Am J Physiol Heart Circ Physiol 305: H29 H40, 2013.

First published May 3, 2013; doi:10.1152/ajpheart.00298.2013.

Mechanisms of rapid vasodilation after a brief contraction in human skeletal

muscle
Anne R. Crecelius,1 Brett S. Kirby,3 Gary J. Luckasen,4 Dennis G. Larson,4 and Frank A. Dinenno1,2
1
Human Cardiovascular Physiology Laboratory, Department of Health and Exercise Science, Colorado State University,
Fort Collins, Colorado; 2Vascular Physiology Research Group, Department of Biomedical Sciences, Colorado State
University, Fort Collins, Colorado; 3Department of Medicine, Division of Hematology and Division of Pulmonary, Allergy,
Critical Care Medicine, Duke University Medical Center, Durham, North Carolina; and 4Medical Center of the Rockies
Foundation, University of Colorado Health, Loveland, Colorado
Submitted 4 April 2013; accepted in final form 30 April 2013

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Crecelius AR, Kirby BS, Luckasen GJ, Larson DG, Dinenno FA.
Mechanisms of rapid vasodilation after a brief contraction in human
skeletal muscle. Am J Physiol Heart Circ Physiol 305: H29H40, 2013.
First published May 3, 2013; doi:10.1152/ajpheart.00298.2013.A mono-
phasic increase in skeletal muscle blood flow is observed after a brief
single forearm contraction in humans, yet the underlying vascular
signaling pathways remain largely undetermined. Evidence from ex-
perimental animals indicates an obligatory role of vasodilation via
K-mediated smooth muscle hyperpolarization, and human data sug-
gest little to no independent role for nitric oxide (NO) or vasodilating
prostaglandins (PGs). We tested the hypothesis that K-mediated
vascular hyperpolarization underlies the rapid vasodilation in humans
and that combined inhibition of NO and PGs would have a minimal
effect on this response. We measured forearm blood flow (Doppler
ultrasound) and calculated vascular conductance 10 s before and for
30 s after a single 1-s dynamic forearm contraction at 10%, 20%, and
40% maximum voluntary contraction in 16 young adults. To inhibit
K-mediated vasodilation, BaCl2 and ouabain were infused intra-
arterially to inhibit inwardly rectifying K channels and Na-K-
ATPase, respectively. Combined enzymatic inhibition of NO and PG
synthesis occurred via NG-monomethyl-L-arginine (L-NMMA; NO
synthase) and ketorolac (cyclooxygenase), respectively. In protocol 1
(n 8), BaCl2 ouabain reduced peak vasodilation (range: 30 45%,
P 0.05) and total postcontraction vasodilation (area under the curve,
5575% from control) at all intensities. Contrary to our hypothesis,
L-NMMA ketorolac had a further impact (peak: 60% and area
under the curve: 80% from control). In protocol 2 (n 8), the order
of inhibitors was reversed, and the findings were remarkably similar.
We conclude that K-mediated hyperpolarization and NO and PGs, in
combination, significantly contribute to contraction-induced rapid
vasodilation and that inhibition of these signaling pathways nearly
abolishes this phenomenon in humans.
hyperemia; exercise; potassium
THE REGULATION of skeletal muscle hyperemia during muscle
contractions is complex and involves a variety of signals that
control both the arteriovenous perfusion pressure gradient and
arteriolar caliber (10, 13). In an attempt to isolate the local
mechanisms underlying exercise hyperemia, early experiments
used a single brief muscle contraction to allow for contraction-
induced hyperemia without the continuous interruption of the
blood flow response or further stimulus for hyperemia, as
occurs with repeated contractions (16). In this regard, the
single contraction model can serve as a tool to examine
Address for reprint requests and other correspondence: F. A. Dinenno,
Colorado State Univ., 220 Moby-B Complex, Fort Collins, CO 80523-1582
(e-mail: Frank.Dinenno@colostate.edu).
http://www.ajpheart.org 0363-6135/13 Copyright 2013 the American Physiological Society
feedforward mechanisms of hyperemia that are largely inde-
pendent of changes in tissue oxidative metabolism (48). The
typical response is characterized by an intensity-dependent,
rapid, monophasic increase in blood flow that occurs immedi-
ately (within one cardiac cycle) after contraction, achieves full
magnitude in approximately five cardiac cycles, and then
declines toward baseline. To date, the essential underlying
mechanisms for this rapid hyperemia, and thus feedforward
regulation of muscle blood flow, have yet to be determined in
humans.
Given the rapid nature of single contraction-induced hyper-
emia, some investigators have suggested that this response is
due to changes in the arteriovenous perfusion pressure gradient
(45, 62, 65); however, several lines of evidence now clearly
demonstrate that vasodilation is obligatory to observe this
hyperemic phenomenon (33, 34, 64) and that a portion of this
response is attributable to the mechanical compression of the
vasculature that occurs during muscle contraction (14, 40).
Over the last century, a multitude of vasodilating factors have
been suggested to contribute to contraction-induced vasodila-
tion, yet in humans, none have been found obligatory to
observe a significant increase in muscle blood flow (12, 38).
Interestingly, using the hamster cremaster preparation, Arm-
strong and colleagues (3) demonstrated that K, likely released
during muscle contractions, evokes vascular smooth muscle
cell hyperpolarization and the subsequent rapid vasodilation. In
this study, K-mediated vasodilation occurred through both
inwardly rectifying K (KIR) channels and Na-K-ATPase,
similar to the mechanisms of K-mediated vasodilation in
humans (17, 21). Furthermore, these animal data substantiated
early observations showing that changes in interstitial K
concentration after brief muscle contraction have the appropri-
ate magnitude and time course to have a significant involve-
ment in the hyperemic response (26, 35, 39, 50, 53, 54) and
that smooth muscle vascular hyperpolarization may be essen-
tial to observe rapid vasodilation (33). Whether K-mediated
vasodilation contributes to contraction-induced rapid vasodila-
tion in humans is unknown.
In an attempt to understand the contributing vasoactive
pathways to rapid vasodilation in human subjects, investigators
have targeted ACh released from motor nerves as well as
traditional substances synthesized by the vascular endothe-
lium. In this context, ACh spillover from motor neurons is not
obligatory to observe the vasodilator response, (5), and, fur-
thermore, independent inhibition of nitric oxide (NO) (5) or
vasodilating prostaglandin (PG) synthesis (63) does not impact
rapid vasodilation. However, a considerable interplay is known
H29
H30 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA
to occur between NO and PGs during a variety of stimuli (46,
56, 61, 66), and thus blockade of these pathways in combina-
tion often reveals an unrecognized role (46, 61). To date, no
studies have determined whether combined NO and PG inhi-
bition reduces the rapid vasodilation in response to single
contractions.
Given this information as background, we sought to deter-
mine the underlying signaling mechanisms of muscle contrac-
tion-induced rapid vasodilation in humans. We tested the
hypothesis that K-stimulated vascular hyperpolarization un-
derlies the vasodilation after a brief single contraction in
humans and that, even in combination, there is little to no role
for NO and PGs in this response.
METHODS
Subjects
With Institutional Review Board approval and after written in-
formed consent, a total of 16 young healthy adults (13 men and 3
women, age: 23 1 yr old, weight: 72.3 2.4 kg, height: 176 2
cm; body mass index: 23.4 0.5 kg/m2; means SE) participated in
the present study. All subjects were sedentary to moderately active,
nonsmokers, nonobese, normotensive (resting blood pressure
140/90 mmHg), and not taking any medications. Experiments were
performed after an overnight fast and 24-h abstention from caffeine
and exercise. Subjects were in the supine position with the experi-
mental arm abducted to 90 and slightly elevated above the heart level
upon a tilt-adjustable table. Female subjects were studied during the
early follicular phase of their menstrual cycle or placebo phase of oral
contraceptive use to minimize any potential cardiovascular effects of
sex-specific hormones. All experiments were performed according to
the Declaration of Helsinki.
Arterial Catheterization, Arterial Blood Pressure, and Heart Rate
A 20-gauge, 7.6-cm catheter was placed in the brachial artery of the
nondominant arm under aseptic conditions after local anesthesia (2%
lidocaine) for the local administration of study drugs and blood
sampling. The catheter was connected to a three-port connector as
well as a pressure transducer for mean arterial pressure (MAP)
measurement and continuously flushed at 3 ml/h with heparinized
saline. The two side ports were used for drug infusions of vasoactive
drugs (20, 42). Heart rate (HR) was determined using a three-lead
ECG (Cardiocap/5, Datex-Ohmeda, Louisville, CO).
Forearm Blood Flow and Vascular Conductance
A 4-MHz pulsed Doppler probe (model 500M, Multigon Indus-
tries, Mt. Vernon, NY) was used to measure brachial artery mean
blood velocity (MBV) with the probe securely fixed to the skin over
the brachial artery proximal to the catheter insertion site, as previously
described by our laboratory (23, 40). The probe insonation angle
relative to the skin was 45. A linear 12-MHz echo Doppler ultra-
sound probe (GE Vingmed Ultrasound Vivid7, Horten, Norway) was
placed in a holder securely fixed to the skin immediately proximal to
the velocity probe to measure brachial artery diameter. Brachial artery
diameter was measured in triplicate before any contractions under all
experimental conditions, as we and others (11, 64) have shown that
brachial diameter does not change in response to this stimulus.
Forearm blood flow (FBF) was calculated as follows: FBF MBV
(brachial artery diameter/2)2 60, where FBF was measured in
milliliters per minute, MBV was measured in centimeters per second,
brachial diameter was measured in centimeters, and 60 was used to
convert from milliliters per second to milliliters per minute. Forearm
vascular conductance (FVC) was calculated as follows: FVC
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org
(FBF/MAP) 100, and was expressed as milliliters per minute per
100 mmHg.
Single Dynamic Forearm Contractions
Maximum voluntary contraction (MVC) was determined for the
experimental arm as the average of three maximal squeezes of a
handgrip dynamometer (Stoelting, Chicago, IL) that were within 3%
of each other. Brief, dynamic forearm contractions were performed at
10%, 20%, and 40% of the subjects MVC using a handgrip pulley
system attached to weights corresponding to each workload. The
weight was lifted 4 5 cm over the pulley for a single 1-s dynamic
contraction, as previously described (11). These mild-to-moderate
contraction intensities were chosen to limit the contribution of sys-
temic hemodynamics to forearm vasodilator responses and to elimi-
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nate reflex increases in sympathetic nervous system activity and thus
isolate the local effects of muscle contraction on vascular tone (11). At
least 1.5 min of relaxation were given between each contraction to
allow continuous measures of forearm hemodynamics postcontraction
as well as ample time for hemodynamics to return to baseline values
(11, 40, 64). Workload intensity was randomized and counterbalanced
across subjects to eliminate any order effect, and trials were per-
formed in triplicate to calculate an average response for each subject.
Pilot studies in our laboratory have determined that MVC is not
affected by any of the vasoactive substances, particularly BaCl2 and
ouabain, administered in this study (n 12, pre: 41 2 kg vs. post:
40 2 kg, P 0.43).
Vasoactive Drug Infusions
All drug infusions occurred via the brachial artery catheter to create
a local effect in the forearm. To inhibit K-mediated hyperpolariza-
tion and vasodilation, both ouabain octahydrate (no. 03125, Sigma, St.
Louis, MO) and BaCl2 [10% (wt/vol) BDH-3238, EMD Chemicals,
Gibbstown, NJ] were administered intra-arterially as previously de-
scribed (17). Ouabain was infused at 2.7 nmol/min for 15 min as a
loading dose to inhibit Na-K-ATPase, and BaCl2, was infused at
0.45 moldl forearm volume1min1 with a minimum dose of 4
mol/min to a maximum dose of 5 mol/min for 3 min as a loading
dose to inhibit KIR channels (9, 17, 21, 27, 37). This dose of BaCl2 has
been adjusted to forearm volume compared with our previous study
(17) to maximize efficacy while still remaining within doses safe for
human administration and specific for KIR channels (21, 36). Ouabain
and BaCl2 were prepared in saline and confirmed sterile and free of
fungus/endotoxin and particulate matter with a standard microbiology
report (JCB-Analytical Research Labs, Wichita, KS) before use. To
inhibit traditional endothelium-derived vasodilators that have not
independently been shown to be playing a role in rapid vasodilation,
NG-monomethyl-L-arginine (L-NMMA; Bachem, Germany) was ad-
ministered to inhibit NO synthase-mediated production of NO in
combination with ketorolac (Hospira, Lake Forest, IL) to inhibit
cyclooxygenase-mediated synthesis of PGs. Loading doses of L-
NMMA and ketorolac were 25 mg (5 mg/min for 5 min) and 6 mg
(600 g/min for 10 min), respectively (17, 19). Depending on the
protocol, maintenance doses of either BaCl2 (0.45 moldl forearm
volume1min1), ouabain (2.7 nmol/min), L-NMMA (1.25 mg/min),
or ketorolac (150 g/min) were infused for 3 min before each set of
single contractions to ensure continuous blockade (see Experimental
Protocols below). Forearm volume used for the normalization for
specific vasoactive drugs was determined from regional analysis of
whole body dual-energy X-ray absorptiometry scans (QDR series
software, Hologic, Bedford, MA). Three single contractions at the
respective workload were performed at 15, 30, and 45 s of the 3-min
loading infusion before each set of single contractions to facilitate
drug delivery to the active tissue.
 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA
Experimental Protocols
Two separate groups of eight subjects were studied, with the
primary difference being the order in which pharmacological inhibi-
tors were administered. The experimental timeline is shown in Fig. 1.
To establish control contraction-induced rapid vasodilatory responses,
subjects performed single brief forearm contractions in triplicate at
10%, 20%, or 40% MVC for 1 s with a minimum of 1.5 min of rest
between contractions. Between contraction intensities, saline was
infused for 3 min before the first contraction (Fig. 1).
Protocol 1. To address our primary hypothesis regarding K-
stimulated vascular hyperpolarization as the predominant signaling
pathway involved in rapid vasodilation, in eight subjects (MVC: 52
3 kg) after control responses, single contraction bouts were performed
at each exercise intensity after the infusion of BaCl2 ouabain. Next,
in an attempt to further elucidate the underlying signaling of rapid
H31
bined contribution of NO and PGs, respectively, to contraction-
induced rapid vasodilation. The third set of single contractions was
performed after inhibition of K-mediated vascular hyperpolarization
(BaCl2 ouabain) in the presence of combined NO and PG inhibi-
tion.
Control experiments. In a subset of subjects (n 6), sodium nitro-
prusside (SNP; Nitropress, Hospira) was infused at 2 g100 ml forearm
volume1min1 for 5 min (41) in control (saline) conditions and after
prior administration of all four antagonists (BaCl2 ouabain L-
NMMA ketorolac) as a negative control to confirm the intact capacity
of the forearm resistance vasculature to vasodilate.
In a different subset of subjects (n 4), before any pharmacolog-
ical inhibition, phenylephrine (PE; Baxter, Irvine, CA) was infused at
6.25 ng100 ml forearm volume1min1 for 2 min to preconstrict the
vasculature (42) before the performance of a bout of 40% MVC single

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vasodilation, we addressed endothelium-dependent vasodilators. The
combined contribution of NO and PGs was assessed with the admin-
istration of L-NMMA ketorolac, respectively, and single contrac-
tions were repeated.
Protocol 2. Given the unexpected findings from protocol 1 regard-
ing an effect of combined inhibition of NO and PGs on rapid
vasodilation (see RESULTS), in eight different subjects (MVC: 42 4
kg), the order of inhibition was reversed so that after control responses
were obtained, L-NMMA ketorolac was infused to assess the com-
contractions to determine the impact of reduced basal vascular tone
per se on forearm rapid vasodilation. This dose of PE was selected to
reduce basal FVC to a similar level (20 30%) as we typically
observed with the infusion of the antagonists used in the experimental
protocols (17, 18).
Time-control experiments were not performed in this study as we
have previously demonstrated that rapid vasodilation to a single
contraction is repeatable over the course of an experiment of similar
duration (23 h) (43).

A GENERAL EXPERIMENTAL TIMELINE

Trial 1: Control 40% MVC
20% MVC
10% MVC
Saline Saline Saline

3 min 3 min 3 min

Trial 2: Dual Block 40% MVC Fig. 1. Experimental timeline. A: each exper-
20% MVC imental protocol consisted of three trials of
Ba/Ouab Ba/Ouab Ba/Ouab
10% MVC single dynamic forearm contractions at three
OR OR OR different intensities [10%, 20%, 40% maximal
L-NMMA/Ket L-NMMA/Ket L-NMMA/Ket voluntary contraction (MVC)], all performed
in triplicate. In the first trial, saline was in-
fused via a brachial artery catheter for 3 min
before each set of contractions. In the second
3 min 3 min 3 min trial, depending on the experimental protocol,
either BaCl2 ouabain (Ba/Ouab; n 8;
protocol 1) or NG-monomethyl-L-arginine
Trial 3: Quad Block ketorolac (L-NMMA/Ket; n 8; protocol 2)
40% MVC was administered for 3 min before contrac-
20% MVC tions. In the third trial, all subjects received all
Ba/Ouab/ 10% MVC Ba/Ouab/ Ba/Ouab/ antagonists (Ba/Ouab/L-NMMA/Ket) before
each set of contractions. B: for each intensity,
L-NMMA/Ket L-NMMA/Ket L-NMMA/Ket
three contractions were performed, each last-
ing 1 s. Between each contraction, at least
1.5 min of rest was provided.
3 min 3 min 3 min

B Single Contraction Bout for a Given % MVC

Contraction Contraction Contraction

~1sec ~1sec ~1sec

Rest Rest Rest Rest
3 min 1.5 min 1.5 min 3 min

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H32 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA
Data Acquisition and Analysis
Data were collected, stored on a computer at 250 Hz, and analyzed
offline with signal-processing software (WinDaq, DATAQ Instru-
ments, Akron, OH). Baseline FBF, FVC, MAP, and HR represent an
average of the last 10 s of the resting time period before muscle
contraction. The postcontraction data represent beat-by-beat analysis
beginning with the first unimpeded cardiac cycle immediately after
the release of the contraction for a total of 30 cardiac cycles (11, 40,
64). The data presented for SNP trials represent an average of the final
30 s of predrug and postdrug infusion. Percent changes in FVC were
calculated as follows: [(FVC post FVC pre)/(FVC pre)] 100 as
this tracks changes in blood vessel radius independent of the initial
level of vascular tone and is therefore the most appropriate index of
changes in vasomotor tone (6). The total contraction-induced vasodi-
lator response [area under the curve (AUC)] was calculated as the sum
of absolute FVC (in ml/min) after contraction for 30 cardiac cycles
minus precontraction FVC.
Statistics
All values are reported as means SE. Baseline hemodynamics
and total vasodilation (AUC) values for each intensity (10%, 20%, and
40% MVC) were assessed by one-way repeated-measures ANOVA for
drug condition. We chose to analyze each intensity separately due to
differences in the magnitude of these values and to limit our analysis
to only the relevant comparisons. For the change in peak vasodilation,
two-way (condition intensity) repeated-measures ANOVA was
used. Student-Newman-Keuls post hoc testing was performed when a
significant F value was observed. Comparisons in the control proto-
cols were made with paired Students t-tests. Significance was set at
P 0.05.
RESULTS
Protocol 1
Baseline hemodynamics for both experimental protocols are
shown in Table 1. Figure 2 shows the dynamic absolute FVC
(A, C, and E) and the vasodilatory response (%FVC; B, D,
and F) to single muscle contractions at 10%, 20%, and 40%
MVC. These responses followed the typical temporal pattern
of vasodilation, composed of an immediate rise in FVC in all
trials that peaked in an intensity-dependent manner within
approximately four to five cardiac cycles and then returned to
baseline levels. It is these dynamic responses from which we
calculate our main variables of interest (peak vasodilation and
total vasodilation). We have not performed statistical analysis
on these dynamic curves but present them in an effort to be
comprehensive.
Table 1. Baseline hemodynamics
Heart Rate,
beats/min
Protocol 1: BaCl2 ouabain first
Preblockade 56 2
BaCl2 ouabain 54 2
BaCl2 ouabain L-NMMA ketorolac 53 1
Protocol 2: L-NMMA ketorolac first
Preblockade 55 3
L-NMMA ketorolac 51 2
L-NMMA ketorolac BaCl2 ouabain 52 3
Values are means SE; n 8 subjects/protocol. FVC, forearm vascular conductance; L-NMMA, NG-monomethyl-L-arginine. *P 0.05 vs. preblockade;
P 0.05 vs. BaCl2 ouabain; P 0.05 vs. L-NMMA ketorolac.
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org
Inhibition of K-mediated vasodilation via BaCl2 ouabain
infusion reduced resting FVC (Table 1 and Fig. 2) and signifi-
cantly reduced the magnitude of the peak vasodilatory response at
all intensities (range: 30 45%, P 0.05; Fig. 3). Similarly, total
postcontraction vasodilation (AUC) was reduced from control
after BaCl2 ouabain infusion for all intensities (10%: 74
8%, 20%: 59 10%, and 40%: 55 4%, P 0.05; Fig. 4).
Additional inhibition of NO and PGs via L-NMMA ketoro-
lac, respectively, tended to further reduce baseline FVC (P
0.12; Table 1 and Fig. 2) and, contrary to our hypothesis, also
attenuated the vasodilatory response after a single contraction
(Fig. 2). The peak postcontraction vasodilatory response was
further reduced by L-NMMA ketorolac for 20% and 40% MVC
(P 0.05; Fig. 3) but only approached significance at 10% MVC
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(P 0.065). Similarly, L-NMMA ketorolac further reduced
total postcontraction vasodilation from the BaCl2 ouabain
condition at 20% and 40% (P 0.05) but not at 10% (P 0.2;
Fig. 4). The presence of all inhibitors (BaCl2 ouabain
L-NMMA ketorolac) reduced peak vasodilation by 60% and
the total vasodilatory response (AUC) by 80% on average for
the three contraction intensities, thus explaining nearly all of the
rapid vasodilation in response to a single muscle contraction. In all
conditions and exercise intensities, systemic hemodynamics did
not change postcontraction.
Protocol 2
Given the findings of protocol 1 regarding an effect of com-
bined inhibition of NO and PGs, we reversed the order of
pharmacological inhibition in protocol 2 to address the combined
role of these pathways without prior inhibition of K-mediated
vasodilation. As anticipated, combined inhibition of NO and PG
synthesis via L-NMMA ketorolac, respectively, significantly
reduced baseline FBF and FVC (Table 1). Figure 5 shows dy-
namic absolute FVC (A, C, and E) and the vasodilatory response
(%FVC; B, D, and F) to single muscle contractions at 10%,
20%, and 40% MVC for protocol 2. Similar to protocol 1, these
responses followed the typical temporal pattern of vasodilation
observed, and prior combined inhibition of NO and PGs signifi-
cantly reduced the magnitude of the peak vasodilatory response at
all intensities (range: 2734%, P 0.05; Fig. 6). Total postcon-
traction vasodilation (AUC) was also reduced from preblockade
after L-NMMA ketorolac at all intensities (10%: 34 14%,
20%: 25 12%, 40%: 40 7%, P 0.05; Fig. 7).
Additional inhibition of K-mediated vasodilation via BaCl2
ouabain further reduced baseline FVC (Table 1) and also attenu-
Mean Arterial Pressure, Forearm Blood Flow, FVC, ml min1 100
mmHg ml/min mmHg1
88 2 30.5 3.8 34.5 4.0
92 2* 22.6 1.4* 24.4 1.4*
97 1* 20.2 0.8* 19.4 1.4*
87 3 21.1 2.5 24.1 2.8
88 2 17.0 1.3* 19.2 1.5*
91 3* 14.1 1.6* 15.5 1.7*
 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA H33

A B

Forearm Vascular Conductance (%)

100 10% MVC 175 10% MVC
Forearm Vascular Conductance

150
Pre-Blockade Pre-Blockade
Ba+Ouab Ba+Ouab
75 125
(ml/min/100 mmHg)

Ba+Ouab+L-NMMA+Ket Ba+Ouab+L-NMMA+Ket

100

50 75

50

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25 25

0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Cardiac Cycle Post-Contraction Cardiac Cycle Post-Contraction

C 150 20% MVC D 300 20% MVC

Forearm Vascular Conductance (%)
Forearm Vascular Conductance

125 Pre-Blockade 250 Pre-Blockade

Ba+Ouab
Ba+Ouab
(ml/min/100 mmHg)

Ba+Ouab+L-NMMA+Ket
200 Ba+Ouab+L-NMMA+Ket
100

150
75

100
50
50
25
0
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Cardiac Cycle Post-Contraction Cardiac Cycle Post-Contraction

E 200 40% MVC F 500

40% MVC
Forearm Vascular Conductance (%)
Forearm Vascular Conductance

Pre-Blockade
400 Pre-Blockade
Ba+Ouab
150 Ba+Ouab
(ml/min/100 mmHg)

Ba+Ouab+L-NMMA+Ket
Ba+Ouab+L-NMMA+Ket
300

100
200

50 100

0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

Cardiac Cycle Post-Contraction Cardiac Cycle Post-Contraction

Fig. 2. Protocol 1: effect of BaCl2 ouabain and BaCl2 ouabain L-NMMA ketorolac on dynamic vasodilator responses to single contractions. Absolute
forearm vascular conductance (FVC; A, C, and E) and relative changes in FVC (%FVC; B, D, and F) are shown for 10%, 20%, and 40% MVC single
contractions, respectively. Across all intensities, prior infusion of BaCl2 ouabain reduced the postcontraction vasodilatory response compared with the
preblockade (saline) condition. Additional infusion of L-NMMA ketorolac further inhibited these responses.

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org

H34
Workload (%MVC)
10 20
Peak Forearm Vascular Conductance (%)
-20
-40
-60
-80
*
Fig. 3. Protocol 1: effect of BaCl2 ouabain and BaCl2 ouabain
L-NMMA ketorolac on the peak postcontraction vasodilator response.
Infusion of BaCl2 ouabain significantly reduced peak postcontraction FVC
(P 0.05 vs. zero) at all contraction intensities. The addition of L-NMMA
ketorolac further inhibited this response at 20% and 40% MVC. *P 0.05 vs.
zero; P 0.05 vs. BaCl2 ouabain.
ated the vasodilatory response after a single contraction (Fig. 5).
The peak postcontraction vasodilatory response was significantly
impacted by the addition of BaCl2 ouabain, approximately
doubling the effect from L-NMMA ketorolac at all contraction
intensities (Fig. 6). Similarly, inhibition of K-mediated vasodi-
lation further reduced total postcontraction vasodilation from the
L-NMMA ketorolac condition at all intensities (P 0.05; Fig.
7). Remarkably similar to protocol 1, the presence of all inhibitors
(BaCl2 ouabain L-NMMA ketorolac) reduced peak
vasodilation by 60% and the total vasodilatory response (AUC)
by 80% on average for the three contraction intensities, again
explaining nearly all of the rapid vasodilation in response to a
single muscle contraction. In all conditions and exercise intensi-
ties, systemic hemodynamics did not change postcontraction.
Control Experiments
To confirm preserved vasodilator capacity after the admin-
istration of BaCl2 ouabain L-NMMA ketorolac, SNP
was administered preblockade and at the end of the experimen-
tal protocol in a subgroup of six subjects. SNP caused signif-
icant vasodilation that was unaffected by BaCl2 ouabain
L-NMMA ketorolac, despite a reduction in baseline FVC
(Table 2).
As stated above and shown in Table 1, changes in baseline
FVC were observed after administration of the experimental
antagonists. To determine whether there was a direct effect of
reduced baseline FVC per se on the vasodilator response to a
single contraction, we preconstricted the forearm vasculature with
PE (1-adrenergic agonist) and had four subjects perform single
contractions at 40% MVC and compared this with the control
(preblockade) condition. As shown in Fig. 8A, we were successful
in reducing baseline FVC to a similar extent as occurred in our
experimental protocols (30%). In contrast to what was observed
in our experimental conditions (BaCl2 ouabain, L-NMMA
ketorolac, and BaCl2 ouabain L-NMMA ketorolac),
preconstriction with PE did not reduce the dynamic vasodilator
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org
SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA
response to a single contraction (Fig. 8B), nor did it impact total
postcontraction vasodilation (control: 1,585 258 ml/100 mmHg
40
vs. preconstriction: 1,497 188 ml/100 mmHg, P 0.7).
DISCUSSION
The purpose of the present study was to determine the primary
vasodilator signaling pathways involved in the response to a
single muscle contraction. Specifically, based on prior work, we
were interested in the contribution of K-stimulated vascular
hyperpolarization and the combined contribution of NO and PGs.
The primary novel findings of the present study support a signif-
icant role for K-mediated vasodilation in all facets of the rapid
vasodilator response. Inhibition of K-mediated vascular hyper-
polarization significantly reduced peak and total vasodilation in
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response to three increasing intensities of single muscle contrac-
tions. Additionally, and contrary to our original hypothesis, we
Ba+Ouab
Ba+Ouab+L-NMMA+Ket demonstrated that combined inhibition of NO and PG synthesis
significantly reduced peak and total vasodilation after a single
contraction. When all signaling pathways were inhibited, peak
and total vasodilatory responses were reduced remarkably by 60
and 80%, respectively, compared with control conditions, thus
explaining the majority of the response. Our collective findings
identify, for the first time, that inhibition of K-mediated vascular
hyperpolarization, along with NO and PG synthesis, nearly abol-
ishes the rapid vasodilator response to a single muscle contraction
and that these pathways largely explain this feedforward aspect of
muscle blood flow regulation in humans.
Contraction-Induced Rapid Vasodilation and Contributing
Signaling Pathways
A previous study (1) investigating muscle blood flow regu-
lation in response to muscle contractions appreciated the rapid
nature with which hyperemia occurs after even a brief contrac-
tion. Many different theories of what contributed to the rapid
response were put forth, including contributions of a mechan-
ical effect of the muscle pump to alter perfusion pressure (29,
1600
Vascular Conductance (ml/100mmHg)
Pre-Blockade
Total Post-Contraction Forearm
1400 Ba+Ouab
Ba+Ouab+L-NMMA+Ket
1200
1000
800
600 *
400 *
*
200
* * *
0
10 20 40
Workload (%MVC)
Fig. 4. Protocol 1: effect of BaCl2 ouabain and BaCl2 ouabain
L-NMMA ketorolac on total postcontraction vasodilation. The area under the
dynamic response curves was calculated to determine total postcontraction FVC.
Infusion of BaCl2 ouabain significantly reduced the total vasodilatory
response from preblockade conditions at all contraction intensities. The addi-
tion of L-NMMA ketorolac further attenuated this response at 20% and
40% MVC but not at 10% MVC. *P 0.05 vs. preblockade; P 0.05 vs.
BaCl2 ouabain.
 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA H35

A 70 10% MVC B
175 10% MVC

Forearm Vascular Conductance (%)

Forearm Vascular Conductance

60 150
Pre-Blockade
Pre-Blockade
(ml/min/100 mmHg)

50 L-NMMA+Ket 125 L-NMMA+Ket

L-NMMA+Ket+Ba+Ouab
L-NMMA+Ket+Ba+Ouab
100
40

75
30
50
20
25

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10
0
0
0 5 10 15 20 25 30
0 5 10 15 20 25 30
Cardiac Cycle Post-Contraction Cardiac Cycle Post-Contraction

C 100 20% MVC D 300

20% MVC
Forearm Vascular Conductance (%)
Forearm Vascular Conductance

250 Pre-Blockade
80 Pre-Blockade
L-NMMA+Ket
(ml/min/100 mmHg)

L-NMMA+Ket
200 L-NMMA+Ket+Ba+Ouab
L-NMMA+Ket+Ba+Ouab
60
150

40 100

50
20
0

0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

Cardiac Cycle Post-Contraction Cardiac Cycle Post-Contraction

E 150 40% MVC F 40% MVC

500
Forearm Vascular Conductance (%)
Forearm Vascular Conductance

120 Pre-Blockade 400 Pre-Blockade

L-NMMA+Ket
(ml/min/100 mmHg)

L-NMMA+Ket
L-NMMA+Ket+Ba+Ouab
L-NMMA+Ket+Ba+Ouab
90 300

200
60

100
30

0
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

Cardiac Cycle Post-Contraction Cardiac Cycle Post-Contraction

Fig. 5. Protocol 2: effect of L-NMMA ketorolac and L-NMMA ketorolac BaCl2 ouabain on dynamic vasodilator responses to single contractions.
Absolute FVC (A, C, and E) and relative changes in FVC (B, D, and F) are shown for 10%, 20%, and 40% MVC single contractions, respectively. Across all
intensities, prior infusion of L-NMMA ketorolac reduced the postcontraction vasodilatory response compared with the preblockade (saline) condition.
Additional infusion of BaCl2 ouabain further inhibited these responses.

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H36 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA

Workload (%MVC) Table 2. Subgroup experiment: forearm hemodynamics

10
during SNP infusion
20 40
Peak Forearm Vascular Conductance (%)

0 Baseline SNP (2 g dl forearm

Condition FVC volume1 min FVC1) %FVC

Preblockade 16.2 2.4 138.5 25.5 877 276

-20 L-NMMA ketorolac 12.2 1.4* 117.5 12.6 912 124
BaCl2 ouabain
Values are means SE; n 6 subjects. SNP, sodium nitroprusside. *P
-40
0.05 vs. preblockade.

studies in humans designed to understand the signaling mech-

-60 anisms have yielded largely negative results. Typically per-
formed in the forearm, human studies have shown little to no

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independent role for ACh (5), NO (5), or PGs (63) in the

-80 mediation of rapid vasodilation. To date, the most convincing
evidence regarding the mechanism of rapid vasodilation comes
*
L-NMMA+Ket
L-NMMA+Ket+Ba+Ouab

Fig. 6. Protocol 2: effect of L-NMMA ketorolac and L-NMMA ketorolac A 250

BaCl2 ouabain on the peak postcontraction vasodilator response. Infusion of
L-NMMA ketorolac significantly reduced peak postcontraction FVC (P
Forearm Vascular Conductance
Control
0.05 vs. zero) at all contraction intensities. The addition of BaCl2 ouabain 200
Phenylephrine Pre-Constricted
significantly augmented this inhibition at all contraction intensities. *P 0.05 (ml/min/100 mmHg)
vs. zero; P 0.05 vs. L-NMMA ketorolac.
150
30, 45), direct mechanically induced vasodilation via arteriole
compression/distortion (14, 32, 40, 49), neurally mediated
100
vasodilation (7, 8, 68), and metabolic vasodilation (31). Near
the end of the 20th century, a key study by Tschakovsky and
colleagues (65) eloquently demonstrated that mechanical ef- 50
fects of a contraction and resultant changes in perfusion pres-
sure could not fully explain the hyperemic response and that
0
vasodilation of the vasculature did in fact occur in response to
a single contraction in humans. These findings were confirmed 0 5 10 15 20 25 30
by studies (3, 33, 47, 67) in experimental animals where Cardiac Cycle Post-Contraction
changes in arteriolar diameter were directly determined. De-
spite the acceptance of this immediate rapid vasodilation,
B 600
Forearm Vascular Conductance (%)

Control
Phenylephrine Pre-Constricted
1200 500
Vascular Conductance (ml/100mmHg)
Total Post-Contraction Forearm

Pre-Blockade
1000 L-NMMA+Ket 400
L-NMMA+Ket+Ba+Ouab

800 300

600
* 200

100
400
* *
0
200
* *
* 0 5 10 15 20 25 30
0
Cardiac Cycle Post-Contraction
10 20 40
Workload (%MVC) Fig. 8. Subgroup experiment: effect of reducing baseline forearm vascular tone
on the dynamic vasodilator responses to a single contraction at 40% MVC.
Fig. 7. Protocol 2: effect of L-NMMA ketorolac and L-NMMA ketorolac A: absolute FVC. Infusion of the vasoconstrictor phenylephrine (1-adrenergic
BaCl2 ouabain on total postcontraction vasodilation. The area under the agonist) reduced forearm vascular tone before contraction (cardiac cycle 0)
dynamic response curves was calculated to determine total postcontraction FVC. compared with the control (saline) condition. The postcontraction changes in
Infusion of L-NMMA ketorolac significantly reduced the total vasodilatory FVC mimicked those of the control condition but were shifted to a lower
response from preblockade conditions at all contraction intensities. The addition of absolute FVC. B: the relative vasodilator response (%FVC) after a single
BaCl2 ouabain further attenuated this response at all contraction intensities. contraction was unaffected by preconstriction with phenylephrine compared
*P 0.05 vs. preblockade; P 0.05 vs. L-NMMA ketorolac. with the control condition.

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org

 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA
from animal models and suggests that K most likely from
muscle released during contraction diffuses to vascular smooth
muscle to activate both KIR channels and Na-K-ATPase and
cause hyperpolarization and subsequent rapid vasodilation (3,
33). However, before the present study, this hypothesized
mechanism of K-mediated vascular hyperpolarization had not
been tested in humans.
Recently, we (17) demonstrated the ability to abolish exog-
enous K-mediated (intra-arterial KCl) vascular hyperpolar-
ization and vasodilation with combined BaCl2 to inhibit KIR
channels and ouabain to inhibit Na-K-ATPase. Thus, build-
ing on this established pharmacology and recent findings in
experimental animals, we tested the hypothesis that K-stim-
ulated vascular hyperpolarization contributes to contraction-
induced rapid vasodilation in humans. In protocol 1, we dem-
onstrated that combined blockade of K-mediated hyperpolar-
ization significantly reduced both peak and total rapid
vasodilatory responses (Figs. 2 4). In addition to the 30
45% reduction in peak change in vascular conductance (Fig. 3),
we observed an 5575% attenuation in total vasodilation
after a single muscle contraction (Fig. 4). Taken together, the
impact of inhibition of K-mediated hyperpolarization on total
vasodilation occurring postcontraction was profound and was
by far the largest in magnitude of any prior pharmacological
inhibition of the rapid vasodilatory response in humans (5, 63).
In protocol 1, given prior observations in humans indicating
little to no independent role for NO or PGs in single contrac-
tion-induced rapid vasodilation (5, 63), we were somewhat
surprised to observe a significant reduction in the response
with combined inhibition of these substances (Figs. 3 and 4).
Specifically, we demonstrated that combined inhibition of NO
and PGs further reduced the peak dilatory response from the
BaCl2 ouabain condition (55 65% from control; Fig. 3) as
well as total vasodilation (80% from control; Fig. 4). These
results contrast previous findings in response to brief muscle
contractions when each substance was independently inhibited
(5, 63), but, on the other hand, are consistent with what has
been demonstrated during continuous steady-state exercise (51,
61). Based on the findings from protocol 1, we next sought to
determine the combined role of NO and PGs in contraction-
induced rapid vasodilation before inhibition of K-mediated
vascular hyperpolarization.
In protocol 2, we reversed the order of drug infusions and
found that combined inhibition of NO and PGs reduced the
peak response by 2535%, whereas the total vasodilatory
response was reduced by 20 40%. These data are the first to
clearly demonstrate a combined active role for these substances
in contraction-induced rapid vasodilation in humans and is
consistent with the previously observed increased role of NO/
PGs when these pathways are inhibited in combination com-
pared with inhibited independently (46, 61). When BaCl2
ouabain was infused after inhibition of NO and PGs, the peak
response was further attenuated (60 70% from control; Fig.
6) as was the total vasodilatory response (80% from control;
Fig. 7). Thus, in both protocols 1 and 2, the impact of all
inhibitors on the rapid vasodilatory response was profound,
and our data provide clear evidence for the combined impor-
tance of K-mediated vascular hyperpolarization and NO and
PGs in mediating this response in healthy human subjects.
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org
H37
Potential Stimuli for Vasodilation After a Single Muscle
Contraction
Evidence from the present study clearly supports a profound
effect of K-mediated vasodilation and NO and PGs on con-
traction-induced rapid vasodilation; however, the exact stimu-
lus for the activation of these signaling pathways is unclear.
Armstrong and colleagues (3) demonstrated that both BaCl2
and ouabain can independently inhibit the arteriolar vasodila-
tion observed in response to muscle contractions evoked via
electrical stimulation and that these pathways were likely
activated by K efflux from skeletal muscle into the interstitial
space; however, direct interstitial measurements K were not
made. In this model, they were able to pharmacologically
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inhibit voltage-gated K channels to specifically address skel-
etal muscle K as the stimulus to activate both KIR channels
and Na-K-ATPase. Unfortunately, in our human forearm
model, we were not able to inhibit K efflux from contracting
muscle, and thus we are limited in the conclusions we are able
to make regarding the source of K, as it could derive from
skeletal muscle, as suggested by Armstrong and colleagues, or
alternatively from small- and intermediate-conductance Ca2-
activated K channels on endothelial cells (28).
K is an attractive candidate for the stimulus of rapid
vasodilation as the time course is appropriate, it causes hyper-
polarization via stimulation of both KIR and Na-K-ATPase,
and, if originating from contracting skeletal muscle, would
serve as a feedforward mechanism that couples rapid vasomo-
tor responses with muscle activation (9, 22, 53). Additionally,
an animal study (9) has reported that K-mediated vasodilation
is most often transient in nature, and this may in part contribute
to the distinct temporal pattern of the response.
While K is one candidate for stimulating both KIR channels
and Na-K-ATPase and supports our primary hypothesis
regarding the mechanisms of rapid vasodilation, K (specifi-
cally that from skeletal muscle) would not explain the unex-
pected combined involvement of NO and PGs that we observed
(17). Recently, extracellular production of adenosine, resulting
from the degradation of adenine nucleotides via ecto 5=-
nucleotidase, has been shown to contribute to rapid vasodila-
tion after electrically stimulated contraction of the hamster
cremaster muscle (59). Previous evidence also suggests that
adenosine is capable of stimulating NO and PG production in
humans (52, 57). It is also possible that ACh, released at the
neuromuscular junction, could diffuse to nearby capillaries and
stimulate NO and PG production (25). However, data in
humans using atropine to inhibit muscarinic receptors have
shown little role for ACh in rapid vasodilation (5), and, thus,
this latter possibility is unlikely. Importantly, endothelial cell
changes in intracellular Ca2 concentration may be directly
sensitive to the mechanical compression/distortion of the vas-
culature or changes in shear stress resulting from muscle
contraction and could stimulate NO and PG production (14, 32,
40, 44, 58). Endothelial cell Ca2 concentration changes can
also activate Ca2-activated K channels to stimulate both
direct and K-mediated hyperpolarization and consequently
could explain the observed effects of inhibition of K-medi-
ated vasodilation via BaCl2 ouabain as well as the inhibition
of combined NO and PG synthesis via L-NMMA ketorolac.
H38 SIGNALING OF IMMEDIATE EXERCISE HYPEREMIA
Experimental Considerations
In several experimental conditions, alterations in baseline
forearm vascular tone occurred as a result of the antagonists we
administered. While we present our primary data as a relative
change from baseline, as this appropriately tracks changes in
arteriolar caliber from conditions of altered baseline blood flow
(6), we felt it was necessary to more directly address whether
increased basal vascular tone per se may impact the vasodilator
response to a single contraction. In a subgroup of subjects, we
preconstricted the forearm vasculature to a similar magnitude
as observed in our experimental conditions with local infusions
of the 1-adrenergic agonist PE. Despite starting from a re-
duced level of FVC, there was no impact of preconstriction on
the vasodilator response after a brief contraction at 40% MVC
(Fig. 8). Thus, although the mechanisms by which PE causes
an increase in basal vascular tone may differ from those of our
inhibitors, we do not believe our primary conclusions regard-
ing K-mediated hyperpolarization and NO and PGs are sim-
ply due to a direct effect of the inhibitors on basal vascular
tone.
Given the magnitude of the effect on vasodilation that we
observed, it is reasonable to question whether vasodilator capacity
per se was impaired in our subjects throughout the experimental
trials. To address this potential concern, in a subgroup of subjects,
we administered SNP, an endothelium-independent vasodilator,
and demonstrated a preserved response after the administration of
all of our antagonists (Table 2). This is consistent with recent
findings from our laboratory (17) demonstrating that combined
BaCl2 ouabain administration does not impair ACh-mediated
vasodilation in humans. Thus, the present observations do not
reflect a generalized impairment in the forearm vasculature to
respond to vasodilator stimuli (i.e., our findings are specific to
muscle contractions).
While we have previously shown that BaCl2 and ouabain
inhibit KCl-mediated vasodilation (17) and a multitude of work
supports that activation of KIR channels and Na-K-ATPase
leads to vascular hyperpolarization in vitro (9, 28, 55), we are
inherently limited in our human in vivo model in that we are
unable to directly determine cellular membrane potential and thus
demonstrate vascular hyperpolarization, nor are we able to inhibit
K release from contracting muscle. It should also be acknowl-
edged that although we did not determine the efficacy of our
inhibitors, we have clearly demonstrated efficacy of all drugs in
various previous studies in the human forearm (17, 24, 61). It is
possible that any potential intensity-dependent differences in the
magnitude of the observed responses was due to incomplete
blockade and the ability of high-intensity (i.e., 40% MVC) con-
tractions to somewhat override our inhibitors. This is particularly
true for BaCl2, which can be overridden by high concentrations of
K (2, 37). Similarly, the remaining minimal vasodilation ob-
served could be explained by a lack of complete blockade or,
alternatively, other additional mechanisms that may contribute to
rapid vasodilation in response to a brief muscle contraction.
Perspectives
As recognized in early studies and clearly appreciated in more
recent investigations, the model of a single muscle contraction
allows for the investigation of feedforward mechanisms of exer-
cising blood flow regulation. Whereas much work has been done
attempting to understand the underlying vasomotor signaling
AJP-Heart Circ Physiol doi:10.1152/ajpheart.00298.2013 www.ajpheart.org
pathways of the metabolic feedback mechanisms of exercise
hyperemia, less attention has been paid to the feedforward re-
sponse (15). We (11, 43) have previously shown that rapid
vasodilation is impaired in older healthy adults, and recent evi-
dence suggests that obese individuals also demonstrate attenuated
rapid vasodilation (4). The underlying mechanisms of this impair-
ment in these populations are currently unknown but, based on the
present findings, are most likely related to decreased K-mediated
hyperpolarization and/or potentially attenuated NO and PG bio-
availability (60).
Conclusions
Rapid vasodilation occurs after a brief skeletal muscle con-
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traction, and, to date, the underlying vasomotor signaling
pathways involved in this response have yet to be determined.
In the present study, we demonstrated that K-stimulated
vascular hyperpolarization and the subsequent vasodilation
significantly contribute to contraction-induced rapid vasodila-
tion, as do NO and PGs, in combination. Collective blockade
of these pathways nearly abolishes this phenomenon in hu-
mans, thus remarkably explaining the vast majority of rapid
vasodilation. Future studies designed to determine whether
vascular hyperpolarization via these pathways, independently
and in tandem with NO and PGs, continues to regulate exercise
hyperemia when muscle contractions are repeated in humans
are warranted.
ACKNOWLEDGMENTS
The authors thank the subjects who volunteered to participate and Jennifer
C. Richards, Leora J. Garcia, and Devin V. Dinenno for assistance in conduct-
ing these experiments and preparation of this manuscript.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-102720 (to F. A. Dinenno).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: A.R.C., B.S.K., and F.A.D. conception and design of
research; A.R.C., B.S.K., G.J.L., D.G.L., and F.A.D. performed experiments;
A.R.C. and B.S.K. analyzed data; A.R.C., B.S.K., G.J.L., D.G.L., and F.A.D.
interpreted results of experiments; A.R.C. prepared figures; A.R.C., B.S.K.,
and F.A.D. drafted manuscript; A.R.C., B.S.K., and F.A.D. edited and revised
manuscript; A.R.C., B.S.K., G.J.L., D.G.L., and F.A.D. approved final version
of manuscript.
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