© 2000 American Heart Association, Inc.
Cellular Biology |
G
i2 but Not Gi3 Is Required for Muscarinic Inhibition of Contractility and Calcium Currents in Adult Cardiomyocytes
From the Whitaker Cardiovascular Institute, Cardiac Muscle Research Laboratory (K.N., M.J., R.L.), Boston University School of Medicine, Boston, Mass; Department of Physiology and Medicine (Endocrine) (R.M.M.), University of Michigan Medical School, Ann Arbor, Mich; and Vascular Division, Departments of Pathology (D.S.M) and Medicine (C.Y.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Richard M. Mortensen, Department of Physiology and Medicine (Endocrine), University of Michigan Medical School, 7726 Medical Science II, Ann Arbor, MI 48109-0622. E-mail rmort@umich.edu or rliao{at}bu.edu
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Abstract |
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Abstract—Parasympathetic stimulation of the heart acts through M2-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins G
i/o, the specific
signal transduction requirements of Gi2 and
Gi3 in mediating muscarinic regulated L-type
calcium currents (ICa, L),
intracellular calcium, and cell contractility remain to be determined.
Adult ventricular myocytes were isolated from
Gi2-null mice,
Gi3-null mice, and their wild-type
littermates. Cell shortening, intracellular calcium levels, and
ICa, L were all measured in
response to isoproterenol, a ß-adrenergic receptor agonist, and
carbachol, a cholinergic receptor agonist. With isoproterenol
stimulation, myocytes from all groups demonstrated a marked increase in
calcium currents, correlating with augmented intracellular calcium
transient amplitude and cell shortening. Carbachol significantly
attenuated the isoproterenol response in wild-type and
Gi3-null cells but had no effect in
Gi2-null cells. This study demonstrates that
Gi2, but not Gi3,
is required for muscarinic inhibition of the ß-adrenergic response in
adult murine ventricular myocytes.
Key Words: Gi proteins • muscarinic receptor • myocyte • contractility • intracellular calcium
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Parasympathetic stimulation of the heart acts through M2-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent cardiac chronotropic and inotropic status.1 2 In mammalian ventricular myocardium, M2 activation is associated with little effect on basal cardiac function but a significant attenuation of the contractile response to ß-adrenergic (ßAR) stimulation, termed indirect inhibitory action.3 The stimulatory ßAR response is initiated via G
s protein activation of adenylyl cyclase and
subsequent protein kinase A (PKA)-mediated phosphorylation of L-type
calcium channels, troponin I, and phospholamban, resulting in increased
calcium influx and augmented contractility (inotropy) as well as
increased calcium reuptake and enhanced relaxation
(lusitropy).4 The
antiadrenergic response of the muscarinic receptor is mediated via
pertussis toxin (PTX)-sensitive G proteins
Gi/o.5 6 7
Although Go, Gi2,
and Gi3 are all known to couple the
M2 receptor, the mechanisms by which
M2-muscarinic activation influences L-type
calcium channel (ICa, L)
activity and corresponding intracellular calcium and myocyte
contractility remain unclear.
Recently, Go has been shown to
mediate L-type calcium channel activity in response to
M2
stimulation.8 We have
previously shown in spontaneously contracting nodal and atrial-like
cells that, in addition to Go, muscarinic
regulation of ICa, L requires
both Gi2 and Gi3
for normal calcium
kinetics.9
Significant differences in the M2 response and
regulation exist between atrial and ventricular
myocardium.3 The
specific signal-transduction requirements of
Gi2 and Gi3 in
mediating muscarinic-regulated L-type calcium channel activity,
intracellular calcium, and cell contractility in adult ventricular
myocytes remain to be determined. Therefore, we examined the ability of
the M2 receptor agonist carbachol to inhibit
isoproterenol-stimulated
ICa, L, intracellular calcium,
and cell contractility in ventricular myocytes from
Gi2- and Gi3-null
mice.
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Materials and Methods |
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Animal Model
Using previously published constructs for gene targeting,10 11 the
i2 and i3 genes
were inactivated in J1 cells cultured on mouse embryo fibroblasts and
injected into C57BL/6 blastocysts. Resulting chimeras passed the
targeted mutation in the germline bred to C57Bl/6. Heterozygotes were
mated to obtain littermates that were either wild type (WT) or
homozygous for the gene inactivation. The inactivation of the targeted
gene was confirmed by polymerase chain reaction. There was no
difference between littermate controls for
Gi2-null and
Gi3-null mice; therefore, data were combined
and presented as WT. Gi2-null and
Gi3-null mice displayed no gross animal
phenotype or cardiac morphological differences. All animal handling and
procedures strictly conformed with the Guide for the Care and
Use of Laboratory Animals (National Institutes of Health
publication number 85-23, 1996).
Adult Ventricular Myocyte Isolation
Gi2-null and
Gi3-null mice and their WT littermates were
intraperitoneally heparinized with 200 U heparin and anesthetized with
ketamine (150 mg/kg) and xylazine (15 mg/kg). Left ventricular myocytes
were dissociated using a modified protocol described
previously.12
Briefly, hearts were quickly excised, cannulated via the aorta, and
perfused in the Langendorff mode with a constant perfusion pressure of
80 mm Hg. The hearts were first perfused for 5 minutes at 37°C with
1.8 mmol/L Ca2+ Tyrode (in mmol/L: NaCl 137,
KCl 5.4, CaCl2 1.8, MgCl2
0.5, HEPES 10, and glucose 10, pH 7.4), followed by
Ca2+-free Tyrode for an additional 5
minutes. They were then perfused with a digestion solution containing
0.06% collagenase D (Boehringer Mannheim) and 0.01% protease XIV
(Sigma Chemical Co). After the hearts were palpably flaccid, the
digestion solution was washed out with
Ca2+-free Tyrode solution for 30 seconds.
The left ventricle (including the septum) was cut into small pieces and
gently agitated, allowing the myocytes to be dispersed in the KB
solution (in mmol/L: KOH 85, KCl 30,
KH2PO4 30,
MgSO4 3, EGTA 0.5, HEPES 10, 1-glutamic acid 50,
taurine 20, and glucose 10, pH 7.4). After 60 minutes, the cells were
resuspended in calcium-containing buffers, with
Ca2+ concentrations gradually increasing
from 0.2 to 0.6 and, finally, to 1.2 mmol/L
Ca2+. Left ventricular myocytes were aliquot
into 2 portions and resuspended in their appropriate buffers for
electrophysiological and myocyte contractility
measurements.
Myocytes included in the study met the following criteria: an overall rod-shape with a clear striation pattern (without granulation and without cauliflower-shaped cell edges); quiescent in the absence of electrical stimulation; and stable mechanical behavior at 5 Hz and 37°C for 5 to 10 minutes. No cells were included past 6 hours after isolation.
Myocyte Cell Shortening
Cells were electrically paced via platinum wires
placed in the myocyte bath and connected to a commercially available
stimulator. Cell length and contractile amplitude of myocytes were
recorded in real time on a Pentium 120-MHz personal computer with a
video edge detector and specialized data acquisition software (SoftEdge
Acquisition System and IonWizard, IonOptix Inc), as previously
described.12
Twitch amplitude was expressed as the difference between diastolic and peak systolic cell lengths. Percent cell shortening (%CS) was expressed as the ratio of absolute twitch amplitude to diastolic cell length. Maximum derivatives of both cell contraction and relaxation were measured as previously described.12 13 14
Intracellular Calcium Measurement
Cytosolic calcium was measured by the fluorescent
calcium indicator fura-2 (Molecular Probes) using a dual fluorescence,
calcium ion sensing system (Ion Optix), as previously
described.12 Freshly
dissociated ventricular myocytes were incubated in 1.2 mmol/L
Ca2+ Tyrode solution containing 1 µmol/L
of membrane permeate fura-2 for 15 minutes at room temperature. After
washing out the fura-2 in the loading solution, an additional 40
minutes were allowed for the deesterification of the fura-2 ester in
the cells. Throughout this procedure, 500 µmol/L probenecid was
included to prevent the leakage of fura-2 from the cells.
Fura-2–loaded myocytes were alternately excited with a xenon lamp at
wavelengths of 360 and 380 nm. The emission fluorescence was collected
by the objective and reflected through barrier filter (510±15 nm) to a
photomultiplier tube. A subset of myocytes from each group was used to
calibrate the fura-2 fluorescence ratio to intracellular calcium
concentrations in situ, as previously
described.12
Myocyte Protocol
Myocytes were stimulated at a physiological rate of
300 bpm at 37°C throughout entire protocol. After 15 minutes of
baseline stabilization period, cell shortening was recorded as baseline
data. A concentration of 1 µmol/L of isoproterenol was then
superperfused, and data were collected when the reaction reached steady
state after 3 minutes. Added to the isoproterenol was 10 µmol/L
carbachol superperfusate, and measurements were recorded after another
3 minutes. Cell shortening and fura-2 fluorescence ratio were
simultaneously recorded at each time point.
Electrophysiological Measurements
Isolated ventricular myocytes were attached to 1%
gelatin-coated tissue culture dishes. Media was changed gradually to
increase the calcium concentration to 1.8 mmol/L. All recordings were
performed at ambient temperature (21°C to 23°C). Calcium currents
(ICa, L) were recorded using a
conventional whole-cell voltage clamp
recording.15 The
internal solution contained (in mmol/L) CsCl 120, EGTA 10, Mg-ATP 3,
and HEPES 10, pH 7.3, adjusted by CsOH. The bath solution contained (in
mmol/L) NaCl 137, CsCl 5.4, CaCl2 1.8,
MgCl2 0.5, HEPES 10, and glucose 10, pH 7.4.
Baseline and stimulated currents were measured in the presence and
absence of 0.5 mmol/L GTP, which showed no effect of added GTP. In
routine protocols, the cells were depolarized every 6 seconds from a
holding potential of -80 to 0 mV for 200 ms after a 50-ms prepulse to
-50 mV. This prepulse, together with the application of 30 µmol/L
tetrodotoxin, was used to eliminate the fast sodium currents. Potassium
currents were eliminated by substituting Cs+
for K+ in both pipette and bath
solutions.
For the determination of current-voltage relations, currents were elicited between -50 and +50 mV from a holding potential of -80 mV. Voltage-clamp pulses of 200 ms after a prepulse at -50 mV (50 ms) were applied every 6 seconds. To measure the steady-state ICa inactivation, a double-pulse voltage-clamp protocol was used. A conditioning pulse from -80 mV to a voltage varying from -80 to +20 mV for 2 seconds was followed by a pulse at -50 mV for 20 ms and then a fixed test pulse at 0 mV for 200 ms. Current was obtained at the test potential of 0 mV by measuring the difference between peak inward current and the leak current at the end of the 2s pulse.
In the studies of ICa, L in response to isoproterenol or carbachol, 2 to 3 minutes after a stable whole-cell configuration was formed, the basal ICa, L was recorded for 3 minutes. Then isoproterenol (1 µmol/L) was added to the bath solution and recordings were made every minute for 4 minutes. Carbachol (10 µmol/L) was added, and the recordings continued for at least 7 minutes. In some experiments, isoproterenol and carbachol were washed out from the bath, with the current returning to baseline. Isoproterenol was then added, and the stimulated currents were recorded to assure that there was no significant current rundown.
Cell membrane capacitance (Cm) was measured by a voltage ramp with a slope (dV/dt) of 10 V/s from -80 to -60 mV. Cm was calculated as Cm=(Iramp-Iss)/(dV/dt), where Iramp is the current at the end of the ramp pulse and Iss is the steady state level of whole cell current at -80 mV.
Calculations
Current-voltage curve was plotted as current per unit
membrane capacitance (pS/pF) versus the membrane potential (mV). Steady
state inactivation data were plotted as I/Imax
against voltage (mV), and the voltage shown is the conditioning
potential. The voltage dependence of calcium current data was curve
fitted by a Boltzmann distribution equation using Graphpad Prism
software (Graphpad, Inc, San Diego, Calif) on a Macintosh G3
computer.
Statistical Analysis
Statistical differences between the mean values for 2
groups were evaluated by the Student’s unpaired t
test. Measurements made sequentially were compared by 2- or 3-factor
ANOVA, with repeated measures where appropriate, using standard
statistical software. Data are expressed as mean±SEM.
P<0.05 was considered statistically
significant.
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Results |
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Myocyte Contractility
The indirect inhibitory effect of M2 stimulation on the ßAR response was investigated in isolated myocytes from adult WT (27 cells; 6 animals), G
i2-null (27 cells; 5 animals), and
Gi3-null mice (27 cells; 4 animals). Cell
contractility and intracellular calcium was simultaneously measured in
the same batch of isolated myocytes in which electrophysiological
parameters were determined. Representative recordings of cell
shortening in response to isoproterenol and carbachol in adult
ventricular myocytes isolated from WT,
Gi2-null, and
Gi3-null mice are shown in
Figure 1A
. Diastolic cell length was similar among WT,
Gi2-null, and
Gi3-null myocytes at baseline (120±3,
118±4, and 124±3 µm, respectively; P=NS). Baseline
%CS (as seen in
Figure 1) was 3.65±0.22%, 3.28±0.23%, and 3.37±0.22%
for WT, Gi2-null, and
Gi3-null myocytes, respectively, and suggests
similar baseline contractile function among groups. With isoproterenol
stimulation, myocytes had an 
2.5-fold increase in %CS that was not
significant among groups. The addition of carbachol in the presence of
isoproterenol resulted in a significant attenuation of the ßAR
response in WT and Gi3-null myocytes of
50% relative to isoproterenol alone. In
Gi2-null cells, however, the addition of
carbachol did not decrease %CS.
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Table 1 shows the maximum rate of contraction
(-dL/dtmax) and the maximum rate of cell
relengthening, or relaxation (+dL/dtmax) at
baseline as well as during infusion of isoproterenol and isoproterenol
plus carbachol. As with cell shortening, all groups had similar
baseline maximum rate of contraction, which was markedly increased with
isoproterenol. The addition of carbachol reduced
-dL/dtmax in WT and
Gi3-null myocytes but had no affect on
maximum rate of contraction in Gi2-null
cells. Both cell shortening and maximum rate of contraction suggest
that the inhibitory effects of the muscarinic system on
ßAR-stimulated contractility are mediated via
Gi2 but not Gi3. In
addition, to increased inotropy, ßAR stimulation also results in
enhanced lusitropy, or relaxation, presumably via PKA-mediated
phosphorylation of troponin I and phospholamban. In our isolated
myocytes, isoproterenol stimulation resulted in a significant increase
in maximum rate of relaxation in all groups. Carbachol inhibited the
rise in +dL/dtmax in WT and
Gi3-null cells but not in
Gi2-null cells, suggesting that the
inhibitory effects of M2 stimulation on
lusitropy are also mediated via
Gi2.
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Intracellular
Ca2+
The inhibitory inotropic and lusitropic effects of
M2 stimulation are believed to be directed by
alterations in intracellular calcium influx and reuptake, and,
therefore, we determined simultaneous intracellular calcium
concentrations with calcium-sensitive probe fura-2.
Representative recordings of intracellular calcium transients in
response to isoproterenol and carbachol in adult ventricular myocytes
isolated from WT, Gi2-null, and
Gi3-null mice are shown in
Figure 2A. WT, Gi2-null, and
Gi3-null cells had similar diastolic
intracellular calcium concentrations at baseline of 185±19, 204±25,
and 190±23 nmol/L, respectively (P=NS). The
amplitudes of the intracellular calcium transient at baseline,
isoproterenol infusion, and isoproterenol plus carbachol infusion are
shown in
Figure 2B. At baseline, all groups had similar calcium
transient amplitudes. With isoproterenol infusion, WT,
Gi2-null, and
Gi3-null cells had similar significant
increases in intracellular calcium transients of 2.5-fold,
consistent with the increase in contractility from ßAR stimulation.
Carbachol attenuated the increase in calcium transient amplitude in WT
and Gi3-null myocytes but had no effect on
intracellular calcium in Gi2-null cells,
resembling the contractile response to M2
stimulation. The maximum rate of intracellular calcium influx and
reuptake (+dCa/dtmax and
-dCa/dtmax, respectively) were also determined
as an assessment of intracellular calcium kinetics. As indicated in
Table 2, isoproterenol caused an increase in the rate of
calcium influx, consistent with augmented calcium transient amplitude
and cell shortening in all groups. M2
stimulation with carbachol reduced the max
+dCa/dtmax in WT and
Gi3 cells but had no effect on
Gi2 cells. Intracellular calcium efflux was
also increased with isoproterenol stimulation in all cells, in
accordance with enhanced cellular relaxation. Carbachol treatment
inhibited the lusitropic effect of ßAR stimulation in WT and
Gi3 cells. Max
-dCa/dtmax was unchanged in
Gi2-null myocytes with carbachol, suggesting
that in addition to the calcium transient amplitude, calcium influx and
efflux kinetics were also unaffected by carbachol treatment in
Gi2 cells.
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Characterization of
ICa, L
Augmented intracellular calcium transients and
subsequent increased cell contractility are believed to be activated by
PKA-stimulated phosphorylation of the L-type calcium channel.
Therefore, to additionally investigate the mechanism underlying altered
M2 response in
Gi2-null myocytes, calcium currents were
recorded in ventricular myocytes from WT,
Gi2-null, and
Gi3-null mice. The current-voltage
relationship and inactivation showed no differences in the calcium
channel characteristics
(Figure 3). In whole-cell configuration, mean cell membrane
capacitance (Cm) of these ventricular myocytes
was 96.8±4.8 for WT (n=8), 92.6±6.7 for
Gi2-null (n=7), and 93.7±8.4 for
i3-null (n=6) pF, with no significantly
differences among the genotypes. The inactivation curve showed a
half-maximal response (Vh) in the range of 23.1
to 24 mV and slope factors of -7.1 to -7.5 mV at baseline. These
results are similar to previously reported characteristics of L-type
calcium channels in
cardiomyocytes.16 17
and showed that major characteristics of these channels were not
altered by gene inactivation.
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,
G
Representative recordings of
ICa, L in response to
isoproterenol and carbachol in adult ventricular myocytes isolated from
WT, Gi2-null, and
Gi3-null mice are shown in
Figure 4A. As shown in
Figure 4B, the time course and degree of
ICa, L in response to
isoproterenol stimulation was similar among groups. The
inhibition of the response to isoproterenol stimulation exerted by
carbachol was rapid and returned to baseline current within 3 minutes
after the addition of carbachol in both WT and
Gi3-null myocytes. However, the response to
carbachol was severely blunted in Gi2–null
myocytes, consistent with the effects on intracellular calcium
transients and cell shortening.
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G |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The antagonistic effects of the muscarinic system on ßAR stimulation have long been recognized,18 although the mechanisms responsible for the antiadrenergic effect of the M2 receptor remain unclear. This study demonstrates that G
i2 but not
Gi3 is required for the indirect inhibitory
action of muscarinic receptors on cell contractility and L-type calcium
currents in adult ventricular myocytes.
Gi2 Required for
Muscarinic Response
Mice with selective knockout of the
Gi2 subunit exhibited a marked attenuation of
the antiadrenergic M2 effect on ßAR-stimulated
increases in intracellular calcium and corresponding myocyte cell
shortening as well as calcium influx kinetics and rate of contraction,
in contrast to WT and Gi3-null mice.
Furthermore, patch-clamp experiments determined that myocytes deficient
in Gi2 had little or no
M2-mediated inhibition of L-type calcium
currents. In addition to a reduction of the inotropic response,
carbachol stimulation decreased the lusitropic response to
isoproterenol, diminishing both calcium uptake and cellular relaxation
in WT and Gi3-null cells but not in
Gi2-null myocytes. Taken together, our data
suggest that both the calcium and contractile inotropic and lusitropic
responses to ßAR stimulation are inhibited by activation of the
M2 receptor via a Gi2
signal transduction pathway.
Mechanism of Gi2
Signal Transduction
Although Gi2 is essential for
M2-muscarinic response, the downstream signal
transduction pathway still remains somewhat unclear. Because the
calcium and contractile inotropic and lusitropic effects of ßAR
activation are mediated though stimulation of adenylyl cyclase and
subsequent PKA-mediated phosphorylation of intracellular proteins,
factors that affect adenylyl cyclase activity, cAMP levels, or protein
phosphorylation remain obvious candidates for M2
activity. Stimulation of calcium currents through exogenous cAMP or
nonhydrolyzable analogs of cAMP is not inhibited by muscarinic activity
or through the use of nonhydrolyzable GTP analogs, suggesting that
M2 inhibition occurs at the level of
cAMP.7 18 19
Importantly, we have previously shown that selective knockout of the
Gi2 subunit does not alter protein levels of
Gi3 or Gß in our
mice,20 suggesting
that in adult ventricular myocytes, Gi2
rather than Gß regulates muscarinic response through the proposed
inhibition of adenylyl cyclase activity.
Alternative Go and
Gi2 Pathways
Previously, Go has been shown
to be required for M2-stimulated changes in
L-type calcium channel activity in adult myocytes, and the level at
which the Go and
Gi2 pathways converge is not presently known.
Whereas Gi2 has been shown to mediate
muscarinic inhibition of adenylyl
cyclase,21
Go inactivation does not affect adenylyl
cyclase activity.22
Although NO production is reported to be required for muscarinic
inhibition of ICa, L in nodal
or nodal-like
cells,9 23 24
there is still considerable controversy as to the role of NO in atrial
cardiocytes and adult ventricular
myocytes.25 26
In rabbit sino atrial and atrioventricular nodal cells, an inhibitor of
NOS was shown to block muscarinic inhibition of L-type calcium channels
and an NO producer to reproduce the
effects.23 24
In murine atrial and nodal-like cardiocytes derived in vitro from
embryonic stem cells, we have shown a similar dependence on NO
generation.9 However,
in adult atria and ventricular cells using NOS3 knockout animals, both
a dependence26 and
an independence25 of
NOS have been reported. In addition, it is has been suggested that
muscarinic-stimulated phosphatase
activity,27 28 29
as well as cGMP
phosphodiesterases,30 31
could potentially contribute to the inhibition of PKA phosphorylation,
although the role of these enzymes in either the
Gi2 or Go
muscarinic pathways remains unclear.
The muscarinic system and inhibitory
Gi proteins play important roles in the
pathophysiology of various cardiac phenotypes, from
aging32 33 34 35
to cardiac
failure,36 37 38
and have been proposed to primarily influence cardiac function through
altered ßAR response. Characterization and understanding of the
signal transduction pathway responsible for the muscarinic effect are
crucial to influencing these phenotypes. In adult ventricular myocytes,
our data demonstrate an independent pathway via the
Gi2 subunit that is essential for functional
signal transduction of the M2-muscarinic
receptor.
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Acknowledgments |
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This work was supported by the National Institutes of Health (National Research Service Award 1F32HL09531 to C.Y., K14H03377 to R.L., and R01 GM49122 and HL58606 to R.M.M.) and the American Heart Association (Established Investigator Award to R.M.M.).
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Footnotes |
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1 Both authors contributed equally to this study.
Received July 25, 2000; revision received August 24, 2000; accepted September 14, 2000.
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