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Background The role of cardiac sympathetic nerves in regulating coronary blood flow is controversial. We sought to determine the degree to which cardiac efferent sympathetic signals modulate coronary blood flow. The heterogeneous sympathetic reinnervation in transplanted hearts provides a model for studying the vasomotor responses to adrenergic stimulation in reinnervated and denervated coronary territories of the same heart.
Methods We studied 14 cardiac-transplant recipients who had normal coronary arteries and no evidence of rejection and 8 normal subjects. We used positron-emission tomography with [11C]hydroxyephedrine, an analogue of norepinephrine, to delineate sympathetic innervation. Using [13N]ammonia, we measured myocardial blood flow at rest, during adenosine-induced hyperemia, and in response to sympathetic stimulation induced by cold pressor testing.
Results In the transplant recipients, the uptake of [11C]hydroxyephedrine was greater in the territory served by the left anterior descending artery (mean ±SE, 0.15±0.01) than in those served by the right c oronary artery (0.07±0.01, P<0.001) or the circumflex artery (0.09±0.01, P< 0.001). The basal flow was similar in all three regions, as was the percent increase in flow during hyperemia. However, the increase in flow in response to cold pressor testing was higher in the territory of the left anterior descending artery (46±10 percent) than in those of the right coronary artery (16±5 percent, P = 0.01) or the circumflex artery (23±6 percent, P = 0.06), although the changes in hemodynamics and levels of circulating catecholamines were similar. No such regional differences were observed in the normal subjects.
Conclusions Increases in coronary blood flow in response to sympathetic stimulation correlated with the regional norepinephrine content in the cardiac sympathetic-nerve terminals. These findings suggest that cardiac adrenergic signals play an important part in regulating myocardial blood flow.
The transplanted heart provides a unique model for studying adrenergic control of the coronary circulation. Cardiac transplantation results in total cardiac denervation due to the sectioning of the postganglionic neural axons that innervate the heart.6 Sympathetic reinnervation has been reported after cardiac transplantation,7,8 but it is only a regional process, favoring the territory of the left anterior descending coronary artery.8,9 Thus, it is possible to study the degree to which cardiac efferent sympathetic signals alter coronary vasomotion by comparing reinnervated and denervated territories in the same heart independently of changes in hemodynamic factors and circulating catecholamines that modulate coronary blood flow. These regions can be defined noninvasively with [11C]hydroxyephedrine, an analogue of norepinephrine, and positron-emission tomographic (PET) imaging.8 Myocardial blood flow can also be quantified by PET with [13N]ammonia,10 a method that allows detailed study of adrenergic control of vascular resistance in the heart.
We sought to study the role of cardiac efferent sympathetic signals in regulating coronary blood flow. We used PET imaging to delineate cardiac sympathetic innervation and measure regional myocardial blood flow in the territories of reinnervated and denervated coronary arteries after cardiac transplantation. Myocardial blood flow was measured at rest, during maximal vasodilatation due to an infusion of adenosine, and in response to sympathetic-nerve stimulation by a cold pressor test. The blood-flow response in the territories of the reinnervated coronary arteries in transplant recipients was compared with that in denervated territories in the same patients.
Methods
Study Population
We studied 14 patients a mean (±SE) interval of 7±1 years (range, 5 to 10) after orthotopic heart transplantation. The patients were selected on the basis of angiographically normal coronary arteries on recent arteriography (performed within the preceding 10±6 months) and the absence of acute rejection as determined by endomyocardial biopsy at the time of the study. The heart transplantations were performed to treat ischemic heart disease in five patients and to treat idiopathic cardiomyopathy in nine. There were 2 women and 12 men (age, 55±9 years). All had normal left ventricular function as assessed by contrast left ventriculography and echocardiography.
Eight healthy volunteers (three men and five women; age, 28±6 years) were studied who were matched for age to the transplanted hearts (age at the time of the study, 31±10 years), and had a low likelihood of coronary artery disease on the basis of the absence of symptoms and risk factors, a normal resting electrocardiogram, and a normal maximal treadmill exercise test. This group was used to establish regional patterns of cardiac sympathetic innervation and to characterize the relations between regional sympathetic efferent signals and myocardial blood flow.
Study Design
The study design was approved by the Human Investigation Committee of Wayne State University, and all the study subjects gave written informed consent. Each subject made two visits to the hospital, during which cardiac sympathetic innervation and regional myocardial blood flow were assessed with a whole-body PET scanner (CTI EXACT HR, Siemens, Knoxville, Tenn.).
All the subjects refrained from drinking caffeine-containing beverages and taking theophylline-containing medications for 24 hours before the PET study. Calcium-channel blockers and beta-blockers were withheld for 24 hours before the study, and arterial vasodilators were withheld for 12 hours. None of the patients received medications known to interfere with catecholamine uptake in presynaptic nerve terminals. All the subjects were studied while fasting.
Assessment of Cardiac Sympathetic-Nerve Terminals
The presence and extent of catecholamine uptake in cardiac sympathetic-nerve terminals were evaluated with [11C]hydroxyephedrine, an analogue of norepinephrine that has the same mechanisms of uptake and storage as the naturally occurring neurotransmitter.11 A 15-minute transmission scan was acquired for the correction of photon attenuation by soft tissue. Beginning with the intravenous bolus administration of [11C]hydroxyephedrine (0.286 mCi per kilogram of body weight), we acquired serial images for 40 minutes (six images for 30 seconds each, two for 60 seconds, two for 150 seconds, two for 300 seconds, and two for 600 seconds). Heart rate and blood pressure were monitored continuously throughout the study.
Assessment of Myocardial Blood Flow
Myocardial blood flow was measured at rest, during a standard intravenous infusion of adenosine (0.14 mg per kilogram per minute), and during cold pressor testing, with [13N]ammonia used as a flow tracer. A 15-minute transmission scan was acquired for correction of photon attenuation. Beginning with the intravenous bolus administration of [13N]ammonia (0.286 mCi per kilogram), images were acquired serially for 20 minutes (12 images for 10 seconds, 3 for 60 seconds, and 3 for 300 seconds). Thirty minutes later, intravenous adenosine was infused for four minutes. Two minutes into the adenosine infusion, a second dose of [13N]ammonia was injected and images were recorded in the same sequence. Thirty minutes later, a cold pressor test was performed by immersing the patient's hand and forearm in ice water for three minutes. Ninety seconds into the cold pressor test, a third dose of [13N]ammonia was injected, and images were recorded in the same acquisition sequence. The patient's movement was minimized by fastening a Velcro strap across his or her chest. The heart rate, systemic blood pressure, and a 12-lead electrocardiogram were recorded at base line and every 60 seconds during and after the infusion of adenosine and the cold pressor test.
Analysis of Data
In each study, the 47 tomographic slices were reoriented into 12 short-axis slices extending from the apex to the base of the left ventricle. To quantify the regional myocardial storage of catecholamines, regions of interest in sectors encompassing the territories of the left anterior descending, circumflex, and right coronary arteries were automatically assigned to each of four midventricular short-axis slices of the [11C]hydroxyephedrine images. An additional small circular region of interest was manually placed in the center of the left ventricular blood pool to quantify the arterial input. The regions of interest were then applied to the entire 40-minute sequence of [11C]hydroxyephedrine images, and regional time–activity curves for myocardial tissue and the blood pool were obtained. In each coronary-artery territory, the fraction of [11C]hydroxyephedrine that was retained was calculated by dividing the concentration in myocardial tissue by the integral of the concentration in arterial blood.
To quantify regional myocardial blood flow, the same regions of interest (the [11C]hydroxyephedrine images) were automatically assigned to each of four midventricular short-axis slices of the [13N]ammonia images, as described previously.12 To ensure that the placement of these regions was identical, the same angle on the circumferential profile was used as the starting point for the sectors of interest on each of the four sets of images studied in each subject. An additional small circular region of interest was manually placed in the center of the left ventricular blood pool to quantify the arterial input. The regions of interest were then copied to the entire sequence of [13N]ammonia images, and regional time–activity curves for myocardial tissue and the blood pool were obtained. In each vascular territory, a single time–activity curve was obtained by averaging the corresponding [13N]ammonia data in adjacent ventricular planes. The curves were then fitted with the use of a previously validated kinetic model of the tracer.10 An index of coronary vascular resistance was calculated as the ratio between the mean aortic blood pressure and the myocardial blood flow. The coronary vasodilator reserve was defined as the ratio between the hyperemic and the basal myocardial blood flows.
Measurement of Circulating Catecholamines
Venous-blood samples for the measurement of circulating catecholamines were obtained from an indwelling venous catheter with the patient at rest (with the patient supine and with little stimulation) and during cold pressor testing. Plasma concentrations of norepinephrine, epinephrine, and dopamine (in picograms per milliliter) were measured by high-performance liquid chromatography.13
Statistical Analysis
Data are presented as means ±SE. Differences between groups were assessed by paired or unpaired Student's t-tests, as appropriate. Differences between multiple groups were studied by a single-factor analysis of variance and by Tukey's test. P values of less than 0.05 were considered to indicate statistical significance. P values of less than 0.10 are also reported, because they were considered to indicate trends toward significance.
Results
Regional Uptake and Storage of Catecholamines
In the transplant recipients, the myocardial uptake of [11C]hydroxyephedrine was consistently lower in the territories of the right coronary artery (0.07± 0.01) and the circumflex artery (0.09±0.01) than in that of the left anterior descending artery (0.15± 0.01; P<0.001 for both comparisons) (Figure 1A and Figure 1B). No such differences between regions were noted in the normal subjects.
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The heart rate and the rate–pressure product increased with cold pressor testing and adenosine in both groups of subjects. Systolic and mean aortic blood pressure increased during the cold pressor test but remained unchanged during the infusion of adenosine (Table 1).
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Both the transplant recipients and the normal subjects had significant increases in plasma norepinephrine in response to the cold pressor test (Table 2). Epinephrine levels rose only slightly in the transplant recipients (Table 2).
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Base-Line Measurements
The base-line blood flow in the transplant recipients was similar in all the coronary territories despite the differences in sympathetic reinnervation. The base-line flow was higher in the transplant recipients than in the normal subjects, reflecting the differences in cardiac work and oxygen demand as measured by the rate–pressure product (Table 3).
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During the cold pressor test, blood flow increased significantly in all coronary territories (Table 3). However, the magnitude of the increase in flow differed among regions: it was higher in the territory of the left anterior descending artery (46±10 percent) than in the territories of the right coronary artery (16±5 percent, P = 0.01) and the circumflex artery (23±6 percent, P = 0.06) (Figure 2), even though changes in levels of circulating catecholamines, the heart rate, and blood pressure produce global effects that should affect all the regions equally. The index of coronary vascular resistance decreased, although slightly, only in the territory of the left anterior descending artery. The magnitude of the increase in flow during cold pressor testing exactly mirrored the uptake of [11C]hydroxyephedrine (Table 4). These differences were not observed in the normal subjects (Table 4).
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During hyperemia, blood flow increased and coronary vascular resistance decreased to a similar degree in all the coronary-artery territories in both groups of patients. Although the peak myocardial blood flow was similar in the transplant recipients and the normal subjects, the estimates of coronary vasodilator reserve tended to be higher in the normal subjects because of a lower base-line flow (the denominator of the calculation of coronary flow reserve) (Table 3).
Discussion
Coronary blood flow is regulated to a large extent by adrenergic mechanisms, through the direct activation of adrenergic receptors and indirectly by changes in metabolic autoregulation and endothelial function. However, the importance of cardiac efferent sympathetic signals, as compared with systemic adrenergic influences, in regulating myocardial perfusion remains controversial. Our findings provide evidence that the increase in coronary flow in response to sympathetic stimulation correlates with the magnitude of regional stores of norepinephrine in cardiac sympathetic-nerve terminals. In this study, blood flow increased by 46 percent in the territory of the left anterior descending artery (which had the highest uptake of [11C]hydroxyephedrine) and by only 16 percent in the territory of the right coronary artery (which had the lowest uptake of [11C]hydroxyephedrine) during the cold pressor test. This difference in flow was largely independent of changes in circulating levels of catecholamines and changes in hemodynamics (i.e., heart rate and blood pressure), since we compared reinnervated and denervated coronary territories in the same heart. These findings suggest that cardiac efferent adrenergic signals play an important part in modulating myocardial blood flow during activation of the sympathetic nervous system.
Exactly how the activation of cardiac sympathetic-nerve terminals may cause coronary vasodilatation cannot be determined from this study. Several potential mechanisms could explain our findings, however. It is possible that the increased density of sympathetic-nerve endings in reinnervated regions (i.e., the territory of the left anterior descending artery) caused relatively greater increases in regional contractility and oxygen demand after sympathetic activation, which in turn produced more metabolic vasodilatation. However, Zeiher et al.4 reported similar increases in blood flow in normal coronary arteries in response to the cold pressor test before and after an intracoronary 
-adrenergic blockade with propranolol. This would suggest that changes in regional contractility (mediated by 1-adrenoceptors) may not be the determinant dilatory mechanism of resistance vessels during sympathetic stimulation.
Another possibility is that coronary vasodilatation in response to neurally released norepinephrine (a mixed 1- and 
-adrenergic agonist) may result from the direct activation of -adrenergic receptors on smooth muscle and endothelial cells in the vessel wall.3,14 The reported exacerbation of pain after propranolol therapy in patients with classic stable angina or vasospastic angina would support this hypothesis.15,16 However, the findings of Zeiher et al.4 showing that increases in blood flow in response to the cold pressor test are similar before and after treatment with intracoronary propranolol would argue against this mechanism. Finally, coronary vasodilatation may also result from the direct stimulation of 2-adrenergic receptors in intact endothelial cells and the release of nitric oxide,17,18 presumably through the activation of local kinin synthesis.18 Indeed, removing the vascular endothelium of isolated and intact canine arteries enhances the constrictor response to norepinephrine.19,20 In addition, patients with endothelial dysfunction have impaired microvascular dilatation in response to sympathetic stimulation.5 Furthermore, -adrenergic vasoconstriction is potentiated by the inhibition of nitric oxide synthesis in coronary arteries in both dogs and humans.21
Another important finding in this study is that the basal flows in the transplant recipients were similar in all coronary territories despite the differences in sympathetic innervation, an observation that suggests that resting coronary flow is not substantially affected by either humoral or neural adrenergic influences. This finding is in agreement with the results of studies in animals22 and humans.23 In addition, the maximal vasodilator response to adenosine was similar to that observed in the normal subjects and was not limited by regional differences in sympathetic innervation. This is consistent with the findings of Hodgson et al. demonstrating that coronary flow reserve, as assessed by the use of intracoronary papaverine, was similar in normally innervated and denervated transplant recipients and was unchanged after blockade with either - or -adrenergic receptors.23
Data obtained by the noninvasive method of assessing myocardial blood flow in vivo with PET imaging have been shown to be both accurate and reproducible.10,24 Evaluating the presence and severity of intimal disease was not part of our study design. Although it is possible that regional vasomotor dysfunction in territories with transplant-related vasculopathy that was not detected by coronary arteriography may have affected our results, such an effect is not very likely, because transplant-associated atherosclerosis is a diffuse rather than a regional process.25 Furthermore, recent evidence shows that coronary vasomotor function may be preserved in long-term survivors of cardiac transplantation despite the presence of intimal disease.26
Studies have shown the importance of endothelial function in modulating the coronary vasomotor response to increased sympathetic stimulation.5 We have now demonstrated that the response of coronary blood flow to such stimulation is related to the norepinephrine content of cardiac sympathetic-nerve terminals and is largely independent of changes in hemodynamics and levels of circulating catecholamines. These findings suggest that signals from cardiac efferent sympathetic nerves play an important part in modulating the ability of the coronary vasculature to dilate and thus increase the flow of blood to the myocardium during periods of activation of the sympathetic nervous system, such as occurs during exercise, exposure to cold, and mental stress.
This novel mechanism of regulating myocardial perfusion may have several important implications. In patients with progressive transplant-associated atherosclerosis, an inadequate dilator response of resistance vessels distal to the stenosis could further limit the supply of blood to the myocardium and contribute to myocardial ischemia during periods of stress. Such vasomotor dysfunction could accentuate the alterations in myocardial perfusion caused by endothelial dysfunction27 and contribute to the vascular complications of transplant-associated atherosclerosis.28
Furthermore, studies of laboratory animals have shown that brief episodes of reversible ischemia can induce sustained abnormalities in the function of cardiac sympathetic nerves in reperfused myocardium.29,30,31 Similar findings have also been reported in patients after myocardial infarction.32,33 These observations suggest that severe ischemia may cause regional "denervation" of ischemically injured but viable myocardium. This effect may be important in patients with unstable angina and myocardial infarction. Transient episodes of thrombotic vessel occlusion at the site of plaque rupture are frequent in unstable angina, and coronary occlusion is often intermittent in myocardial infarction.34,35 In addition, the release of vasoactive substances by platelets and vasoconstriction due to endothelial dysfunction may contribute to reduced coronary flow.36 These transient episodes of severe ischemia distal to the site of coronary thrombosis would lead to regional dysfunction of efferent sympathetic nerves, which in turn could reduce the dilator capacity of resistance vessels and influence the extent of myocardial damage. This abnormal vasomotor response may also be present in patients who have diabetic autonomic neuropathy that involves efferent sympathetic pathways. In such patients, impaired coronary vasodilation due to cardiac efferent adrenergic dysfunction may contribute to the pathogenesis of myocardial ischemia and possibly to left ventricular dysfunction.37,38,39
Supported in part by a grant from the Community Foundation for Southeastern Michigan, Detroit.
We are indebted to Galina Rabkin, Teresa Jones, and Benjamin Lathrop for their expert technical assistance in performing the PET studies; to Drs. M.E. Landa, D. Chugani, J.D. Marsh, and R.J. Spears for their valuable comments; and to Medco Research, Inc., and Fujisawa USA, Inc., for kindly supplying the adenosine.
Source Information
From the Division of Cardiology, Department of Internal Medicine (M.F.D.), and the Department of Radiology (M.F.D., T.M., O.M., P.C.), Wayne State University School of Medicine; and the Henry Ford Heart and Vascular Institute (M.C.T., A.B.L., T.B.L.) — both in Detroit.
Address reprint requests to Dr. Di Carli at the Division of Cardiology, Harper Hospital, 3990 John R. St., Detroit, MI 48201.
References
1- and 2-adrenergic constriction in the canine epicardial coronary microcirculation. Circulation 1993;87:1264-1274. - and -blockade on resting and hyperemic coronaryblood flow in conscious, unstressed humans. Circulation 1989;79:797-809.
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