Am J Physiol Heart Circ Physiol 307: H1046–H1055, 2014.
First published August 8, 2014; doi:10.1152/ajpheart.00421.2014.
Metabolic hyperemia requires ATP-sensitive K⫹ channels and H2O2 but not
adenosine in isolated mouse hearts
Xueping Zhou,1,2 Bunyen Teng,1,2 Stephen Tilley,3 Catherine Ledent,4 and S. Jamal Mustafa1,2
1
Department of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia; 2Center for
Cardiovascular and Respiratory Sciences, West Virginia University, Morgantown, West Virginia; 3Department of Medicine,
University of North Carolina, Chapel Hill, North Carolina; and 4Universite Libre de Bruxelles, Brussels, Belgium
Submitted 17 June 2014; accepted in final form 4 August 2014
coronary metabolic hyperemia; adenosine receptor knockouts; A2A
adenosine receptor; A2B adenosine receptor; hydrogen peroxide;
ATP-sensitive K⫹ channels
is dependent on a constant O2 supply
from the coronary circulation to myocytes. Myocardial O2
consumption (mvO2) increases whenever there is enhanced
cardiac work. Because the myocardium has a very limited
anerobic capacity with a high O2 extraction (75%) (46) under
resting conditions, the increased O2 delivery during exercise
mostly relies on increased coronary blood flow, termed as
metabolic hyperemia. Numerous mechanisms are responsible
for coronary metabolic hyperemia, allowing a balance between
MYOCARDIAL FUNCTION
Address for reprint requests and other correspondence: S. J. Mustafa, Dept.
of Physiology and Pharmacology, West Virginia Univ., 1 Medical Center
Drive, PO Box 9104, Morgantown, WV 26506-9104 (e-mail: sjmustafa@hsc.
wvu.edu).
H1046
myocardial O2 demand and supply. Disturbance of these mechanisms results in decreased cardiac output, hypotension, arrhythmia, heart failure, and death.
Metabolic coronary vasodilation has been found to be regulated by both local metabolic feedback and adrenergic feedforward mechanisms (46). For decades, adenosine, a locally
released metabolite from the myocardium, has been postulated
as one of the important mechanisms responsible for metabolic
coronary hyperemia (3, 4, 6, 16, 17, 19, 28). However, recent
evidence suggests that adenosine is not required for metabolic
coronary vasodilation (4, 14, 47– 49), and its role in regulating
resting coronary flow (CF) still remains controversial (14,
25–27, 49). The discrepancy may be due to different animal
models, differences in species, and/or the different agonists and
antagonists applied in these studies. It is well established that
adenosine exerts its effect through activation of four receptor
subtypes, namely, A1 and A3 adenosine receptors (ARs), which
exert constrictive effects, and A2A and A2BARs, which have
dilatory effects (31). The majority of these studies to characterize the functional role of adenosine in coronary hyperemia
have relied heavily on adenosine-related pharmacological compounds, which may lead to misinterpretation of the data due to
the mixed specificity of the agonists and antagonists for the
four different ARs. In addition, the complexity of the combined
intrinsic (e.g., myogenic, shear-mediated, metabolic, and endothelium-originated mediators) with extrinsic (neural hormonal regulatory mechanism) blood flow control mechanisms
make whole animal study a nonoptimal model for the mechanistic exploration in local metabolic coronary blood flow regulation. Therefore, there is a need to reexamine the functional
role of adenosine in coronary metabolic hyperemia using
combined AR knockout (KO) mice with traditional pharmacological agents in isolated Langendorff-perfused hearts.
Increasing evidence suggests that H2O2, an endogenous
metabolite, acts as an important mediator responsible for metabolic coronary vasodilation (37, 42, 53, 54). We (42, 57) have
previously demonstrated that A2AAR-mediated H2O2 production opens ATP-sensitive K⫹ (KATP) channels in coronary
smooth muscle cells, contributing to the coronary ischemic
vasodilation (42, 57). While the KATP channel is known to be
important for coronary reactive hyperemia (1, 2, 10, 11, 42), it
remains controversial regarding its role in metabolic hyperemia
(13, 15, 16, 32, 34, 35, 47, 51). Therefore, the present study
further examined whether H2O2 and/or KATP channels play an
important role in coronary metabolic hyperemia using isolated
Langendorff-perfused mouse hearts in the presence of catalase
(an enzyme that decomposes H2O2 to water) or glibenclamide
(a selective KATP channel blocker).
0363-6135/14 Copyright © 2014 the American Physiological Society
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Zhou X, Teng B, Tilley S, Ledent C, Mustafa SJ. Metabolic
hyperemia requires ATP-sensitive K⫹ channels and H2O2 but not
adenosine in isolated mouse hearts. Am J Physiol Heart Circ
Physiol 307: H1046 –H1055, 2014. First published August 8, 2014;
doi:10.1152/ajpheart.00421.2014.—We have previously demonstrated that adenosine-mediated H2O2 production and opening of
ATP-sensitive K⫹ (KATP) channels contributes to coronary reactive
hyperemia. The present study aimed to investigate the roles of
adenosine, H2O2, and KATP channels in coronary metabolic hyperemia (MH). Experiments were conducted on isolated Langendorffperfused mouse hearts using combined pharmacological approaches
with adenosine receptor (AR) knockout mice. MH was induced by
electrical pacing at graded frequencies. Coronary flow increased
linearly from 14.4 ⫾ 1.2 to 20.6 ⫾ 1.2 ml·min⫺1·g⫺1 with an increase
in heart rate from 400 to 650 beats/min in wild-type mice. Neither
non-selective blockade of ARs by 8-(p-sulfophenyl)theophylline (8SPT; 50 ␮M) nor selective A2AAR blockade by SCH-58261 (1 ␮M)
or deletion affected MH, although resting flow and left ventricular
developed pressure were reduced. Combined A2AAR and A2BAR
blockade or deletion showed similar effects as 8-SPT. Inhibition of
nitric oxide synthesis by N-nitro-L-arginine methyl ester (100 ␮M) or
combined 8-SPT administration failed to reduce MH, although resting
flows were reduced (by ⬃20%). However, glibenclamide (KATP
channel blocker, 5 ␮M) decreased not only resting flow (by ⬃45%)
and left ventricular developed pressure (by ⬃36%) but also markedly
reduced MH by ⬃94%, resulting in cardiac contractile dysfunction.
Scavenging of H2O2 by catalase (2,500 U/min) also decreased resting
flow (by ⬃16%) and MH (by ⬃24%) but to a lesser extent than
glibenclamide. Our results suggest that while adenosine modulates
coronary flow under both resting and ischemic conditions, it is not
required for MH. However, H2O2 and KATP channels are important
local control mechanisms responsible for both coronary ischemic and
metabolic vasodilation.
ADENOSINE IN CORONARY FLOW REGULATION
MATERIALS AND METHODS
adenosine agonist)-induced CF responses], SCH-58261 (selective
A2AAR antagonist, Ki ⫽ 1.3 nM, 1 ␮M, Tocris Bioscience), CVT6883 (selective A2BAR antagonist, Ki ⫽ 22 nM, 1 ␮M, Tocris
Bioscience), catalase (the enzyme that decomposes H2O2 to water,
2,500 U/ml, Sigma), or glibenclamide (KATP channel blocker, 5 ␮M,
Sigma) were delivered into the aortic perfusion line for 15 min using
a microinjection pump (Harvard Apparatus, Holliston, MA) as 1% of
CF. In the presence of each pharmacological reagent, a second
pacing-induced functional hyperemia was elicited. Time-matched
control experiments with two consecutive inductions of functional
hyperemia showed no differences in baseline function as well as the
magnitude of pacing-induced changes in CF (n ⫽ 3 animals).
Data analysis and statistics. All data collected were analyzed by
LabChart 7.0 software. Baseline CF, LVDP, and dP/dt were calculated as mean values after each heart was paced at 400 beats/min for
10 min. Mean CF was calculated during the 2-min pacing at each
frequency. Because absolute CF rates changed proportionally with
heart mass, CF was normalized by heart mass and presented as
milliliters per gram of wet heart weight per minute. Because mvO2 is
a function of HR, cardiac contractility, ventricular wall tension, and
muscle shortening (9, 12, 20), it is represented by the rate-pressure
product (RPP). To examine the relationship between mvO2 and RPP,
PO2 of the inflow (Pi{O2}; an indication of arterial O2 tension) and
outflow perfusate (Po{O2}; an indication of venous O2 tension collected through a tube inserted in the right atrium close to the coronary
sinus) were measured using a GEM Premier 4000 gas analyzer (n ⫽
2). Because of the absence of hemoglobin in the perfusion buffer, O2
content is dependent solely on the O2 solubility in the buffer at 37°C,
which was 0.023 ml O2·ml buffer⫺1·atm⫺1 (39). mvO2 was calculated
using the following equation: mvO2 ⫽ CF ⫻ (Pi{O2} ⫺ Po{O2}) ⫻
0.023/760, as previously described (39). As expected, mvO2 linearly
correlated with RPP as hearts were paced at graded frequencies (mvO2
⫽1.49 ⫻ RPP ⫹ 101.5, R2 ⫽ 0.9596, P ⬍ 0.01), indicating that mvO2
is a function of the HR and LVDP product.
Least-square linear regression analysis was conducted between HR
or RPP, and CF to calculate the slope of the relationship. The slope of
each curve was compared before and after drug treatment or between
groups. All data are presented as means ⫾ SE; n represents the
number of animals unless otherwise indicated. Paired t-test was used
for paired data analysis. One-way ANOVA was used to compare the
data between groups. The effect of treatment on the relation between
two variables was analyzed by analysis of covariance using GraphPad
Prism5. P values of ⬍0.05 were considered as statistically significant.
RESULTS
Baseline function from WT, A2A KO, A2B KO, and A2A/2B
DKO mouse hearts. Baseline functional parameters of isolated
mouse hearts were recorded at 10 min of pacing (400 beats/
min) after 20-min stabilization. There were no statistically
significant differences in the heart-to-body weight ratio and
maximum ⫺dP/dt among all animal strains, although ⫹dP/dt
had a trend to be less in A2A KO, A2B KO, or A2A/2B DKO
mice compared with WT mice. Baseline CF as well as LVDP
were significantly lower in KO mice compared with WT mice
(P ⬍ 0.05 vs. WT mice; Table 1).
Pacing-induced coronary hyperemia in the absence or presence of 8-SPT. The CF response in isolated hearts of WT mice
was measured before and after the addition of 8-SPT (50 ␮M,
a nonselective adenosine antagonist, n ⫽ 6). Under control
conditions, mean CF increased significanlty from 14.4 ⫾ 1.2 to
16.8 ⫾ 1.1, 18.5 ⫾ 1.3, 19.5 ⫾ 0.9, and 20.6 ⫾ 1.2
ml·min⫺1·g⫺1 when hearts were paced from 400 to 500, 550,
600, and 650 beats/min, respectively (Fig. 1A). The increased
CF linearly correlated with the enhanced mvO2, indicated as
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Animals. The Institutional Animal Care and Use Committee of
West Virginia University School of Medicine approved all experimental protocols. We followed guidelines set forth by the American
Physiological Society and National Institutes of Health regarding the
care and use of laboratory animals. A2AAR and A2BAR single KO
mice (A2A KO and A2B KO mice, respectively) were generously
provided by Dr. C Ledent (Universite Libre de Bruxelles, Brussels,
Belgium) and Stephen Tilley (University of North Carolina, Chapel
Hill, NC), respectively. A2A and A2B KO mice, both backcrossed 12
generations to the wild-type (WT) C57BL/6 background (Jackson
Laboratory, Bar Harbor, ME), were bred to generate A2A/A2B double-KO (DKO) heterozygotes. Double heterozygotes were intercrossed, and 1/16 of the offspring were A2A/A2B DKO mice. A2A/A2B
DKO breeding pairs were then established. Mice were caged in a
12:12-h light-dark cycle with free access to standard chow and water.
The absence of A2AAR and A2BAR at both mRNA and protein levels
in A2A KO, A2B KO, and A2A/2B DKO mice has been confirmed by
our previous studies using PCR, Western blot analysis, and immunohistochemistry in the aorta, mesenteric arteries, and coronary arteries
(30, 41, 43– 45, 57). Our functional data also confirmed the lack of a
CGS-21680 (a selective A2AAR agonist)-induced CF response in A2A
KO and A2A/2B DKO mice, the lack of a BAY 60-6583 (selective
A2BAR agonist) response in A2B KO and A2A/2B DKO mice, and the
lack of an adenosine response in A2A/2B DKO mice (41, 45).
Langendorff-perfused mouse heart preparations. Mice (14 –17 wk)
of either sex (equal ratio) were anesthetized with pentobarbital sodium
(50 mg/kg body wt ip), and hearts were excised into heparinized (5
U/ml) ice-cold Krebs-Henseleit buffer containing (in mM) 119 NaCl,
11 glucose, 22 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5
CaCl2, 2 pyruvate, and 0.5 EDTA. After removal of the lung and
surrounding tissue, the aorta was rapidly cannulated with a 20-gauge,
blunt-ended needle and continuously perfused with 37°C buffer bubbled with 95% O2 and 5% CO2 at a constant perfusion pressure of 80
mmHg. The left atrium was removed, and a fluid-filled balloon made
of plastic wrap was inserted into the left ventricle (LV) across the
mitral valve. The balloon was connected to a pressure transducer for
the continuous measurement of LV developed pressure (LVDP). The
heart was then immersed in a water-jacketed perfusate bath maintained at 37°C and allowed to beat spontaneously. LV diastolic
pressure was adjusted to 2–5 mmHg. CF was continuously measured
with an ultrasonic flow probe (Transonic Systems, Ithaca, NY) placed
in the aortic perfusion line. A Power Lab Chart data-acquisition
system (AD Instruments, Colorado Springs, CO) was used for data
acquisition. After stabilization for 20 min, hearts were then paced at
400 beats/min by two platinum wires connected to the stainless steel
cannula and through the apical myocardium. Function was allowed to
stabilize for another 10 min before baseline functional data were
collected. The heart was then paced at gradually increasing frequencies (500, 550, 600, and 650 beats/min) to induce metabolic hyperemia. Hearts with persistent arrhythmias or LVDP of ⬍80 mmHg
were excluded from the study.
Metabolic hyperemia protocol. After stabilization, hearts were
subjected to a 2 min of pacing at each frequency of 500, 550, 600, and
650 beats/min, respectively. The reason why we paced the heart from
400 to 650 beats/min (1.62-fold increase) was based on the fact that
heart rate (HR) has been reported to increase by ⬃1.9-, 1.8-, 1.8-, and
1.7-fold in humans, swine, dogs, and mice, respectively, upon maximum exercise (13, 17, 29). At the end of each pacing, the heart was
allowed to stabilize for 5 min at a HR of 400 beats/min before the
heart was paced at a different frequency. 8-(p-Sulfophenyl)theophylline (8-SPT; nonselective adenosine receptor antagonist, 50 ␮M,
Sigma), N-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO)
synthase inhibitor, 100 ␮M, Sigma, based on our previous study (45)
showing that L-NAME at ⱖ10⫺5 reached its maximum inhibition on
resting CF and 5=-(N-ethylcarboxamido)adenosine (nonselective
H1047
H1048
ADENOSINE IN CORONARY FLOW REGULATION
Table 1. Baseline functional data in isolated WT, A2A KO, A2B KO, and A2A/2B DKO mouse hearts
No. of animals
Age, wk
Body weight, g
Heart weight, mg
Heart-to-body weight ratio, %
Coronary flow, ml·min⫺1·g⫺1
Left ventricular developed pressure, mmHg
⫹dP/dt, mmHg/s
⫺dP/dt, mmHg/s
WT
A2A KO
A2B KO
A2A/2B DKO
23
14.8 ⫾ 0.17
24.3 ⫾ 0.82
112.4 ⫾ 2.74
0.46 ⫾ 0.005
17.1 ⫾ 0.54
105 ⫾ 4.0
4,704 ⫾ 233.7
3,582 ⫾ 169.5
12
14.8 ⫾ 0.20
25.1 ⫾ 1.14
109 ⫾ 3.9
0.43 ⫾ 0.010
14.4 ⫾ 0.56*
90 ⫾ 3.9*
4,158 ⫾ 278.6
3,743 ⫾ 272.5
6
14.3 ⫾ 0.25
22.3 ⫾ 0.96
103 ⫾ 4.7
0.46 ⫾ 0.001
14.1 ⫾ 1.78*
93 ⫾ 5.3
3,878 ⫾ 313.9
3,167 ⫾ 164.1
6
15.0 ⫾ 0.10
25.0 ⫾ 1.49
113 ⫾ 8.1
0.45 ⫾ 0.020
13.4 ⫾ 0.52*
88 ⫾ 2.5*
4,078 ⫾ 249.0
3,245 ⫾ 404.4
All values are means ⫾ SE. WT, wild type; A2A KO, A2A adenosine receptor knockout; A2B KO, A2B adenosine receptor knockout; A2A/AB DKO, A2A/2B
adenosine receptor double knockout. *P ⬍ 0.05 compared with WT.
To confirm the antagonistic ability of 8-SPT against adenosine, we examined the effect of 8-SPT at the same concentration (50 ␮M) on adenosine (10⫺9⬃10⫺5 M)-induced CF responses. 8-SPT significantly decreased both baseline CF (from
13.2 ⫾ 0.55 to 10.8 ⫾ 0.52 ml·min⫺1·g⫺1, n ⫽ 3, P ⬍ 0.05)
as well as the adenosine-induced CF increase (maximal coronary flow was reduced from 35 ⫾ 1.4 to 15.4 ⫾ 1.5
ml·min⫺1·g⫺1, P ⬍ 0.05; Fig. 1D).
Combined effect of L-NAME and 8-SPT on pacing-induced
coronary hyperemia. Baseline CF was significantly decreased
by L-NAME (10⫺4 M) from 18.0 ⫾ 0.86 to 14.8 ⫾ 0.78
ml·min⫺1·g⫺1 (P ⬍ 0.05, n ⫽ 8; Fig. 2A). However, the
pacing-induced CF increase was not affected by L-NAME
(CF increased linearly from 14.8 ⫾ 0.78 to 22.9 ⫾ 1.53
ml·min⫺1·g⫺1 when hearts were paced from 400 to 650 beats/
min; Fig. 2, A and B). The slope of the relationship between
HR or RPP and CF was not significantly changed before or
after the addition of L-NAME (P ⬎ 0.05), and the linear curve
was shifted downward in a parallell manner. Concomitant with
the decreased baseline flow, LVDP was signficantly diminshed
by L-NAME from 110 ⫾ 5.9, 130 ⫾ 4.9, 136 ⫾ 3.5, 135 ⫾ 3.5,
Fig. 1. Effect of 8-(p-sulfophenyl)theophylline (8-SPT; 50 ␮M) on the pacing-induced
coronary flow (CF) response. A–C: relationship between heart rate [HR; in beats/min
(bpm)] and CF (A), rate-pressure product
(RPP) and CF (B), and HR and left ventricular developed pressure (LVDP; C) before
and after the addition of 8-SPT (n ⫽ 6).
D: adenosine (10⫺9–10⫺5M)-induced coronary response before and after the infusion
of 8-SPT (50 ␮M, n ⫽ 3). *P ⬍ 0.05,
significant difference between the curves.
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RPP, which increased from 41 ⫾ 5.1 to 56 ⫾ 5.1, 65 ⫾ 6.7,
70 ⫾ 6.1, and 75 ⫾ 7.0 ⫻103 beats·min⫺1·mmHg, respectively
(P ⬍ 0.05; Fig. 1B). LVDP increased significanlty from 102 ⫾
12.7 to 112 ⫾ 10.0, 117 ⫾ 12.1, 117 ⫾ 10.0, and 115 ⫾ 10.7
mmHg, respectively (P ⬍ 0.05; Fig. 1C). The linear relationship between CF and RPP suggests a balance between O2
delivery and consumption in pacing-induced metabolic hyperemia in isolated Langendorff-perfused mouse hearts.
In the presence of 8-SPT, resting CF significantly decreased
from 14.4 ⫾ 1.20 to 10.8 ⫾ 0.91 ml·min⫺1·g⫺1 (P ⬍ 0.05 by
paired t-test). The decreased CF was associated with a correlated decrease in baseline LVDP from 102 ⫾ 12.7 to 80 ⫾ 6.5
mmHg (P ⬍ 0.05; Fig. 1C). However, the pacing-induced
increase in CF was not prevented by 8-SPT, although the CF at
each given HR or RPP was decreased (Fig. 1, A and B). CF
increased from 10.8 ⫾ 0.91 to 16.0 ⫾ 0.61 ml·min⫺1·g⫺1 when
hearts were paced from 400 to 650 beats/min, and the linear
curve of the relationship between HR or RPP and CF was
shifted downward in a parallell manner without significant
changes in the slope before or after 8-SPT treatment (0.187 ⫾
0.001 vs. 0.185 ⫾ 0.002, P ⬎ 0.05; Fig. 1, A and B).
A
30
Coronary Flow (ml/min/g)
ADENOSINE IN CORONARY FLOW REGULATION
25
y = 0.0319x+ 5.4
R²= 0.9929
*
20
y = 0.0264x+ 4.3671
R²= 0.9133
15
10
Control
y = 0.0316x+ 1.9969
R²= 0.959
H1049
CF, nor did it affect pacing-induced hyperemia (P ⬎ 0.05 vs.
L-NAME; Fig. 2, A–C).
Effect of A2AAR blockade or deletion on pacing-induced
coronary hyperemia. The role of A2AARs in coronary functional hyperemia was examined in WT mice (n ⫽ 6) in the
presence of SCH-58261 (1 ␮M, a selective A2AAR antagonist).
SCH-58261 significantly decreased baseline CF from 17.5 ⫾
1.54 to 11.3 ⫾ 0.70 ml·min⫺1·g⫺1 (P ⬍ 0.05; Fig. 3, A and B)
and LVDP from 101 ⫾ 3.5 to 70 ⫾ 5.8 mmHg, respectively
L-NAME
5
L-NAME+8-SPT
0
300
400
500
600
700
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Heart Rate (bpm)
Coronary Flow (ml/min/g)
B
30
y = 0.1817x+ 9.8399
R²= 0.9956
25
20
*
y = 0.1791x+ 8.1662
R²= 0.9095
y = 0.1871x+ 8.0763
R²= 0.937
15
10
Control
L-NAME
5
L-NAME+8-SPT
0
20
40
60
80
100
RPP (mmHg/min*1000)
C
160
Control
L-NAME
L-NAME+8-SPT
LVDP (mmHg)
140
120
100
*
80
*
*
*
*
60
300
400
500
600
700
Heart Rate (bpm)
Fig. 2. Effect of N-nitro-L-arginine methyl ester (L-NAME) on the pacinginduced CF response in the absence or presence of 8-SPT. A–C: relationship
between HR and CF (A), RPP and CF (B), and HR and LVDP (C) under
control conditions (n ⫽ 8), in the presence of L-NAME (nitric oxide synthase
inhibitor, 100 ␮M, n ⫽ 4), and in the combined presence of 8-SPT (50 ␮M)
and L-NAME (100 ␮M, n ⫽ 4). *P ⬍ 0.05, significant difference from the
control curve (A and B) and versus control (C).
and 134 ⫾ 3.4 to 88 ⫾ 6.1, 104 ⫾ 6.4, 112 ⫾ 44.8, 112 ⫾ 4.8,
and 116 ⫾ 4.1 mmHg when hearts were paced at 400, 500,
550, 600, and 650 beats/min, respectively (P ⬍ 0.05; Fig. 2C).
In four of eight animals, 8-SPT (50 ␮M) was added in the
presence of L-NAME. 8-SPT did not further decrease baseline
Fig. 3. Effect of A2A adenosine receptor (AR) blockade or deletion on
pacing-induced coronary hyperemia. A–C: relationship between HR and CF
(A), RPP and CF (B), and HR and LVDP (C) in control wild-type (WT) mice
(n ⫽ 6), A2AAR knockout (A2A KO) mice (n ⫽ 9), and WT mice treated with
SCH-58261 (selective A2AAR antagonist, 1 ␮M, n ⫽ 6). *P ⬍ 0.05, significant
difference between the curves or significant difference versus control; #P ⬍
0.05, significant difference versus A2A KO mice.
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H1050
ADENOSINE IN CORONARY FLOW REGULATION
beats/min was decreased from 7.4 ⫾ 0.12 to 4.6 ⫾ 0.49
ml·min⫺1·g⫺1, P ⬍ 0.05). The slope of the relationship between HR and/or RPP and CF in the presence of catalase was
significantly lower than that under control conditions (P ⬍
0.05 by paired t-test; Fig. 5, A and B).
Effect of glibenclamide on pacing-induced coronary
hyperemia. The role of KATP channels in pacing-induced coronary hyperemia was determined in isolated WT mouse hearts
(n ⫽ 6) in the absence or presence of glibenclamide (KATP
channel blocker, 5 ␮M). In the absence of glibenclamide, CF
increased by 6.2 ml·min⫺1·g⫺1 after hearts were paced from
400 to 650 beats/min (Fig. 6, A and B). After glibenclamide
infusion, CF decreased significantly by ⬃ 45% (from 20 ⫾ 1.6
to 11 ⫾ 0.9 ml·min⫺1·g⫺1, P ⬍ 0.05; Fig. 6, A and B) and
LVDP was reduced by ⬃36% (from 112 ⫾ 13.3 to 72 ⫾ 7.0
mmHg, P ⬍ 0.05; Fig. 6C). Moreover, pacing-induced hyperemia was dramatically decreased by ⬃94% (the net CF increase before and after glibenclamide was 6.1 and 0.39
ml·min⫺1·g⫺1, respectively; Fig. 6, A and B). The dramatic
reduction in functional hyperemia was concomitant with a
significant decrease in LVDP during pacing (LVDP decreased
from 72 ⫾ 7.0 to 63 ⫾ 9.0, 57 ⫾ 4.8, and 59 ⫾ 9.2 mmHg
when hearts were paced at 550, 600, and 650 beats/min,
respectively; Fig. 6C), suggesting compromised heart function
as a result of an imbalance between O2 supply and demand
after KATP channel blockade.
DISCUSSION
In the present study, using targeted AR KO mice combined
with pharmacological approaches, we further examined the
role of adenosine, H2O2, and KATP channels in coronary
metabolic hyperemia. In isolated Langendorff-perfused mouse
hearts from A2A KO, A2B KO, and A2A/2B DKO mice, we
demonstrated that adenosine and its receptor subtypes (A2A
and A2B) are not required for pacing-induced coronary metabolic hyperemia, although they exert important roles in maintaining CF under resting conditions. Similarly, broad pharmacological blockade of ARs by 8-SPT in the absence or presence
of a NO synthase inhibitor or selective A2AAR and A2BAR
blockade did not affect pacing-induced increase in CF, although baseline flow was reduced. However, KATP channel
blockade by glibenclamide remarkably reduced both resting
flow and pacing-induced hyperemia, resulting in a dramatic
decrease in LVDP. Scavenging of H2O2 by catalase also
decreased resting flow and pacing-induced coronary hyperemia
but to a lesser extent than glibenclamide. Our results suggest
that while adenosine modulates coronary vascular tone under
both resting and ischemic conditions, it is not required for
metabolic hyperemia even when the NO pathway is inhibited.
However, H2O2 and KATP channels are important local control
mechanisms responsible for both coronary ischemic and metabolic vasodilation.
Since Berne (6) proposed that adenosine is an important
metabolite that links the changes in CF to mvO2, there have
been numerous studies that have examined the adenosine
hypothesis in coronary metabolic hyperemia (3, 4, 7, 14, 21,
47– 49). Early studies supported this hypothesis in that the
augmentation in cardiac interstitial adenosine concentration
was observed to be correlated with increased mvO2 in chronically instrumented dogs (3, 19, 21) and pharmacological block-
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(P ⬍ 0.05; Fig. 3C). However, after A2AAR blockade, CF still
increased from 11.3 ⫾ 0.70 to 16.3 ⫾ 0.54 ml·min⫺1·g⫺1 when
hearts were paced from 400 to 650 beats/min. The slope of the
relationship between HR or RRP and CF was not different
before or after A2AAR blockade (P ⬎ 0.05; Fig. 3, A and B).
We further examined the effect of A2AAR deletion on
pacing-induced coronary hyeremia using A2A KO mice (n ⫽
9). Baseline CF and LVDP were 14.4 ⫾ 0.56 ml·min⫺1·g⫺1
and 90 ⫾ 3.9 mmHg, respectively, which were significantly
lower than those in WT mice (Table 1 and Fig. 3) but were
higher than those in WT mice treated with SCH-58261 (P ⬍
0.05; Fig. 3, A and C). Consistent with the effect of SCH58261, A2AAR deletion did not affect the pacing-induced CF
increase, although receptor deletion resulted in a parallel
downward shift of the relationship between RPP or HR and CF
(Fig. 3, A and B).
Effect of double blockade or deletion of A2AAR and A2BAR
on pacing-induced coronary hyperemia. Both A2AARs and
A2BARs are involved in coronary vasodilation (8, 41, 45).
Therefore, we examined the effect of double blockade or
deletion of both ARs on pacing-induced coronary hyperemia.
In A2A KO mice (n ⫽ 4), CVT-6883 (1 ␮M) further decreased
baseline CF from 13.5 ⫾ 1.03 to 11.6 ⫾ 0.75 ml·min⫺1·g⫺1
(P ⬍ 0.05) and LVDP from 91 ⫾ 2.3 to 71 ⫾ 5.7 mmHg (P ⬍
0.05; Fig. 4D). Similarly, in A2B KO mice, which have lower
baseline CF (14.1 ⫾ 1.78 ml·min⫺1·g⫺1, n ⫽ 6) and LVDP
(93 ⫾ 5.3 mmHg) than WT mice (P ⬍ 0.05; Table 1 and Fig.
4D), combined blockade of A2AARs by SCH-58261 (1 ␮M)
further decreased baseline CF and LVDP to 10.6 ⫾ 0.65
ml·min⫺1·g⫺1 and 74 ⫾ 3.3 mmHg, respectively (P ⬍ 0.05;
Fig. 4D). However, in both cases, pacing-induced hyperemia
was not affected (CF increased from 11.0 ⫾ 0.58 and 10.6 ⫾
0.65 to 16.0 ⫾ 0.81 and 15.7 ⫾ 0.77 ml·min⫺1·g⫺1, respectively when hearts were paced from 400 to 650 beats/min; Fig.
4, A–C). The slope of the relationship between HR and/or RPP
and CF under each experimental condition was not significantly different from WT mice (Fig. 4, A–C).
To confirm the role of A2AARs and A2BARs in coronary
metabolic hyperemia, we further examined the hyperemic
response in A2A/2B DKO mice (n ⫽ 6). Baseline CF (13.4 ⫾
0.52 ml·min⫺1·g⫺1) and LVDP (88 ⫾ 2.5 mmHg) were lower
than those in WT mice (P ⬍ 0.05) and were less than those in
either A2A KO or A2B KO mice, although they did not reach
statistical significance (Table 1 and Fig. 4, E and F). However,
the pacing-induced hyperemic response was not affected (CF
increased from 13.3 ⫾ 0.52 to 19.9 ⫾ 0.73 ml·min⫺1·g⫺1,
linearly correlated with the increased HRs from 400 to 650
beats/min; Fig. 4E). The slope of the relationship between HR
and/or RPP and CF in A2A/2B DKO mice was not significantly
different from that in WT mice, suggesting that neither
A2AARs nor A2BARs are required for the pacing-induced
increase in CF.
Effect of catalase on pacing-induced coronary hyperemia.
The role of H2O2 in pacing-induced hyperemia was examined
in WT mice (n ⫽ 6) before and after the addition of catalase
(2,500 U/ml). Catalase significantly decreased CF from 16.5 ⫾
1.22 to 13.8 ⫾ 1.40 ml·min⫺1·g⫺1 (Fig. 5A) and significantly
decreased LVDP from 96 ⫾ 7.4 to 88 ⫾ 7.1 mmHg (Fig. 5C),
respectively (P ⬍ 0.05 by paired t-test). Additionally, catalase
significantly attenuated the pacing-induced increase in CF by
⬃37% (the net increase in CF of hearts paced from 400 to 650
H1051
ADENOSINE IN CORONARY FLOW REGULATION
Coronary Flow (ml/min/g)
B
WT
A2A KO
A2A KO + CVT6883
A2B KO
A2B KO + SCH58261 y = 0.0231x + 6.5815
30
25
R² = 0.9909
y = 0.0208x + 5.1812
R² = 0.9957
20
*
*
15
*
y = 0.0225x + 5.1867
R² = 0.9871
10
y = 0.0201x + 2.8216
y = 0.0212x + 2.7759
R² = 0.9836
R² = 0.9854
5
Coronary Flow (ml/min/g)
A
0
300
A2A KO + CVT6883
*
20
15
10
y = 0.2244x + 5.3261
R² = 0.9269
y = 0.2553x + 3.5972
R² = 0.9709
5
0
700
20
25
WT
y = 0.2533x + 5.3272
R² = 0.9608
A2B KO
A2B KO + SCH58261
20
*
15
60
70
40
50
60
70
80
RPP (bpm*mmHg*1000)
F
35
30
y = 0.0303x + 4.6649
R² = 0.9964
25
*
20
15
y = 0.0268x + 2.7989
R² = 0.9877
WT
A2A/A2B DKO
0
300
400
*
*
500
600
*
*
*
80
700
HR (bpm)
Coronary Flow (ml/min/g)
30
*
*
*
20
Coronary Flow (ml/min/g)
50
*
y = 0.2035x + 4.5458
R² = 0.9902
5
5
40
y = 0.2322x + 4.948
R² = 0.9575
10
10
30
RPP (bpm*mmHg*1000)
0
E
y = 0.2533x + 5.3272
R² = 0.9608
A2A KO
25
D
30
Coronary Flow (ml/min/g)
500
600
Heart Rate (bpm)
WT
30
y = 0.1949x + 9.03
R² = 0.9939
25
*
20
15
y = 0.2061x + 5.4711
R² = 0.9918
10
5
WT
A2A/2B DKO
0
20
40
60
80
100
RPP (mmHg/min*1000)
Fig. 4. Effect of double blockade or deletion of A2AARs and A2BARs on the pacing-induced CF response. A–D: relationship between HR and CF (A), RPP and
CF (B and C), and HR and LVDP (D) in WT mice (n ⫽ 6), A2A KO mice (n ⫽ 4), A2BAR knockout (A2B KO) mice (n ⫽ 6), A2A KO mice with CVT-6883
(selective A2BAR antagonist, 1 ␮M, n ⫽ 4), and A2B KO mice with SCH-58261 (1 ␮M, n ⫽ 6). E and F: the relationship between HR and CF (E) and the
relationship between RPP and CF (F) were compared between WT mice (n ⫽ 6) and A2A/2BAR double-knockout (A2A/2B DKO) mice (n ⫽ 6). *P ⬍ 0.05,
significant difference between the curves or significant different versus control.
ade of ARs was shown to decrease coronary metabolic hyperemia in isolated guinea pig hearts (21). However, more recent
studies in awake dogs have reported that there was no significant increase in cardiac interstitial adenosine levels that was
sufficient to cause coronary vasodilation (49, 50, 52) and that
AR blockade (49, 50) or triple blockade of adenosine, KATP
channels, and NO (47, 48) did not affect exercise or norepinephrine-induced coronary hyperemia, suggesting that adenosine is not required in coronary functional hyperemia. Interest-
ingly, Duncker and colleagues (16, 17) suggested that adenosine
is an important mediator for coronary metabolic hyperemia only
when the KATP channel is blocked or under conditions where
coronary perfusion pressure is reduced. The discrepancies
among these studies may be due to the mixed drug specificity
or to the complexity of mixed local and neuronal control
mechanisms responsible for coronary blood flow regulation
under in vivo conditions. Using targeted AR KO mice combined with an isolated heart preparation to solve the specificity
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C
400
30
H1052
A
Coronary Flow (ml/min/g)
ADENOSINE IN CORONARY FLOW REGULATION
30
Control
25
Catalase
*
20
15
10
5
0
300
400
500
600
700
selective A2AAR or A2BAR blockade by SCH-58261 (1 ␮M)
or CVT-6883 (1 ␮M) also resulted in a significant reduction
(by ⬃25%, ⬃35%, and ⬃18%, respectively) in resting CF
(Figs. 1, 3, and 4). In agreement with our findings, other studies
in humans (18), pigs (14), dogs (49), and isolated guinea pig
hearts (21) have also reported that adenosine is involved in
modulating CF under resting condition. Of note, a lower
reduction in CF was observed in A2A KO mice (by ⬃18%)
versus SCH-58261-treated animals (by ⬃35%; Fig. 3, A and
C). The lower reduction in baseline CF might be due to
upregulated A2BARs in A2A KO mice compared with WT
mice, as previously reported (45). Regarding the downstream
B
30
Control
25
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
Coronary Flow (ml/min/g)
HR (bpm)
Catalase
*
20
15
10
5
0
20
30
40
50
60
70
RPP (mmHg/min*1000)
LVDP (mmHg)
C
160
Control
Catalase
140
120
100
80
*
**
*
500
600
60
300
400
700
HR (bpm)
Fig. 5. Effect of catalase on pacing-induced coronary hyperemia. A–C: relationship between HR and CF (A), RPP and CF (B), and HR and LVDP (C)
before and after the addition of catalase (the enzyme that decomposes H2O2 to
water, 2,500 U/ml, n ⫽ 6). *P ⬍ 0.05, significant difference in the slope of the
linear curves (A and B) and significant difference versus control (C, paired
t-test).
issue as well as to exclude the neurohormonal effect on CF
regulation, the present study showed that A2AARs and A2BARs
are important in modulating coronary vascular tone under
resting conditions because baseline CF in A2A KO, A2B KO,
and A2A/2B DKO mice was reduced (by ⬃16%, ⬃18%, and
⬃24%, respectively) compared with WT mice (Figs. 3 and 4).
Of interest, although double deletion of A2AARs and A2BARs
further reduced baseline CF from either A2A KO or A2B KO
mice, the extent of reduction in A2A/2B DKO mice (by ⬃22%)
was much less than the summed effect of A2AAR and A2BAR
deletion (⬃34%), suggesting that other unknown compensatory mechanisms might be turned on to maintain resting CF
when both ARs are deleted. Consistent with the results observed in AR KO mice, broad AR blockade by 8-SPT and
Fig. 6. Effect of ATP-sensitive K⫹ channel blockade on pacing-induced
coronary hyperemia. A–C: relationship between HR and CF (A), RPP and CF
(B), and HR and LVDP (C) before and after the addition of glibenclamide
(ATP-sensitive K⫹ channel blocker, 5 ␮M, n ⫽ 6). *P ⬍ 0.05, significant
difference in the slope of the linear curves (A and B) and significant difference
versus control; #P ⬍ 0.05 vs. LVDP at a HR of 400 beats/min.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00421.2014 • www.ajpheart.org
ADENOSINE IN CORONARY FLOW REGULATION
levels after the addition of sodium nitroprusside reversed the
cardiac dysfunction, suggesting that KATP channels exert essential roles in maintaining resting CF and that the decreased
CF upon KATP channel blockade in the coronary vasculature
causes ischemia-induced cardiac contractile dysfunction (15).
It is important to note, however, that KATP channels at concentrations of ⬎10 ␮M have been reported to inhibit other K⫹
and Ca2⫹ channels (5, 36). The concentration we used in the
present study (5 ␮M) is within its selectivity range (5, 36), thus
strengthening our conclusion that the KATP channel is important in modulating resting CF. Interestingly, in disagreement
with the majority of studies showing that blockade of KATP
channels did not attenuate the increase in CF during treadmill
exercise or paired cardiac pacing in dogs and pigs (13, 15, 34,
35, 47), the present study on isolated mouse hearts demonstrated that blockade of KATP channels not only reduced resting
flow but also markedly inhibited pacing-induced functional
hyperemia by ⬃94% (Fig. 6, A and B). The discrepancy among
the studies could be due to 1) species differences (mice vs. pigs
and dogs) or 2) the difference in animal models (isolated hearts
vs. whole animals). In isolated hearts from mice deficient in
Kir6.1 (one of the KATP subunits), isoprenaline-induced coronary vasodilation was significantly attenuated, indicating an
important role of KATP channels in coronary metabolic hyperemia (55). Additionally, the redundant mechanisms responsible
for CF regulation in whole animals, including local and neurohormonal control mechanisms, may complicate the explanation of the data, e.g., the reduction of coronary metabolic
hyperemia by the KATP channel blocker might be compensated
by an enhanced neurohormonal effect or other unknown mechanisms to maintain the balance between O2 supply and demand, thus resulting in unchanged metabolic hyperemia in
vivo even after KATP channel blockade. Concomitant with the
reduced metabolic hyperemia in isolated mouse hearts, we also
observed dramatic cardiac contractile dysfunction. In contrast
to significant increases in LVDP upon pacing in control condition, KATP channel blockade resulted in a significant reduction in LVDP upon pacing (Fig. 6C), suggesting potential
ischemic cell damage related to a decrease in CF.
It is well established the KATP channels play important roles
in adenosine-mediated coronary reactive hyperemia (1, 8, 22,
41, 42, 57). Our recent studies (42, 58) have demonstrated that
A2AAR-mediated H2O2 production opens KATP channels, partially contributing to coronary reactive hyperemia. In these
studies, although adenosine-induced KATP current was reduced
in A2A KO and A2A/2B DKO animals, pinacidil (KATP opener)induced current was not affected after deletion of both A2AARs
and A2BARs, suggesting that A2AARs and A2BARs are not
required for KATP activation and that the absence of ARs does
not affect channel expression, as we previously reported (33,
42). Additionally, we demonstrated that KATP channel blockade inhibited coronary reactive hyperemia (57) to a higher
extent than A2AAR blockade, indicating that other factors,
including adenosine, can also activate KATP channels, thus
contributing to coronary vasodilation. In the present study,
KATP channel blockade dramatically decreased metabolic hyperemia, whereas only a parallel downward shift of the relationship between CF and RPP was observed upon A2AAR
and/or A2BAR blockade or ablation, suggesting that other
unknown mediators than adenosine released during increased
cardiac metabolic demand can activate KATP channels, contrib-
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mechanisms of adenosine in modulating resting CF, our results
(45) and those of others (56) suggest that A2AAR-mediated NO
production may be responsible for resting tone regulation
because 8-SPT (nonselective adenosine antagonist; Fig. 2) or
SCH-58261 (selective A2AAR antagonist) (56) failed to further
decrease baseline CF in the presence of a NO synthase inhibitor. Concomitant with the decreased baseline flow, a significant reduction in LVDP (Table 1 and Figs. 1C, 3C, and 4D)
and a relatively but not statistically lower ⫹dP/dt (Table 1)
were observed upon blockade of adenosine either pharmacologically or genetically. The correlated decrease in cardiac contraction
with the decreased CF suggest that adenosine is an important
mediator involved in modulating resting CF and that the decreased cardiac function is associated with the decreased CF.
Despite the decrease in resting CF, pacing-induced hyperemia was not affected after AR blockade or deletion, in as
much as the linear curve of the relationship between HR and/or
RPP and CF was downward shifted in a parallel manner and
the slope of the curve was not altered before or after AR
blockade or deletion (Figs. 1, 3, and 4). These results, obtained
from targeted AR KO mice, add further evidence to the
previous findings that an AR blocker failed to inhibit exerciseinduced coronary hyperemia in chronically instrumented dogs,
although it lowered the balance between O2 supply and delivery under resting conditions (47, 49). The approach of using
targeted AR KO mice allowed us to rule out the possibility that
increased endogenous adenosine overcomes the competitive
receptor blockade, thus masking the direct AR blockade effect.
Additionally, in the presence of a NO synthase inhibitor, 8-SPT
did not further decrease resting flow as well as pacing-induced
coronary hyperemia, indicating both NO and adenosine act as
tonic vasodilator but are not required for coronary metabolic
hyperemia.
Increasing evidence suggests that H2O2, an endogenous
metabolite, acts as a pivotal mediator responsible for metabolic
coronary vasodilation (37, 42, 53, 54). In support of this idea,
our results showed that catalase significantly decreased resting
CF (by ⬃16%) as well as the pacing-induced increase in CF
(by ⬃37%; Fig. 5, A and B). It should be pointed out that
although A2AAR-mediated H2O2 production in the coronary
endothelium and smooth muscle cells has been previously
reported to play an important role in coronary ischemic hyperemia (42, 57), the failure of AR blockade or deletion to reduce
metabolic hyperemia (discussed above) suggest that other
mechanisms than adenosine-mediated signaling are responsible for H2O2 production, thus contributing to coronary metabolic vasodilation. Regarding the source of H2O2, we speculate
that mitochondrial electron transport in cardiomyocytes might be
an important source for pacing-induce H2O2 production (37).
The KATP channel has been reported to be involved in
coronary ischemic vasodilation (24, 41, 42, 57), although its
role in metabolic vasodilation remains controversial (13, 15,
23, 35, 47, 55). In isolated mouse hearts, the present study
showed that glibenclamide (5 ␮M) significantly reduced CF
(by ⬃45%; Fig. 6A) and LVDP (by ⬃36%; Fig. 6C) at
rest. In agreement with our results, studies in dogs have
reported that intracoronary infusion of glibenclamide (50 – 80
␮g·kg⫺1·min⫺1) significantly decreased baseline flow (by
⬃20 –50%) (38) and was associated with cardiac contractile
dysfunction (systolic wall thickening decreased by ⬃43%)
(15). Importantly, recovery of the CF rate to preglibenclamide
H1053
H1054
ADENOSINE IN CORONARY FLOW REGULATION
ing of KATP channels, leading to coronary metabolic vasodilation in isolated mouse hearts.
A compromised coronary vasodilation may not necessarily
cause ischemia under resting condition. However, during increased myocardial demand, e.g., exercise, the coronary vascular dysfunction will lead to a mismatch between O2 demand
and supply, manifesting the clinical symptom known as angina.
Although mice differ significantly from humans in many aspects, which may limit the extrapolation of present study to
clinical situations, our model, using paced mouse hearts to
gradually increase rates that are within the range of HR change
during exercise in humans, will provide an important basis for
further mechanistic studies in humans. The different mechanisms regarding the roles of adenosine, H2O2, and KATP
channels in CF regulation during ischemic versus metabolic
hyperemia may add new knowledge to the understanding of
pathophysiology of coronary artery diseases.
GRANTS
This work was supported by National Institutes of Health Grants HL027339, HL-09444, HL-071802, and U54-GM-104942.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: X.Z. and S.J.M. conception and design of research;
X.Z. performed experiments; X.Z. analyzed data; X.Z. interpreted results of
experiments; X.Z. prepared figures; X.Z. drafted manuscript; X.Z. and S.J.M.
edited and revised manuscript; X.Z., B.T., S.L.T., C.L., and S.J.M. approved
final version of manuscript.
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It should be pointed out that there are some limitations
regarding the translation of our findings based on our present
experimental model into the clinical applications regarding the
pathophysiology of CF regulation. First, although the effects of
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buffer-perfused isolated hearts did not allow us to clearly
separate the contribution of shear- and/or pressure-induced CF
changes from local metabolic effects. Second, the continuously
oxygenated (95% O2) Krebs buffer used in isolated hearts has
much less O2 carrying capacity than blood, which might cause
hypoxia and relatively lower cardiac contractile function compared with blood-perfused hearts (40). However, in bufferperfused hearts paced from 400 to 600 beats/min, we observed
a correlated increase in mvO2 from 160 to 230 ␮l·min⫺1·g⫺1 as
well as CF (increased by ⬃40%). More importantly, CF
increased more than twofold over baseline after 15-s flow
occlusion (42, 57), indicating that buffer-perfused hearts have
a minimum hypoxia and coronary vessels have the capacity to
further dilate. However, due to the relatively lower myoglobin
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in modulating resting coronary blood flow in our model. Future
studies are needed to compare adenosine levels in bufferversus blood-perfused hearts. Finally, to extend our study to an
atherosclerotic model, where blunted metabolic hyperemia has
been reported, it should be noted that other factors, such as
phenotypic changes of coronary vessels (more stiff and fibrotic) and hemodynamic alterations due to collateral circulation and/or coronary steal (less flow to the ischemic region
where the vascular network is already maximally dilated), need
to be considered for studies.
In conclusion, blockade of ARs, in particular, A2AARs and
A2B ARs using combined pharmacological compounds with
targeted gene deletion, decreased CF at rest but failed to reduce
the pacing-induced increase in CF. Inhibition of NO synthase
decreased resting CF without an effect on pacing-induced
hyperemia, which was not altered by combined blockade of
ARs, suggesting both NO and adenosine are not required for
coronary metabolic hyperemia. The mechanisms responsible
for pacing-induced coronary hyperemia involve mostly KATP
channels and, to a lesser extent, H2O2, since blockade of KATP
channels by glibenclamide almost abolished pacing-induced
coronary vasodilation, whereas scavenging of H2O2 by catalase
only partially inhibited the response. Thus, although adenosine-mediated H2O2 production and the subsequent opening of
KATP channels in the coronary vasculature play an important
role in coronary ischemic vasodilation, other mechanisms, but
not adenosine, are responsible for H2O2 production and open-
ADENOSINE IN CORONARY FLOW REGULATION
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