© 2001 American Heart Association, Inc.
Integrative Physiology |
Embryonic Epinephrine Synthesis in the Rat Heart Before Innervation
Association With Pacemaking and Conduction Tissue Development
From the Department of Pharmacology, Georgetown University Medical Center (S.N.E.), Washington, DC, and Department of Cell Biology and Anatomy, Medical University of South Carolina (R.P.T.), Charleston, South Carolina.
Correspondence to Steven N. Ebert, PhD, Department of Pharmacology, Georgetown University Medical Center, Medical-Dental Building, SE 402, 3900 Reservoir Rd NW, Washington, DC 20007. E-mail eberts{at}gunet.georgetown.edu
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Abstract |
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Abstract—Epinephrine is a potent neurotransmitter and hormone that can influence cardiac performance beginning shortly after the first myocardial contractions occur in developing vertebrate embryos. In the present study, we provide evidence that the heart itself may produce epinephrine during embryonic development. Using antibodies that selectively recognize the catecholamine biosynthetic enzymes, tyrosine hydroxylase, dopamine ß-hydroxylase, and phenylethanolamine N-methyltransferase, we used coimmunofluorescent staining techniques to identify cardiac cells that have the capability of producing catecholamines. Initially, cells expressing catecholamine biosynthetic enzymes were found interspersed throughout the myocardium, but by embryonic day 11.5 (E11.5), they became preferentially localized to the dorsal venous valve and atrioventricular canal regions. As development proceeded, catecholamine biosynthetic enzyme expression decreased in these regions but became quite strong along the crest of the interventricular septum by E16.5. This expression pattern was also transient, decreasing in the ventricular septum by E19.5. These data are consistent with a transient and progressive association of catecholamine-producing cells within regions of the heart that become the sinoatrial node, atrioventricular node, and bundle of His. This is the first evidence demonstrating that intrinsic cardiac adrenergic cells may be preferentially associated with early pacemaking and conduction tissue development.
Key Words: phenylethanolamine N-methyltransferase • dopamine ß-hydroxylase • tyrosine hydroxylase • adrenergic
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epinephrine and norepinephrine, the primary peripheral catecholamines in most vertebrates, are neurotransmitters and hormones that have profound influences on cardiac function beginning almost as soon as the heart starts to beat and continuing through all remaining stages of development. In adult mammals, peripheral catecholamines are primarily produced in the adrenal medullae and sympathetic ganglia by the enzymatic conversion of l-tyrosine, as illustrated in Figure 1
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Recent evidence suggests that the mammalian heart may be a source of catecholamines at early developmental stages.1 2 Catecholamine biosynthetic enzymes have been detected in the embryonic heart long before either sympathetic innervation of the heart or production of catecholamines in adrenal chromaffin cells.1 2 In rats, for example, the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT) is expressed in the heart as early as embryonic day 9.5 (E9.5), whereas the earliest appearance of this enzyme in the adrenal gland does not occur until E15.5 to E16.5.1 3 Similarly, sympathetic nerves do not appear in the developing rat heart until E16 to E17.4 Parasympathetic innervation of the heart occurs much earlier than sympathetic innervation5 but does not begin to appear until E12 in the rat.6 By this stage of development (E12), the relative abundance of cardiac PNMT mRNA is already starting to decrease, having reached peak levels between E10 and E11.1 Studies with chick and human embryos have likewise demonstrated the expression of catecholamine biosynthetic enzymes in the heart before sympathetic innervation.2 7 8 Thus, the embryonic heart seems to have the capability of producing catecholamines at stages of development that precede production of catecholamines in the adrenal gland and cardiac sympathetic nerves.
Although chromaffin-like and small intensely fluorescent catecholaminergic cells have been found in and around adult hearts from many species,9 10 11 12 13 typically in association with cardiac nerves, few studies have examined catecholamine production in the prenatal heart. Consequently, the goal of the present study was to identify and map the location of cells expressing the major catecholamine biosynthetic enzymes (PNMT, dopamine ß-hydroxylase [DBH], and tyrosine hydroxylase [TH]) in the developing embryonic rat heart.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
All animal studies conformed with the guidelines prescribed by the Georgetown University Animal Care and Use Committee and the National Institutes of Health. Timed pregnant Sprague-Dawley rats were obtained from Taconic Labs (Germantown, NY). Embryos were isolated, staged, and dissected as described previously.1
Immunofluorescence Histochemistry
Double- and single-immunofluorescent staining of
developing rat hearts was performed essentially as described
previously.14 Anti-PNMT and
anti-DBH antisera are polyclonal rabbit antisera raised against PNMT
and DBH purified from bovine adrenal chromaffin
cells15 16 and
were kindly provided by Dr D.L. Wong (Harvard University Medical
School, Boston, Mass). The anti-TH and anti–
-actinin antisera are
mouse monoclonal antibodies (ascites) obtained from Sigma Chemical Co.
Secondary antibodies (FITC-conjugated donkey anti-rabbit IgG,
TRITC-conjugated donkey anti-mouse IgG, Texas Red–conjugated donkey
anti-rabbit IgG, and Texas Red–conjugated donkey anti-mouse IgG) were
obtained from Jackson Labs and used at a dilution of
1:200.
Epinephrine Radioimmunoassay
Embryonic samples were prepared by sonication for 10
seconds in 0.1 mol/L HCl, followed by microcentrifugation
(14 000g, 10 minutes) to
remove residual debris. Protein concentrations were determined for the
supernatants using the Bio-Rad protein assay, and equivalent amounts of
each sample (1 µg protein diluted in 1 mL of 0.1 mol/L HCl) were
assayed using a commercially available radioimmunoassay (ALPCO Labs,
Inc).
An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.
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Results |
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Transient expression of the gene encoding the epinephrine biosynthetic enzyme, PNMT, has previously been demonstrated in the preinnervation-stage embryonic rat heart using enzymatic and RNase protection assays.1 To identify the cells that express PNMT within the heart, we initiated immunofluorescent histochemical staining using a rabbit anti-PNMT antiserum15 and an FITC-conjugated anti-rabbit IgG secondary antibody. At relatively early stages of development (eg, E10.5), PNMT expression was detected throughout the developing myocardium (Figure 2A
). Specifically, PNMT expression was observed in
the truncus arteriosus as well as the common atrial and ventricular
chambers at this stage of development (E10.5,
Figures 2A through 2F). In contrast, no staining for PNMT was
seen in nearby aortic arch tissue
(Figures 2A through 2C and 2F, small arrows). To visualize
cardiac cells expressing PNMT relative to those expressing
muscle-specific proteins, we performed coimmunofluorescent staining
with antibodies that selectively recognize either PNMT or the
muscle-specific protein sarcomeric
-actinin.18 As shown in
Figures 2A and 2B, a similar but partially nonoverlapping
pattern of expression was found in the E10.5 rat heart for these 2
proteins. This can be observed with greater clarity in the higher
magnification photomicrographs shown in
Figures 2D and 2E. To facilitate comparison, the arrow
depicted in
Figures 2A through 2F points to the same region of the
truncus arteriosus. Although it appears that some overlapping
expression of PNMT and -actinin occurs (compare
Figures 2A and 2B), there are distinct regions of
nonoverlapping expression (compare
Figures 2D and 2E). In particular, whenever we observed
bright, intense staining for -actinin, we did not see coexpression
of PNMT in those cells, although adjacent or nearby cells often were
intensely stained for PNMT. Many blood cells found in the atrial
chamber shown in
Figures 2A through 2C were also fluorescently labeled when
viewed with either filter set, but this staining was not specific,
because it could also be observed in the absence of both primary
antibodies (data not shown). For orientation, the embryo sections were
stained with hematoxylin and eosin (H&E) after the fluorescent staining
procedure, and photomicrographs of these H&E sections are shown in the
rightmost column of
Figure 2.
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Two days later in development (E12.5), PNMT immunostaining
remained strong at the atrioventricular (AV) junction
(Figures 2G
through 2L, arrow) but was decreased considerably
in the truncus arteriosus and most other regions of the heart, although
limited expression was seen interspersed throughout the myocardium.
Endocardial-cushion tissue development was prominent at E12.5
(Figures 2I and 2L), but neither -actinin nor PNMT
expression was detected in the cushions. The remaining panels of
Figure 2 depict control immunofluorescent staining. For
example,
Figures 2M through 2O illustrate cardiac muscle fibers from a
postnatal day 8 (P8) rat ventricle. No PNMT expression was observed in
this region at this stage of development
(Figure 2M), but -actinin staining was strong and produced
a characteristic Z-banding pattern
(Figure 2N), as expected for a sarcomeric protein.
Conversely, PNMT expression was robust and localized to the medulla of
a P8 adrenal gland section
(Figure 2P), whereas -actinin staining was completely
absent from the adrenal section
(Figure 2Q). These results demonstrate the specificity of the
PNMT and -actinin antibodies and show that there is no
cross-reactivity or bleed-through staining of the immunofluorescent
signals.
Endogenous production of epinephrine typically requires the
coexpression of DBH and TH in the same cells that express PNMT (see
Figure 1). Therefore, we extended our immunofluorescent histochemical
staining analysis of the developing embryonic rat heart to include
antibodies that specifically recognize DBH and TH. Because the anti-DBH
antiserum was produced in rabbits while the anti-TH ascites fluid was
produced in mice, we were able to perform double-immunofluorescent
labeling experiments with these antibodies. In our initial experiments
with these antibodies, we optimized antibody concentrations and
verified the specificity of immunofluorescent labeling using adult rat
adrenal gland sections (see the online data supplement available at
http://www.circresaha.org). To provide anatomical orientation, we
initially captured low-magnification images of a mid-sagittal E11.5 rat
heart section coimmunofluorescently labeled for PNMT and -actinin
(Figures 3A and 3B, respectively). In this section, PNMT
immunofluorescent labeling was largely concentrated in the
caudal-dorsal portion of the atrium (arrow), immediately adjacent to
the venous inlet. Other regions of this section show limited staining
for PNMT, although we typically observed some staining in the region
immediately caudal to the heart and rostral to the hepatic primordium.
The PNMT-stained cells in this region showed no costaining for
-actinin and were located outside of the cardiac chamber walls.
Nevertheless, cells in this region are thought to subsequently give
rise to the epicardium.19
Within the heart, the cluster of fluorescent cells in the atrial wall
immediately adjacent to the dorsal venous valve represents a
predominant site of PNMT expression at this stage of development. Note
that -actinin staining intensity in this region appears to be
inversely proportional to the PNMT staining in the same section
(Figures 3A and 3B). Higher-magnification images of this cell
cluster are shown in
Figures 3C and 3D (arrow). At this increased magnification,
it is clear that some individual cells are stained for both markers,
whereas others stain for either PNMT or -actinin but not both. DBH
and TH immunostaining in an adjacent section from this same E11.5 rat
heart produced a pattern of staining that was very similar to that
shown for PNMT
(Figures 3E through 3H). A high concordance of immunostaining
was observed for DBH and TH
(Figures 3E and 3F, respectively) as well as for PNMT and TH
(Figures 3G and 3H, respectively) in these sections,
indicating that expression of catecholamine biosynthetic enzymes was
specifically concentrated in a clustering of cells located in the
dorsal-caudal region of the primitive atrial wall immediately adjacent
to the venous valve at E11.5. Because the sinoatrial (SA) node
has also been described in this region at similar stages of development
in the mouse,20 our data
suggest an association between catecholamine biosynthetic enzyme
expression and development of the SA node in the E11.5 rat
heart.
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In other sections from E11.5 rat embryos, we have observed
clusters of cells expressing catecholamine biosynthetic enzymes in the
AV canal region. For example, the confocal images depicted in
Figure 4 show DBH (green) and -actinin (red) expression
in another mid-sagittal section of an E11.5 rat heart. The yellow
regions represent areas where DBH and -actinin are colocalized
(Figure 4A, arrows). As shown in
Figure 3, there is a concentration of DBH staining in the
dorsal venous-valve region of the atrium and sporadic staining in the
walls of the atrium, ventricle, and outflow tract
(Figure 4A). In addition, prominent DBH staining was observed
in the AV canal region
(Figure 4, bracketed region). This staining is particularly
evident in the higher-magnification image shown in
Figure 4B. To gain a more 3-dimensional perspective, we used
the confocal microscope to capture optical sections (2 µm/section)
through the AV canal region. We then reconstructed these images by
stacking the inner
(Figure 4C) and outer
(Figure 4D) optical layers. The cardiac muscle fibers in this
region have well-developed Z-bands, and they appear to form a lattice
network, with isolated clusters of small roundish DBH-expressing cells
found within this lattice. There seems to be very little overlapping
expression for DBH and -actinin in the same cells within the AV
canal region. Similar patterns of staining were observed for TH and
PNMT in other sections
(Figure 3 and unpublished data, February 1998). Thus,
these data show that there are two regions within the E11.5 rat heart
where catecholamine biosynthetic enzyme expression is concentrated: AV
canal and atrial wall adjacent to the dorsal venous valve
cusp.
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We also examined cells that express catecholamine
biosynthetic enzymes at later stages of embryonic development and found
a dramatic shift in their distribution within the heart. Notably, at
E16.5 we observed a major concentration of putative
epinephrine-producing cells along the crest of the interventricular
septum
(Figures 5 and 6). For example, a concentrated cluster of
cells displaying intense immunofluorescent staining for PNMT expression
was observed in the anterior portion of the interventricular septum,
with isolated sporadic cell staining occurring in the middle and lower
regions of the ventricular septum
(Figure 5A). To provide a reference for cardiac muscle cells
in this region, -actinin immunostaining was simultaneously
performed. Intense -actinin immunofluorescent staining was observed
in muscle throughout this region
(Figure 5B). As we observed at earlier developmental stages,
-actinin staining intensity was inversely proportional to PNMT
staining intensity in these sections (compare
Figures 5A and 5B). DBH and TH staining patterns were very
similar to those observed for PNMT at this stage of development, and
these enzymes appear to be colocalized in a knot of cells within the
upper portion of the ventricular septum
(Figures 5C and 5D). This pattern of expression resembles the
developing bundle of His and, to a lesser extent, ventricular
conduction tissue (ie, Purkinje fibers) that has been described at
analogous stages of development in mouse
embryos.21
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To examine this expression in greater detail, we used
laser-scanning confocal fluorescence microscopy after DBH
immunofluorescent labeling of transverse sections through this region
of the heart. Again, we observed a concentrated cluster of positively
labeled cells at the crest of the interventricular septum
(Figure 6). Note that the images shown in
Figures 6A and 6B are 
100 µm anterior to the sections
shown in
Figures 6C and 6D. The DBH-expressing cells were clearly more
concentrated in the anterior portion of the ventricular septum, as was
observed in
Figure 6. At higher magnification
(Figures 6B and 6D), individual cell staining could be
discriminated. The pattern of staining at the single-cell level
appeared to be predominantly cytoplasmic, although the distribution
within the cytoplasm was splotchy and nonuniform. Although not shown in
these images, relatively little staining for catecholamine biosynthetic
enzymes was observed in the SA- and AV-node regions at E16.5 and later
stages of development, although a few positively labeled cells were
observed in these regions. In addition, at somewhat later stages of
development (eg, E19.5), we did not observe this intense cell labeling
along the crest of the interventricular septum after immunofluorescent
staining for catecholamine biosynthetic enzyme expression (data not
shown). These data suggest a transient association between cardiac
catecholamine-producing cells and pacemaking and conduction tissue
development. A summary of these results is provided in the
Table.
At each of the developmental stages indicated, we observed
approximately 200 to 500 cardiac cells that were immunofluorescently
stained for catecholamine biosynthetic enzymes.
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The coexpression of the major catecholamine
biosynthetic enzymes in embryonic cardiac cells before innervation
suggests that epinephrine is produced in the embryonic rat heart. To
determine the epinephrine content of the embryonic rat heart, we
isolated hearts from head and trunk regions of E11.5 rat embryos, as
described previously,1 and
used this material for epinephrine radioimmunoassays. The combined
results of 5 separate experiments are shown in
Figure 7A, where low but detectable concentrations of
epinephrine were found in the heart, head, and trunk regions of E11.5
rat embryos. On average, there was 30% more epinephrine found in
the heart than in either the head or the trunk of E11.5 rat embryos,
but these differences were not significant
(P>0.05). However, the amount
of epinephrine detected in each of these regions was significantly
greater than background
(P<0.001). We estimated the
amount of epinephrine in the E11.5 rat heart on a per-cell basis and
compared it with the amount of epinephrine found on a per-cell basis in
an adult rat adrenal chromaffin cell. A conservative estimate, on the
basis of data generated in the present study, is
3.5x10-8
µmol epinephrine per adult rat adrenal chromaffin cell. This estimate
is similar to the amount of epinephrine reported per adult rat adrenal
chromaffin cell
(1.4x10-7
µmol) by Tomlinson et al22
and translates to 5x1010
molecules epinephrine per adult adrenal chromaffin cell. By comparison,
we calculated that, on average, there were at least
1.6x106 molecules
(2.7x10-12
µmol) epinephrine per E11.5 rat catecholaminergic cardiac cell.
Although the amount of epinephrine present in the embryonic rat heart
was substantially less than that found in a typical adrenal gland
(50 000-fold fewer molecules of epinephrine on a per-cell basis),
our results nevertheless demonstrate that epinephrine and the enzymatic
machinery necessary for its synthesis are present in the
preinnervation-stage embryonic rat heart.
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To assess epinephrine concentrations in the developing rat
embryo at different developmental stages, we measured the amount of
epinephrine present relative to the total amount of protein present in
extracts from heart, head, and trunk regions from pooled tissue samples
isolated at various stages of embryonic development. As shown in
Figure 7B, cardiac epinephrine concentrations were
relatively low at early stages of development but slowly began to
increase at E13.5 and continued through at least E19.5. These
results show that epinephrine was present in the embryonic rat heart
beginning at very early stages of cardiac development and, in contrast
to cardiac PNMT mRNA expression, gradually increased at later stages of
prenatal development.
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Discussion |
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The conduction system of the mammalian heart consists of the SA node, AV node, bundle of His, and ventricular Purkinje fibers.23 Our results show that cardiac cells expressing catecholamine biosynthetic enzymes are present in the embryonic heart, where they seem to be progressively associated with each of these regions of the cardiac conduction system. Beginning
E11.5 in the rat, catecholamine biosynthetic enzymes were
expressed in cardiac cells preferentially localized to the atrial wall
adjacent to the dorsal venous valve cusp and the AV canal. Cells within
these regions of the heart develop into the SA and AV nodes,
respectively.20 21 24
Thus, catecholamine-producing cells were concentrated in regions of the
embryonic heart that later become the SA and AV nodes. A similar
phenomenon was observed several days later in embryonic development
(E16.5), when cells expressing catecholamine biosynthetic enzymes were
found clustered in the developing bundle of His and, to a lesser
extent, in the ventricular septum where the branching bundles and
Purkinje fibers of the ventricular conduction system would be expected
to appear.
The association between catecholamine-producing cells and
the development of the cardiac conduction system was essentially
transient, because early expression (before E11.5) did not seem to be
specifically localized to any particular region of the developing
heart. Moreover, the clusters of adrenergic cells in regions of the
heart that give rise to the SA and AV nodes were not found in these
regions after E12.5, and the labeled cells found concentrated in the
bundle of His region at E16.5 were no longer concentrated in this
region by E19.5. These data suggest two windows of development when
adrenergic cells are preferentially associated with pacemaking and
conduction tissue. The first window occurs between E10.5 and E13.5 and
primarily involves atrial components (future SA and AV nodes), whereas
the second, occurring between E13.5 and E19.5, involves ventricular
components (bundle of His and ventricular septum) of the cardiac
conduction system. It is perhaps because of this limited and transient
pattern of expression that these cells were overlooked in previous
studies. Consequently, this is the first report that identifies a
population of putative catecholamine-producing cells that are
progressively and transiently associated with embryonic cardiac
conduction tissue development. Interestingly, such increasingly
restricted localization to early conduction tissue of reactivity
initially dispersed throughout the tubular heart has been observed with
several other markers, including atrial natriuretic
peptide,25 26
leu-7,27 28 and
embryonic avian
polypeptide.29
The specific origin of cardiac catecholamine-producing cells is not known, but they do not seem to be neuronal, because they do not have a neuronal morphology and they are present in the heart several days before the first appearance of nerve-like cells in and around the heart.6 In addition, unlike the catecholamine-producing cells of the adrenal medulla, the cardiac cells that express catecholamine biosynthetic enzymes do not seem to be derived from the neural crest. Fate-mapping and ablation studies have shown that cardiac neural crest cells migrate into the heart and contribute to outflow septation, formation of cardiac ganglia, and development of aortic arteries.30 Because the pattern of expression observed for catecholamine biosynthetic enzymes in the developing rat heart was completely different from the reported distribution of neural crest cells within the embryonic heart, it seems unlikely that the adrenergic cells identified in the present study were of neural crest origin.
Instead, these adrenergic cells seem to be intrinsically
derived from the primitive myocardium. In support of this hypothesis,
we demonstrated that some embryonic cardiac cells coexpressed
catecholamine biosynthetic enzymes and the muscle-specific marker
-actinin. However, most of the cells that we observed expressed
either -actinin or catecholamine biosynthetic enzymes. These data
suggest that cardiac cells expressing these 2 biomarkers could be
derived from common progenitor cells in the developing cardiac
primordium and are consistent with the findings of Huang et
al,2 who recently described a
population of intrinsic cardiac adrenergic cells in rat and human
hearts.
We have shown that low concentrations of epinephrine are
present in the embryonic rat heart at least as early as E11.5. We also
detected epinephrine in the head and trunk regions in E11.5 rat
embryos. However, because the heart is the primary endogenous tissue
source for the major epinephrine-producing enzymes (TH, DBH, and PNMT)
at this stage of embryonic rat development, we hypothesize that most of
the epinephrine detected in E11.5 rat embryos was synthesized in the
heart. It could have been secreted from embryonic
catecholamine-producing cells in the heart, although it is not clear
whether this would have occurred in vivo or during the isolation and
dissection procedures. In addition, contributions from maternal sources
and other embryonic tissues, such as developing lung buds (see Figure 2
online, available at http://www.circresaha.org), cannot be ruled out.
Interestingly, epinephrine concentrations actually increase in the
heart at later stages of prenatal development despite declining PNMT
mRNA concentrations in the heart after
E12.5.1 At these later stages
of prenatal development (E13.5 through E19.5), the appearance of
adrenergic nerves in the central and peripheral nervous
systems5 may contribute to
some of the increases in epinephrine content observed in the heart as
development proceeds. Similarly, adrenal chromaffin cells begin to
produce epinephrine beginning E15.5 to E16.5 and likely contribute
to the rise in peripheral epinephrine concentrations observed at this
and later stages of
development.3
Studies dating back to the 1930s have demonstrated that
explanted embryonic chick hearts are responsive (increased heart rate)
to exogenously administered epinephrine soon after the heart begins to
beat.31 32
However, it is clear that very early stage intrinsically beating hearts
(eg, E2) do not generally respond to epinephrine until 1 day later
(ie, E3).31 32 In
the developing rat embryo, increased heart rate after isoproterenol
treatment has been demonstrated as early as
E10.5.33 34
Similar to the chick studies, full responsiveness and coupling of
ß-adrenergic receptors to second messengers and ion channel
activities seem to mature at least 1 or 2 days after the initiation of
spontaneous myocardial contractions and the appearance of
ß-adrenergic receptor expression in developing cardiac
cells.35 Consequently,
because the heart is responsive to the actions of catecholamines at
relatively early developmental
stages,33 local production
of catecholamines in the heart could play a significant role in
regulating the beating activity or differentiation of embryonic
myocardial cells. This hypothesis is supported by the finding that
intrinsic heart rates of transgenic mouse embryos lacking the ability
to produce catecholamines were significantly slower than their
age-matched wild-type counterparts and typically died in utero from
cardiovascular
failure.36 37 38
The vast majority of these deaths occurred between E11.5 and E15.5,
which roughly corresponds to the developmental period between E13 and
E17 in the rat. Because we observed expression of intrinsic cardiac
catecholamine biosynthetic enzyme expression in the heart during this
period as well as during the period immediately preceding this window
of development, intrinsic production of catecholamines locally within
the heart could play an important role in the development of cardiac
function. One might expect that the conduction system in these mouse
hearts may not have developed properly and that this could have
contributed to the bradycardia and eventual cardiovascular failure.
Additional functional studies are necessary to test this and other
hypotheses regarding the role of intrinsic catecholamine production in
the developing embryonic
heart.
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Acknowledgments |
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This work was supported by start-up funds from the Institute for Cardiovascular Studies at Georgetown University Medical Center (to S.N.E.) and National Institutes of Health grant HL50582-06 (to R.P.T.).
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Footnotes |
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Original received June 9, 2000; revision received November 14, 2000; accepted November 15, 2000.
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References |
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