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Laboratory of Cardiovascular Physiology, Vall d'Hebron Hospitals, and Cardiology Service, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, 08025 Barcelona, Spain
Submitted 4 February 2003 ; accepted in final form 3 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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arteries; receptors; methacholine; cholinergic agonists
During the acute phase of myocardial infarction, some patients may present episodes of recurrent ischemia. Postinfarction angina is a serious clinical condition with a potentially unfavorable outcome due to progression of the ischemic process, leading to reinfarction or increased mortality (4, 8, 15). The mechanism of postinfarction angina is not fully understood. In most instances, rethrombosis of the culprit plaque has been documented (1, 28). However, in other circumstances, an increase in coronary reactivity has been alluded (3, 9, 23, 26). In this regard, it is not known whether the infarct-related coronary artery may react in a normal fashion to vasoconstrictor stimuli elicited in the acute phase of myocardial infarction.
The aim of this work was to analyze whether ischemia-reperfusion alters coronary smooth muscle reactivity to vasoconstrictor stimuli such as those elicited by an adventitial muscarinic stimulation with methacholine. Possible mechanisms of the observed in vivo dysfunction were also studied.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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In Vitro Studies
Eighteen pigs were submitted to a midsternotomy under sodium thiopental anesthesia (30 mg/kg iv) preceded by azaperone (1–2 mg/kg im). In 6 of the 18 pigs, the left anterior descending (LAD) coronary artery was occluded below the first diagonal branch for a period of 2 h followed by 45 min of reperfusion (study group), whereas in the remaining 12 pigs the coronary artery was not occluded (control group). The hearts were removed, and the LAD was dissected and placed in oxygenated (95% O2-5% CO2) Krebs solution at 4°C (in mM: 120 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 10 glucose; pH 7.4). The LAD was divided in three thirds (proximal, middle, and distal), and two rings (3–4 mm width) were taken from each third to assess regional differences in coronary reactivity. LAD rings above the site of the ligature were excluded from the study. In all cases, the endothelium was removed by rubbing the luminal coronary surface with a thin forceps, and denudation was verified by histological examination. Coronary rings were placed in 10-ml organ baths containing oxygenated Krebs solution at 37°C and were connected to horizontal force transducers (FSG-01, Experimetria, London, UK). Changes in tension were amplified (SG-M DC bridge amplifier module, Experimetria) and stored in a computer for later analysis. All rings were equilibrated at a resting tension of 2 g for 1 h. Tissues were contracted three or four times with 40 mM KCl, every 10 min, until the amplitude of the contractile response was similar in magnitude (29). Constriction concentration-response curves to methacholine (10-8 to 10-5 M, spaced 30 min) were studied in rings from all 18 pigs. Methacholine chloride was obtained from Sigma Chemical (St. Louis, MO) and dissolved in saline.
Data analysis. Changes in tension induced by methacholine were expressed as percentage of the contraction to KCl 40 mM. Response of duplicate rings of the same coronary segment was averaged. Data were fitted to a sigmoid function to determine the maximal effect (Emax) and the concentration of agonist necessary to produce half-maximal response (EC50 value). All values are expressed as means ± SD. Tension responses induced in the three coronary thirds of control pigs were compared by use of the ANOVA and Tukey's tests. Differences in Emax and EC50 between control and ischemic-reperfused rings were assessed by Student's t-test.
In Vivo Studies
Forty-two pigs were submitted to a midsternotomy under general anesthesia with 
-chloralose (100 mg/kg iv) preceded by premedication with azaperone (1–2 mg/kg im) and sodium metomidate (4 mg/kg iv) or sodium thiopental (7–9 mg/kg iv). Pulmonary ventilation was maintained with a pressure respirator. The thorax was opened through a midsternotomy, and the heart was exposed. In experimental series 1 (29 pigs), we assessed LAD reactivity to adventitial methacholine. In these cases the LAD was looped below the first diagonal branch with a 3-0 silk snare, and two dissections were made, respectively, at 10 and 15 mm below the occluding snare. The upper dissection was used for adventitial muscarinic stimulation with 1% methacholine, and the lower dissection allowed placement of a coronary blood flow probe (5, 6). In experimental series 2 (13 pigs), we assessed the endothelial-dependent vasodilator response induced by intracoronary administration of bradykinin (22). A small diagonal branch of the LAD was cannulated by use of a 24G Abbocath-T (Venisystems, Abbott, Sligo, Ireland), and the tip of the cannula was gently introduced into the lumen of the LAD to administer bradykinin. A 3-0 silk snare was placed around the LAD, just below the catheterized arterial branch, and flow measurements were performed 10 mm distal to the occluding snare. Conventional ECG was recorded with a seven-channel Elema ink-jet polygraph (Mingograf 710 System, Siemens, Sweden). Left ventricular (LV) pressure and LV change in pressure over time (dP/dt) were measured with a Millar SPC-350 catheter transducer (Millar Instruments, Houston, TX). Blood flow at the LAD and aortic root were measured with ultrasonic probes (Transonic T206, Transonic Systems, Ithaca, NY). Regional myocardial shortening at the apical and basal regions of the LV was assessed by ultrasonic crystals (System 6, Triton Technology, San Diego, CA) as described elsewhere (5, 6). All signals were recorded in a thermal-array Nihon Kohden RTA 1200 polygraph and digitally stored for later analysis.
Study protocol. EXPERIMENTAL SERIES 1 (METHACHOLINE). Pigs included in this series underwent two 15-s methacholine applications to the adventitia of the LAD, by use of a 5-mm2 piece of gauze soaked in 1% methacholine chloride, spaced 3 h. In this model, methacholine adventitial application induces a marked and reproducible reduction in coronary blood flow due to a severe local vasoconstriction as denoted by coronary angiography (5).
The following subsets of animals were created. 1) Sham methacholine controls (group 1a, n = 6): no maneuvers were performed between the two methacholine applications. 2) In group 1b, the second methacholine test was performed after 2-h occlusion and 45 min reperfusion of the LAD to analyze reactivity of infarct-related coronary artery to muscarinic stimulation. 3) Deendothelization (group 1c, n = 11): The second methacholine test was performed after LAD endothelial denudation to assess the role of endothelium in the coronary response to methacholine. Endothelial denudation was provoked by a 3.5-Fr shaft angioplasty catheter with a balloon of 3.5-mm diameter and 20-mm length (Schneider Europe) introduced percutaneously under X-ray control. The balloon was inflated at 10 atm and then withdrawn for the entire coronary artery length. Deendothelization procedure lasted always 30–40 s. Endothelial denudation was confirmed by histological examination.
EXPERIMENTAL SERIES 2 (BRADYKININ). Pigs included in this series were used to verify that LAD occlusion (2 h) and reperfusion (45 min) caused endothelial dysfunction in this model. Bradykinin has been shown to induce an endothelial-dependent vasodilation (22) and thus is a good tool to assess the functional integrity of endothelium. Two subgroups were created. 1) sham bradykinin controls (group 2a, n = 5): two intracoronary injections of bradykinin (0.2 ml of a 3 x 10-6 M solution, dissolved in 0.9% NaCl) were administered as previously described (22), spaced 3 h but with no occlusion of the LAD. 2) Coronary occlusion (group 2b, n = 8): reactivity of the LAD to bradykinin was analyzed before and after 2-h occlusion-45 min reperfusion.
To exclude active effects of the bradykinin solution vehicle, 0.2 ml of 0.9% sodium chloride solution were injected in all 13 pigs to the LAD. Moreover, the systemic effects of 0.2 ml of 3 x 10-6 M of bradykinin injected in the femoral vein were tested in four pigs of group 2a at the end of the experiment.
Data analysis. Changes in heart rate, ST segment potential in ECG lead V1, LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), peak (+) and (-) of the LV dP/dt, mean blood flow at the LAD and aortic root, and systolic segment shortening ratio induced by methacholine or bradykinin, before and after each maneuver, were compared by repeated-measures ANOVA test. These variables were analyzed on samples taken at baseline and at 120, 180, 240, and 300 s after methacholine application, or at baseline and at 25, 35, 45, and 80 s after bradykinin administration. To better assess the sequential coronary blood flow changes induced by methacholine, the early vasoconstrictor phase was assessed at baseline and at 30, 60, 90, and 120 s, whereas the ensuing recovery phase was evaluated at baseline and at 60, 270, 300, and 330 s. Similarly, coronary blood flow changes induced by bradykinin were assessed at baseline and at 15, 20, 25, and 40 s for the early vasodilator response and at baseline and at 40, 70, 120, and 180 s for the latest. Baseline values before and after each maneuver were compared by Student's t-test. Data are expressed as mean ± SD. A P value < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Effects of methacholine. Stimulation of control rings with 40 mM KCl induced higher increases in tension as we moved in the proximal direction in the coronary artery (1.88 ± 0.55, 1.39 ± 0.54 and 0.93 ± 0.44 g in proximal, middle and distal rings, respectively, ANOVA, P < 0.001). Despite this higher response to KCl, the methacholine Emax in all three segments was similar (83 ± 21, 89 ± 39, and 101 ± 41% in proximal, middle, and distal, respectively), suggesting that the same proportion of muscarinic receptors is present in all areas. All coronary rings showed a concentration-dependent increase in tension after exposure to methacholine. In contrast with Emax values, proximal LAD segments of the 12 control pigs showed a significantly lower EC50 (8.0 ± 2.5 x 10-7 M, ANOVA, P < 0.05) than middle (1.2 ± 0.4 x 10-6 M) and distal (1.3 ± 0.5 x 10-6 M) coronary segments.
Data from middle and distal segments with similar reactivity to methacholine were pooled and used to assess the reactivity of coronary rings preexposed to ischemia-reperfusion. LAD regions above the ligature were not analyzed. LAD arteries from six pigs submitted to coronary occlusion and reperfusion showed a similar response to methacholine to those from 12 control animals (Fig. 1). In addition, no changes in the response to 40 mM KCl were observed in rings previously submitted to ischemia-reperfusion (1.20 ± 0.55 and 1.08 ± 0.47 g in control and ischemic-reperfused segments, respectively).
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In Vivo Coronary Reactivity
Our in vitro results did not show differences in reactivity after muscarinic stimulation between endothelium-denuded controls and infarct-related coronary arteries. We then evaluated the integrity of vascular smooth muscle reactivity in an in vivo situation that mimics early coronary reperfusion and endothelial dysfunction in patients with acute myocardial infarction.
Experimental series 1 (methacholine). SHAM METHACHOLINE CONTROLS (GROUP 1A). LAD adventitial methacholine application was followed by a marked drop in regional coronary blood flow associated with depression of LV hemodynamic parameters and abnormal regional myocardial shortening in the region perfused by the LAD. Figure 2 shows a representative recording showing the effects of methacholine in baseline conditions. The methacholine-induced reduction in coronary blood flow was accompanied by a 12% drop in LVSP, 25% fall in LV (+)dP/dt, 23% reduction in LV (-)dP/dt, and 80% attenuation of regional segment shortening in the apical region. These changes were associated with small increases in heart rate and with ST segment elevation. Methacholine did not induce appreciable changes in aortic blood flow, LVEDP, or regional segment shortening in the proximal region. The second methacholine test in the six sham control pigs induced comparable LV hemodynamic changes.
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As depicted in Fig. 3A, sham controls showed a reduction of coronary blood flow from 7.7 ± 1.5 to 0.6 ± 1.5 ml/min (ANOVA, P < 0.01), which recovered spontaneously 4–5 min after drug application, giving rise to an hyperemic reaction (16.7 ± 7.0 ml/min). The second methacholine application performed 3 h later induced a similar coronary blood flow reduction (from 8.2 ± 2.5 to 0.5 ± 0.5 ml/min) and a comparable hyperemic reaction (19.5 ± 8.4 ml/min).
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REACTIVITY OF INFARCT-RELATED CORONARY ARTERY TO METHACHOLINE (GROUP 1B). Pigs submitted to 2 h LAD occlusion followed by reperfusion, thus having acute myocardial infarction, showed a recovery of the hemodynamic variables but regional systolic shortening at the LV apical region remained severely depressed (Fig. 4). The effects of the first methacholine test on LAD coronary blood flow were comparable to those induced in the sham group (Fig. 3B). Coronary blood flow decreased from 9.6 ± 4.6 to 1.9 ± 2.6 ml/min (ANOVA, P < 0.01), and this was followed by an hyperemic reaction (17.9 ± 8.0 ml/min). Adventitial application of methacholine to the infarct-related coronary artery induced a severe and persistent coronary blood flow decay but with a significantly delayed recovery (P < 0.001) and no appreciable hyperemic reaction (Fig. 3B). Heart rate and LV-derived changes induced by methacholine application after ischemia-reperfusion were comparable to those elicited by the first methacholine test (Fig. 4).
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During coronary occlusion, 6 of 12 pigs developed episodes of ventricular fibrillation (VF), which were terminated by internal direct-current countershocks of 15 W/s. Cardioversion did not modify the response to methacholine after ischemia-reperfusion, with no hyperemic reaction in any case (from a baseline flow of 11.2 ± 6.9 ml/min to a maximal postmethacholine flow of 9.83 ± 6.8 ml/min in animals with VF, and from 8.2 ± 7.6 to 6.4 ± 5.1 ml/min in animals without VF).
REACTIVITY OF ENDOTHELIUM-DENUDED CORONARY ARTERY TO METHACHOLINE (GROUP 1C). As shown in Fig. 3C, baseline coronary blood flow was not affected by the deendothelization maneuver. Methacholine application to endothelium-denuded LAD showed a similar vasoconstriction as previously described and a significantly reduced hyperemic reaction (ANOVA, P = 0.035).
Experimental series 2 (bradykinin). SHAM BRADYKININ CONTROLS (GROUP 2A). Figure 5A shows that LAD bradykinin injection induced a marked increase in coronary blood flow (ANOVA, P < 0.01) with a biphasic pattern: a first peak was reached after 20 s and a second peak value was achieved 60–80 s after bradykinin administration. Significant changes in LV parameters were observed 25–45 s after bradykinin administration. These included a 6% reduction in LV pressure (P < 0.05), 24% decrease in LV (-)dP/dt, 13% drop in LVEDP (P < 0.05), 12% increase in LV (+)dP/dt (P < 0.01), 7% increase in heart rate (P < 0.05), 15% increase in aortic blood flow (P < 0.05), and 9% augmentation of systolic segment shortening in the LV apical region (P < 0.05). The second bradykinin test performed 3 h later in the five sham control pigs induced a similar biphasic increase in coronary blood flow (first peak, 168 ± 155%; second peak, 188 ± 96%) (Fig. 5A).
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Intracoronary administration of 0.9% sodium chloride solution did not induce significant effects. Intravenous administration of bradykinin caused a significant (P < 0.05) decrease in coronary blood flow, LV pressure, and LV dP/dt and an increase in aortic blood flow and systolic segment shortening at both apical and basal LV regions. The trend of this hemodynamic response is comparable to that observed 25–45 s after intracoronary bradykinin administration, thus suggesting that the late hemodynamic effects observed after an intracoronary bradykinin administration may reflect systemic actions of the peptide.
REACTIVITY OF INFARCT-RELATED CORONARY ARTERY TO BRADYKININ (GROUP 2B). Compared with the baseline test, intracoronary administration of bradykinin into the infarct-related coronary artery only induced a mild increase in coronary blood flow (52 ± 34 vs. 180 ± 149%, P < 0.001) (Fig. 5B). The two bradykinin injections induced comparable changes in heart rate and LV-derived parameters.
During coronary occlusion, five of eight pigs developed episodes of VF, which were terminated by internal direct-current countershocks of 15 W/s. As occurred in methacholine group 1b, cardioversion did not modify the response to bradykinin after ischemia-reperfusion.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Coronary arteries exposed to reversible interruption of coronary blood flow show endothelial function impairment as denoted by their attenuated response to the endothelium-dependent vasodilator substances acetylcholine (7, 18, 21, 22, 24, 27, 32), ADP (12, 24), or bradykinin (12, 16, 22). The endothelial dysfunction appears to be caused by oxygen-derived free radicals released during the reperfusion period (31) and may contribute to the incompetence of the microvasculature to reperfuse the myocardium ("no reflow" phenomenon) (13, 17). Most of the studies analyzing the effects of ischemia-reperfusion on coronary vascular function have focused on the endothelium, and the involvement of other vascular structures such as the smooth muscle has been ill defined.
Studies analyzing the intrinsic reactivity of coronary smooth muscle after ischemia-reperfusion have not afforded conclusive data. It has been reported that coronary arteries preexposed to ischemia-reperfusion depict a normal response to endothelium-independent vasodilators like NO donors, adenosine, or isoproterenol (12, 16, 24, 32), but others have found an attenuated vasodilation after in vivo exposure to endothelial-independent papaverine (2). These differences may be due to the fact that in most studies it is not possible to subtract the net effects caused by the simultaneous exposure of the endothelium to the drug. Indeed, the increased vasoconstrictor effect induced by KCl in some studies (12, 16, 32), although not in others (11, 24), has been attributed to the presence of endothelial dysfunction leading to a diminished release of endothelium-derived vasodilators (12, 16, 32). In favor of the role of the endothelial function, coronary rings devoid of endothelium develop a comparable response to KCl before and after exposure to ischemia (16, 24).
The present study delineates the effects of ischemia-reperfusion on coronary smooth muscle reactivity both in vivo and in vitro in a model that avoids simultaneous stimulation of endothelium. Adventitial application of methacholine induces a specific muscarinic-mediated local constriction of the coronary artery in pigs, as denoted by its inhibition with atropine and by interruption of flow at angiography (5). In the in vivo model, we assessed the reactivity of conduit coronary arteries to adventitial muscarinic stimulation with methacholine before and after 2 h of ischemia followed by reperfusion in the same preselected coronary segment, mimicking the pathophysiological condition of an infarct-related coronary artery exposed to a heightened cardiac parasympathetic neural drive. Our in vitro data evidence similar responses to methacholine and KCl in both control and ischemic-reperfused endothelium-denuded rings. In addition, our in vivo data reveal that methacholine application to the LAD induces a severe coronary vasoconstriction leading to a virtual cessation of coronary blood flow, both at baseline conditions and after 2 h of LAD ligature followed by 45 min of reperfusion. Thus both in vivo and in vitro data demonstrate that smooth muscle reactivity is not affected in infarct-related coronary artery. However, in vivo we observed that blood flow recovery after methacholine administration in infarct-related coronary artery was markedly delayed and was not accompanied by an hyperemic reaction as occurred during the preischemic baseline methacholine test. In light of these findings, it is possible to speculate that imbalance between sympathetic and parasympathetic nervous systems after myocardial infarction may play a significant role in the genesis of postinfarction angina. Our data support a prolonged effect of muscarinic stimulation after ischemia-reperfusion.
The well-known loss of endothelial-dependent relaxation reported in other studies after ischemia-reperfusion (12, 13, 16, 18, 22, 27) may theoretically render the infarct-related coronary artery less prone to recover from vasoconstrictor stimuli. Indeed, in this study we have confirmed the existence of endothelial dysfunction after exposure of the LAD to reversible coronary occlusion because the coronary blood flow response to the endothelial-dependent vasodilator bradykinin was attenuated. Moreover, we have observed that endothelial denudation of the LAD by using an angioplasty balloon catheter prolongs the vasoconstriction effects of methacholine.
Although we have not carried out binding studies, the fact that both the EC50 and Emax values for methacholine were similar in vitro between control and ischemic-reperfused rings may exclude the possibility of an increased affinity for methacholine or an increased number of muscarinic receptors in infarct-related coronary arteries. Preservation of vascular smooth muscle reactivity after ischemia-reperfusion may depend on appropriate blood supply from vasa vasorum. This hypothesis, nonetheless, needs further investigation.
One could think that the modified response to both methacholine and bradykinin after ischemia-reperfusion was an artifact caused by the manipulation of the coronary artery necessary to place the silk snare occluder. However, we can rule out this possibility, because control animals were submitted to the same maneuver, placing a silk snare around the coronary artery but without occluding it. As can be seen in Figs. 3A and 5A, reproducibility of the response before and after placing the snare was good. In addition, pericoronary nerves are surrounded by a fat layer that protects them from damage (5, 6), and previous studies have demonstrated in pigs that chronic placement of Ameroid constrictors on left circumflex coronary arteries for 3 wk does not modify adrenergic innervation of circumflex-perfused myocardium (25).
Clinical Implications
This study indicates that infarct-related coronary arteries develop a significant and persistent smooth muscle-dependent vasoconstriction in vivo after adventitial muscarinic receptor stimulation. On the basis of the similarities in coronary innervation between pigs and humans (5, 14), our data suggest that, compared with normal coronary vessels, the infarct-related coronary artery would be more prone to develop sustained coronary constriction when exposed to a vagally mediated adventitial release of acetylcholine. This study supports that an altered coronary vascular reactivity to autonomic nervous influences may play a role on the genesis of recurrent ischemia in postinfarction patients. In clinical circumstances other than acute ischemia in which the endothelium is being damaged, as observed in patients with left ventricular hypertrophy (10), atherosclerosis (30), or coronary spastic angina (19), or after bypass surgery (20), the coronary vessel may also be more sensitive to autonomic influences.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1-adrenoceptor contractile responses in the tail artery of spontaneously hypertensive rats. Br J Pharmacol
119: 765-771, 1996.[ISI][Medline]
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) and after preexposure to ischemia-reperfusion (
).
) adventitial applications of 1% methacholine to the LAD in 6 sham control pigs (A), in 12 pigs submitted to 2h of LAD occlusion followed by reperfusion (B), and in 11 pigs submitted to endothelial denudation with an angioplasty balloon (C). Notice the long-lasting recovery of CBF changes after ischemia-reperfusion. P < 0.05, significant differences in the time course of the changes in CBF induced by methacholine between the first and second drug exposures. NS, not significant.
