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1 Cardiology Division, Department of Medicine, and 2 Department of Molecular Pharmacology and Biological Chemistry, 3 Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of ~0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 µM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 µM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.
digoxin; actodigin; digitalis; human heart
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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OVER THE PAST 40 years, there has been much evidence presented in support of the theory that the positive inotropic and toxic actions of cardiac glycosides in the heart arise as the result of binding to and inhibition of the sarcolemmal Na+-K+-ATPase (NKA; for reviews, see Refs. 19 and 20). This scheme states that inhibition of the Na+ pump causes an intracellular accumulation of Na+, thus reducing Ca2+ removal from the cytoplasm and/or promoting reverse mode Ca2+ influx via the Na+/Ca2+ exchanger. This subsequently leads to an increase in Ca2+ accumulation in the sarcoplasmic reticulum (SR) with a resulting positive inotropic effect and, in the case of toxicity, to SR Ca2+ overload.
However, there have also been numerous reports of cardiac glycoside actions that could not be explained by this singular effect on the Na+ pump. For example, there are pronounced differences in action potential characteristics for different glycosides (9, 11, 13) and in the relationship between changes in intracellular Na+ activity, positive inotropy and toxicity for different glycosides (29, 32). These and other studies have been interpreted as suggesting that cardiac glycosides may have additional actions, possibly intracellular and independent of any direct interaction with the Na+ pump, which could contribute to their positive inotropic and/or toxic effects.
One of the possible suggestions for secondary actions in the heart includes a direct effect to promote SR Ca2+ release. Isenberg (10) showed that intracellular application of nanomolar concentrations of glycosides caused positive inotropic effects both in the absence of external Na+ and in the presence of digitalis antibody. Nunez-Duran et al. (26) found evidence suggesting that hydrophilic glycosides must be transported across the sarcolemma before positive inotropy can occur. These recent studies are consistent with older reports (5, 8), demonstrating a direct interaction of glycosides with SR proteins.
Consequently, it was not altogether unexpected that we found activation by glycosides in the nanomolar range of the SR Ca2+ release channel (RyR2) isolated from crude SR vesicles as measured using single-channel recordings in artificial lipid bilayers (27). A subsequent study then provided evidence for direct [3H]digoxin binding to sheep RyR2 and activation of single-channel activity by digoxin as the result of increased number of openings without changes in open time duration and with no discernible voltage dependence to this effect (24). Similar results were obtained with ouabain and digitoxin. These observations offered an additional explanation for glycoside actions in the heart by suggesting that glycosides might also have a direct, high-affinity action to promote SR Ca2+ release in addition to increasing the amount of Ca2+ stores in the SR via Na+ pump inhibition.
The current study was intended to examine the interaction of cardiac glycosides with the RyR2 channel, with an emphasis on the factors that might modulate glycoside actions on single-channel activity. We selected the glycosides digoxin and actodigin because the former is used clinically and is therefore of considerable interest whereas the latter is a semisynthetic agent that possesses very distinct effects on electrophysiological, mechanical, and toxic behavior of cardiac preparations that differ markedly from conventional glycosides (9, 32).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of crude and purified RyR2 protein. Crude cardiac SR vesicles were prepared from left ventricles of 16 dogs according to a modification of Tsushima et al.'s (31) method. Skeletal SR vesicles were prepared from three dogs with the use of the muscle from the hindlimb. Dogs were anesthetized with pentobarbital sodium (35 mg/kg iv) and the heart was quickly removed. Skeletal muscle was removed immediately thereafter. The hearts of eight rats were obtained after removal of the heart under pentobarbital anesthesia (35 mg/kg ip) and combined in a single preparation of crude SR vesicles. Each muscle preparation was homogenized and the heavy SR vesicles were separated from the crude homogenate by ultracentrifugation. Vesicles were stored in 200 mM NaCl solution and snap-frozen in liquid nitrogen where they were stored for subsequent use in single-channel experiments.
Purification of RyR2 channel protein was accomplished by a modification of the method of Anderson et al. (3), as described in an earlier publication (31). Briefly, heavy SR vesicles were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solution. The homogenate was separated with the use of sucrose gradient centrifugation and the purified protein was dialyzed to remove the detergent. Purified protein was suspended in proteoliposomes and stored in liquid nitrogen.Single-channel recordings.
Single-channel events were recorded with the use of the
Muller-Rudin technique of artificial lipid bilayers. Crude vesicular RyR2 channels were recorded with the use of a mixture of 40 µg of
phosphatidylethanolamine and 40 µg of
phosphatidylserine (bovine brain, Avanti Polar Lipids) suspended in 20 µl of n-decane. The cis (cytoplasmic) side of
the bilayer apparatus contained 250 mM Cs-methanesulfonate and the
trans (luminal) side contained 20 mM Cs-methanesulfonate
(both sides buffered to pH 7.2 with 20 mM HEPES). Unless stated
otherwise, the trans chamber also contained 1 mM
CaCl2 to simulate the normally high concentration of
luminal [Ca2+]. The artificial lipid mixture was then
painted over the 200-µm hole separating the two sides, forming a
bilayer. Three microliters of vesicle suspension was then added to the
cis chamber, and the solution was stirred until vesicle
fusion with the bilayer occurred. Each type of experiment was performed
in at least three separate SR preparations, each representing a
different animal. The Cs+ gradient was then abolished by
addition of the appropriate amount of Cs-methanesulfonate from a 3 M
stock. In most experiments, the holding potential was +40 to +50 mV to
measure drug effects when mixed Cs+ and Ca2+
movements are in the physiological direction (lumen to cytoplasm). In
some instances, 
40 mV was chosen to show that drug effects were not
dependent on either transbilayer voltage or the direction of current flow.
200A amplifier and
a DMA interface from Axon Instruments. Data were filtered at 2 KHz with
the use of an eight-pole Bessel filter (model 902; Analog Devices) and
digitized at 5 kHz for 30-s recording intervals. Single-channel
analysis was performed with the use of pCLAMP6 software to obtain
channel open and closed times and to calculate single-channel open
probability (Po). Open and closed events were measured using an algorithm based on half-amplitude threshold detection.
Single-channel recordings of purified protein were performed with the
use of KCl instead of Cs-methanesulfonate because no K+ or
Cl
channels were present. Data files were handled in an
identical manner as described above.
Drugs were added from dilute ethanol stocks directly to the solutions
on either side of the bilayer. Equivalent ethanol concentrations (up to
1%) showed no effect on single-channel activity in separate control
experiments (n = 4).
Statistics. Statistical analyses were performed using SigmaStat software (Jandel). Data are presented as means ± SE. Comparisons between sample means were made using Student's t-tests or a one-way analysis of variance, with secondary comparisons made using a Newman-Keuls test. Differences between means were considered significant if P values were <0.05, unless indicated otherwise.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The effects of digoxin on single-channel activity recorded from
crude vesicular RyR2 channels are shown in Fig.
1. The control recording shows typical
single-channel activity under our experimental conditions (holding
potential +50 mV). With a free [Ca2+] previously measured
at 5 µM (by Ca2+ electrode), brief channel openings
during the course of the recording period gave a
Po of 0.010. After the addition of 0.3 nM
digoxin to the cis chamber, single-channel activity
increased, giving a Po of 0.020. The increase of
digoxin concentrations in a cumulative manner caused a progressive
increase in Po up to 1 nM
(Po = 0.057).
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The concentration dependence of the effects of digoxin on
single-channel activity are summarized in Fig.
2A. Activation of Po occurred beginning above 0.1 nM digoxin and
reached a maximum at ~1 nM. The fitted line shows the best sigmoidal
fit to the data (Hill coefficient of 1.2), and a concentration at which
50% of maximal effect (EC50) was ~0.2 nM. When the data
are presented as percentage of control Po (Fig.
2B), the results indicate that digoxin produces an increase
in single-channel activity of about fivefold at maximal concentrations.
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The effects of the semisynthetic agent actodigin on single-channel
activity recorded under identical conditions are shown in Fig.
3. Increasing drug concentration
cumulatively on the cis side of the bilayer from 0 to 0.6 nM
resulted in progressive increase in Po from
0.002 in control to 0.022 at the highest concentration of actodigin.
These results are similar to those obtained with digoxin in Fig. 1.
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Figure 4A summarizes the
concentration dependence of the effects of actodigin on
Po. The concentration dependence of channel activation is similar to that of digoxin, with an
EC50 of ~0.3 nM (Hill coefficient of 1.7). The maximal
effect of actodigin produces about a sixfold increase in
Po (Fig. 4B). These results demonstrate that the effect of glycosides to activate the RyR2 channel
occurs at low concentrations that are quite close to the nanomolar
concentrations of digoxin found in plasma of patients undergoing
glycoside therapy (14).
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Cardiac glycoside effects and MgATP. One of the most important physiological regulators of the RyR2 channel is Mg2+-ATP on the cytoplasmic face of the channel. We examined the effect of glycosides in the presence of MgATP (2 mM) to determine whether or not the activation of RyR2 channels by digoxin was affected by this important regulator. In this series of experiments, MgATP was present under control conditions. In seven experiments [5 with 10 nM digoxin and 2 with 10 nM actodigin, initial Po = 0.02 ± 0.03; not significant (NS) compared with experiments in the absence of MgATP] single channels exposed to glycoside caused a 196 ± 18.0% increase in Po in the presence of 2 mM MgATP (P < 0.05). These results demonstrate that glycoside activation of the channel occurs independently from cis MgATP, although the activating effect appears to be somewhat diminished.
Species differences in cardiac RyR2 channel activation by digoxin.
To determine if a high sensitivity of RyR2 to digoxin is shared by
other glycoside-sensitive species, we also performed a series of
experiments in human cardiac RyR2 channels isolated from three normal
hearts. As shown in Fig. 5,
single-channel Po increased from 0.03 to 0.10 during exposure to 1 nM digoxin. In a total of four experiments,
Po increased from 0.059 ± 0.018 to 0.111 ± 0.039 (P < 0.02), indicating that human
cardiac RyR2 channels are activated by low concentrations of digoxin
just as in dog heart. These observations are particularly important
because both species are known to be sensitive to the inotropic and
toxic effects of glycosides. Therefore, we would expect high
sensitivity of the RyR2 channel to the effects of glycosides if this
effect contributes to a change in cardiac cell contraction and/or
development of toxicity.
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Addition of digoxin to luminal side of bilayer. To determine the location of the binding site for digoxin on the dog cardiac RyR2 channel, we performed several experiments, in which glycoside (10 nM) was added to the trans side of the bilayer. In six experiments, Po was 0.021 ± 0.005 in control and 0.020 ± 0.004 (NS) during exposure to digoxin. These results indicate that the site of digoxin activation resides on the cytoplasmic face of the channel and that drug application to the lumen does not allow access to that site. Thus it is more likely that the binding site is present in the cytoplasmic domain of the channel protein than in either the transmembrane or luminal portions of the protein sequence.
Effects of glycosides on purified cardiac RyR2 and crude skeletal
RyR1.
We next measured the effects of digoxin on single-channel activity in
purified RyR2 channels from the dog heart to determine whether or not
the purification process might alter sensitivity to glycosides. Figure
7A shows that
Po in control was 0.195; after addition of
digoxin (1 nM) to the cis side, there was little change in
Po (0.230). In six experiments,
Po was 0.04 ± 0.03 in control and
0.05 ± 0.04 after addition of digoxin (NS). These data indicate that, in contrast to the crude vesicular membrane-bound form of the
channel, the purified RyR2 channel bound to proteoliposomes is not
activated by digoxin. One possible explanation is that the binding site
of digoxin to the channel is removed by the purification process and
thus may reside on an associated protein. Alternatively, the occupied
receptor on the RyR2 channels may be unable to cause channel
activation, possibly because the internal (luminal)
Ca2+-sensitive binding sites are no longer available to
induce activation. Other possible explanations exist but must await
further examination.
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Luminal Ca2+ and glycoside activation of RyR2. All of the experiments to this point included 1 mM Ca2+ on the luminal side of the bilayer. We examined the possibility that this condition might be required for glycoside action.
The effect of digoxin on single-channel activity at different concentrations of luminal (trans) Ca2+ is shown in Fig. 8. Under control conditions (holding potential +40 mV), single-channel Po doubled at 30 µM and then declined slightly as [Ca2+] was increased over the concentration range of 300 µM to 10 mM, giving a slight bell shape to the [Ca2+]-Po relationship. These results differ somewhat from those reported by several other investigators (21, 30) who found a greater reliance of Po on luminal [Ca2+] but under quite different experimental conditions. In a separate series of experiments, the same protocol was repeated with 10 nM digoxin. After control recordings were obtained ([Ca2+] = 5 µM), addition of digoxin (10 nM) had little effect on single-channel activity. Subsequent increases in trans [Ca2+] to 100 µM caused identical changes in Po to those found in the absence of digoxin. However, at [Ca2+]
300 µM and up to 10 mM, there was a significant increase in Po. Thus, in the presence of digoxin, there is
little effect at [Ca2+] <300 µM but a significant
increase in Po at 0.3-10 mM. These data
suggest that the ability of digoxin to activate the RyR2 channel is
related to SR Ca2+ load. In control, there may be a slight
increase in RyR2 channel activity in the physiological range of luminal
[Ca2+]. However, when Na+ pump inhibition
causes an increased SR Ca2+ load, the ability of the RyR2
to release that stored Ca2+ is also enhanced.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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It is well known that cardiac glycosides inhibit the sarcolemmal NKA and that this action is thought to underlie both the positive inotropic (therapeutic) and arrhythmogenic (toxic) actions of these agents. However, a single cellular mechanism of action for all such agents is unlikely to explain the numerous reports over the past 40 years detailing the variety of actions of different cardiac glycosides in whole heart (1) and at the cellular level. Most notably, Isenberg (10) reported that intracellular application of nanomolar concentrations of digoxin or ouabain produced a positive inotropic action in bovine cardiac myocytes, despite the absence of Na+ or the presence of saturating extracellular concentrations of digoxin antibody. Subsequently, it was reported that hydrophilic glycosides (e.g., ouabain) failed to produce a positive inotropic action in guinea pig atria when membrane transport inhibitors were present whereas the effects of lipophillic agents were unaffected (26). This result was interpreted as suggesting that hydrophilic agents required active transmembrane transport to an intracellular site of action whereas lipophillic agents were capable of passive diffusion to that site. These interesting results recalled earlier suggestions that glycosides might have an intracellular site of action, possibly involving the sarcoplasmic reticulum (5, 8).
With this potential target in mind, we investigated the effects of low concentrations of glycosides on RyR2 channels and found that cardiac glycosides activated single-channel activity of dog RyR2 channels in artificial lipid bilayers (27). Subsequently, McGarry and Williams (24) found that digoxin increased single-channel Po in sheep cardiac RyR2 channels with an estimated EC50 of ~650 pM. In addition, they found that digoxin acted to increase the sensitivity of RyR2 channels to cytoplasmic Ca2+ in the range of 1-10 µM, thus suggesting a mechanism by which glycosides increase RyR2 channel activity over the physiological range of cytosolic [Ca2+]. These investigators also reported specific binding of [3H]digoxin to heavy SR vesicles, demonstrating both high- and low-affinity sites on the cardiac RyR (23).
The present results demonstrate that as in another glycoside-sensitive species (sheep), low concentrations of digoxin and actodigin activate the RyR2 channel in the dog and human heart. Glycoside activation takes the form of increased number of openings with little change in either open or closed time durations. We have also measured the EC50 to be ~200-300 pM, values quite close to that estimated at 650 pM by McGarry and Williams (24). In addition, this effect does not require the presence of Mg2+ and/or ATP on the cytoplasmic side of the channel, suggesting that glycoside actions occur independent of the interactions of these two ligands with their receptors. Just as with sheep heart (24), we also found that digoxin activated only the cardiac isoform of the channel and had no effect on the skeletal channel in dog.
Interestingly, both digoxin and actodigin had very similar effects on concentration dependency and maximal extent of stimulation of RyR2 activity. This is in contrast to the pronounced differences in cellular actions that have been reported between these agents (9, 32). Because both agents produce similar effects to stimulate RyR2 activity, the differences at the cellular level must therefore not be due to the quality of interaction between these drugs and the RyR2, which appears to be similar for each. The difference between agents may be related to differences in lipid solubility and therefore access of each drug to this intracellular site (26) rather than simply to efficacy of sarcolemmal Na+ pump inhibition, with the balance of these two actions causing the apparent distinctions in drug effects on cardiac preparations in vitro.
Species sensitivities of glycoside actions.
One of the most striking effects of cardiac glycosides is the species
difference in sensitivity to the inotropic and toxic actions of these
agents. It is well known that rat heart is generally insensitive to
glycoside actions, requiring at least 10-fold and often far greater
drug concentrations, depending on experimental conditions (2,
18). This was thought to be the result of a selective expression
of a glycoside-insensitive isoform of the NKA (
1) in the
adult rat rather than the more common glycoside-sensitive isoform
(3) predominantly present in sensitive species
(28). However, it is also possible that different amino
acid sequences may exist in a single isoform, which may also impart
varying degrees of glycoside sensitivity to that isoform
(22).
Role of luminal [Ca2+] on glycoside-induced activation of RyR2. It is well known that the functional unit regulating SR Ca2+ release in both skeletal and cardiac SR includes several Ca2+ binding proteins aside from the RyR protein. The high-affinity Ca2+ binding protein calsequestrin is located in the SR lumen and has long been thought to serve as a major mechanism of concentrating Ca2+ in the vicinity of the RyR thus ensuring that sufficient Ca2+ is available for effective release (7, 25). Junctin and triadin are thought to serve as anchoring proteins that stabilize calsequestrin at the inner face of the RyR (4, 12, 15, 16, 25).
This functional unit is responsible for ensuring that sufficient concentrations of Ca2+ are available at the mouth of the RyR to promote contraction, for coupling the luminal Ca2+ source (calsequestrin) to the RyR and for allowing fine regulation of RyR activity and, therefore, of SR Ca2+ release. Our results also suggest a pharmacological role for this arrangement. The effect of digitalis requires that luminal Ca2+ be in the physiological range to regulate RyR2 channel operation. This result suggests that the functional release unit must be intact for digitalis to be effective. Alternatively, it is possible that at low luminal [Ca2+], the glycoside may no longer bind to a cytoplasmic receptor. This observation might also suggest the mechanism by which to explain the lack of effectiveness of digoxin on the purified cardiac RyR2 channel; in the absence of associated proteins, the glycoside cannot activate the channel. Even though the same concentration of Ca2+ (1 mM) is present on the luminal side of the channel, digoxin can no longer modulate activity. Several possibilities might explain this effect: 1) purification might remove the glycoside binding site from the RyR2 channel protein itself or from one of the associated proteins with access to the cytoplasm (junctin or triadin); 2) removal of the ancillary Ca2+-binding proteins might prevent glycoside binding to its receptor; or 3) receptor occupation may no longer be capable of translation to the effector system responsible for increased RyR2 channel activity. It is not possible to discriminate between these possibilities at this time because all we know is that purification of the cardiac isoform of the RyR2 channel removes these proteins, with the exception of the integral protein FK-506 binding protein (FKBP12.6) which remains associated with the channel during purification (17).Possible mechanism for cardiac glycoside action in heart.
The requirement of digoxin activation of RyR2 channels on physiological
concentrations of Ca2+ suggests a mechanism for glycoside
actions on RyR2 channel activity (Fig.
9). There appears to be a range of
luminal [Ca2+] in which the RyR2 channel is most
sensitive to glycoside activation. In the top panel (at normal SR
Ca2+ load), there is little effect of glycoside on SR
Ca2+ release even though its receptor on the RyR2 channel
is occupied, as might occur at drug concentrations below the level of
significant inhibition of NKA. When the NKA is inhibited (Fig. 9,
bottom), not only does the increased SR Ca2+
load make more Ca2+ available for release but it also
promotes the ability of the glycosides to activate the RyR2 channel
directly, amplifying SR release beyond that expected simply from the
increase in SR load secondary to NKA inhibition alone. Thus the ability
of glycoside to activate RyR2 and stimulate SR Ca2+ release
is a result of both direct and indirect actions on the RyR2; the action
is direct because it occurs as the result of a high-affinity binding
interaction to stimulate single-channel activity and indirect in that
it also relies on the ability of NKA inhibition to increase SR
Ca2+ load. This mechanism, combining increased SR load (via
NKA inhibition) with direct increase in SR release, could explain how
the action of glycosides to increase release could contribute to a
sustained positive inotropic action without depleting the SR
(6).
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-30724.
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FOOTNOTES |
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Present address of R. G. Tsushima: Department of Medicine, University of Toronto, Medical Sciences Building, Room 7308, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
Address for reprint requests and other correspondence: J. A. Wasserstrom, Division of Cardiology (S203), Northwestern Univ. Medical School, 303 E. Chicago Ave. Chicago, IL 60611 (E-mail: ja-wasserstrom{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpheart.00700.2001
Received 7 August 2001; accepted in final form 8 November 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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