Am J Physiol Heart Circ Physiol
281: H2097–H2104, 2001.
Hyperglycemia reduces coronary collateral blood flow
through a nitric oxide-mediated mechanism
JUDY R. KERSTEN,1 WOLFGANG G. TOLLER,5 JOHN P. TESSMER,1
PAUL S. PAGEL,1,2,4 AND DAVID C. WARLTIER1,2,3,4
Departments of 1Anesthesiology, Pharmacology, and 2Toxicology, and 3Division of Cardiovascular
Diseases, Department of Medicine, Medical College of Wisconsin; 4Clement J. Zablocki Veterans
Affairs Medical Center, Milwaukee, Wisconsin 53226; and 5Department of Anesthesiology,
University of Graz, A-8036 Graz, Austria
Received 27 April 2001; accepted in final form 24 July 2001
remain to be defined. Hyperglycemia impairs endothelium-dependent vasodilation in vitro (30) and coronary
microvascular responses to myocardial ischemia in
vivo (18). Human coronary vasculature may also demonstrate attenuated vasodilation during hyperglycemia (26). Coronary collateral perfusion is an important
determinant of the extent of myocardial ischemic injury, but whether hyperglycemia affects collateral
blood flow is unknown. We tested the hypothesis that
hyperglycemia alters retrograde coronary collateral
blood flow by a nitric oxide-dependent mechanism in a
canine Ameriod constrictor model of enhanced coronary collateral development.
METHODS
HYPERGLYCEMIA may be an important contributor to and
independent predictor of increases in short- and longterm cardiovascular mortality. A strong correlation
between blood glucose concentrations at the time of
hospital admission and long-term mortality was recently observed in a study of diabetic patients with
acute myocardial infarction (28). A metaregression
analysis of data published in 20 studies of more than
95,000 patients also demonstrated a relationship between fasting blood glucose concentration and the relative risk of sustaining a cardiovascular event (6). The
mechanisms responsible for these increases in risk
All experimental procedures and protocols used in this
investigation were reviewed and approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. All procedures conformed to the “Guiding
Principles in the Care and Use of Animals” of the American
Physiological Society and were performed in accordance with
the Guide for the Care and Use of Laboratory Animals by the
National Institutes of Health (Revised, 1996).
Implantation of Ameriod constrictors. Conditioned mongrel
dogs were fasted overnight. Anesthesia was induced with intravenous propofol (5 mg/kg). After tracheal intubation, anesthesia
was maintained with isoflurane (1.5 to 2%) in 100% oxygen
using positive-pressure ventilation. A left thoracotomy was performed using sterile technique, and a segment (1.0–1.5 cm) of
the left anterior descending (LAD) coronary artery immediately
distal to the first diagonal branch was isolated. An Ameriod
constrictor (Research Instruments and Manufacturing; Corvallis, OR) was placed around the vessel. The diameter of the
internal lumen of the constrictor was 2.0–3.0 mm, and its size
was chosen for a snug fit around the vessel without producing
visible stenosis. The chest was closed in layers, and the pneumothorax was evacuated with a chest tube. Each dog received
antibiotics [cefazolin (40 mg/kg) and gentamicin (4.5 mg/kg)]
and analgesics [epidural morphine (0.2 mg/kg) and fentanyl (2
␮g/kg)].
General preparation. Twelve weeks after implantation of
Ameriod constrictors, dogs were anesthetized with pentobar-
Address for reprint requests and other correspondence: J. R. Kersten, Medical College of Wisconsin, Dept. of Anesthesiology, M4280,
8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
collateral circulation; myocardial ischemia; reperfusion injury
http://www.ajpheart.org
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
H2097
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Kersten, Judy R., Wolfgang G. Toller, John P. Tessmer, Paul S. Pagel, and David C. Warltier. Hyperglycemia reduces coronary collateral blood flow through a nitric
oxide-mediated mechanism. Am J Physiol Heart Circ Physiol
281: H2097–H2104, 2001.—We tested the hypothesis that
hyperglycemia alters retrograde coronary collateral blood
flow by a nitric oxide-mediated mechanism in a canine Ameriod constrictor model of enhanced collateral development.
Administration of 15% dextrose to increase blood glucose
concentration to 400 or 600 mg/dl decreased retrograde blood
flow through the left anterior descending coronary artery to
78 ⫾ 9 and 82 ⫾ 8% of baseline values, respectively. In
contrast, saline or L-arginine (400 mg 䡠 kg⫺1 䡠 h⫺1) had no
effect on retrograde flow. Coronary hypoperfusion and 1 h of
reperfusion decreased retrograde blood flow similarly in saline- or L-arginine-treated dogs (76 ⫾ 11 and 89 ⫾ 4% of
baseline, respectively), but these decreases were more pronounced in hyperglycemic dogs (47 ⫾ 10%). L-Arginine prevented decreases in retrograde coronary collateral blood flow
during hyperglycemia (100 ⫾ 5 and 95 ⫾ 6% of baseline at
blood glucose concentrations of 400 and 600 mg/dl, respectively) and after coronary hypoperfusion and reperfusion
(84 ⫾ 14%). The results suggest that hyperglycemia decreases retrograde coronary collateral blood flow by adversely affecting nitric oxide availability.
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HYPERGLYCEMIA REDUCES CORONARY COLLATERAL BLOOD FLOW
tubing at equal pressure to delineate the normal and collateral-dependent regions, respectively. Transmural tissue
samples were selected from the collateral-dependent (distal
to the Ameriod constrictor) and normal (LCCA perfused)
regions and subdivided into subepicardial, midmyocardial,
and subendocardial layers of approximately equal thickness
and weight. Tissue blood flow (in ml 䡠 min⫺1 䡠 g⫺1) was calculated as Q̇r 䡠 Cm 䡠 Cr⫺1 where Q̇r is the rate of withdrawal of the
reference blood flow sample (ml/min), Cm is the activity
[counts/min (cpm)/g] of the myocardial tissue sample, and Cr
is the activity (cpm) of the reference blood flow sample.
Transmural blood flow was considered as the average of
subepicardial, midmyocardial, and subendocardial blood
flows. Tissue blood flow to the collateral-dependent myocardium is a measure of microvascular not total collateral blood
flow. Coronary perfusion pressure was determined as the
difference between end-diastolic arterial pressure and LV
end-diastolic pressure. Retrograde collateral conductance
was calculated as the ratio of retrograde blood flow to coronary perfusion pressure.
Experimental protocol. Hemodynamics were recorded, radioactive microspheres were injected, and LAD retrograde
blood flow was measured 30 min after completion of the acute
surgical preparation. Retrograde blood flow was assessed by
collecting blood from the LAD cannula into a graduated
cylinder for 90 s while the cannula tip was held at the level of
the left atrium. Measurements were performed in triplicate
and the results averaged. Microsphere injections were performed with the retrograde flow cannula open so that retrograde and LAD transmural (microvascular collateral) blood
flow were measured simultaneously (15). Microvascular collateral flow was measured during baseline conditions, after
60 min of interventions, during prolonged diversion of retrograde flow (hypoperfusion), and 1 h after antegrade LAD flow
was reestablished (reperfusion). Thus all determinations of
microvascular collateral flow were made with the retrograde
flow cannula open for either brief or prolonged periods of
time.
The effects of acute hyperglycemia on coronary collateral
blood flow before or after a 60-min period of coronary hypoperfusion were studied in four separate experimental groups
(Fig. 1). Retrograde coronary collateral blood flow and pressure were measured under steady-state hemodynamic conditions after instrumentation during intravenous infusion of
0.9% saline (3 times at 30-min intervals), during 60 min of
Fig. 1. Experimental protocol.
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bital sodium (15 mg/kg) and sodium barbital (200 mg/kg) and
ventilated using positive pressure with oxygen-enriched air
(fractional inspired oxygen concentration ⫽ 0.40) after tracheal intubation. A dual, micromanometer-tipped catheter
was inserted into the aorta and left ventricle (LV) through
the left carotid artery to measure arterial and LV pressures,
respectively. Heparin-filled catheters were inserted into the
right femoral vein and artery for administration of intravenous fluids and withdrawal of reference arterial blood samples used in the microsphere technique, respectively. The
arterial catheter was advanced to the level of the descending
thoracic aorta. A thoracotomy was performed in the left fifth
intercostal space, the lung gently retracted, and the heart
suspended in a pericardial cradle. A heparin-filled catheter
was inserted in the left atrium for injection of radioactive
microspheres. Descending thoracic aortic and inferior vena
cava snares were placed to facilitate control of arterial pressure. Segments of the left circumflex coronary artery (LCCA;
proximal to the first marginal branch) and LAD (distal to the
Ameriod constrictor) were dissected free from surrounding
myocardium. A transit time flow probe was positioned
around the LCCA to measure coronary blood flow (ml/min).
Each dog was anticoagulated with heparin (500 U/kg). Largebore polyethylene cannulas attached to Silastic tubing were
inserted in the right carotid artery and left jugular vein. The
proximal LAD was ligated, a large-bore metal cannula positioned and secured in the distal arterial segment, and a
carotid artery-to-LAD shunt established. Perfusion in the
LAD was restored within 3 min after ligation. A segment of
tubing perpendicular to the metal cannula was used to measure retrograde coronary flow and pressure during interruption of antegrade coronary flow through the carotid artery
shunt to the LAD. Patency of the distal LAD perfusion
cannula between measurements of retrograde blood flow was
assessed. Reduced perfusion of collateral-dependent myocardium was achieved by diverting retrograde collateral blood
flow to the left jugular vein. Hemodynamics were monitored
continuously on a polygraph and digitized using a computer
interfaced with an analog-to-digital converter.
Measurement of myocardial perfusion. Carbonized plastic
microspheres labeled with 141Ce, 103Ru, 51Cr, or 95Nb were
used to measure myocardial perfusion as previously described (20). At the conclusion of each experiment, 10 ml of
Patent blue dye were injected into the LCCA simultaneously
with saline infused intracoronary into the LAD perfusion
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HYPERGLYCEMIA REDUCES CORONARY COLLATERAL BLOOD FLOW
Table 1. Hemodynamics during control (saline) experiments
Time, min
HR, beats/min
MAP, mmHg
LVSP, mmHg
LVEDP, mmHg
RF, ml/min
RP, mmHg
RC, ml 䡠 min⫺1 䡠 mmHg⫺1 ⫻ 102
CBF, ml/min
RPP, beats/min 䡠 mmHg ⫻ 10⫺3
Baseline
30
60
90
145 ⫾ 8
102 ⫾ 4
107 ⫾ 4
8⫾1
64 ⫾ 10
71 ⫾ 5
78 ⫾ 14
41 ⫾ 10
15.9 ⫾ 0.9
142 ⫾ 8
102 ⫾ 4
108 ⫾ 4
10 ⫾ 2
63 ⫾ 9
70 ⫾ 4
81 ⫾ 13
38 ⫾ 9
15.5 ⫾ 0.7
141 ⫾ 7
103 ⫾ 4
108 ⫾ 4
9⫾1
64 ⫾ 10
71 ⫾ 5
80 ⫾ 14
41 ⫾ 9
15.6 ⫾ 0.9
138 ⫾ 7
103 ⫾ 4
109 ⫾ 5
9⫾1
65 ⫾ 10
70 ⫾ 4
78 ⫾ 13
39 ⫾ 8
15.2 ⫾ 0.08
Hypoperfusion
132 ⫾ 7
102 ⫾ 5
107 ⫾ 6
9⫾1
70 ⫾ 13
86 ⫾ 17
45 ⫾ 9
14.2 ⫾ 0.08
Reperfusion
126 ⫾ 6*
103 ⫾ 5
110 ⫾ 5
10 ⫾ 2
47 ⫾ 10*
64 ⫾ 4
63 ⫾ 14
37 ⫾ 7
14.0 ⫾ 0.9
Data are means ⫾ SE; n ⫽ 10 dogs. HR, heart rate; MAP, mean arterial pressure; LVSP and LVEDP, left ventricular systolic and
end-diastolic pressures, respectively; RF, retrograde blood flow; RP, retrograde pressure; RC, retrograde conductance; CBF, left circumflex
coronary artery blood flow; RPP, rate-pressure product. * Significantly (P ⬍ 0.05) different from baseline.
RESULTS
Forty-eight dogs were instrumented with Ameriod
constrictors to obtain 43 successful experiments. The
LAD cannulation was unsuccessful in five dogs.
Hemodynamics during control experiments. Saline
did not alter systemic hemodynamics, LCCA blood
flow, or retrograde collateral blood flow, conductance,
and pressure (Table 1). Diversion of retrograde flow to
produce hypoperfusion of the collateral-dependent region did not affect hemodynamics. A significant (P ⬍
0.05) decrease in retrograde collateral blood flow (76 ⫾
11% of baseline) was observed after 1 h of reperfusion.
Effects of hyperglycemia. Hemodynamics were unchanged during administration of 15% dextrose (Table
2). Dextrose decreased retrograde collateral blood flow
(Fig. 2), retrograde conductance, and retrograde pressure (Table 2). Retrograde flow (Fig. 3) and conductance were lower during coronary hypoperfusion (83 ⫾
9 and 77 ⫾ 10% of baseline, respectively) and reperfusion (47 ⫾ 10 and 44 ⫾ 10% of baseline, respectively) in
hyperglycemic compared with saline-treated dogs
(107 ⫾ 6 and 108 ⫾ 8% of baseline during hypoperfusion and 76 ⫾ 11 and 81 ⫾ 13% of baseline during
reperfusion, respectively).
Effects of L-arginine. L-Arginine did not alter systemic hemodynamics, LCCA blood flow, or retrograde
flow (Table 3). Increases in retrograde blood flow (Fig.
3) and LCCA blood flow were observed during hypoperfusion of the collateral-dependent region in dogs receiving L-arginine. After reperfusion, retrograde flow decreased to a similar extent in the presence of L-arginine
compared with dogs receiving saline.
Effects of L-arginine during hyperglycemia. L-Arginine decreased heart rate but did not alter mean arterial pressure during graded increases in blood glucose
concentration (Table 4). L-Arginine prevented decreases in retrograde collateral blood flow (Fig. 2),
Table 2. Hemodynamics during hyperglycemia
Time, min
HR, beats/min
MAP, mmHg
LVSP, mmHg
LVEDP, mmHg
RF, ml/min
RP, mmHg
RC, ml 䡠 min⫺1 䡠 mmHg⫺1 ⫻ 102
CBF, ml/min
RPP, beats 䡠 min⫺1 䡠 mmHg ⫻ 10⫺3
Blood glucose, mg/dl
Baseline
30
60
90
Hypoperfusion
Reperfusion
151 ⫾ 7
94 ⫾ 5
102 ⫾ 5
7⫾1
59 ⫾ 11
74 ⫾ 6
78 ⫾ 14
36 ⫾ 5
15.7 ⫾ 1.1
74 ⫾ 3
148 ⫾ 9
101 ⫾ 5
108 ⫾ 5
7⫾1
55 ⫾ 11
70 ⫾ 4
64 ⫾ 12
39 ⫾ 6
16.3 ⫾ 1.3
218 ⫾ 15*
144 ⫾ 8
103 ⫾ 5
109 ⫾ 6
8⫾2
47 ⫾ 10*
66 ⫾ 5*
56 ⫾ 13*
34 ⫾ 4
16.0 ⫾ 1.4
424 ⫾ 9*
145 ⫾ 9
101 ⫾ 4
108 ⫾ 5
8⫾1
49 ⫾ 10*
63 ⫾ 4*
60 ⫾ 13
37 ⫾ 5
15.7 ⫾ 1.4
584 ⫾ 14*
143 ⫾ 10
100 ⫾ 5
105 ⫾ 5
9⫾2
53 ⫾ 14
145 ⫾ 11
99 ⫾ 5
105 ⫾ 6
10 ⫾ 2*
31 ⫾ 10*
57 ⫾ 5*
35 ⫾ 11*
42 ⫾ 6
15.3 ⫾ 1.2
571 ⫾ 24*
Data are means ⫾ SE; n ⫽ 11 dogs. * Significantly (P ⬍ 0.05) different from baseline.
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61 ⫾ 15
47 ⫾ 10
15.3 ⫾ 1.3
579 ⫾ 17*
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coronary hypoperfusion, and after 1 h of reperfusion. A
second group of dogs received intravenous 15% dextrose
in water to increase blood glucose concentrations to 200,
400, and 600 mg/dl (30 min at each concentration) before
coronary hypoperfusion and reperfusion. Blood glucose
concentration was maintained at 600 mg/dl during 60 min
of coronary hypoperfusion and reperfusion. In two final
groups of experiments, dogs received intravenous L-arginine
(400 mg 䡠 kg⫺1 䡠 h⫺1) in the absence or presence of acute hyperglycemia with and without coronary hypoperfusion and
reperfusion. Arterial pressure was maintained constant
throughout experimentation by partial aortic or vena cava
constriction. Arterial blood gas tensions were maintained
within a physiological range by adjusting tidal volume and
respiratory rate and by administering sodium bicarbonate as
necessary.
Statistical analysis. Statistical analysis of data within and
between groups was performed with multiple analysis of
variance for repeated measures followed by application of
Student-Newman-Keuls test. Changes within and between
groups were considered statistically significant when P ⬍
0.05. Data are expressed as means ⫾ SE.
H2100
HYPERGLYCEMIA REDUCES CORONARY COLLATERAL BLOOD FLOW
conductance, and pressure that occurred during dextrose-induced increases in blood glucose concentration.
Retrograde collateral blood flow (84 ⫾ 14% of baseline)
and conductance (91 ⫾ 15% of baseline) were significantly greater 1 h after reperfusion in dogs receiving
dextrose in the presence compared with the absence of
L-arginine.
Microvascular collateral and normal zone blood flow.
There were no differences in microvascular collateral
blood flow among groups (Table 5) under control conditions, after 60 min of saline or dextrose administration, or during prolonged diversion of retrograde flow
(hypoperfusion). Microvascular collateral flow increased after reperfusion in dogs receiving dextrose
and L-arginine compared with those treated with
L-arginine alone. Perfusion of normal myocardium
(Table 6) was similar among groups under baseline
conditions, after 60 min of saline or dextrose administration, and after reperfusion. Transmural blood flow
to normal myocardium increased during hypoperfusion
in dogs receiving dextrose and L-arginine compared
with those treated with L-arginine alone.
DISCUSSION
Nitric oxide and coronary collaterals. Chronic imbalances of myocardial oxygen supply and demand produced by a coronary artery stenosis or occlusion stimulate growth of the coronary collateral circulation. This
collateralization increases oxygen delivery to myocardium at risk for ischemic injury and may prevent
infarction. The functional response of the coronary
collateral circulation to various physiological and pharmacological stimuli may also be a critical factor that
influences the extent of ischemic injury. Coronary colAJP-Heart Circ Physiol • VOL
Fig. 3. Retrograde coronary collateral blood flow (expressed as a
percentage of baseline) in dogs receiving saline alone, 15% dextrose
in water to increase blood glucose concentration to 600 mg/dl (Hyp),
saline and L-Arg (400 mg 䡠 kg⫺1 䡠 h⫺1), or L-Arg in presence of hyperglycemia (Hyp ⫹ L-Arg). Retrograde flow was measured during 60
min of coronary hypoperfusion (by diversion of retrograde collateral
blood flow) and after 1 h of reperfusion. *Significantly (P ⬍ 0.05)
different from baseline; †Significantly (P ⬍ 0.05) different from
saline alone; §Significantly (P ⬍ 0.05) different from L-Arg; ¶Significantly (P ⬍ 0.05) different from Hyp ⫹ L-Arg.
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Fig. 2. Retrograde coronary collateral blood flow (expressed as a
percentage of baseline) in dogs receiving saline alone, 15% dextrose
in water to increase blood glucose concentration to 200, 400, and 600
mg/dl (Hyp), saline and L-arginine (400 mg 䡠 kg⫺1 䡠 h⫺1; L-Arg), or
L-Arg in the presence of hyperglycemia (Hyp ⫹ L-Arg). *Significantly
(P ⬍ 0.05) different from baseline; †Significantly (P ⬍ 0.05) different
from saline alone; §Significantly (P ⬍ 0.05) different from L-Arg;
¶Significantly (P ⬍ 0.05) different from Hyp ⫹ L-Arg.
lateral vessels respond to endothelium-dependent and
-independent vasodilators in vitro (10) and in vivo (2).
Nitric oxide has a tonic vasodilating effect on the coronary collateral circulation. Administration of a nitric
oxide synthase (NOS) inhibitor increased coronary collateral resistance in models of enhanced collateral development produced by repetitive coronary artery occlusion or Ameriod constrictor implantation. This
action was partially reversed by the nitric oxide precursor L-arginine (11). Nitric oxide contributes to maintenance of basal coronary blood flow (4, 25) and also
maintains coronary collateral blood flow responsiveness during exercise (36). Moreover, nitric oxide may
be a critically important regulator of coronary blood
flow during myocardial ischemia and reperfusion (5, 8,
9). Inhibition of NOS blunts reactive hyperemia, increases the critical coronary perfusion pressure below
which coronary flow becomes pressure dependent, and
further reduces flow at pressures below the lower limit
of autoregulation (32). These findings indicate that
nitric oxide modulates coronary resistance adjustments required for the regulation of myocardial perfusion during and after ischemia. The availability of NOS
substrate may influence this regulatory process. Endothelial dysfunction early (20 min) after coronary artery
occlusion and reperfusion is reversible with the provision of excess substrate (L-arginine) for NOS (27). However, defects in L-arginine membrane transfer or the
nitric oxide synthetic system may not be surmountable
by excess L-arginine later during reperfusion (27).
Effects of acute hyperglycemia. The present results
confirm and extend previous findings demonstrating a
role of nitric oxide in the modulation of coronary col-
H2101
HYPERGLYCEMIA REDUCES CORONARY COLLATERAL BLOOD FLOW
Table 3. Hemodynamics during L-arginine
Time, min
HR, beats/min
MAP, mmHg
LVSP, mmHg
LVEDP, mmHg
RF, ml/min
RP, mmHg
RC, ml 䡠 min⫺1 䡠 mmHg⫺1 ⫻ 103
CBF, ml/min
RPP, beats/min 䡠 mmHg ⫻ 10⫺3
Baseline
30
60
90
Hypoperfusion
Reperfusion
137 ⫾ 5
97 ⫾ 4
105 ⫾ 3
5⫾1
60 ⫾ 10
68 ⫾ 6
73 ⫾ 11
45 ⫾ 8
14.7 ⫾ 0.9
136 ⫾ 7
98 ⫾ 4
105 ⫾ 4
5⫾1
60 ⫾ 10
70 ⫾ 5
69 ⫾ 10
46 ⫾ 7
14.5 ⫾ 1.1
132 ⫾ 6
97 ⫾ 4
104 ⫾ 4
5⫾1
56 ⫾ 9
69 ⫾ 5
64 ⫾ 9
44 ⫾ 6
14.0 ⫾ 1.0
125 ⫾ 6
99 ⫾ 4
104 ⫾ 4
5⫾1
59 ⫾ 9
65 ⫾ 6
67 ⫾ 9
43 ⫾ 7
13.3 ⫾ 1.0
123 ⫾ 7
100 ⫾ 7
105 ⫾ 7
6⫾1
67 ⫾ 10*
116 ⫾ 6*
98 ⫾ 4
105 ⫾ 4
6⫾1
53 ⫾ 8*
60 ⫾ 5
63 ⫾ 10
45 ⫾ 7
12.5 ⫾ 1.0*
77 ⫾ 13
61 ⫾ 9*
13.3 ⫾ 1.5
Data are means ⫾ SE; n ⫽ 11 dogs. L-Arginine dose was 400 mg 䡠 kg⫺1 䡠 h⫺1. * Significantly (P ⬍ 0.05) different from baseline.
neous in different-sized vessels (31). Retrograde coronary blood flow primarily reflects flow derived from
epicardial collateral vessels with a small contribution
from intramural collaterals. In contrast, microvascular
collateral flow (measured with radioactive microspheres during retrograde flow collection) reflects continuing blood flow through microvascular intramural
collaterals (7). Microvascular collateral flow accounts
for ⬃50% of the total collateral blood flow (15). Total
collateral blood flow available to perfuse dependent
myocardium is the sum of retrograde flow derived from
large interarterial collateral vessels and microvascular
collateral blood flow. Hyperglycemia decreased interarterial (retrograde) collateral conductance but did not
appreciably affect vasomotor tone of microvascular collaterals or resistance vessels in collaterally dependent
myocardium, because ongoing tissue blood flow to this
region remained essentially constant. These results
suggest that hyperglycemia produces greater effects to
alter vasomotor tone of large interarterial compared
with microvascular collaterals and are consistent with
findings that NOS inhibition decreases retrograde coronary collateral blood flow by ⬃30% (23). Decreases in
retrograde collateral blood flow during hyperglycemia
may also result from increases in vasomotor tone of
epicardial coronary arteries, proximal to the origin of
interarterial collateral vessels. The absence of changes
in perfusion of normal myocardium during hyperglyce-
Table 4. Hemodynamics during hyperglycemia and L-arginine
Time, min
HR, beats/min
MAP, mmHg
LVSP, mmHg
LVEDP, mmHg
RF, ml/min
RP, mmHg
RC, ml 䡠 min⫺1 䡠 mmHg⫺1 ⫻ 102
CBF, ml/min
RPP, beats/min 䡠 mmHg ⫻ 10⫺3
Blood glucose, mg/dl
Baseline
30
60
90
Hypoperfusion
Reperfusion
145 ⫾ 6
92 ⫾ 3
99 ⫾ 4
6⫾2
62 ⫾ 9
66 ⫾ 5
80 ⫾ 12
49 ⫾ 5
14.4 ⫾ 0.8
88 ⫾ 9
135 ⫾ 6*
96 ⫾ 3
103 ⫾ 4
8⫾2
60 ⫾ 9
66 ⫾ 6
76 ⫾ 12
53 ⫾ 5
13.8 ⫾ 0.7
194 ⫾ 9*
129 ⫾ 6*
96 ⫾ 4
104 ⫾ 5
8⫾2
60 ⫾ 9
67 ⫾ 5
78 ⫾ 13
57 ⫾ 5
13.3 ⫾ 0.8
398 ⫾ 22*
123 ⫾ 4*
97 ⫾ 3
104 ⫾ 5
9⫾2
58 ⫾ 9
66 ⫾ 5
75 ⫾ 12
56 ⫾ 4
12.8 ⫾ 0.7
578 ⫾ 16*
128 ⫾ 6*
96 ⫾ 3
103 ⫾ 4
9⫾2
68 ⫾ 9
119 ⫾ 5*‡
94 ⫾ 3
103 ⫾ 3
11 ⫾ 3*
48 ⫾ 8
60 ⫾ 4
65 ⫾ 11
66 ⫾ 7*†‡§
12.2 ⫾ 0.7*
588 ⫾ 9*
89 ⫾ 13
79 ⫾ 8*†
13.2 ⫾ 0.9
592 ⫾ 12*
Data are means ⫾ SE; n ⫽ 11 dogs. L-Arginine dose was 400 mg 䡠 kg⫺1 䡠 h⫺1. * Significantly (P ⬍ 0.05) different from baseline;
† Significantly (P ⬍ 0.05) different from corresponding value in control experiments (Table 1); ‡ Significantly (P ⬍ 0.05) different from
corresponding value in hyperglycemic dogs (Table 2); § Significantly (P ⬍ 0.05) different from corresponding value in dogs receiving
L-arginine alone (Table 3).
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lateral vasomotor tone. More importantly, we show
that acute hyperglycemia alters the permissive action
of nitric oxide on regulation of blood flow in the coronary collateral circulation. Retrograde collateral blood
flow was reduced by hyperglycemia before and after
hypoperfusion and reperfusion, and nitric oxide played
an important role in this process. Mild hyperglycemia
(200 mg/dl) alone had no effect on retrograde flow, but
moderate (400 mg/dl) and profound (600 mg/dl) hyperglycemia produced similar reductions (⬃20%) in retrograde flow. Hyperglycemia also reduced retrograde collateral blood flow to ⬍50% of baseline values after 1 h
of reperfusion. Administration of L-arginine completely
abolished these hyperglycemia-induced decreases in
retrograde flow before, during, or after myocardial hypoperfusion but did not ameliorate the less pronounced
reductions in retrograde flow during reperfusion observed in normoglycemic dogs. The results confirm that
nitric oxide is at least partially responsible for the
maintenance of blood flow through coronary collateral
vessels. The results also indicate that hyperglycemia
attenuates these nitric oxide-induced effects on collateral blood flow. Taken together, the data support the
contention that hyperglycemia decreases retrograde
collateral blood flow by interference with nitric oxide.
Coronary collateral blood flow was differentiated on
the basis of collateral size because coronary vascular
responses to pharmacological stimuli are heteroge-
H2102
HYPERGLYCEMIA REDUCES CORONARY COLLATERAL BLOOD FLOW
Table 5. Microvascular collateral blood flow
Baseline
Reperfusion
0.78⫾0.21 0.87⫾0.22 0.85⫾0.18 0.98⫾0.15
0.76⫾0.14 0.72⫾0.15 0.87⫾0.22 0.93⫾0.22
0.57⫾0.13 0.53⫾0.13 0.51⫾0.11 0.53⫾0.14
0.94⫾0.20 0.99⫾0.19 1.00⫾0.22 1.18⫾0.13
0.53⫾0.14 0.62⫾0.20 0.57⫾0.13 0.68⫾0.14
0.68⫾0.30 0.46⫾0.10 0.59⫾0.16 0.61⫾0.15
0.45⫾0.13 0.42⫾0.11 0.45⫾0.14 0.39⫾0.12
0.66⫾0.15 0.71⫾0.14 0.63⫾0.13 0.73⫾0.09
0.45⫾0.07 0.52⫾0.13 0.52⫾0.08 0.53⫾0.08
0.42⫾0.06 0.46⫾0.08 0.56⫾0.12 0.60⫾0.14
0.49⫾0.14 0.48⫾0.12 0.51⫾0.16 0.46⫾0.15
0.53⫾0.11 0.57⫾0.11 0.51⫾0.12 0.89⫾0.23
0.59⫾0.14 0.67⫾0.18 0.64⫾0.13 0.73⫾0.11
0.62⫾0.15 0.55⫾0.10 0.67⫾0.16 0.71⫾0.15
0.50⫾0.13 0.47⫾0.11 0.49⫾0.13 0.46⫾0.13
0.71⫾0.14 0.76⫾0.14 0.71⫾0.15 0.94⫾0.07*
Data are means ⫾ SE (in ml 䡠 min⫺1 䡠 g⫺1). * Significantly (P ⬍ 0.05)
different from the respective value during experiments with L-arginine alone.
mia, however, suggests that epicardial coronary arterial vasomotor activity was unaffected by hyperglycemia in the present investigation.
Hyperglycemia and nitric oxide. Acute hyperglycemia has previously been demonstrated to impair
responses to endothelium-dependent but not -independent vasodilators (3, 30, 37). Moderate hyperglycemia (270 mg/dl) reduced leg blood flow and increased
platelet aggregation and blood viscosity in healthy
volunteers. These detrimental effects were abolished
by intravenous administration of L-arginine (12). Interestingly, administration of a NOS inhibitor produced
very similar vascular abnormalities, indirectly suggesting hyperglycemia may act by decreasing nitric
oxide availability (12). Substrate availability for NOS
may become rate limiting during hyperglycemia because of impaired arginine uptake, increased utilization, or reduced enzyme affinity (16, 34). Hyperglycemia also increases superoxide anion formation (13, 29),
and production of oxygen-derived free radicals inactivates nitric oxide (14). Ischemia and reperfusion are
associated with disrupted architecture of the constitutively expressed NOS enzyme (17), an action that may
result in enhanced production of superoxide anion and
decreased nitric oxide formation (17). The present results suggest that hyperglycemia and L-arginine may
exacerbate and ameliorate this response, respectively.
L-Arginine prevented reduction in retrograde flow after
coronary hypoperfusion and reperfusion in hyperglycemic dogs in the present investigation, findings that
appear to be very similar to those observed during
AJP-Heart Circ Physiol • VOL
Table 6. Transmural myocardial blood flow in
normal region
Baseline
Control
Hyperglycemia
L-Arginine
Hyperglycemia ⫹
L-arginine
60 Min
Hypoperfusion Reperfusion
1.13 ⫾ 0.11 1.32 ⫾ 0.16 1.09 ⫾ 0.10
1.01 ⫾ 0.11 1.03 ⫾ 0.12 1.09 ⫾ 0.16
1.17 ⫾ 0.12 0.95 ⫾ 0.05 0.99 ⫾ 0.08
1.34 ⫾ 0.18
1.46 ⫾ 0.21
1.03 ⫾ 0.07
1.29 ⫾ 0.23 1.45 ⫾ 0.22 1.50 ⫾ 0.16* 1.52 ⫾ 0.14
Data are means ⫾ SE (in ml 䡠 min⫺1 䡠 g⫺1). * Significantly (P ⬍ 0.05)
different from the respective value during experiments with L-arginine alone.
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Subepicardium
Control
Hyperglycemia
L-Arginine
Hyperglycemia⫹
L-arginine
Midmyocardium
Control
Hyperglycemia
L-Arginine
Hyperglycemia⫹
L-arginine
Subendocardium
Control
Hyperglycemia
L-Arginine
Hyperglycemia⫹
L-arginine
Transmural
Control
Hyperglycemia
L-Arginine
Hyperglycemia⫹
L-arginine
60 Min
Prolonged
Diversion of
Retrograde
Flow
ischemia and reperfusion injury in skeletal muscle
(17). L-Arginine treatment decreased superoxide anion
production, increased nitric oxide accumulation, and
prevented vasoconstriction in this previous study (17),
but whether L-arginine produces such beneficial actions in myocardium is unknown.
Limitations. The results of the present investigation
should be interpreted within the constraints of several
potential limitations. Prostaglandins (1) and ATP-sensitive potassium (KATP) channels (24) have been implicated in the tonic vasodilation of coronary collateral
vessels. We did not specifically evaluate the interactions between hyperglycemia and prostaglandins or
KATP channels. However, elevated glucose concentrations have been previously shown to enhance the production of vasoconstrictor prostanoids (35). We recently demonstrated that hyperglycemia abolishes
endogenous activation of KATP channels during ischemic preconditioning (21) and also impairs pharmacological activation of mitochondrial KATP channels by
diazoxide (19). Whether hyperglycemia specifically impairs KATP channel-regulated coronary collateral vascular responsiveness in the presence or absence of
coronary hypoperfusion remains to be determined.
However, our findings indicate that hyperglycemia is
not only deleterious for ischemic myocardium by inhibition of endogenous cardioprotective pathways. Hyperglycemia can also impact ischemic myocardium by
reducing oxygen supply via collateral perfusion.
We have previously shown that acute administration
of dextrose is also associated with hyperinsulinemia
(22). Hyperinsulinemia may contribute to coronary vasospasm and sudden cardiac death in patients with
coronary artery disease (33). Topical hyperglycemia in
the absence of systemic hyperinsulinemia impaired
nitric oxide-mediated vasodilation of rat arterioles (3),
and the severity of hyperglycemia was directly related
to myocardial infarct size independent of plasma insulin concentration in dogs (22). These findings suggest
that hyperglycemia alone, and not concomitant hyperinsulinemia, is probably responsible for the impairment of nitric oxide-regulated coronary collateral blood
flow observed in the present investigation. Nevertheless, a role for hyperinsulinemia alone in this process
cannot be strictly excluded from the analysis. It also
appears unlikely that hyperosmolality was responsible
for decreases in retrograde blood flow observed during
hyperglycemia. Incubation of endothelial cells with 33
HYPERGLYCEMIA REDUCES CORONARY COLLATERAL BLOOD FLOW
The authors thank David Schwabe for technical assistance and
Mary Lorence-Hanke for preparation of the manuscript.
This work was supported in part by an American Heart Association Grant-in-Aid 97–50634 (to J. R. Kersten); American Diabetes
Association Award (to J. R. Kersten), National Institutes of Health
Grants HL-03690 (to J. R. Kersten), HL-63705 (to J. R. Kersten),
AA-12331 (to P. S. Pagel), and HL-54280 (to D. C. Warltier); and
Anesthesiology Research Training Grant GM-08377 (to D. C. Warltier).
REFERENCES
1. Altman J, Dulas D, and Bache RJ. Effect of cyclooxygenase
blockade on blood flow through well-developed coronary collateral vessels. Circ Res 70: 1091–1098, 1992.
2. Altman J, Dulas D, Pavek T, Laxson DD, Homans DC, and
Bache RJ. Endothelial function in well-developed canine coronary collateral vessels. Am J Physiol Heart Circ Physiol 264:
H567–H572, 1993.
3. Bohlen HG and Lash JM. Topical hyperglycemia rapidly suppresses EDRF-mediated vasodilation of normal rat arterioles.
Am J Physiol Heart Circ Physiol 265: H219–H225, 1993.
4. Chu A, Chambers DE, Lin CC, Kuehl WD, Palmer RM,
Moncada S, and Cobb FR. Effects of inhibition of nitric oxide
formation on basal vasomotion and endothelium-dependent responses of the coronary arteries in awake dogs. J Clin Invest 87:
1964–1968, 1991.
5. Coughlan MG, Kenny D, Kampine JP, Bosnjak ZJ, and
Warltier DC. Differential sensitivity of proximal and distal
coronary arteries to a nitric oxide donor following reperfusion
injury or inhibition of nitric oxide synthesis. Cardiovasc Res 27:
1444–1448, 1993.
6. Coutinho M, Gerstein HC, Wang Y, and Yusuf S. The relationship between glucose and incident cardiovascular events. A
metaregression analysis of published data from 20 studies of
95,783 individuals followed for 12.4 years. Diabetes Care 22:
233–240, 1999.
7. Downey HF, Crystal GJ, and Bashour FA. Functional significance of microvascular collateral anastomoses after chronic
coronary artery occlusion. Microvasc Res 21: 212–222, 1981.
8. Duncker DJ and Bache RJ. Inhibition of nitric oxide production aggravates myocardial hypoperfusion during exercise in the
presence of a coronary artery stenosis. Circ Res 74: 629–640,
1994.
AJP-Heart Circ Physiol • VOL
9. Engelman DT, Watanabe M, Engelman RM, Rousou JA,
Flack JE, 3rd, Deaton DW, and Das DK. Constitutive nitric
oxide release is impaired after ischemia and reperfusion. J Thorac Cardiovasc Surg 110: 1047–1053, 1995.
10. Flynn NM, Kenny D, Pelc LR, Warltier DC, Bosnjak ZJ,
and Kampine JP. Endothelium-dependent vasodilation of canine coronary collateral vessels. Am J Physiol Heart Circ Physiol
261: H1797–H1801, 1991.
11. Frank MW, Harris KR, Ahlin KA, and Klocke FJ. Endothelium-derived relaxing factor (nitric oxide) has a tonic vasodilating action on coronary collateral vessels. J Am Coll Cardiol 27:
658–663, 1996.
12. Giugliano D, Marfella R, Coppola L, Verrazzo G, Acampora R, Giunta R, Nappo F, Lucarelli C, and D’Onofrio F.
Vascular effects of acute hyperglycemia in humans are reversed
by L-arginine. Evidence for reduced availability of nitric oxide
during hyperglycemia. Circulation 95: 1783–1790, 1997.
13. Graier WF, Posch K, Fleischhacker E, Wascher TC, and
Kostner GM. Increased superoxide anion formation in endothelial cells during hyperglycemia: an adaptive response or initial
step of vascular dysfunction? Diabetes Res Clin Pract 45: 153–
160, 1999.
14. Gryglewski RJ, Palmer RM, and Moncada S. Superoxide
anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454–456, 1986.
15. Hautamaa PV, Dai XZ, Homans DC, and Bache RJ. Vasomotor activity of moderately well-developed canine coronary
collateral circulation. Am J Physiol Heart Circ Physiol 256:
H890–H897, 1989.
16. Honing ML, Morrison PJ, Banga JD, Stroes ES, and Rabelink TJ. Nitric oxide availability in diabetes mellitus. Diabetes Metab Rev 14: 241–249, 1998.
17. Huk I, Nanobashvili J, Neumayer C, Punz A, Mueller M,
Afkhampour K, Mittlboeck M, Losert U, Polterauer P,
Roth E, Patton S, and Malinski T. L-arginine treatment alters
the kinetics of nitric oxide and superoxide release and reduces
ischemia/reperfusion injury in skeletal muscle. Circulation 96:
667–675, 1997.
18. Kersten JR, Brooks LA, and Dellsperger KC. Impaired
microvascular response to graded coronary occlusion in diabetic
and hyperglycemic dogs. Am J Physiol Heart Circ Physiol 268:
H1667–H1674, 1995.
19. Kersten JR, Montgomery MW, Ghassemi T, Gross ER,
Toller WG, Pagel PS, and Warltier DC. Diabetes and hyperglycemia impair activation of mitochondrial KATP channels.
Am J Physiol Heart Circ Physiol 280: H1744–H1750, 2001.
20. Kersten JR, Schmeling TJ, Hettrick DA, Pagel PS, Gross
GJ, and Warltier DC. Mechanism of myocardial protection by
isoflurane. Role of adenosine triphosphate-regulated potassium
(KATP) channels. Anesthesiology 85: 794–807, 1996.
21. Kersten JR, Schmeling TJ, Orth KG, Pagel PS, and Warltier DC. Acute hyperglycemia abolishes ischemic preconditioning in vivo. Am J Physiol Heart Circ Physiol 275: H721–H725,
1998.
22. Kersten JR, Toller WG, Gross ER, Pagel PS, and Warltier
DC. Diabetes abolishes ischemic preconditioning: role of glucose,
insulin, and osmolality. Am J Physiol Heart Circ Physiol 278:
H1218–H1224, 2000.
23. Kinn JW, Altman JD, Chang MW, and Bache RJ. Vasomotor
responses of newly developed coronary collateral vessels. Am J
Physiol Heart Circ Physiol 271: H490–H497, 1996.
24. Lamping KG, Bloom EN, and Harrison DG. Regulation of
native collateral vessel dilation after coronary occlusion in the
dog. Am J Physiol Heart Circ Physiol 266: H769–H778, 1994.
25. Lefroy DC, Crake T, Uren NG, Davies GJ, and Maseri A.
Effect of inhibition of nitric oxide synthesis on epicardial coronary artery caliber and coronary blood flow in humans. Circulation 88: 43–54, 1993.
26. Lickerman A, Grover-McKay M, and Dellsperger KC. Hyperglycemia-induced angina pectoris in a patient with diabetes
mellitus. Clin Cardiol 20: 736–737, 1997.
27. Ma XL, Weyrich AS, Lefer DJ, and Lefer AM. Diminished
basal nitric oxide release after myocardial ischemia and reper-
281 • NOVEMBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 16, 2017
mM (600 mg/dl) glucose increased superoxide anion
production and decreased nitric oxide activity, in contrast to a similar hypertonic concentration of mannitol
(44 mM) (13). In addition, increases in plasma osmolality produced by the nonmetabolizable sugar raffinose
decreased myocardial infarct size in dogs (22), in contrast to increases in infarct size observed during dextrose-induced hyperosmolality.
In summary, the present results demonstrate that
hyperglycemia decreases retrograde coronary collateral blood flow and exacerbates myocardial hypoperfusion and reperfusion-induced collateral vascular dysfunction. Decreases in retrograde collateral blood flow
observed during reperfusion were exaggerated by hyperglycemia and reversed by L-arginine. The results
suggest that nitric oxide plays a role in maintaining
interarterial coronary collateral blood flow. The results
also demonstrate that hyperglycemia impairs the nitric oxide-mediated regulation of interarterial coronary
collateral blood flow. Impairment of collateral blood
flow regulation by hyperglycemia interfering with nitric oxide metabolism may represent a potential mechanism for increased cardiovascular risk during diabetes mellitus.
H2103
H2104
28.
29.
30.
31.
fusion promotes neutrophil adherence to coronary endothelium.
Circ Res 72: 403–412, 1993.
Malmberg K, Norhammar A, Wedel H, and Ryden L. Glycometabolic state at admission: important risk marker of mortality in conventionally treated patients with diabetes mellitus
and acute myocardial infarction: long-term results from the
Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study. Circulation 99: 2626–2632, 1999.
McDonagh PF, Hokama JY, Copeland JG, and Reynolds
JM. The blood contribution to early myocardial reperfusion
injury is amplified in diabetes. Diabetes 46: 1859–1867, 1997.
Pieper GM, Meier DA, and Hager SR. Endothelial dysfunction in a model of hyperglycemia and hyperinsulinemia. Am J
Physiol Heart Circ Physiol 269: H845–H850, 1995.
Sellke FW, Myers PR, Bates JN, and Harrison DG. Influence of vessel size on the sensitivity of porcine coronary microvessels to nitroglycerin. Am J Physiol Heart Circ Physiol 258:
H515–H520, 1990.
Smith TP, Jr and Canty JM, Jr. Modulation of coronary
autoregulatory responses by nitric oxide. Evidence for flow-
AJP-Heart Circ Physiol • VOL
33.
34.
35.
36.
37.
dependent resistance adjustments in conscious dogs. Circ Res 73:
232–240, 1993.
Suzuki M, Nishizaki M, Arita M, Kakuta T, and Numano F.
Impaired glucose tolerance with late hypersecretion of insulin
during oral glucose tolerance test in patients with vasospastic
angina. J Am Coll Cardiol 27: 1458–1463, 1996.
Taylor PD and Poston L. The effect of hyperglycaemia on
function of rat isolated mesenteric resistance artery. Br J Pharmacol 113: 801–808, 1994.
Tesfamariam B, Brown ML, Deykin D, and Cohen RA.
Elevated glucose promotes generation of endothelium-derived
vasoconstrictor prostanoids in rabbit aorta. J Clin Invest 85:
929–932, 1990.
Traverse JH, Kinn JW, Klassen C, Duncker DJ, and Bache
RJ. Nitric oxide inhibition impairs blood flow during exercise in
hearts with a collateral-dependent myocardial region. J Am Coll
Cardiol 31: 67–74, 1998.
Williams SB, Goldfine AB, Timimi FK, Ting HH, Roddy
MA, Simonson DC, and Creager MA. Acute hyperglycemia
attenuates endothelium-dependent vasodilation in humans in
vivo. Circulation 97: 1695–1701, 1998.
281 • NOVEMBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 16, 2017
32.
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