© 1997 American Heart Association, Inc.
Articles |
Changing Activation Sequence in the Embryonic Chick Heart
Implications for the Development of the His-Purkinje System
From the Departments of Pediatrics, Medicine, Biomedical Engineering, and Genetics and the Cardiac Bioelectricity Research and Training Center, Case Western Reserve University, Cleveland, Ohio.
Correspondence to Michiko Watanabe, PhD, Division of Pediatric Cardiology, Department of Pediatrics, Rainbow Babies and Children's Hospital, 11000 Euclid Ave, Cleveland OH 44106-6011. E-mail mxw13{at}po.cwru.edu
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Abstract In the mature heart, impulse propagation through the His-Purkinje system (HPS) is required for efficient ventricular contraction in an apex-to-base direction. However, the embryonic heart begins to contract as a myocardial tube without a specialized conduction system. To identify the developmental stage when the HPS begins to function, we mapped the ventricular depolarization sequence from microvolt-level electrograms recorded from embryonic myocardium using 50-µm extracellular electrodes, high-gain amplification, and signal-processing techniques. Analysis of left ventricular activation in 99 embryonic hearts revealed a transition in the activation sequence that was dependent on developmental stage. As the heart develops, a transition in the activation sequence occurred from the primitive base-to-apex pattern (in 20 of 33 hearts) at early stages (Hamburger-Hamilton stages 25 to 28) to the HPS-like apex-to-base pattern (12 of 17 hearts) late in development (stages 33 to 36). Immunohistological experiments (n=10) also confirm that the expression pattern of two biochemical HPS markers changes in parallel with the change to the mature ventricular activation pattern. These data indicate that the ventricular activation sequence in the chick heart develops to a mature pattern at stages 29 to 31, suggesting that preferential conduction through the HPS begins shortly after ventricular septation is complete.
Key Words: conduction • electrophysiology • heart development • His-Purkinje system
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Introduction |
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The HPS is required for coordinated contraction of the mature heart. Propagation of the cardiac impulse through the specialized and rapidly conducting HPS results in an orderly electrical activation from the apex toward the outflow tracts at the base of the heart, resulting in efficient ejection of blood from the ventricles. However, the HPS is not necessary for the function of the primitive heart during early embryonic development. The heart begins beating as a tube of myocardium at the 9- to 11-somite stage of development1 2 and lacks recognizable specialized conduction pathways.3 Cardiac impulses propagate cranially from the sinus venosus, which lies at the most caudal portion of the tubular heart.2 4 The caudocranial activation of the myocardium persists throughout the morphological transformation of the primitive heart from the tubular stages up to the looped heart configuration, when a second pattern of impulse propagation appears. Alternating regions of slow and fast conduction in the looped heart result in sequential activation of the atrium, ventricle, and outflow tract, but the general sequence of ventricular activation remains inflow to outflow.4 5 6 7 In contrast, in the mature four-chambered avian heart,8 like the mammalian heart,9 10 11 12 13 the ventricular apex activates before the base, because of the presence of a functional HPS.
Because little was known regarding the developmental stage when the HPS begins to function, we have developed a technique for recording and analyzing microvolt-level extracellular electrograms to detect the ventricular activation sequence in developing chick embryonic hearts. Specifically, we sought to identify the developmental stage when the LV activation pattern changes from the primitive base-to-apex sequence to the mature apex-to-base sequence, indicating the presence of a functionally mature HPS.
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Materials and Methods |
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Experimental Preparation
White leghorn chicken (Gallus gallus) embryos were staged according to Hamburger-Hamilton criteria.14 Hearts were carefully removed, placed into a tissue bath, and superfused with oxygenated (95% O2/5% CO2) Tyrode's solution (35±2°C) of the following composition (mmol/L): MgCl2 0.7, NaH2PO4 1.2, CaCl2 1.5, KCl 4.6, dextrose 10, NaHCO3 25, and NaCl 114 (pH 7.6). Fine tungsten wire electrodes (50-µm diameter) insulated with polytetrafluoroethylene, except at the tips, were positioned in specific mapping locations using micromanipulators under microscopic guidance (Nikon SMZ-2T, Nikon Corp). Photographs were taken using a camera mounted on the microscope to record the location of each electrode on the surface of the heart. Electrogram recordings were typically obtained from chick hearts within 30 minutes of excision. Since the ventricular wall is thin (
100 µm) relative to the
adult until stage 33, transmural propagation times between the
epicardial and endocardial surfaces were considered negligible, so that
epicardial activation patterns essentially mirrored activation patterns
of the ventricular wall.
Electrophysiological Recordings
Protocol A
To determine the global activation sequence of the embryonic
ventricle for the looped and septated heart configurations, hearts from
24 embryos between stages 22 and 39 were analyzed. In each
experiment, two unipolar extracellular electrodes were
simultaneously positioned on two of four predefined
ventricular mapping sites: (1) LV base, (2) LV apex, (3) RV
base, and (4) RV apex (Fig 1
). Both pairs
of unipolar electrodes were grounded relative to a common reference
electrode in the tissue bath. The recording electrode pair was
moved sequentially to different mapping sites to determine the relative
timing of activation between two ventricular sites
simultaneously. The distance between electrode pairs varied
between 0.2 and 1.8 mm depending on developmental stage. In this
way, the relative activation sequence among the four
ventricular sites was determined.
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) becomes
apex-to-base (
), and RV base (*) in the mature heart. Sites chosen in
Figs 2
Protocol B
To identify the developmental stage at which the HPS begins to
function, it was necessary to accurately determine the timing of
activation between the LV apex and LV base during multiple stages of
development. A change in LV activation from the immature base-to-apex
activation sequence to the mature apex-to-base activation was used to
identify the emergence of a functional HPS. Signal-processing
techniques were used to identify local depolarizations from
microvolt-level extracellular electrograms recorded in these
preparations (see below). A reference bipolar electrogram was
recorded from electrodes positioned on the atrium and the RV (Fig 2). This electrogram was used to monitor
cardiac rhythm and as a temporal reference of beat-to-beat
ventricular activation. An additional unipolar mapping
electrode was positioned sequentially on the LV apex and base to
measure the timing of local activation at these sites (Fig 2, mapping
electrode). Because the timing of local depolarization at the mapping
electrode site was made relative to the reference signal, care was
taken to precisely maintain all the electrodes in their exact positions
during the course of each experiment. Consistent positioning of
the electrodes to the appropriate morphological positions was carefully
maintained in all the experiments. The distance between LV apex and LV
base recording sites varied from 0.5 to 1 mm, as estimated
using photomicrographs. Seventy-five hearts from embryos between stages
25 and 36 were analyzed using this protocol.
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Protocol C
To determine whether the changes in activation pattern were
directly correlated with changes in the expression pattern of
biochemical markers for the HPS, both
electrophysiological and
histological data were compiled for the same heart.
Electrophysiological recordings were made
on freshly excised hearts from 10 embryos between stages 25 and 36. In
these experiments, unipolar electrograms were recorded
simultaneously from the LV apex and base to determine the
precise sequence of LV activation. These same hearts were then
processed histologically (see
"Histological Analysis").
All recordings in each experimental protocol were made during
spontaneous (ie, sinus) rhythm. Electrograms were recorded with
high-gain (x10 000) low-noise amplifiers (Gould, Inc), which included
a stage of preamplification using separate amplifiers positioned
adjacent to the preparation to minimize ambient noise. The signals were
band-pass–filtered (10 to 300 Hz) and digitized with 12-bit precision
at a sampling rate of 5000 to 10 000 Hz with a UNIX microcomputer
(Concurrent 5450S, Concurrent Computer Corp). Preliminary
analysis on the frequency content of the unipolar signals
recorded from these preparations showed that low-frequency
components were not attenuated by this filtering protocol. Electrograms
were monitored throughout each experiment (1 hour) to ensure the
stability of the cardiac rhythm and the activation sequence.
Data Analysis
Local depolarization was determined from unipolar extracellular
electrograms as the time point of maximum negative derivative
(–[dV/dt]max).17 Derivatives were
calculated from digitized electrograms using successive differences and
a five-sample-point running average. In protocols A and C, the sequence
in which activation occurred was determined by comparing the earliest
depolarization times between electrograms recorded
simultaneously from two mapping sites.
In protocols B and C, the mapping-electrogram recordings from
at least 10 consecutive beats were averaged in order to maximize the
accuracy in detecting local activation time differences between
ventricular mapping sites. For the purposes of averaging,
the mapping electrograms were temporally aligned using a fiducial point
derived from the ventricular deflection of the reference
electrogram (Fig 3, solid inverted
triangles). Consequently, mapping electrograms were recorded with
voltage and temporal resolutions of 1 µV and 200 µs, respectively,
making it possible to analyze microvolt-level electrograms
recorded during each stage of development. Depolarization times
were then determined from the –[dV/dt]max of the
averaged mapping electrogram recorded from the LV apex and LV base
(Fig 3). Determination of activation sequences from unaveraged
electrograms and signal-averaged electrograms obtained using protocol C
was consistent.
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In a series of preliminary studies, electrograms were recorded repeatedly from one ventricular site to determine the reproducibility of estimating activation time using the procedure described under protocol B. The beat-to-beat variability of measuring local depolarization was associated with a standard deviation of 300 µs. Therefore, depolarization time differences between apex and base were considered significant only when the difference was at least three times greater than the uncertainty in the measurement, ie, three times the standard deviation. Consequently, the LV activation sequence was defined as base-to-apex or apex-to-base when the depolarization time from base-to-apex was >1000 µs or <-1000 µs, respectively. The LV activation sequence was classified as concurrent when depolarization of apex and base occurred within 1000 µs of each other.
Histological Analysis
Immunohistochemistry was performed using the following
modifications of methods described previously.18 After
electrograms were recorded by following protocol C, hearts were
dehydrated in graded ethanol and 20% sucrose in PBS solution. Hearts
were fixed in gelatin and then serially cryosectioned at 20-µm
thickness. Sections were incubated in the presence of the following
antibodies diluted in 10% natural goat serum in HBSS: anti-titin
(monoclonal mouse IgG, diluted 1:1000; Sigma Chemical Co) was used to
identify cardiac myocytes19 ; HNK-1 (mouse IgM, 1:1000) and
anti-PSA (clone 5A5, monoclonal mouse IgM, 1:500) are
glycoprotein antibodies that in combination identify the
cardiac conduction system in the developing chick.18
Combined HNK-1 and PSA carbohydrate expression was observed using a
fluorescein-conjugated anti-mouse-IgM antibody (1:1000,
Cappel/Organon Teknika); titin expression was viewed using a
rhodamine-conjugated anti-mouse IgG antibody (1:1000,
Cappel/Organon Teknika). Negative control experiments showed
that HNK-1 and PSA antibodies did not interfere with each other when
simultaneously or sequentially incubated. Furthermore, the
labeled second antibodies were specific for the appropriate antibody
subtypes and did not cross-react with inappropriate subtypes.
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Results |
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Electrogram Characteristics
Electrograms recorded from one representative experiment are shown in Fig 3
. Atrial and ventricular
depolarizations were easily identified from the reference electrogram,
verifying the presence of stable sinus rhythm. The amplitude of
unipolar mapping electrograms varied from 1.5 to 100 µV, and the
value of –[dV/dt]max varied from -30 to -250 mV/s,
depending on developmental stage. As expected, both the amplitude and
–[dV/dt]max of the extracellular electrograms increased
as heart mass increased at later developmental stages. No extreme
beat-to-beat variations in signal amplitude were observed in any one
set of recordings. In addition to using high-gain low-noise
amplification, signal averaging was critical to the analysis of
low-level electrogram signals recorded from embryonic hearts.
Although unprocessed electrograms contained a signal-to-noise ratio
that ranged from 4 to 10, signal-averaging dramatically increased the
signal-to-noise ratio by as much as 10-fold. In the example shown in
Fig 3B, averaging of 26 electrogram signals increased the
signal-to-noise ratio from 6 to 57. Although the unprocessed mapping
electrogram exhibited considerable noise (Fig 3A), after averaging
(Figure 3B), the electrogram contained no visually apparent noise, and
the time point of –[dV/dt]max (arrow) can be clearly
identified from this signal.
Global Ventricular Activation Sequence
Since few studies have characterized electrical activation of the
entire ventricle at various stages of embryonic cardiac morphogenesis,
surface electrograms were analyzed from 23 embryonic hearts
during looped and septated periods of development (stages 22 to 39)
using protocol A. In all studies of RV activation, the RV apex always
depolarized before the RV base, regardless of the age of the embryo.
However, activation of the LV was highly dependent on developmental
stage. In stage-22 to -29 hearts, LV activation always preceded RV
activation, and the activation of the LV base always preceded
activation of the LV apex. This sequence reflects the presence of the
immature activation pattern seen in both the tubular (Fig 1A) and the
looped (Fig 1B) hearts. On the other hand, in the majority (77%) of
stage-31 to -39 hearts, activation was initiated
simultaneously at the LV and RV apices and spread toward
the base of the heart, consistent with the mature activation
sequence (Fig 1C). No beat-to-beat variations in activation sequence
were observed. Since the HPS is responsible for rapid and preferential
propagation, the reversal in LV activation to the mature apex-to-base
sequence was used as a marker for the presence of a functional HPS.
Transition to Mature LV Activation Sequence
To more precisely determine the developmental stage when the
mature LV activation sequence emerges, unipolar electrograms
recorded from 75 chick hearts in stage-25 to -35 embryos were
carefully analyzed using protocol B. Attention was paid to
identifying a relationship between the changes in the LV activation
sequence and the morphology of the embryonic heart. Based on
morphological data, these stages were subdivided into three periods of
cardiac morphogenesis: the preseptation, transitional, and
postseptation periods, corresponding to stages 25 to 28, 29 to 31, and
32 to 36, respectively. The electrograms shown in Fig 4 illustrate the typical patterns of LV
activation as observed in three representative hearts
during embryonic stages 25, 29, and 35. The arrows indicate the
–[dV/dt]max of each electrogram corresponding to the
time point of local activation. Before stage 30 (Fig 4A), activation
spread from the LV base to apex. Of all preseptated hearts exhibiting
this pattern of activation (n=20), basal LV activation preceded apical
LV activation by 3.2±0.5 ms. During the period of
ventricular septation (ie, stages 29 to 31), the LV apex
and base depolarized nearly simultaneously, giving rise to
a concurrent pattern of LV activation (Fig 4B). In contrast,
electrograms recorded from the postseptated embryonic heart (Fig 4C) demonstrated a switch to the apex-to-base LV activation sequence.
Note also that electrograms recorded in these more mature hearts
exhibited larger amplitudes than did the preseptated hearts. Among all
the postseptated hearts exhibiting this pattern of activation (n=12),
apical LV activation preceded basal LV activation by 4.1±0.5 ms.
Again, no beat-to-beat variation in activation sequence was observed
during any of these recordings, and these patterns persisted as
long as 60 minutes after excision of the heart.
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) and base (
) are marked. A,
Base-to-apex pattern from Hamburger-Hamilton
The LV activation sequences measured from all 75 embryos are shown in
the Table. Note that (1) the early stages
of embryonic development are typically associated with base-to-apex LV
activation; (2) there is a transitional period between stages 29 and
31, where the concurrent LV activation pattern predominates; and (3) in
later stages, ie, after stage 31, the apex-to-base pattern of LV
activation predominates. Note also that there were exceptions to this
pattern during each developmental period. These results are summarized
in Fig 5, where the prevalence of each
pattern of LV activation is shown for preseptated, transitional, and
postseptated periods of development. The preseptated heart was most
often characterized by the immature base-to-apex activation sequence.
During the transitional period when the ventricular septum
develops, the LV activation sequence does not follow a clear pattern,
because there was a near equal distribution of apex-to-base,
base-to-apex, and concurrent activation patterns. Postseptated hearts,
however, largely exhibited the apex-to-base LV activation sequence. The
relationship between the prevalence of the immature base-to-apex
activation pattern or the mature apex-to-base activation pattern with
the developmental periods of the embryonic hearts (preseptated and
postseptated hearts, respectively) was found to be highly significant
(
2=21.7, P<.001). Taken together, the
data shown in Fig 5 indicate that the preseptated heart uses a
primitive activation sequence, whereas around the time of
ventricular septation (stages 29 to 31), there is a
transitional period when the mature LV activation sequence emerges,
indicating the developmental stage when the HPS begins to function.
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Immunohistological Correlations with LV
Activation Sequence
As further confirmation that the switch to apex-to-base LV
activation was indeed a marker for functional maturation of the HPS,
electrical activation patterns were compared with the expression of
morphological markers of the HPS. From previous studies, we have
identified two carbohydrate epitopes whose complementary expression
patterns in the embryonic chick heart by stage 32 resemble the
HPS.18 The trabeculae and bundle branches were
immunopositive for PSA,20 a carbohydrate that is expressed
on the cell adhesion molecule NCAM.21 22 In contrast, the
common His bundle expresses the HNK-1 epitope, a carbohydrate moiety
that is expressed on many cell adhesion molecules.23 24 25 26 27
Together, these epitopes appear to delineate the HPS continuously from
the common bundle to the Purkinje fibers lining the
ventricular lumen in hearts from stage 32 or older embryos,
but not at stages before stage 28.18 To determine whether
the changes in the LV activation pattern directly correlate with
dynamic changes in the expression patterns of these carbohydrate
epitopes, embryonic hearts were analyzed both
electrophysiologically and
histologically, using protocol C (see "Materials and
Methods").
Of the 10 hearts analyzed in this fashion,
representative samples of the preseptated (n=4) and
postseptated (n=6) periods of development are shown in Fig 6. In the preseptated heart (stage 25;
Fig 6, top), the signal-averaged electrogram reveals a base-to-apex LV
activation pattern (compare position of arrows in trace b with trace
a). Histological sections from this heart revealed
light and incomplete HNK-1 and PSA antibody staining for HPS components
(section on left) at the atrioventricular junction from
the primitive atria along the dorsal wall to the
ventricular trabeculae. The
myocardium-specific marker titin (section on right)
confirmed that this restricted area of PSA/HNK-1 expression (arrows) is
myocardial and not extraneous staining of the endocardial cushions. In
contrast, the postseptated heart (stage 34; Fig 6, bottom) revealed
apex-to-base LV activation (see bottom tracing). Moreover,
histological sections of this heart reveal continuous
HNK-1/PSA antibody staining in myocardial cells (titin costaining, data
not shown) in a pattern resembling the HPS from the bifurcation
of the His bundles to the ventricular
trabeculae of the LV and the RV. Therefore, the emergence
of a mature apex-to-base LV activation sequence coincides with the
biochemical expression and histological appearance of
HPS tissue, reaffirming that the change in the LV activation sequence
may be indicative of biochemical HPS maturation and that this process
begins after stage 30.
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Discussion |
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Measuring Electrical Activation in Embryonic Heart
In these experiments, extracellular electrograms were microvolt-level signals due to the very small myocardial mass of embryonic chick hearts. The amplitude of electrograms recorded from the embryonic ventricle was typically <40 µV, even during more mature stages of development, when myocardial mass increases. This trend agrees with previous observations made from action potential recordings.28 29 The minute dimensions of the embryonic ventricle further compound the problem of measuring the activation sequence, since activation time differences are so small (several milliseconds) between ventricular mapping sites. Our studies demonstrate that high-gain amplification, signal-averaging techniques, and rapid digital sampling can provide the voltage and temporal resolutions necessary to detect and analyze these small electrograms and to deduce activation patterns in the embryonic chick ventricle. Our analysis assumes that activation patterns on the epicardial surface reflect activation on the endocardium, where the HPS is developing. This is a reasonable assumption, since the ventricular wall does not begin to thicken appreciably until after stage 35.30 Therefore, the conduction time between the endocardial and epicardial surfaces (separated by
100 µm) are negligible compared with conduction between the
LV apex and base (1000-µm separation).
Activation Sequence in Embryonic Heart
The general pattern of cardiac conduction changes while the heart
undergoes morphogenesis. The tubular heart is characterized by slow
homogeneous propagation originating from the primitive
pacemaker in the caudal region (9-somite embryo, stage
10–),15 which results in a peristaltic-like
pattern of contraction (Fig 1A). Regions of alternating slow and fast
conduction have been demonstrated beginning at stage 13, when distinct
atrial and ventricular chambers are
detectable5 6 7 (Fig 1B). Delays of electrical propagation
at the sinoatrial junction, the atrioventricular
junction, and the outflow tract result in sequential contraction of the
atrium, ventricle, and outflow tract. Significant increases in
conduction velocity also occur with increasing developmental
stage.7
The main finding of the present study is that the mature
ventricular apex-to-base activation sequence begins between
stages 29 and 31, a period in heart development characterized by
completion of interventricular septation30 31
and maturation of the coronary circulatory
system.30 This is significant, since before stage 29, the
circulation is in series (atrium, LV, bulboventricular
foramen, RV, and outflow) and therefore functions most optimally by a
correspondingly sequential activation sequence. Beyond this stage, the
circulation is a more parallel circuit; each ventricle now has a
separate inflow and outflow and is better served by an apex-to-base
pattern of ventricular activation to optimize the ejection
of blood. Although we observed a consistent transition in LV
activation sequence across all preparations (Fig 5), the developmental
stage at which the switch to the mature activation pattern occurred
either very early or very late in some embryos. This may be related to
biological variability, errors in staging, or minor variations in
electrode placements between hearts.
de Jong et al7 have suggested that ventricular apex-to-base activation begins at stage 23 and have proposed that "preferential conduction apparently passes through the trabeculae," giving rise to an apex-to-base activation that resembled a mature HPS-like activation pattern. Although conduction system precursors in the ventricular trabeculae may exist at these earlier stages,20 our data suggest that these precursors do not begin to function as a mature HPS until stages 29 to 31. One possible explanation for the apparent discrepancies between our results and those of de Jong et al is that these investigators restricted their analysis of ventricular activation to the RV. In our experiments, the RV activates in an apex-to-base direction during all developmental stages, whereas the LV activation sequence consistently changed to the apex-to-base pattern during a specific period of development, ie, stages 29 to 31. This finding reaffirms the importance of focusing on a detailed analysis of the LV activation sequence to study the development of the HPS.
Implications for Development of the HPS
The development of the HPS has been studied using conventional
histochemical methods and, more recently, by the use of specific
antibodies or mRNAs. The combined results of these analyses
strongly support the idea that HPS precursors arise from a subset of
the myocardium32 33 and that at least the
precursors of the central HPS components (common His bundle and bundle
branches) show differential gene expression at the mRNA or
protein/carbohydrate level well before we have detected evidence for
HPS function.32 33 34 Nonetheless, a number of significant
morphological and biochemical transformations occur at the time of
initiation of HPS function. First, the central components of the HPS
become distinct by conventional histochemical staining from the
"beginning of the middle third of incubation" in the avian
embryo,35 36 which translates to stage 30 for the chick.
The avian HPS at this stage has been reported to have a more compact
texture and a darker staining intensity than the surrounding
myocardium, which probably reflects cellular differences in
composition and organization. The fascicular pattern of the HPS is
probably also accentuated by the development of connective tissue that
may begin to ensheath portions of the conduction system. Second, we
have recently found that the HPS is undergoing a stage of biochemical
maturation at this time by becoming distinctly
immunostained for cell surface markers that are associated
with changes in cell-cell and cell-matrix interactions.18
Our immunohistochemical findings reveal that at stages where both
biochemical markers are not distinctly expressed in presumed HPS
precursors, a base-to-apex activation pattern is observed in the LV.
Conversely, when both biochemical markers distinctly stain myocardial
cells that appear to be part of the HPS, the LV activation pattern is
apex-to-base, resembling HPS conduction.
To date, the functional development of the HPS is not well understood. Conduction through the HPS appears to begin around the same time that muscular connections at the atrioventricular junction are being greatly reduced, severed, and replaced with connective tissue (between stages 29 and 34), so that the only remaining atrioventricular connection for propagation would be the HPS.37 38 39 What mechanisms guide the separation of atrium and ventricle or how this process is coordinated with the onset of HPS activation is unknown. It is possible that the concurrent activation pattern we identified at stages 29 to 31 is due to conduction through both the HPS and residual muscular atrioventricular connections. Last, cellular action potential recordings similar to mature atrioventricular node and His bundle cells have been identified in preseptated chick hearts beginning around stage 28,40 suggesting that components of the HPS are in place but that subsequent maturation is necessary for HPS function in its final form. Future studies will analyze the anatomic processes that may govern HPS functional maturation using both immunohistochemical and electrophysiological techniques that may explain this concurrent activation pattern.
Conclusions
High-fidelity
electrophysiological recordings of
the embryonic chick heart have identified a period in cardiac
development when the activation sequence changes such that conduction
from the atrioventricular junction proceeds directly to
the apex of both ventricles, presumably because of the emergence of a
functional HPS. This period of heart development coincides with stages
29 to 31 and occurs in concert with numerous other morphological and
biochemical changes that may play a role in initiating or facilitating
HPS conduction. These findings should be helpful in future studies
aimed at identifying the molecular mechanisms governing HPS
differentiation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-38172 (Dr Watanabe) and HL-54807 (Dr Rosenbaum) and by the Medical Research Service of the Department of Veterans Affairs, The Whitaker Foundation, and the American Heart Association (Dr Rosenbaum). We are grateful to Dr Steven Girouard, Dr Kenneth Laurita, and Joseph Pastore for their technical assistance with the electrophysiological data analysis.
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Footnotes |
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1 Both authors contributed equally to this work.
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995.
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References |
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