- Email: [email protected]
Effects of neuropeptide Y on coronary artery vasomotion in patients with microvascular angina
Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients With Microvascular Angina Giuseppe M.C. Rosano, Dimitris Tousoulis, Eugene McFadden, John Clarke, Graham J. Davies, Juan Carlos Kaski PII: DOI: Reference:
S0167-5273(17)31520-6 doi:10.1016/j.ijcard.2017.03.024 IJCA 24703
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
19 August 2016 6 March 2017 8 March 2017
Please cite this article as: Rosano Giuseppe M.C., Tousoulis Dimitris, McFadden Eugene, Clarke John, Davies Graham J., Kaski Juan Carlos, Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients With Microvascular Angina, International Journal of Cardiology (2017), doi:10.1016/j.ijcard.2017.03.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients
IP
T
With Microvascular Angina
SC R
Giuseppe M.C. Rosano, MD**, FACC; Dimitris Tousoulis, MD; Eugene McFadden, MD; John Clarke, MD and Graham J. Davies, MD, FRCP, Juan Carlos Kaski, MD,
MA
NU
FACC*
1 Cardiovascular & Cell Science Institute, St George’s Hospitals University of London,
ED
United Kingdom
2 Department of Medical Sciences, IRCCS San Raffaele Roma, Italy
AC CE
PT
3 Hippokration General Hospital , Athens, Greece
Running title: Microvascular angina - Effects of neuropeptide Y
Address for reprints: Giuseppe M.C. Rosano, MD, Cardiovascular & Cell Sciences Institute St George’s Hospital Medical School Cranmer Terrace London SW17 0RE Email: [email protected]
ACCEPTED MANUSCRIPT 2
Abstract Background. Patients with microvascular angina (exertional angina, positive exercise tests
T
and normal coronary arteriograms) usually have a reduced coronary blood flow reserve.
IP
Neuropeptide Y (NPY) is a potent endogenous vasoconstrictor involved in modulation of
SC R
coronary vasomotor tone and may play a role in microvascular angina.
Methods. We compared the effects of NPY (0.2-1.0 pmol/kg, intracoronary) on the vasomotor response of proximal and distal segments of the coronary arteries in 7 patients
NU
with microvascular angina, 9 with chronic stable angina, and 9 control individuals. The
MA
coronary response to the administration of ergonovine was also assessed in 9 other patients with microvascular angina. Computerized coronary artery diameter measurements were
ED
carried out before (baseline) and after the administration of the vasoactive agents. Results. Mean baseline coronary lumen diameters were similar in control, microvascular
PT
angina, and coronary artery disease patients. NPY constricted proximal coronary segments by 8±2%, 5±2% and 6±3% and distal segments by 14±2%, 11±2% and 10±2% in control,
AC CE
microvascular angina, and coronary artery disease patients, respectively (p=NS between groups). In patients with microvascular angina, ergonovine constricted proximal coronary segments by 7±1.5% and distal segments by 12.5±3% (p=NS vs. NPY). During NPY administration four microvascular angina patients developed chest pain, ST segment depression, and a marked lengthening of the contrast medium run off, in the absence of epicardial coronary artery spasm. Control individuals and coronary artery disease patients did not experience chest pain, ST segment shifts, or lengthening of the run off during NPY administration. Ergonovine administration caused chest pain and lengthening of the contrast run-off, in the absence of epicardial coronary artery spasm, in one microvascular angina patient.
ACCEPTED MANUSCRIPT 3 Conclusions. Exogenous NPY causes mild epicardial coronary artery constriction which is similar in patients with non-cardiac chest pain, microvascular angina and coronary artery
T
disease. Myocardial ischemia and marked lengthening of the contrast run off in response to
IP
NPY occurred in microvascular angina patients but not in control or coronary artery disease
SC R
patients. An abnormal constrictor response to NPY at the microcirculation level could be the mechanism underlying the ischemic manifestations observed in patients with microvascular
MA
NU
angina.
Key words: Syndrome X, microvascular angina, coronary reactivity, neuropeptide Y, left
AC CE
PT
ED
ventricular dysfunction
ACCEPTED MANUSCRIPT 4
Condensed Abstract (Table of Contents)
T
Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients
SC R
IP
With Microvascular Angina
The vasomotor response of proximal and distal coronary artery segments was studied in
NU
twenty five patients: 7 microvascular angina, 9 chronic stable angina, and 9 control subjects. Computerized measurements of coronary diameters were carried out before and after the
MA
intracoronary administration of neuropeptide Y (NPY) and ergonovine. Constriction of epicardial arteries in response to NPY was mild and not significantly different in control,
ED
microvascular angina and coronary artery disease patients. Ergonovine-induced epicardial coronary artery constriction was similar to that of NPY. However, NPY caused transient
PT
myocardial ischemia in patients with microvascular angina (probably through constriction of
patients.
AC CE
the small intramyocardial vessels), but not in control subjects or coronary artery disease
ACCEPTED MANUSCRIPT 5
Introduction Patients with "microvascular angina" 1 or "syndrome X"2,3,4 have exertional angina which A
T
may be due to a reduced vasodilatory capacity of the coronary microcirculation. 1-3,5
IP
reduced coronary flow reserve, similar to that observed in patients with left ventricular
SC R
hypertrophy6,7,8 and patients with systemic hypertension,9,10,11 has been reported in patients with microvascular angina.3,12 It has been also shown that in patients with microvascular
NU
angina, ergonovine can exacerbate the limitation of coronary flow reserve (without causing epicardial coronary artery spasm).13 Dynamic small vessel constriction has been suggested to
MA
play a role in the genesis of angina in microvascular angina,4 as a large proportion of microvascular angina patients have angina and ischemia-like ST segment changes at rest.
ED
Recently, it has been reported that in patients with microvascular angina the abnormal vasodilator response affects not only the coronary vascular smooth muscle but also the
PT
systemic vasculature.14 The reason for the abnormal behaviour of the microcirculation in
AC CE
microvascular angina is unknown, but it has been suggested that an increased sympathetic drive may play a pathogenic role.12,15,16 Neuropeptide Y (NPY), a 36 aminoacid peptide17 endogenous to human coronary arteries,18 has been shown to increase coronary vascular resistance in animal models. 19 In humans, the administration of NPY may trigger transient myocardial ischemia20. The action of NPY on the circulation is currently the subject of intense research 21
22
which has been
stimulated by findings of its co-storage and co-operation with noradrenaline in perivascular nerve endings.23
24
Accumulating evidence suggests that sympathetic stimulation releases
both NPY and catecholamines simultaneously.25 NPY appears to exert biological activities which might influence ischemia-induced myocardial damage and arrhythmogenesis,26 and to be a factor involved in the development of cardiac hypertrophy resulting from increased sympathetic activity.27 NPY is a potent inhibitor of cardiac vagal activity,28 and following
ACCEPTED MANUSCRIPT 6 central sympathetic activation it is released in parallel with noradrenaline.29
Recently,
Clarke et al20 suggested that NPY- induced transient myocardial ischemia in man is due to its
T
constrictor effects on the coronary microcirculation.
IP
In this study we assessed, using computerized quantitative coronary arteriography, the
SC R
coronary vasococonstrictor effects of NPY in patients with microvascular angina, patients with chronic stable angina pectoris, and control individuals .
Methods
NU
Patients
MA
The study group comprised 25 patients; nine had atypical non-cardiac chest pain and normal coronary arteriograms (control group), seven had microvascular angina, and nine had
ED
chronic stable angina pectoris with documented atheromatous coronary artery disease. Control group. Seven men and two women, aged 49±7 years, were studied. These patients
PT
had atypical chest pain, negative exercise tests and angiographically normal coronary arteries. All had been referred for coronary arteriography by their treating physician. None of these
AC CE
patients had left ventricular hypertrophy, which was assessed by two-dimensional guided Mmode echocardiograms. Measurements were obtained according to American Society of Echocardiography recommendations.30 Left ventricular wall thickness was calculated as the arithmetic mean of both posterior and interventricular septal thickness, and left ventricular mass was calculated using the method proposed by Troy et al.31 Left ventricular hypertrophy was excluded using gender-specific normal limits from The Framingham Heart Study.32 Systemic hypertension (defined as a blood pressure > 140/90 in repeated readings), coronary artery spasm, cardiomyopathy, valvular heart disease or conduction disturbances were not present in any patient. In this group, chest pain was most probably non cardiac in origin. Coronary blood flow reserve, which was assessed by positron emission tomography (dipyridamole testing and oxygen-15 labelled water measurements33) in 5 patients, was found
ACCEPTED MANUSCRIPT 7 to be normal in all (= 3.8 in every case; range 3.8-4.5, consistent with normal values from our laboratory34 and with results reported by other investigators35, using a similar technique).
T
Microvascular angina. was defined as typical exertional angina, positive exercise testing
IP
(=1 mm of ST segment depression, horizontal or downsloping), and completely normal
SC R
coronary arteriograms. Four men and three women, aged 48±8 years, were included in the study. None of the patients had left ventricular hypertrophy, which was assessed as reported above (left ventricular mass index ranged from 72 to 97 g m-2). Patients with conduction
NU
disturbances, systemic hypertension, coronary artery spasm, cardiomyopathy or valvular heart
depression,
horizontal
or
MA
disease were not included. All 7 microvascular angina patients had episodes of ST segment downsloping,
as
assessed
by
24-hour
ambulatory
electrocardiographic monitoring. Coronary blood flow reserve in response to intravenous
ED
dipyridamole was assessed by positron emission tomography in 5 of the 7 patients and was
technique.
PT
found to be reduced in all 5 (mean 2.4 ±0.4), as found also by Geltman et al 33 using a similar
AC CE
For comparative purposes, nine other microvascular angina patients (4 men, 5 women; aged 44 ±7), selected on the basis of the above criteria, were also included in the study to assess the effects of the coronary vasoconstrictor ergonovine. Coronary artery disease. Nine patients with chronic stable exertional angina , seven men and two women, aged 50±9 years, were studied. All had positive exercise tests and angiographically documented single vessel coronary artery disease (=50 % fixed lumen diameter reduction of the right coronary artery -2 patients-, the left circumflex -2 patients-, or the left anterior descending coronary artery -5 patients-). None of the patients had left ventricular hypertrophy or a history of myocardial infarction, and all had normal baseline electrocardiograms and resting left ventricular function.
ACCEPTED MANUSCRIPT 8 Study Protocol Ethics. The study protocol was approved by the hospital's ethics committee, and the
T
nature of the study was explained to each subject before obtaining written informed consent.
IP
Coronary arteriography. All antianginal medication was discontinued at least 48 hours
SC R
before the study, with the exception of sublingual nitrates, which were allowed for relief of anginal attacks. All patients were fasting and had not smoked or received acute nitrates in the 8 hours preceeding cardiac catheterization. After diagnostic coronary arteriography,
NU
orthogonal views of relevant coronary segments were obtained and an optimal view was
MA
chosen to visualize the artery to be assessed. The position of the camera subsequently remained unchanged throughout the study. The left coronary artery system was used for analysis in every patient. Only segments that appeared angiographically normal were studied
ED
in patients with coronary artery disease. All intracoronary infusions were administered in the
Melsugen).
PT
left coronary artery at room temperature with use of a syringe pump (Perfusor, Braun-
AC CE
Neuropeptide Y. Intracoronary NPY (5-minute infusion of sterile toxin-free NPY, Peninsula Laboratories) was administered to all patients at rates of 0.2, 0.6 and 1.0 pmol/kg per min. The lowest dose was infused before proceeding to the next dose. A single 5-minute infusion of saline was administered in every patient to assess the effects of the vehicle on coronary diameters. Arterial pressure and two electrocardiographic leads, which reflected the territory of the artery under study, were continuously monitored. A control arteriogram was obtained before the administration of NPY and arteriograms were repeated after each 5-minute NPY infusion. Before each angiogram, the catheter was emptied to avoid the effects of bolus injection of NPY. Finally, an intracoronary bolus dose of isosorbide dinitrate (2 mg in 2 ml of 0.9% saline) was administered.
ACCEPTED MANUSCRIPT 9 Ergonovine. In a group of nine patients with microvascular angina, ergonovine maleate (20 µg in 4 ml saline) was administered into the left coronary artery. Arteriograms of the relevant
T
coronary segments were repeated 3 and 5 minutes after the ergonovine infusion. An
IP
intracoronary bolus of 2 mg isosorbide dinitrate was administered after the ergonovine test .
SC R
Measurement of coronary artery diameters.
Our technique to measure coronary artery diameters has been reported in detail previously.36,37,38 Briefly, coronary luminal diameters were measured by an automated edge
NU
contour detection system (Coronary Angiography Analysis System [CAAS]; Pie Data
MA
Medical).39 The stem of the Judkins coronary catheter was used for calibration to determine absolute measurements in millimeters, and correction was made for radiographic pincushion distortion. The accuracy (defined as the signed difference between the measured and true
ED
value) and the precision (defined as the standard deviation of these differences) of the system
PT
had been determined in previous studies. The accuracy was 0.08 mm and the precision was 0.12 mm. Coronary diameters were measured by two independent observers and the
AC CE
angiograms were also re-analyzed blindly at a later time to ascertain the inter and intraobserver variability. Intraobserver variation (standard error of the estimate -SEE-) was 0.11 mm and interobserver variation was 0.10 mm. To assess the effect of the intracoronary interventions, appropriate arterial segments were selected on end-diastolic frames from proximal and distal portions of the left coronary artery, as defined in the American Heart Association Committee Report.40
The percent
vasoconstriction by NPY and ergonovine used to quantify changes of coronary artery diameter were calculated as follows: %Constriction = {(Control diameter - Diameter after constrictor agent) / Control diameter}x 100
ACCEPTED MANUSCRIPT 10 Data analysis Coronary diameter data are presented as mean values ± SEM, and hemodynamic variables
T
as mean ± 1 SD. Differences between proportions were analyzed by Yates corrected chi-
IP
square tests. Paired and unpaired Student's t-tests were used, as appropriate, to analyze
SC R
continuous data. Analysis of variance was used to assess the reproducibility of the angiographic measurements. A p value < 0.05 was considered significant.
Results
NU
Baseline coronary artery diameters
MA
We analyzed a total of 132 coronary artery segments: 37 in the control group, 63 in patients with microvascular angina (27 for assessment of NPY and 36 for assessment of
ED
ergonovine), and 32 in patients with coronary artery disease. Baseline diameters of proximal and distal coronary segments in control subjects, microvascular angina patients and coronary
PT
artery disease patients are presented in Table 1. Mean diameter of proximal segments was: 3.18 ±0.18 mm, 3.14 ±0.18 mm and 2.91±0.19 mm, for control individuals, microvascular
AC CE
angina and coronary artery disease patients, respectively (p=NS, >0.05). Mean distal diameters were: 1.35 ±0.06 mm, 1.36 ±0.08 mm and 1.30 ±0.08 mm, respectively (p=NS, >0.05).
Effects of NPY on coronary artery diameter There were no significant changes in heart rate or systemic blood pressure associated with the administration of saline, NPY or ergonovine. The systemic hemodynamic response to NPY was similar in the three patient groups. The changes observed in the coronary segments we selected for analysis after the administration of NPY are detailed in Table 1. The intracoronary administration of 0.9% saline was not associated with significant changes in epicardial lumen. After NPY administration, proximal coronary segments reduced their calibre by 8 ±2%, 5 ±2% and 6 ±2% in the control group, microvascular angina, and coronary
ACCEPTED MANUSCRIPT 11 artery disease patients, respectively (p=NS between groups). Accordingly, constriction of distal epicardial coronary artery segments in response to NPY was not significantly different
T
between the three patient subgroups. Coronary diameters were reduced by 14 ±2%, 11 ±2%
IP
and 10 ±2%, respectively. In patients with microvascular angina, NPY caused a significantly
SC R
larger coronary diameter reduction in distal segments compared to proximal segments. Although constriction of distal segments in control and coronary artery disease patients was larger than that of proximal segments, the difference was not statistically significant.
NU
Although beyond the scope of the study, in patients with coronary artery disease no
segments and coronary stenoses.
MA
significant differences were observed between the response of angiographically normal
Clinical, electrocardiographic and angiographic findings during NPY administration
ED
Four microvascular angina patients developed typical anginal pain during the
PT
administration of NPY, which was associated with ischemic ST segment depression and a marked lengthening of the contrast medium run off. The flow of contrast in the visualized
AC CE
vessels of these four microvascular angina patients was strikingly slow, resulting in an extraordinarily long delay in the clearance of the contrast medium from the coronary tree. In three of the four patients, the contrast remained “trapped” within the lumen of the coronary artery and was squeezed backwards during several systoles. Epicardial coronary artery spasm was not observed. Chest pain, ST segment changes and run-off velocity returned to baseline after the administration of 2 mg of isosorbide dinitrate (after 2±1, 3.5±1 and 8±3 minutes, respectively). Ostensible marked prolongation of the run-off time after NPY administration, but not during baseline, was a frequent finding in microvascular angina patients (5 out of 7 patients). In these patients, the slow run-off was not altered by repositioning the tip of the catheter and was usually long lasting, even after the administration of nitrate.
ACCEPTED MANUSCRIPT 12 None of the control individuals or coronary artery disease patients developed chest pain or electrocardiographic signs of ischemia in response to NPY. Contrast run-off remained
T
unchanged in control patients but was prolonged in one coronary artery disease patient.
IP
Response to ergonovine
SC R
In the nine microvascular angina patients that underwent ergonovine testing, average diameter change of proximal and distal segments in response to ergonovine was similar to that observed in the other microvascular angina group in response to the administration of
NU
NPY. Proximal segments constricted by 7 ±1% and distal segments by 12.5 ±3%. Only one
MA
of the nine patients in the ergonovine group developed chest pain and mild ST segment depression which were associated with lengthening of the contrast run off. The intracoronary administration of nitrate resulted in prompt relief of symptoms and contrast run off velocity
ED
returned to baseline levels within 3 minutes. None of the remaining eight patients had
AC CE
Discussion
PT
symptoms or angiographic changes associated with ergonovine administration.
This study shows that the administration of NPY, a peptide endogenous to human coronary arteries, causes mild epicardial coronary artery constriction which is not significantly different in control, microvascular angina and coronary artery disease patients. Constriction of epicardial coronary arteries of microvascular angina patients by NPY was not significantly different than that observed with ergonovine. The response of proximal coronary segments to NPY in patients with microvascular angina was similar to that documented in control individuals and coronary artery disease patients. Similarly, constriction of distal coronary segments was not significantly different in the three patient groups.
However,
the
distal
segments of the epicardial coronary arteries of our microvascular angina patients showed an increased constrictor response to NPY compared with the proximal segments. This finding is
ACCEPTED MANUSCRIPT 13 in agreement with observations by Bortone et al.41 who reported exercise-induced distal coronary vasoconstriction in patients with ischemia-like symptoms, normal coronary
T
arteriograms and a reduced dilatory capacity of the resistance coronary vessels. The authors
IP
postulated that the abnormal response of the distal coronary segments in these patients was
SC R
due to an abnormal neurohumoral tone. Coronary adrenergic hyperreactivity and inappropriate constriction of distal coronary artery segments in response to the cold pressor test have been recently reported by Montorsi et al.12 in patients with microvascular angina
NU
who had resting electrocardiographic abnormalities. Whether the abnormal adrenergic
MA
response found in microvascular angina is associated with an increased release of NPY by adrenergic neurons, or an exaggerated response of the vascular smooth muscle to NPY is not yet known, but it has been shown that NPY has the potential to modulate sympathetic drive42 . To know whether the effects of exogenously administered NPY observed in the present
ED
43
PT
study might occur physiologically it is important to determine if the concentration of NPY used in this study could be achieved under physiological circumstances. The dose of NPY
AC CE
used in the present study correspond to blood concentrations of approximately X-X. Derchi et al.44 reported plasma levels of 296 pg x ml-1 under physiological circumstances, and of 652 pg x ml-1 in patients with heart failure. Thus the concentration used in our study can be achieved under different circumstances in patients. The abnormal distal coronary artery response of patients with microvascular angina is also in agreement with our previous observations using intracoronary ergonovine36, and with recent preliminary observations by McFadden et al.45 using intracoronary serotonin, which suggest that a dynamic alteration of the distal epicardial vessels may contribute to the impaired coronary flow reserve present in microvascular angina. An important finding in our study is that angina and ischemic ST segment shifts developed in patients with microvascular angina, despite the mild epicardial coronary constriction
ACCEPTED MANUSCRIPT 14 elicited by NPY, whereas neither control subjects nor patients with coronary artery disease had chest pain or ST segment changes during the infusion of NPY despite similar changes in
T
epicardial coronary diameter. A possible explanation for this finding may be found in our
IP
observation that the flow of contrast in some of the microvascular angina patients
SC R
(particularly those who developed chest pain and ischemic ST segment depression) was strikingly slow during NPY infusion, compared with that of control and coronary artery disease patients. The slow run-off may have been due to the arteriolar vasoconstrictor effect
NU
of NPY, as monitoring of heart rate and central aortic pressure did not reveal bradycardia or a
MA
significant drop in blood pressure which could have been associated to a decrease in the velocity of contrast medium flow. Bradycardia did not develop in any patient in the present series, but bradycardia requiring the insertion of a temporary pacing wire had been observed
ED
by our group in previous studies.20
PT
Abnormal epicardial coronary constriction did not account for the markedly slow run-off found in our microvascular angina patients, as NPY-induced coronary constriction of
AC CE
epicardial segments was mild, and similar in the three patient subgroups. Angina pectoris and slow flow velocity of contrast in the coronary arteries of a group of patients with angina and normal coronary arteriograms had been described twenty years ago by Tambe et al., 46 who postulated that an increased baseline tone of the coronary resistance vessels was present in patients with the syndrome of angina pectoris and normal coronary arteriograms who had a slow run off of the contrast medium during baseline arteriography. Slow run-off of the contrast dye was observed in patients with scleroderma who had structural abnormalities of the coronary microcirculation47. Because epicardial coronary artery spasm did not develop in our microvascular angina patients during NPY administration, a likely explanation for the presence of angina, ST segment changes and a markedly lengthened run-off is that NPY caused coronary vasoconstriction at the arteriolar level which resulted in transient myocardial
ACCEPTED MANUSCRIPT 15 ischemia. This finding is consistent with results of experimental studies, which showed that NPY exerts a predominantly arteriolar constrictor effect19
48 49 50
. Our data confirm and
T
expand previous findings20 that exogenously administered NPY can provoke myocardial
IP
ischemia in patients with angina pectoris. However, in our study, only patients with
SC R
microvascular angina developed anginal pain, ST segment depression and lengthening of the run off in response to NPY, whereas none of the control subjects or patients with coronary artery disease experienced symptoms during the administration of NPY. The reason why the
NU
coronary microcirculation of microvascular angina patients but not that of control individuals
MA
or patients with coronary artery disease exhibit an enhanced constrictor response to NPY is not known. Our microvascular angina patients had "microvascular angina"1, characterized by
ED
an abnormal dilatory capacity of the coronary microcirculation. A patchy pre-arteriolar abnormal constriction has been suggested to play a pathogenetic role in microvascular
PT
angina51, and it might be speculated that particular segments of the coronary microcirculation of these patients have an abnormal sensitivity to NPY. Our findings in microvascular angina
AC CE
suggest that NPY could play a pathogenetic role in disorders associated with intramyocardial or systemic resistance vessel dysfunction (as in microvascular angina). Indeed, NPY is endogenous to the coronary arteries and widely distributed within the myocardium.18 It is conceivable that a baseline alteration of small vessel coronary vasomotor tone may be present in patients with microvascular angina, which can be exarcebated by the administration of a powerful arteriolar constrictor such as NPY. Whether the response to NPY found in our microvascular angina patients represent a specific or a non-specific one is not known. Ergonovine provoked angina and lengthening of the contrast run off in only one of the nine microvascular angina patients in our study, but Cannon et al13 observed that, on average, ergonovine exacerbates the microvascular dysfunction of patients with microvascular angina.
ACCEPTED MANUSCRIPT 16 One limitation of the present study is represented by the low number of patients. However, the results are very consistent within groups and, the presence of prolonged microvascular
IP
T
constriction suggested to proceed with further recruitment of patients.
SC R
Conclusion
The results of our study indicate that, in patients with microvascular angina, the exogenous administration of NPY may trigger myocardial ischemia, which may be associated with the
NU
powerful microcirculatory constrictor effect of NPY, as the response of the large epicardial
AC CE
PT
ED
MA
coronary segments remains within the physiological range.
ACCEPTED MANUSCRIPT 17
References Cannon RO, Epstein SE. "Microvascular angina" as a cause of chest pain with angiographically normal coronary arteries (editorial). Am J Cardiol 1988; 61: 1338-43.
IP
T
1
Kemp HG. Left ventricular function in patients with the anginal syndrome and normal coronary arteriograms. Am J Cardiol 1973; 32: 375-6. 3
SC R
2
Opherk D, Zebe H, Weihe E, Mall G, Durr C, Gravert B, Mehmel HC, Schwarz F, Kübler
Henein MY, Rosano GM, Underwood R, Poole-Wilson PA, Gibson DG. Relations between
MA
4
NU
W. Reduced coronary dilatory capacity and ultrastructural changes of the myocardium in patients with angina pectoris but normal coronary arteriograms. Circulation 1981; 63: 817-25.
resting ventricular long axis function, the electrocardiogram, and myocardial perfusion imaging in syndrome X. Br Heart J. 1994 Jun;71(6):541-7. Cannon RO, Watson RM, Rosing DR, Epstein SE. Angina caused by reduced vasodilator reserve of the small coronary arteries. J Am Coll Cardiol 1983; 1: 359-73
PT
ED
5
AC CE
Cannon RO, Rosing DR, Maron BJ, Leon MB, Bonow RO, Watson RM, Epstein SE. Myocardial ischemia in patients with hypertrophic cardiomyopathy: contribution of inadequate vasodilator reserve and elevated left ventricular filling pressures. Circulation 1985; 71: 234-243. 6
Rosano GM, Peters NS, Kaski JC, Mavrogeni SI, Collins P, Underwood RS, Poole-Wilson PA. Abnormal uptake and washout of thallium-201 in patients with syndrome X and normalappearing scans. Am J Cardiol. 1995 Feb 15;75(5):400-2. 7
Wangler RD, Peters KG, Marcus ML, Tomanek RJ. Effects of duration and severity of arterial hypertension and cardiac hypertrophy on coronary vasodilator reserve. Circ Res 1982; 51: 10-18. 8
Opherk D, Mall G, Zebe H, Schwarz F, Weihe E, Manthey J, Kubler W. Reduction of coronary reserve: a mechanism for angina pectoris in patients with arterial hypertension and normal coronary arteries. Circulation 1984; 69: 1-7. 9
ACCEPTED MANUSCRIPT 18
Brush JE, Cannon RO, Schenke WH, Bonow RO, Leon MB, Maron BJ, Epstein SE. Angina due to coronary microvascular disease in hypertensive patients without left ventricular hypertrophy. N Engl J Med 1988; 319: 1302-1307.
T
10
Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 1990; 323: 22-27. 12
SC R
IP
11
Montorsi P, Fabbiocchi F, Loaldi A, et al. Coronary adrenergic hyperreactivity in patients
with syndrome X and abnormal electrocardiogram at rest. Am J Cardiol 1991; 68: 1698-1703. Cannon RO, Schenke WH, Leon M, Rosing DR, Urqhart J, Epstein SE. Limited coronary flow reserve after dipyridamole in patients with ergonovine -induced coronary vasoconstriction. Circulation 1987; 75: 163-74.
MA
NU
13
14
PT
ED
Sax FL, Cannon RO, Hanson C, Epstein SE. Impaired forearm vasodilator reserve in patients with microvascular angina. Evidence of a generalized disorder of vascular function? N Engl J Med 1987; 317: 1366-70.
AC CE
Kaski JC, Rosano G, Gavrielides S, Chen L. Effects of angiotensin-converting enzyme inhibition on exercise-induced angina and ST segment depression in patients with microvascular angina. J Am Coll Cardiol. 1994 Mar 1;23(3):652-7. 15
Rosano GM, Ponikowski P, Adamopoulos S, Collins P, Poole-Wilson PA, Coats AJ, Kaski JC. Abnormal autonomic control of the cardiovascular system in syndrome X. Am J Cardiol. 1994 Jun 15;73(16):1174-9. 16
Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y: A novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 1982; 296: 659-60. 17
Wharton J, Gulbenkian S, Polak JM. Neuropeptide tyrosine (NPY) in human cardiovascular innervation, in Mutt V, Hokfelt T, Fuxe K, Lundberg JM (eds): Neuropeptide Y. New York, Raven Press Publishers, 1988, pp 83-91. 18
19
Macho P, Perez R, Huidobro-Toro JP, Domenech RJ. Neuropeptide Y (NPY): a coronary
vasoconstrictor and potentiator of catecholamine-induced coronary constriction. Eur J Pharmacol 1989; 167: 67-74.
ACCEPTED MANUSCRIPT 19
Clarke J, Davies GJ, Kerwin R, Hackett D, Larkin S, Dawbarn D, Lee Y, Bloom SR, Yacoub M, Maseri A. Coronary artery infusion of neuropeptide Y in patients with angina pectoris. Lancet 1987; i: 1057-59. Lehmann J. Neuropeptide Y-An overview. Drug Develop Res 1990; 19:329-51.
IP
21
T
20
Waeber B, Aubert JF, Corder R et al. Cardiovascular effects of neuropeptide Y. Am J Hypert 1988; 1: 193-9.
SC R
22
Allen YS, Adrian TE, Allen JM, Tatemoto K, Crow TJ. Neuropeptide Y distribution in the rat brain. Science 1983; 221: 877-79.
NU
23
Ekblad E, Edvinsson L, Wahlestedt C, Sundrez F. Neuropeptide Y co-exists with noradrenaline in perivascular nerve fibres. Regul Pept 1988; 8: 225-35.
MA
24
Hellstrom PM, Olerup O, Tatemoto K. Neuropeptide Y may mediate effects of sympathetic nerve stimulation on colonic motility and blood flow in the cat. Acta Physiol Scand 1985;
ED
25
PT
124: 613-624.
AC CE
Schömig A, Haass M, Richardt G. Catecholamine release and arrhythmias in acute myocardial ischemia. Eur Heart J 1991; 12 (suppl F): 38-47. 26
Woo ND, Sahai A, Anderson WA, Ganguly PK. Modulation of sympathetic activity by brain neuropeptide Y in cardiac hypertrophy. Am Heart J 1991; 122: 1028-1034. 27
28
Warner MR, Levy MN. Neuropeptide Y as a putative modulator of vagal effects on heart
rate. Circ Res 1989; 64: 882-89. Haass M, Cheng B, Richardt G, Land RE, Schömig A. Characterization and presynaptic modulation of exocytotic co-release of noradrenaline and neuropeptide Y from the guinea pig heart. Naunyn-Schmiedeberg’s Arch Pharmachol 1989; 339: 71-8. 29
Sahn DJ, De Maria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978; 58: 1072-83. 30
Troy BL, Pombo J, Rackley CE. Measurement of left ventricular thickness and mass by echocardiography. Circulation 1972; 45: 602-11. 31
ACCEPTED MANUSCRIPT 20
Levi D, Savage DD, Garrison RJ, Anderson KM, Kannel WB, Castelli WP. Echocardiographic criteria for left ventricular hypertrophy: The Framingham Heart Study. Am J Cardiol 1987; 59: 956-60.
IP
T
32
Araujo LI, Lammertsma AA, Rhodes CG, Mc Falls EO, Iida H, Rechavia E, Galassi AR, De Silva R, Jones T, Maseri A. Non invasive quantification of regional myocardial blood flow in normal volunteers and patients with coronary artery disease using oxygen-15 labelled
SC R
33
water and positron emission tomography. Circulation 1991; 83: 875-85. 34
MA
NU
Ponikowski P, Rosano GM, Amadi AA, Collins P, Coats AJ, Poole-Wilson PA, Kaski JC. Transient autonomic dysfunction precedes ST-segment depression in patients with syndrome X. Am J Cardiol. 1996 May 1;77(11):942-7. 35
ED
Geltman EM, Henes G, Senneff MJ, Sobel BE, Bergmann SR. Increased myocardial perfusion at rest and diminished perfusion reserve in patients with angina and angiographically normal coronary arteries. J Am Coll Cardiol 1990; 16: 586-95. 36
AC CE
PT
Hackett D, Larkin S, Chierchia S, Davies G, Kaski JC, Maseri A. Induction of coronary artery spasm by a direct local action of ergonovine. Circulation 1987; 75: 577- 582. McFadden EP, Clarke JG, Davies GJ, Kaski JC, Haider AW, Maseri A. Effect of intracoronary serotonin on coronary vessel in patients with stable angina and patients with variant angina. N Engl J Med 1991; 324: 648-654 37
38
Stec K, Haberka M, Mizia M, Chmiel A, Wierzbicka-Chmiel J, Skowerski M, Gasior Z.
Coronary artery calcium score assessed by a 64 multislice computed tomography and early indexes of functional and structural vascular remodeling in cardiac syndrome X patients. J Nucl Cardiol. 2008 Sep-Oct;15(5):655-62. Reiber JHC, Serruys PW, Kooijman CJ, Wijns W, Slager CJ, Gerbrands JJ, Schuurbiers JC, den-Boer A, Hugenholtz PG. Assessment of short-, medium-, and long-term variations in arterial dimensions, from computer-assisted quantitation of coronary cineangiograms. Circulation 1985; 71 (2): 280-288 39
40
Kidawa M, Krzeminska-Pakula M, Peruga JZ, Kasprzak JD. Arterial dysfunction in syndrome X: results of arterial reactivity and pulse wave propagation tests. Heart. 2003 Apr;89(4):422-6.
ACCEPTED MANUSCRIPT 21
Bortone AS, Hess OM, Eberli FR , Nonogi H, Marolf AP, Grimm J, Krayembeuhl HP. Abnormal coronary vasomotion during exercise in patients with normal coronary arteries and reduced coronary flow reserve. Circulation 1989; 79 (3): 516-52
T
41
Lundberg JM, Terenius L, Hokfelt T. et al. Neuropeptide Y (NPY) like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol Scand 1982; 116: 477-80.
SC R
IP
42
Franco-Cereceda A, Lundberg JM, Dahlof G. Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone. Acta Physiol Scand 1985; 124: 361-69.
NU
43
Derchi G, Dupuis J, de Champlain J, Roleau JL. Paradoxical decrease in circulating neuropeptide Y-like immunoreactivity during mild orthostatic stress in subjects with and
MA
44
without congestive heart failure. Eur Heart J 1993; 14: 34-39. Proudler AJ, Crook D, Godsland IF, Collins P, Rosano GM, Stevenson JC. Serum angiotensin-I-converting enzyme activity in women with cardiological syndrome X: relation
ED
45
PT
to blood pressure and lipid and carbohydrate metabolic risk markers for coronary heart disease. J Clin Endocrinol Metab. 1995 Feb;80(2):696-9.
AC CE
Tambe A.A, Demany M.A, Zimmerman H.A, Mascarenhas E. Angina pectoris and slow flow velocity of dye in coronary arteries-A new angiographic finding. Am Heart J 1972; 84: 66-71. 46
Gupta MP, Zoneraich S, Zeitlin W, Zoneraich O, D’Angelo W. Scleroderma heart disease with slow flow velocity in coronary arteries. Chest 1975; 67: 116-119. 47
Franco-Cereceda A. Endothelin -and neuropeptide Y- induced vasoconstriction of human epicardial coronary arteries in vitro. Br J Pharmacol 1989; 97: 968-72. 48
Emson PC, Dequidt ME. NPY: a new member of the pancreatic polypeptide family. Trends Neurosci 1984; 7: 31-35. 49
Franco-Cereceda A, Lundberg JM. Potent effects of neuropeptide Y and calcitonin generelated peptide on human coronary vascular tone in vitro. Acta Physiol Scand 1987; 131:15950
160. 51 Rosano GM, Lindsay DC, Poole-Wilson PA. Syndrome X: an hypothesis for cardiac pain without ischaemia. Cardiologia. 1991 Nov;36(11):885-95.
S0167-5273(17)31520-6 doi:10.1016/j.ijcard.2017.03.024 IJCA 24703
To appear in:
International Journal of Cardiology
Received date: Revised date: Accepted date:
19 August 2016 6 March 2017 8 March 2017
Please cite this article as: Rosano Giuseppe M.C., Tousoulis Dimitris, McFadden Eugene, Clarke John, Davies Graham J., Kaski Juan Carlos, Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients With Microvascular Angina, International Journal of Cardiology (2017), doi:10.1016/j.ijcard.2017.03.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients
IP
T
With Microvascular Angina
SC R
Giuseppe M.C. Rosano, MD**, FACC; Dimitris Tousoulis, MD; Eugene McFadden, MD; John Clarke, MD and Graham J. Davies, MD, FRCP, Juan Carlos Kaski, MD,
MA
NU
FACC*
1 Cardiovascular & Cell Science Institute, St George’s Hospitals University of London,
ED
United Kingdom
2 Department of Medical Sciences, IRCCS San Raffaele Roma, Italy
AC CE
PT
3 Hippokration General Hospital , Athens, Greece
Running title: Microvascular angina - Effects of neuropeptide Y
Address for reprints: Giuseppe M.C. Rosano, MD, Cardiovascular & Cell Sciences Institute St George’s Hospital Medical School Cranmer Terrace London SW17 0RE Email: [email protected]
ACCEPTED MANUSCRIPT 2
Abstract Background. Patients with microvascular angina (exertional angina, positive exercise tests
T
and normal coronary arteriograms) usually have a reduced coronary blood flow reserve.
IP
Neuropeptide Y (NPY) is a potent endogenous vasoconstrictor involved in modulation of
SC R
coronary vasomotor tone and may play a role in microvascular angina.
Methods. We compared the effects of NPY (0.2-1.0 pmol/kg, intracoronary) on the vasomotor response of proximal and distal segments of the coronary arteries in 7 patients
NU
with microvascular angina, 9 with chronic stable angina, and 9 control individuals. The
MA
coronary response to the administration of ergonovine was also assessed in 9 other patients with microvascular angina. Computerized coronary artery diameter measurements were
ED
carried out before (baseline) and after the administration of the vasoactive agents. Results. Mean baseline coronary lumen diameters were similar in control, microvascular
PT
angina, and coronary artery disease patients. NPY constricted proximal coronary segments by 8±2%, 5±2% and 6±3% and distal segments by 14±2%, 11±2% and 10±2% in control,
AC CE
microvascular angina, and coronary artery disease patients, respectively (p=NS between groups). In patients with microvascular angina, ergonovine constricted proximal coronary segments by 7±1.5% and distal segments by 12.5±3% (p=NS vs. NPY). During NPY administration four microvascular angina patients developed chest pain, ST segment depression, and a marked lengthening of the contrast medium run off, in the absence of epicardial coronary artery spasm. Control individuals and coronary artery disease patients did not experience chest pain, ST segment shifts, or lengthening of the run off during NPY administration. Ergonovine administration caused chest pain and lengthening of the contrast run-off, in the absence of epicardial coronary artery spasm, in one microvascular angina patient.
ACCEPTED MANUSCRIPT 3 Conclusions. Exogenous NPY causes mild epicardial coronary artery constriction which is similar in patients with non-cardiac chest pain, microvascular angina and coronary artery
T
disease. Myocardial ischemia and marked lengthening of the contrast run off in response to
IP
NPY occurred in microvascular angina patients but not in control or coronary artery disease
SC R
patients. An abnormal constrictor response to NPY at the microcirculation level could be the mechanism underlying the ischemic manifestations observed in patients with microvascular
MA
NU
angina.
Key words: Syndrome X, microvascular angina, coronary reactivity, neuropeptide Y, left
AC CE
PT
ED
ventricular dysfunction
ACCEPTED MANUSCRIPT 4
Condensed Abstract (Table of Contents)
T
Effects of Neuropeptide Y on Coronary Artery Vasomotion in Patients
SC R
IP
With Microvascular Angina
The vasomotor response of proximal and distal coronary artery segments was studied in
NU
twenty five patients: 7 microvascular angina, 9 chronic stable angina, and 9 control subjects. Computerized measurements of coronary diameters were carried out before and after the
MA
intracoronary administration of neuropeptide Y (NPY) and ergonovine. Constriction of epicardial arteries in response to NPY was mild and not significantly different in control,
ED
microvascular angina and coronary artery disease patients. Ergonovine-induced epicardial coronary artery constriction was similar to that of NPY. However, NPY caused transient
PT
myocardial ischemia in patients with microvascular angina (probably through constriction of
patients.
AC CE
the small intramyocardial vessels), but not in control subjects or coronary artery disease
ACCEPTED MANUSCRIPT 5
Introduction Patients with "microvascular angina" 1 or "syndrome X"2,3,4 have exertional angina which A
T
may be due to a reduced vasodilatory capacity of the coronary microcirculation. 1-3,5
IP
reduced coronary flow reserve, similar to that observed in patients with left ventricular
SC R
hypertrophy6,7,8 and patients with systemic hypertension,9,10,11 has been reported in patients with microvascular angina.3,12 It has been also shown that in patients with microvascular
NU
angina, ergonovine can exacerbate the limitation of coronary flow reserve (without causing epicardial coronary artery spasm).13 Dynamic small vessel constriction has been suggested to
MA
play a role in the genesis of angina in microvascular angina,4 as a large proportion of microvascular angina patients have angina and ischemia-like ST segment changes at rest.
ED
Recently, it has been reported that in patients with microvascular angina the abnormal vasodilator response affects not only the coronary vascular smooth muscle but also the
PT
systemic vasculature.14 The reason for the abnormal behaviour of the microcirculation in
AC CE
microvascular angina is unknown, but it has been suggested that an increased sympathetic drive may play a pathogenic role.12,15,16 Neuropeptide Y (NPY), a 36 aminoacid peptide17 endogenous to human coronary arteries,18 has been shown to increase coronary vascular resistance in animal models. 19 In humans, the administration of NPY may trigger transient myocardial ischemia20. The action of NPY on the circulation is currently the subject of intense research 21
22
which has been
stimulated by findings of its co-storage and co-operation with noradrenaline in perivascular nerve endings.23
24
Accumulating evidence suggests that sympathetic stimulation releases
both NPY and catecholamines simultaneously.25 NPY appears to exert biological activities which might influence ischemia-induced myocardial damage and arrhythmogenesis,26 and to be a factor involved in the development of cardiac hypertrophy resulting from increased sympathetic activity.27 NPY is a potent inhibitor of cardiac vagal activity,28 and following
ACCEPTED MANUSCRIPT 6 central sympathetic activation it is released in parallel with noradrenaline.29
Recently,
Clarke et al20 suggested that NPY- induced transient myocardial ischemia in man is due to its
T
constrictor effects on the coronary microcirculation.
IP
In this study we assessed, using computerized quantitative coronary arteriography, the
SC R
coronary vasococonstrictor effects of NPY in patients with microvascular angina, patients with chronic stable angina pectoris, and control individuals .
Methods
NU
Patients
MA
The study group comprised 25 patients; nine had atypical non-cardiac chest pain and normal coronary arteriograms (control group), seven had microvascular angina, and nine had
ED
chronic stable angina pectoris with documented atheromatous coronary artery disease. Control group. Seven men and two women, aged 49±7 years, were studied. These patients
PT
had atypical chest pain, negative exercise tests and angiographically normal coronary arteries. All had been referred for coronary arteriography by their treating physician. None of these
AC CE
patients had left ventricular hypertrophy, which was assessed by two-dimensional guided Mmode echocardiograms. Measurements were obtained according to American Society of Echocardiography recommendations.30 Left ventricular wall thickness was calculated as the arithmetic mean of both posterior and interventricular septal thickness, and left ventricular mass was calculated using the method proposed by Troy et al.31 Left ventricular hypertrophy was excluded using gender-specific normal limits from The Framingham Heart Study.32 Systemic hypertension (defined as a blood pressure > 140/90 in repeated readings), coronary artery spasm, cardiomyopathy, valvular heart disease or conduction disturbances were not present in any patient. In this group, chest pain was most probably non cardiac in origin. Coronary blood flow reserve, which was assessed by positron emission tomography (dipyridamole testing and oxygen-15 labelled water measurements33) in 5 patients, was found
ACCEPTED MANUSCRIPT 7 to be normal in all (= 3.8 in every case; range 3.8-4.5, consistent with normal values from our laboratory34 and with results reported by other investigators35, using a similar technique).
T
Microvascular angina. was defined as typical exertional angina, positive exercise testing
IP
(=1 mm of ST segment depression, horizontal or downsloping), and completely normal
SC R
coronary arteriograms. Four men and three women, aged 48±8 years, were included in the study. None of the patients had left ventricular hypertrophy, which was assessed as reported above (left ventricular mass index ranged from 72 to 97 g m-2). Patients with conduction
NU
disturbances, systemic hypertension, coronary artery spasm, cardiomyopathy or valvular heart
depression,
horizontal
or
MA
disease were not included. All 7 microvascular angina patients had episodes of ST segment downsloping,
as
assessed
by
24-hour
ambulatory
electrocardiographic monitoring. Coronary blood flow reserve in response to intravenous
ED
dipyridamole was assessed by positron emission tomography in 5 of the 7 patients and was
technique.
PT
found to be reduced in all 5 (mean 2.4 ±0.4), as found also by Geltman et al 33 using a similar
AC CE
For comparative purposes, nine other microvascular angina patients (4 men, 5 women; aged 44 ±7), selected on the basis of the above criteria, were also included in the study to assess the effects of the coronary vasoconstrictor ergonovine. Coronary artery disease. Nine patients with chronic stable exertional angina , seven men and two women, aged 50±9 years, were studied. All had positive exercise tests and angiographically documented single vessel coronary artery disease (=50 % fixed lumen diameter reduction of the right coronary artery -2 patients-, the left circumflex -2 patients-, or the left anterior descending coronary artery -5 patients-). None of the patients had left ventricular hypertrophy or a history of myocardial infarction, and all had normal baseline electrocardiograms and resting left ventricular function.
ACCEPTED MANUSCRIPT 8 Study Protocol Ethics. The study protocol was approved by the hospital's ethics committee, and the
T
nature of the study was explained to each subject before obtaining written informed consent.
IP
Coronary arteriography. All antianginal medication was discontinued at least 48 hours
SC R
before the study, with the exception of sublingual nitrates, which were allowed for relief of anginal attacks. All patients were fasting and had not smoked or received acute nitrates in the 8 hours preceeding cardiac catheterization. After diagnostic coronary arteriography,
NU
orthogonal views of relevant coronary segments were obtained and an optimal view was
MA
chosen to visualize the artery to be assessed. The position of the camera subsequently remained unchanged throughout the study. The left coronary artery system was used for analysis in every patient. Only segments that appeared angiographically normal were studied
ED
in patients with coronary artery disease. All intracoronary infusions were administered in the
Melsugen).
PT
left coronary artery at room temperature with use of a syringe pump (Perfusor, Braun-
AC CE
Neuropeptide Y. Intracoronary NPY (5-minute infusion of sterile toxin-free NPY, Peninsula Laboratories) was administered to all patients at rates of 0.2, 0.6 and 1.0 pmol/kg per min. The lowest dose was infused before proceeding to the next dose. A single 5-minute infusion of saline was administered in every patient to assess the effects of the vehicle on coronary diameters. Arterial pressure and two electrocardiographic leads, which reflected the territory of the artery under study, were continuously monitored. A control arteriogram was obtained before the administration of NPY and arteriograms were repeated after each 5-minute NPY infusion. Before each angiogram, the catheter was emptied to avoid the effects of bolus injection of NPY. Finally, an intracoronary bolus dose of isosorbide dinitrate (2 mg in 2 ml of 0.9% saline) was administered.
ACCEPTED MANUSCRIPT 9 Ergonovine. In a group of nine patients with microvascular angina, ergonovine maleate (20 µg in 4 ml saline) was administered into the left coronary artery. Arteriograms of the relevant
T
coronary segments were repeated 3 and 5 minutes after the ergonovine infusion. An
IP
intracoronary bolus of 2 mg isosorbide dinitrate was administered after the ergonovine test .
SC R
Measurement of coronary artery diameters.
Our technique to measure coronary artery diameters has been reported in detail previously.36,37,38 Briefly, coronary luminal diameters were measured by an automated edge
NU
contour detection system (Coronary Angiography Analysis System [CAAS]; Pie Data
MA
Medical).39 The stem of the Judkins coronary catheter was used for calibration to determine absolute measurements in millimeters, and correction was made for radiographic pincushion distortion. The accuracy (defined as the signed difference between the measured and true
ED
value) and the precision (defined as the standard deviation of these differences) of the system
PT
had been determined in previous studies. The accuracy was 0.08 mm and the precision was 0.12 mm. Coronary diameters were measured by two independent observers and the
AC CE
angiograms were also re-analyzed blindly at a later time to ascertain the inter and intraobserver variability. Intraobserver variation (standard error of the estimate -SEE-) was 0.11 mm and interobserver variation was 0.10 mm. To assess the effect of the intracoronary interventions, appropriate arterial segments were selected on end-diastolic frames from proximal and distal portions of the left coronary artery, as defined in the American Heart Association Committee Report.40
The percent
vasoconstriction by NPY and ergonovine used to quantify changes of coronary artery diameter were calculated as follows: %Constriction = {(Control diameter - Diameter after constrictor agent) / Control diameter}x 100
ACCEPTED MANUSCRIPT 10 Data analysis Coronary diameter data are presented as mean values ± SEM, and hemodynamic variables
T
as mean ± 1 SD. Differences between proportions were analyzed by Yates corrected chi-
IP
square tests. Paired and unpaired Student's t-tests were used, as appropriate, to analyze
SC R
continuous data. Analysis of variance was used to assess the reproducibility of the angiographic measurements. A p value < 0.05 was considered significant.
Results
NU
Baseline coronary artery diameters
MA
We analyzed a total of 132 coronary artery segments: 37 in the control group, 63 in patients with microvascular angina (27 for assessment of NPY and 36 for assessment of
ED
ergonovine), and 32 in patients with coronary artery disease. Baseline diameters of proximal and distal coronary segments in control subjects, microvascular angina patients and coronary
PT
artery disease patients are presented in Table 1. Mean diameter of proximal segments was: 3.18 ±0.18 mm, 3.14 ±0.18 mm and 2.91±0.19 mm, for control individuals, microvascular
AC CE
angina and coronary artery disease patients, respectively (p=NS, >0.05). Mean distal diameters were: 1.35 ±0.06 mm, 1.36 ±0.08 mm and 1.30 ±0.08 mm, respectively (p=NS, >0.05).
Effects of NPY on coronary artery diameter There were no significant changes in heart rate or systemic blood pressure associated with the administration of saline, NPY or ergonovine. The systemic hemodynamic response to NPY was similar in the three patient groups. The changes observed in the coronary segments we selected for analysis after the administration of NPY are detailed in Table 1. The intracoronary administration of 0.9% saline was not associated with significant changes in epicardial lumen. After NPY administration, proximal coronary segments reduced their calibre by 8 ±2%, 5 ±2% and 6 ±2% in the control group, microvascular angina, and coronary
ACCEPTED MANUSCRIPT 11 artery disease patients, respectively (p=NS between groups). Accordingly, constriction of distal epicardial coronary artery segments in response to NPY was not significantly different
T
between the three patient subgroups. Coronary diameters were reduced by 14 ±2%, 11 ±2%
IP
and 10 ±2%, respectively. In patients with microvascular angina, NPY caused a significantly
SC R
larger coronary diameter reduction in distal segments compared to proximal segments. Although constriction of distal segments in control and coronary artery disease patients was larger than that of proximal segments, the difference was not statistically significant.
NU
Although beyond the scope of the study, in patients with coronary artery disease no
segments and coronary stenoses.
MA
significant differences were observed between the response of angiographically normal
Clinical, electrocardiographic and angiographic findings during NPY administration
ED
Four microvascular angina patients developed typical anginal pain during the
PT
administration of NPY, which was associated with ischemic ST segment depression and a marked lengthening of the contrast medium run off. The flow of contrast in the visualized
AC CE
vessels of these four microvascular angina patients was strikingly slow, resulting in an extraordinarily long delay in the clearance of the contrast medium from the coronary tree. In three of the four patients, the contrast remained “trapped” within the lumen of the coronary artery and was squeezed backwards during several systoles. Epicardial coronary artery spasm was not observed. Chest pain, ST segment changes and run-off velocity returned to baseline after the administration of 2 mg of isosorbide dinitrate (after 2±1, 3.5±1 and 8±3 minutes, respectively). Ostensible marked prolongation of the run-off time after NPY administration, but not during baseline, was a frequent finding in microvascular angina patients (5 out of 7 patients). In these patients, the slow run-off was not altered by repositioning the tip of the catheter and was usually long lasting, even after the administration of nitrate.
ACCEPTED MANUSCRIPT 12 None of the control individuals or coronary artery disease patients developed chest pain or electrocardiographic signs of ischemia in response to NPY. Contrast run-off remained
T
unchanged in control patients but was prolonged in one coronary artery disease patient.
IP
Response to ergonovine
SC R
In the nine microvascular angina patients that underwent ergonovine testing, average diameter change of proximal and distal segments in response to ergonovine was similar to that observed in the other microvascular angina group in response to the administration of
NU
NPY. Proximal segments constricted by 7 ±1% and distal segments by 12.5 ±3%. Only one
MA
of the nine patients in the ergonovine group developed chest pain and mild ST segment depression which were associated with lengthening of the contrast run off. The intracoronary administration of nitrate resulted in prompt relief of symptoms and contrast run off velocity
ED
returned to baseline levels within 3 minutes. None of the remaining eight patients had
AC CE
Discussion
PT
symptoms or angiographic changes associated with ergonovine administration.
This study shows that the administration of NPY, a peptide endogenous to human coronary arteries, causes mild epicardial coronary artery constriction which is not significantly different in control, microvascular angina and coronary artery disease patients. Constriction of epicardial coronary arteries of microvascular angina patients by NPY was not significantly different than that observed with ergonovine. The response of proximal coronary segments to NPY in patients with microvascular angina was similar to that documented in control individuals and coronary artery disease patients. Similarly, constriction of distal coronary segments was not significantly different in the three patient groups.
However,
the
distal
segments of the epicardial coronary arteries of our microvascular angina patients showed an increased constrictor response to NPY compared with the proximal segments. This finding is
ACCEPTED MANUSCRIPT 13 in agreement with observations by Bortone et al.41 who reported exercise-induced distal coronary vasoconstriction in patients with ischemia-like symptoms, normal coronary
T
arteriograms and a reduced dilatory capacity of the resistance coronary vessels. The authors
IP
postulated that the abnormal response of the distal coronary segments in these patients was
SC R
due to an abnormal neurohumoral tone. Coronary adrenergic hyperreactivity and inappropriate constriction of distal coronary artery segments in response to the cold pressor test have been recently reported by Montorsi et al.12 in patients with microvascular angina
NU
who had resting electrocardiographic abnormalities. Whether the abnormal adrenergic
MA
response found in microvascular angina is associated with an increased release of NPY by adrenergic neurons, or an exaggerated response of the vascular smooth muscle to NPY is not yet known, but it has been shown that NPY has the potential to modulate sympathetic drive42 . To know whether the effects of exogenously administered NPY observed in the present
ED
43
PT
study might occur physiologically it is important to determine if the concentration of NPY used in this study could be achieved under physiological circumstances. The dose of NPY
AC CE
used in the present study correspond to blood concentrations of approximately X-X. Derchi et al.44 reported plasma levels of 296 pg x ml-1 under physiological circumstances, and of 652 pg x ml-1 in patients with heart failure. Thus the concentration used in our study can be achieved under different circumstances in patients. The abnormal distal coronary artery response of patients with microvascular angina is also in agreement with our previous observations using intracoronary ergonovine36, and with recent preliminary observations by McFadden et al.45 using intracoronary serotonin, which suggest that a dynamic alteration of the distal epicardial vessels may contribute to the impaired coronary flow reserve present in microvascular angina. An important finding in our study is that angina and ischemic ST segment shifts developed in patients with microvascular angina, despite the mild epicardial coronary constriction
ACCEPTED MANUSCRIPT 14 elicited by NPY, whereas neither control subjects nor patients with coronary artery disease had chest pain or ST segment changes during the infusion of NPY despite similar changes in
T
epicardial coronary diameter. A possible explanation for this finding may be found in our
IP
observation that the flow of contrast in some of the microvascular angina patients
SC R
(particularly those who developed chest pain and ischemic ST segment depression) was strikingly slow during NPY infusion, compared with that of control and coronary artery disease patients. The slow run-off may have been due to the arteriolar vasoconstrictor effect
NU
of NPY, as monitoring of heart rate and central aortic pressure did not reveal bradycardia or a
MA
significant drop in blood pressure which could have been associated to a decrease in the velocity of contrast medium flow. Bradycardia did not develop in any patient in the present series, but bradycardia requiring the insertion of a temporary pacing wire had been observed
ED
by our group in previous studies.20
PT
Abnormal epicardial coronary constriction did not account for the markedly slow run-off found in our microvascular angina patients, as NPY-induced coronary constriction of
AC CE
epicardial segments was mild, and similar in the three patient subgroups. Angina pectoris and slow flow velocity of contrast in the coronary arteries of a group of patients with angina and normal coronary arteriograms had been described twenty years ago by Tambe et al., 46 who postulated that an increased baseline tone of the coronary resistance vessels was present in patients with the syndrome of angina pectoris and normal coronary arteriograms who had a slow run off of the contrast medium during baseline arteriography. Slow run-off of the contrast dye was observed in patients with scleroderma who had structural abnormalities of the coronary microcirculation47. Because epicardial coronary artery spasm did not develop in our microvascular angina patients during NPY administration, a likely explanation for the presence of angina, ST segment changes and a markedly lengthened run-off is that NPY caused coronary vasoconstriction at the arteriolar level which resulted in transient myocardial
ACCEPTED MANUSCRIPT 15 ischemia. This finding is consistent with results of experimental studies, which showed that NPY exerts a predominantly arteriolar constrictor effect19
48 49 50
. Our data confirm and
T
expand previous findings20 that exogenously administered NPY can provoke myocardial
IP
ischemia in patients with angina pectoris. However, in our study, only patients with
SC R
microvascular angina developed anginal pain, ST segment depression and lengthening of the run off in response to NPY, whereas none of the control subjects or patients with coronary artery disease experienced symptoms during the administration of NPY. The reason why the
NU
coronary microcirculation of microvascular angina patients but not that of control individuals
MA
or patients with coronary artery disease exhibit an enhanced constrictor response to NPY is not known. Our microvascular angina patients had "microvascular angina"1, characterized by
ED
an abnormal dilatory capacity of the coronary microcirculation. A patchy pre-arteriolar abnormal constriction has been suggested to play a pathogenetic role in microvascular
PT
angina51, and it might be speculated that particular segments of the coronary microcirculation of these patients have an abnormal sensitivity to NPY. Our findings in microvascular angina
AC CE
suggest that NPY could play a pathogenetic role in disorders associated with intramyocardial or systemic resistance vessel dysfunction (as in microvascular angina). Indeed, NPY is endogenous to the coronary arteries and widely distributed within the myocardium.18 It is conceivable that a baseline alteration of small vessel coronary vasomotor tone may be present in patients with microvascular angina, which can be exarcebated by the administration of a powerful arteriolar constrictor such as NPY. Whether the response to NPY found in our microvascular angina patients represent a specific or a non-specific one is not known. Ergonovine provoked angina and lengthening of the contrast run off in only one of the nine microvascular angina patients in our study, but Cannon et al13 observed that, on average, ergonovine exacerbates the microvascular dysfunction of patients with microvascular angina.
ACCEPTED MANUSCRIPT 16 One limitation of the present study is represented by the low number of patients. However, the results are very consistent within groups and, the presence of prolonged microvascular
IP
T
constriction suggested to proceed with further recruitment of patients.
SC R
Conclusion
The results of our study indicate that, in patients with microvascular angina, the exogenous administration of NPY may trigger myocardial ischemia, which may be associated with the
NU
powerful microcirculatory constrictor effect of NPY, as the response of the large epicardial
AC CE
PT
ED
MA
coronary segments remains within the physiological range.
ACCEPTED MANUSCRIPT 17
References Cannon RO, Epstein SE. "Microvascular angina" as a cause of chest pain with angiographically normal coronary arteries (editorial). Am J Cardiol 1988; 61: 1338-43.
IP
T
1
Kemp HG. Left ventricular function in patients with the anginal syndrome and normal coronary arteriograms. Am J Cardiol 1973; 32: 375-6. 3
SC R
2
Opherk D, Zebe H, Weihe E, Mall G, Durr C, Gravert B, Mehmel HC, Schwarz F, Kübler
Henein MY, Rosano GM, Underwood R, Poole-Wilson PA, Gibson DG. Relations between
MA
4
NU
W. Reduced coronary dilatory capacity and ultrastructural changes of the myocardium in patients with angina pectoris but normal coronary arteriograms. Circulation 1981; 63: 817-25.
resting ventricular long axis function, the electrocardiogram, and myocardial perfusion imaging in syndrome X. Br Heart J. 1994 Jun;71(6):541-7. Cannon RO, Watson RM, Rosing DR, Epstein SE. Angina caused by reduced vasodilator reserve of the small coronary arteries. J Am Coll Cardiol 1983; 1: 359-73
PT
ED
5
AC CE
Cannon RO, Rosing DR, Maron BJ, Leon MB, Bonow RO, Watson RM, Epstein SE. Myocardial ischemia in patients with hypertrophic cardiomyopathy: contribution of inadequate vasodilator reserve and elevated left ventricular filling pressures. Circulation 1985; 71: 234-243. 6
Rosano GM, Peters NS, Kaski JC, Mavrogeni SI, Collins P, Underwood RS, Poole-Wilson PA. Abnormal uptake and washout of thallium-201 in patients with syndrome X and normalappearing scans. Am J Cardiol. 1995 Feb 15;75(5):400-2. 7
Wangler RD, Peters KG, Marcus ML, Tomanek RJ. Effects of duration and severity of arterial hypertension and cardiac hypertrophy on coronary vasodilator reserve. Circ Res 1982; 51: 10-18. 8
Opherk D, Mall G, Zebe H, Schwarz F, Weihe E, Manthey J, Kubler W. Reduction of coronary reserve: a mechanism for angina pectoris in patients with arterial hypertension and normal coronary arteries. Circulation 1984; 69: 1-7. 9
ACCEPTED MANUSCRIPT 18
Brush JE, Cannon RO, Schenke WH, Bonow RO, Leon MB, Maron BJ, Epstein SE. Angina due to coronary microvascular disease in hypertensive patients without left ventricular hypertrophy. N Engl J Med 1988; 319: 1302-1307.
T
10
Panza JA, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 1990; 323: 22-27. 12
SC R
IP
11
Montorsi P, Fabbiocchi F, Loaldi A, et al. Coronary adrenergic hyperreactivity in patients
with syndrome X and abnormal electrocardiogram at rest. Am J Cardiol 1991; 68: 1698-1703. Cannon RO, Schenke WH, Leon M, Rosing DR, Urqhart J, Epstein SE. Limited coronary flow reserve after dipyridamole in patients with ergonovine -induced coronary vasoconstriction. Circulation 1987; 75: 163-74.
MA
NU
13
14
PT
ED
Sax FL, Cannon RO, Hanson C, Epstein SE. Impaired forearm vasodilator reserve in patients with microvascular angina. Evidence of a generalized disorder of vascular function? N Engl J Med 1987; 317: 1366-70.
AC CE
Kaski JC, Rosano G, Gavrielides S, Chen L. Effects of angiotensin-converting enzyme inhibition on exercise-induced angina and ST segment depression in patients with microvascular angina. J Am Coll Cardiol. 1994 Mar 1;23(3):652-7. 15
Rosano GM, Ponikowski P, Adamopoulos S, Collins P, Poole-Wilson PA, Coats AJ, Kaski JC. Abnormal autonomic control of the cardiovascular system in syndrome X. Am J Cardiol. 1994 Jun 15;73(16):1174-9. 16
Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y: A novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 1982; 296: 659-60. 17
Wharton J, Gulbenkian S, Polak JM. Neuropeptide tyrosine (NPY) in human cardiovascular innervation, in Mutt V, Hokfelt T, Fuxe K, Lundberg JM (eds): Neuropeptide Y. New York, Raven Press Publishers, 1988, pp 83-91. 18
19
Macho P, Perez R, Huidobro-Toro JP, Domenech RJ. Neuropeptide Y (NPY): a coronary
vasoconstrictor and potentiator of catecholamine-induced coronary constriction. Eur J Pharmacol 1989; 167: 67-74.
ACCEPTED MANUSCRIPT 19
Clarke J, Davies GJ, Kerwin R, Hackett D, Larkin S, Dawbarn D, Lee Y, Bloom SR, Yacoub M, Maseri A. Coronary artery infusion of neuropeptide Y in patients with angina pectoris. Lancet 1987; i: 1057-59. Lehmann J. Neuropeptide Y-An overview. Drug Develop Res 1990; 19:329-51.
IP
21
T
20
Waeber B, Aubert JF, Corder R et al. Cardiovascular effects of neuropeptide Y. Am J Hypert 1988; 1: 193-9.
SC R
22
Allen YS, Adrian TE, Allen JM, Tatemoto K, Crow TJ. Neuropeptide Y distribution in the rat brain. Science 1983; 221: 877-79.
NU
23
Ekblad E, Edvinsson L, Wahlestedt C, Sundrez F. Neuropeptide Y co-exists with noradrenaline in perivascular nerve fibres. Regul Pept 1988; 8: 225-35.
MA
24
Hellstrom PM, Olerup O, Tatemoto K. Neuropeptide Y may mediate effects of sympathetic nerve stimulation on colonic motility and blood flow in the cat. Acta Physiol Scand 1985;
ED
25
PT
124: 613-624.
AC CE
Schömig A, Haass M, Richardt G. Catecholamine release and arrhythmias in acute myocardial ischemia. Eur Heart J 1991; 12 (suppl F): 38-47. 26
Woo ND, Sahai A, Anderson WA, Ganguly PK. Modulation of sympathetic activity by brain neuropeptide Y in cardiac hypertrophy. Am Heart J 1991; 122: 1028-1034. 27
28
Warner MR, Levy MN. Neuropeptide Y as a putative modulator of vagal effects on heart
rate. Circ Res 1989; 64: 882-89. Haass M, Cheng B, Richardt G, Land RE, Schömig A. Characterization and presynaptic modulation of exocytotic co-release of noradrenaline and neuropeptide Y from the guinea pig heart. Naunyn-Schmiedeberg’s Arch Pharmachol 1989; 339: 71-8. 29
Sahn DJ, De Maria A, Kisslo J, Weyman A. Recommendations regarding quantitation in M mode echocardiography: results of a survey of echocardiographic measurements. Circulation 1978; 58: 1072-83. 30
Troy BL, Pombo J, Rackley CE. Measurement of left ventricular thickness and mass by echocardiography. Circulation 1972; 45: 602-11. 31
ACCEPTED MANUSCRIPT 20
Levi D, Savage DD, Garrison RJ, Anderson KM, Kannel WB, Castelli WP. Echocardiographic criteria for left ventricular hypertrophy: The Framingham Heart Study. Am J Cardiol 1987; 59: 956-60.
IP
T
32
Araujo LI, Lammertsma AA, Rhodes CG, Mc Falls EO, Iida H, Rechavia E, Galassi AR, De Silva R, Jones T, Maseri A. Non invasive quantification of regional myocardial blood flow in normal volunteers and patients with coronary artery disease using oxygen-15 labelled
SC R
33
water and positron emission tomography. Circulation 1991; 83: 875-85. 34
MA
NU
Ponikowski P, Rosano GM, Amadi AA, Collins P, Coats AJ, Poole-Wilson PA, Kaski JC. Transient autonomic dysfunction precedes ST-segment depression in patients with syndrome X. Am J Cardiol. 1996 May 1;77(11):942-7. 35
ED
Geltman EM, Henes G, Senneff MJ, Sobel BE, Bergmann SR. Increased myocardial perfusion at rest and diminished perfusion reserve in patients with angina and angiographically normal coronary arteries. J Am Coll Cardiol 1990; 16: 586-95. 36
AC CE
PT
Hackett D, Larkin S, Chierchia S, Davies G, Kaski JC, Maseri A. Induction of coronary artery spasm by a direct local action of ergonovine. Circulation 1987; 75: 577- 582. McFadden EP, Clarke JG, Davies GJ, Kaski JC, Haider AW, Maseri A. Effect of intracoronary serotonin on coronary vessel in patients with stable angina and patients with variant angina. N Engl J Med 1991; 324: 648-654 37
38
Stec K, Haberka M, Mizia M, Chmiel A, Wierzbicka-Chmiel J, Skowerski M, Gasior Z.
Coronary artery calcium score assessed by a 64 multislice computed tomography and early indexes of functional and structural vascular remodeling in cardiac syndrome X patients. J Nucl Cardiol. 2008 Sep-Oct;15(5):655-62. Reiber JHC, Serruys PW, Kooijman CJ, Wijns W, Slager CJ, Gerbrands JJ, Schuurbiers JC, den-Boer A, Hugenholtz PG. Assessment of short-, medium-, and long-term variations in arterial dimensions, from computer-assisted quantitation of coronary cineangiograms. Circulation 1985; 71 (2): 280-288 39
40
Kidawa M, Krzeminska-Pakula M, Peruga JZ, Kasprzak JD. Arterial dysfunction in syndrome X: results of arterial reactivity and pulse wave propagation tests. Heart. 2003 Apr;89(4):422-6.
ACCEPTED MANUSCRIPT 21
Bortone AS, Hess OM, Eberli FR , Nonogi H, Marolf AP, Grimm J, Krayembeuhl HP. Abnormal coronary vasomotion during exercise in patients with normal coronary arteries and reduced coronary flow reserve. Circulation 1989; 79 (3): 516-52
T
41
Lundberg JM, Terenius L, Hokfelt T. et al. Neuropeptide Y (NPY) like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function. Acta Physiol Scand 1982; 116: 477-80.
SC R
IP
42
Franco-Cereceda A, Lundberg JM, Dahlof G. Neuropeptide Y and sympathetic control of heart contractility and coronary vascular tone. Acta Physiol Scand 1985; 124: 361-69.
NU
43
Derchi G, Dupuis J, de Champlain J, Roleau JL. Paradoxical decrease in circulating neuropeptide Y-like immunoreactivity during mild orthostatic stress in subjects with and
MA
44
without congestive heart failure. Eur Heart J 1993; 14: 34-39. Proudler AJ, Crook D, Godsland IF, Collins P, Rosano GM, Stevenson JC. Serum angiotensin-I-converting enzyme activity in women with cardiological syndrome X: relation
ED
45
PT
to blood pressure and lipid and carbohydrate metabolic risk markers for coronary heart disease. J Clin Endocrinol Metab. 1995 Feb;80(2):696-9.
AC CE
Tambe A.A, Demany M.A, Zimmerman H.A, Mascarenhas E. Angina pectoris and slow flow velocity of dye in coronary arteries-A new angiographic finding. Am Heart J 1972; 84: 66-71. 46
Gupta MP, Zoneraich S, Zeitlin W, Zoneraich O, D’Angelo W. Scleroderma heart disease with slow flow velocity in coronary arteries. Chest 1975; 67: 116-119. 47
Franco-Cereceda A. Endothelin -and neuropeptide Y- induced vasoconstriction of human epicardial coronary arteries in vitro. Br J Pharmacol 1989; 97: 968-72. 48
Emson PC, Dequidt ME. NPY: a new member of the pancreatic polypeptide family. Trends Neurosci 1984; 7: 31-35. 49
Franco-Cereceda A, Lundberg JM. Potent effects of neuropeptide Y and calcitonin generelated peptide on human coronary vascular tone in vitro. Acta Physiol Scand 1987; 131:15950
160. 51 Rosano GM, Lindsay DC, Poole-Wilson PA. Syndrome X: an hypothesis for cardiac pain without ischaemia. Cardiologia. 1991 Nov;36(11):885-95.