hz. All diameters were normalized to the passive value at 60 mm Hg. Normalized mean diameter was compared between the vessels with basal tone and the vessels with induced tone by unpaired t test. Comparisons of mean diameters between conditions with and without pulsation and between conditions with and without endothelium were made by two-way ANOVA, followed by a post hoc Scheffe´'s multiple comparison test. Mean internal diameter was compared by paired t test before versus after bradykinin administration. Percent diameter increase as a result of pulsation and bradykinin administration was calculated and compared between vessels with endothelium and vessels without endothelium by paired t test.
Data are reported as the mean±SD. Where appropriate, figures are given as box plots to indicate the distribution of the data. A value of P<.05 was considered to be statistically significant.
Effect of Pulsatile Pressure on Coronary Arteriolar CSA
Fig 1⇑ shows a representative response of a preconstricted coronary arteriole to an increase in amplitude of the transmural pulse pressure. In response to a stepwise increase in the transmural pressure pulse amplitude, the CSA increased immediately and then decreased to a new steady level, which was higher than the initial level. Fig 2⇓ shows a representative response of a preconstricted coronary arteriole to decreases in the amplitude of the transmural pressure pulse. In response to a stepwise decrease in transmural pressure pulse, the CSA decreased to a new steady level.
We analyzed the relationship between amplitude of the pressure pulse and mean CSA at steady state. Fig 3⇓ shows percent changes in the mean CSA at a fixed mean transmural pressure under passive and preconstricted active conditions at various pressure pulse amplitudes (range, 0 to 100 mm Hg). When vascular tone was abolished, the mean CSA decreased upon increasing the pressure pulse amplitude (y=0.023−0.004x−0.001x2, P<.0001). In contrast, preconstricted vessels dilated with increasing pressure pulse amplitude (y=0.154−0.158x+0.005x2, P<.0001). This difference in effect of pressure pulse on dilated and preconstricted vessels is further demonstrated in Fig 4⇓, where data are grouped into low (range, 0 to 50 mm Hg) and high (range, 50 to 100 mm Hg) pressure pulse ranges. Under active conditions, the mean CSA at steady state in the high pressure pulse range was larger than that in the low pressure pulse range, whereas under passive conditions the mean CSA at steady state in the high pressure pulse range was smaller than that in the low pressure pulse range (P<.0001).
Fig 5⇓ shows a representative response of a preconstricted active coronary arteriole to a decrease in mean transmural pressure at a fixed amplitude of the transmural pressure pulse. In response to a stepwise decrease in mean transmural pressure, the CSA first decreased immediately and then increased to a new steady level, which was larger than the initial level. The relationships between the mean pressure and mean CSA at steady state are shown in Fig 6⇓. Under passive conditions, the mean CSA increased with mean pressure throughout the range of pressures tested. On the other hand, under active conditions the mean CSA did not increase further at mean pressures higher than 50 mm Hg. Upon increasing the mean pressure in the pressure range of coronary autoregulation (50 to 100 mm Hg), the mean CSA increased when vascular tone was abolished (P<.001), but not in the preconstricted vessels (P=.56).
Fig 7⇓ shows the mean CSA in two pressure groups, ie, low mean pressure (range, 50 to 75 mm Hg) and high mean pressure (range, 75 to 100 mm Hg). Under passive conditions, the mean CSA at high mean pressure was larger than that at low mean pressure (P<.05), whereas under active conditions, there was no significant difference in the mean CSA between the two pressure groups.
Role of Endothelial Cells in Pulsation-Induced Vasodilation
We tested the involvement of the endothelial cells in the response to pulse pressure by comparing this response in vessels before and after removal of endothelial cells. These experiments were performed on six vessels with basal tone and nine vessels with 3 μmol/L U46619–induced tone. In order to test whether the endothelium was indeed removed by the rubbing procedure, we tested the effect of 10−8 mol/L bradykinin, which is an endothelium-dependent dilator. The results are depicted in Fig 8⇓ for basal tone and Fig 9⇓ for induced tone. The levels of tone before removal of the endothelium were comparable for these two groups: normalized diameter was 63.0±13.0 for basal tone (Fig 8⇓) versus 58.0±13.9 for induced tone (Fig 9⇓, P=.49). Although basal tone redeveloped to a somewhat less extent after removal of the endothelial cells, the difference was not statistically significant (P=.39, Fig 8⇓). U46619-induced tone was unaffected by the endothelial denudation (P=.99, Fig 9⇓).
Bradykinin dilated the vessels with basal tone to a great extent when endothelium was present (P<.0001) but only slightly when endothelium was absent (P<.05, Fig 8⇑). Endothelial denudation greatly decreased the bradykinin-induced percentage increase in diameter from 67.4±46.0 to 6.3±4.6 (P<.001). Pulsatile pressure increased the diameter in the vessels with endothelium (Fig 8⇑, P<.0001). This vasodilating effect of pulsatile pressure was still present after removal of the endothelium (P<.001). There were no significant differences in pulsation-induced diameter changes in vessels before versus after endothelial denudation: the percentage increase in diameter was 16.9±6.4 versus 12.2±7.5, respectively (P=.27).
Fig 9⇑ shows the effects of pulsatile pressure and bradykinin on the normalized mean internal diameters of the preconstricted vessels before and after endothelial denudation. Bradykinin dilated only arterioles with endothelium and failed to significantly dilate vessels without endothelium (Fig 9⇑). Pulsatile pressure increased the mean diameter in these vessels before and after endothelial denudation (P<.0001 and P<.001, respectively, Fig 9⇑). There was no significant difference in pulsation-induced diameter change between the vessels with endothelium and the vessels without endothelium: the percentage increase in diameter was 23.1±10.0 versus 23.1±6.1, respectively (P=.99).
The major findings of the present study are as follows: (1) With increasing amplitude of the transmural pressure pulse, mean CSA at steady state increased under active conditions, whereas it decreased under passive conditions. (2) This effect was not dependent on the endothelial cells. (3) With continuous pressure pulsation, mean CSA at steady state under passive condition increased with mean pressure, whereas under active conditions it did not increase further when mean pressure was raised above 50 mm Hg.
The finding that at full dilation and steady state the mean CSA decreased with increasing pressure pulse amplitude (Figs 3 and 4B⇑⇑) can be explained by the nonlinear pressure-CSA relation of a vessel (Fig 10⇓). The slope of this relation, which reflects the compliance of the vessel, becomes smaller at higher transmural pressures.12 As a result, the mean CSA in the presence of a pulsatile pressure is lower than the stationary CSA in the absence of pulsation (Fig 10⇓). However, when the vessels had been activated, the mean CSA at steady state increased with increasing pressure pulse amplitude (Figs 1, 3, and 4A⇑⇑⇑).
In the present study, the vessels were pressurized through both pipettes to prevent flow and subsequent flow-dependent vasodilation.15 Therefore, it is unlikely that a net flow effect caused the dilatation. It still could have been possible that a phasic shear rate component that is due to a capacitive flow component is involved. Also, other endothelium-dependent mechanisms might have been involved in the pulsation-induced dilation. For that reason, we compared the effect of pulsatile pressure in vessels before and after removal of the endothelium. In vessels that were preconstricted with U46619, removal of the endothelium, which fully inhibited the vasodilating effect of bradykinin, had no effect at all on the dilating response to pulse pressure. Also, in vessels with basal tone, removal of the endothelium, which greatly inhibited the effect of bradykinin, did not abolish pulsation-induced vasodilation. Thus, the pulsation-induced dilation of the coronary arterioles cannot be attributed to endothelial mechanisms.
A recent report on the vascular effect of pulsatile flow amplitude also supports the view that the dilating effect of pulse pressure does not depend on the endothelium. Hutcheson and Griffith16 have studied the effect of amplitude of pulsatile flow on flow-induced release of endothelium-derived relaxing factor (EDRF) in rat aortic segments. Using a cascade bioassay technique, they showed that an increase in the amplitude of the pressure pulse actually depresses rather than stimulates release of EDRF.
Although the present data demonstrate a pulsation-induced change in vascular smooth muscle tone, they do not allow us to determine the mechanism by which pulsatile pressure could be transduced into a signal for vascular smooth muscle tone. Although this aspect is beyond the scope of the present study, pulsation-related changes in ionic conductances of the vascular smooth muscle cell membrane17 may, at least partly, be involved. Further studies are needed to disclose the signaling pathways involved in pulsation-induced relaxation in the coronary resistance vessels.
Speden and Warren18 investigated the effect of pulse pressure on diameters of the rabbit ear central arteries. That study showed an initial dilatory response similar to ours when pressure was switched from steady to pulsatile. However, unlike in the present study, there was no steady state effect. Apart from the species and tissue difference, a possible reason for this divergence might be the limitation of the pressure pulses to 40 mm Hg. Thus, it remains to be established whether pulsation-induced dilation is a specific adaptation mechanism of coronary vessels to the rhythmic compression by cardiac contraction19 20 or a more general property of blood vessels.
In the present study, the diameter change of the isolated arterioles during pulsation, estimated from the CSA, was ≈5% (Fig 2⇑). Recently, Yada et al4 observed subendocardial and subepicardial arterioles in the beating porcine hearts using a portable needle-probe charge-coupled device (CCD) videomicroscope. In systole, the diameter of the subendocardial arterioles decreased by ≈20% and that of the subepicardial arterioles decreased only ≈2% compared with diastole.4 Hiramatsu et al21 reported that in dogs the phasic diameter change in intramural arterioles during cardiac cycle was ≈10%. Therefore, the amplitude of diameter changes in the present study is in the physiological range.
We have attempted to cannulate subendocardial vessels, but we were only successful in one case. The major technical problems were formed by the size of the arterioles and the large number of side branches. The only subendocardial vessel that we studied behaved in a manner similar to the subepicardial and midmyocardial vessels. It was demonstrated by Kuo et al2 that the development of basal tone is of the same order of magnitude in subendocardial and subepicardial vessels, namely, a reduction to 70% to 80% of the relaxed diameter at 60 cm H2O. Furthermore, myogenic responsiveness to pressure changes was present in vessels from both layers, although there was some difference in strength. Thus, in those respects, subendocardial and subepicardial vessels behave similarly. Therefore, we suggest that the presently found dilating effect of pulse pressure is also a general property of coronary resistance vessels. If this is true, then the dilator response under physiological circumstances will be larger in the subendocardium because of the larger pulsatile diameter changes found in this layer. Such a dilator response would compensate partly for the extra impeding effect of cardiac contraction on subendocardial perfusion. However, more conclusive evidence for a role of pulsation-induced dilation for flow control in the subendocardium awaits further experiments.
Myogenic responsiveness may be involved in the autoregulation of flow.22 23 The present study demonstrates that the myogenic responsiveness of isolated coronary small arteries remains present in a pulsatile pressure regime. Hence, these results are consistent with a role for myogenic responsiveness in coronary autoregulation in the beating heart.
In conclusion, pulsatile pressure dilates coronary arterioles through an endothelium-independent mechanism. The vasodilating effect of the pulsation may compensate for the compressing effect of cardiac contraction on the vascular resistance.
This study was funded in part by the Netherlands Heart Foundation.
Reprint requests to Prof Dr Ir. Jos A.E. Spaan, Department of Medical Physics, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam Z0, The Netherlands.
- Received March 26, 1996.
- Accepted August 6, 1996.
- © 1996 American Heart Association, Inc.
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