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Am J Physiol Heart Circ Physiol
280: H1889–H1895, 2001.
Optical mapping of activation patterns
in an animal model of congenital heart block
MARK RESTIVO,1,2 DMITRY O. KOZHEVNIKOV,1 AND MOHAMED BOUTJDIR1,2
Molecular and Cellular Cardiology Program, Veterans Affairs New York Harbor Health Care
System, and 2State University of New York Health Science Center, Brooklyn, New York 11209
1
Received 18 May 2000; accepted in final form 8 November 2000
(8) and McCue et al. (18) noted more
than 20 years ago that women who gave birth to
children with congenital heart block (CHB) often had
autoimmune diseases. It is now well established that
CHB detected before or at birth, in the absence of
structural cardiac abnormalities, is strongly associated
with maternal antibodies to SSA/Ro and/or SSB/La
ribonucleoproteins (6). Though more prevalent in the
presence of autoimmune disorders, CHB in offspring is
independent of whether the mother has systemic lupus
erythematosus, Sjögren’s syndrome, or is totally
asymptomatic (6, 26). Damage to the cardiac conduction system occurs in an otherwise normal fetus and is
presumed to arise from transplacental passage of these
maternal autoantibodies (immunoglobulin; IgG) (25).
Other neonatal abnormalities affecting the skin, liver,
and blood cells have also been reported (26) and are
associated with these maternal IgG. Although varying
degrees of conduction block can occur, third-degree
atrioventricular (AV) block, which is irreversible, carries substantial morbidity and mortality that approaches 30%. More than 60% of the children affected
by AV block require lifelong pacemakers (26). Noncardiac manifestations are transient, resolving ⬃6 mo
after birth, coincident with the disappearance of maternal IgG from infant circulation (7).
The candidate antigens and their cognate antibodies
have been extensively characterized at the molecular
level. The 60-kDa SSA/Ro contains a putative zinc
finger and an RNA-binding protein consensus motif (2),
both of which could account for its direct interaction
with small cytoplasmic hY-RNAs, a class of low-molecular-weight molecules of 83–112 bases, which are
small uncapped cytoplasmic RNA (16). This protein
may function as part of a novel quality control or
discard pathway for 5S rRNA precursors in Xenopus
oocytes (22). Many sera, which recognize the 60-kDa
SSA/Ro protein, also react with another protein of 52
kDa (1). Anti-SSB/La antibodies recognize a 48-kDa
polypeptide, which does not share antigenic determinants with either 52-kDa or 60-kDa SSA/Ro (9).
SSB/La facilitates the maturation of RNA polymerase
III transcripts, directly binds a spectrum of RNAs, and
associates at least transiently with 60-kDa SSA/Ro
(13).
We recently (3, 4) provided evidence that IgG-enriched fractions and anti-52-kDa SSA/Ro antibodies
affinity purified from sera of mothers whose children
have CHB induce complete AV block in Langendorff
perfused hearts and inhibit the L-type Ca2⫹ current
(ICa,L). In addition, immunization of female BALB/C
mice with recombinant ribonucleoproteins generated
high-titer antibodies, which crossed the placenta during pregnancy and were associated with varying degrees of AV conduction abnormalities in the pups,
including complete AV block (4, 17, 20). We unexpectedly found significant sinus bradycardia in mouse pups
born to mothers injected with human maternal anti-
Address for reprint requests and other correspondence: M. Restivo,
Dept. of Veterans Affairs, New York Harbor Health Care System,
Cardiology Division (111A), 800 Poly Pl., Brooklyn, NY 11209
(E-mail: [email protected]).
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.
cardiac electrophysiology; autoantibodies; atrioventricular
node
CHAMEIDES ET AL.
http://www.ajpheart.org
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Restivo, Mark, Dmitry O. Kozhevnikov, and Mohamed Boutjdir. Optical mapping of activation patterns in
an animal model of congenital heart block. Am J Physiol
Heart Circ Physiol 280: H1889–H1895, 2001.—Congenital
heart block (CHB) is associated with high mortality and
affects children of mothers with autoantibodies (IgG) to ribonucleoproteins SSB/La and SSA/Ro. IgG from mothers of
children with CHB (positive IgG) was used to assess activation patterns in both the right atrium (RA) and right ventricle (RV) of Langendorff-perfused young rabbit hearts. Optical
action potentials (AP) were obtained by using a 124-site
photodiode array with 4-[-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium. Optical APs were recorded to simultaneously image activation patterns from the RA and RV.
Perfusion of positive IgG (800–1,200 ␮g/ml) resulted in sinus
bradycardia and varying degrees of heart block. Activation
maps revealed marked conduction delay at the sinoatrial
junction but only minor changes in overall atrial and ventricular activation patterns. No conduction disturbances were
seen in the presence of IgG from mothers with healthy
children. In conclusion, besides atrioventricular (AV) block,
positive IgG induces sinus bradycardia. These results establish that the sequelae of CHB are associated with impaired
intrasinus and/or sinoatrial conduction. The findings raise
the possibility that sinus bradycardia in the developing heart
may indicate the potential for AV conduction disturbances.
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OPTICAL MAPPING AND CONDUCTION ABNORMALITIES
METHODS
Surgical procedure and experimental setup. The rabbit
model was chosen for several reasons. First, the dimensions
of the heart are well suited for the spatial resolution of the
optical system and permit imaging of large planar surfaces of
the heart and specific cardiac structures (15, 24). Second, the
optical resolution of the system can be adjusted from 400 to
Fig. 1. Experimental setup. A: view of right atrium and
ventricle. B: superimposition of optical grid on heart. 1,
left ventricle; 2, right ventricle (RV); 3, left atrium; 4,
right atrium (RA); 5, superior vena cava (SVC); 6,
inferior vena cava (IVC); and 7, aorta.
1,800 ␮M/pixel, depending on the dimension of the region of
interest, and electrophysiological observations can be correlated with high resolution with the anatomic features of the
heart (10). Third, the electrophysiological properties of the
ionic currents that constitute the rabbit action potential (AP)
have been well characterized and may better represent the
human heart rather than smaller animals (21).
Young New Zealand White rabbits of either sex, weighing
1.5–2 kg, were anesthetized by intravenous injection of fentanyl citrate (100 ␮g/kg ⫹ 15 ␮g 䡠 kg⫺1 䡠 h⫺1) with heparin
sodium (1,000 U/kg). A tracheotomy was performed and the
animal intubated with an endotracheal tube. The rabbit was
ventilated with room air via a positive pressure ventilator
(MD Industries; Mobile, AL). A midline thoracotomy was
performed and the heart was exposed in a pericardial cradle.
The caval veins were isolated and loose ligatures were placed
around the inferior and superior vena cavae. The heart was
rapidly excised and placed in cold oxygenated Tyrode solution containing 1,500 U/l heparin sodium. The heart was
then cannulated and retrogradely perfused in a modified
Langendorff setup and optical perfusion chamber.
The animal procedures conformed to the principles embodied in the Declaration of Helsinki. All of the experimental
protocols were approved by the Animal Studies Subcommittee of the Research and Development Department of the
Department of Veterans Affairs, New York Harbor Healthcare System, and all procedures related to animal use comply
with the Guiding Principles for Research Involving Animals
and Human Beings.
The perfused hearts were positioned in a fluid-filled Plexiglas chamber, as previously described (24). Placing the heart
against an optically clear glass surface minimizes spatial
aberration and provides for the highest fidelity signals while
minimizing motion artifacts. A plastic cannula was inserted
through the right atrium from the inferior vena cava to the
superior vena cava (see Fig. 1A). Ligatures at the caval veins
and a ligature at the tip of the right atrial appendage were
affixed to three posts within the chamber. This provided for a
smooth surface for optical imaging. Figure 1B shows the
perfused heart setup. The heart was situated against the
imaging surface by positioning a Lexan plunger posterior to
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bodies (17) or immunized with recombinant antigens
(4, 20). These findings brought clinical attention not
only to AV nodal disorders but also to sinoatrial (SA)
nodal conduction abnormalities. In this regard, Brucato et al. (5) recently reported similar sinus bradycardia in infants born to mothers seropositive to antiSSA/Ro antibodies, suggesting that the spectrum of
conduction abnormalities associated with maternal
IgG extend beyond the AV node.
The electrophysiological mechanisms by which maternal antibodies affect electrical propagation throughout the intact heart remain unclear. This may be due in
part to the inability to map electrical activity patterns
by using conventional microelectrode techniques in isolated multicellular preparations (3). Furthermore,
whereas the primary electrophysiological consequence
of maternal antibodies is AV block, little, if anything, is
known about the effects of these maternal IgG on both
atrial and ventricular activation and repolarization
patterns. Sinus bradycardia might well be an early
manifestation of CHB and may prove to be a critical
clinical marker preceding the onset of advanced AV
conduction disorders in autoimmune-associated CHB.
Driven by these observations, we mapped cardiac activation patterns in Langendorff-perfused young rabbit
hearts (atria and ventricles) exposed to maternal IgG.
Optical mapping techniques, which reveal the twodimensional spread of activation, were used to study
propagation abnormalities by using a 124-element photodiode array during sinus and paced rhythms.
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OPTICAL MAPPING AND CONDUCTION ABNORMALITIES
immunoblot (3, 4, 17, 20). This will be referred to in the text
as positive IgG. Control IgG (negative IgG) was purified from
sera of healthy mothers (n ⫽ 3) with healthy children who
tested negative for anti-SSA/Ro and anti-SSB/La antibodies
by ELISA and immunoblot, as described above (3, 4, 17, 20).
An IgG concentration of 800–1,200 ␮g/ml was used in the
experiments. The selected doses were based on our previous
observations (3), in which a complete AV block was induced
at 800 ␮g/ml in the rat heart.
Data analysis. All data are presented as means ⫾ SE.
Statistical tests were performed by using Systat for Windows, version 8 (SPSS; Chicago, IL). AP characteristics were
compared by Student’s t-test for paired and unpaired data
where appropriate. Differences in activation time (total atrial/ventricular activation time) and cardiac intervals (P-R
interval and R-R interval) were compared by ANOVA. Data
before and after IgG application were compared by using
ANOVA for repeated measures. If ANOVA failed to reach
statistical significance, Bonferroni’s correction for paired
data was also applied to test for effect.
Optical APs were mapped by using a photodiode array (124
sites) and the fluorescent dye di-4-ANEPPS. The time of
atrial or ventricular activation for each optical AP in the grid
corresponded to the peak first temporal derivative of the
upstroke (phase 0). The rate of change of fluorescence (dF/dt)
was computed by using a four-point central difference formula and was applied to the normalized optical signal. Repolarization corresponded to 90% recovery of the normalized
optical AP duration (APD90) (11). For this study, APD90 is
defined as the difference between these intervals. Peak dF/dt
and APD90 were computed by using a sampling interval of
0.64 ms. Isochronal activation maps were constructed with
contours drawn at 1- to 3-ms intervals. Analysis was both
descriptive and quantitative.
RESULTS
In the present study, 15 isolated hearts were used
with 3 hearts used for sham study. Ten experimental
hearts were exposed to 800 ␮g/ml positive IgG, and in
two experiments a dose of 1,200 ␮g/ml positive IgG was
used. Of the 10 hearts exposed to 800 ␮g/ml positive
IgG, 4 hearts were studied by using a high data acquisition rate (0.64 ms/sample) to compare dF/dt and
APD90 before and after IgG. In the sham experiments,
hearts were exposed to 1,200 ␮g/ml negative IgG (i.e.,
control IgG obtained from healthy mothers with
healthy children) and considered as controls for positive IgG. As shown in Table 1, there was no significant
change in ECG parameters from control in these
hearts. In 9 of 12 hearts exposed to positive IgG,
complete heart block (3° AV block) was obtained. Only
Table 1. Summary of electrocardiographic
measurements in hearts with and without IgG
n
Interval, in ms
Control
IgG
Positive IgG†
9
Negative IgG
3
R-R
P-R
R-R
P-R
380 ⫾ 25
63 ⫾ 6
337 ⫾ 26
55 ⫾ 4
781 ⫾ 67*
121 ⫾ 9*
344 ⫾ 18
56 ⫾ 6
Values are means ⫾ SE; n, no. of hearts. All IgG measurements
were made 15 min after start of perfusion. * P ⬍ 0.05 compared with
control. † Three rabbits developed 3° atrioventricular block before
electrocardiographic measurements could be performed.
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the imaged plane. The plunger also served to seal the chamber, and the exterior of the heart was bathed in the effused
Tyrode solution. A probe was positioned within the chamber
to monitor temperature. With this experimental setup, epicardial temperature gradients were ⬍0.2°C (15).
Hearts were perfused with oxygenated (95% O2-5% CO2)
modified Tyrode solution containing (in mM) 130 NaCl, 25
NaHCO3, 1.20 MgSO4, 4.75 KCl, 10 dextrose, and 1 CaCl2
(pH 7.4, 37°C). Perfusion pressure was maintained at 70–80
mmHg by adjusting the flow rate of a constant flow peristaltic pump. Hearts were stained with a voltage-sensitive dye,
4-[-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4ANEPPS, Molecular Probes; Eugene, OR) by injection of
150–200 ␮l (from a 3 mM stock solution) into an air bubble
trap (5-ml volume) immediately proximal to the cannulated
heart. The dye was infused over a 5-min period. Data were
acquired after a 15-min stabilization period.
The surface of the heart was illuminated with the use of
two halogen light sources. Light was passed through 520 ⫾
20-nm interference filters, collimated, and focused on the
heart surface with an incidence angle of ⬃45°. Fluoresced
and scattered light was collected from the stained heart with
the use of a high numerical aperture, epi-illumination lens
(50 mm, 1:1.4 E series, Nikon; Garden City, NJ). Collected
light was passed through a 645-nm cutoff filter (model RG645; Schott Glass) and focused onto a 12 ⫻ 12 photodiode
array (Centronics; Newbury Park, CA). The physical dimensions of the photodiode array were 18 ⫻ l8 mm; the dimension was 1.4 ⫻ l.4 mm per diode with a 0.1-mm dead space
between each diode. At ⫻1.5, the signal from each diode
corresponded to an area of ⬃1 ⫻ 1 mm. The depth of field was
⬃200 ␮m (15). Other details of the optic arrangement can be
found elsewhere (15, 24).
Amplified signals were multiplexed and analog to digital
converted and transferred to a Pentium computer (Dimension XPS-T550, Dell Computers; Round Rock, TX). Data were
filtered at a band-pass range of 0.05–1,000 Hz. Because
optical signals cannot be monitored continuously and because the occurrence of spontaneous events cannot be predicted a priori, a circular memory buffer was used to acquire
data with an adjustable pretrigger of up to 40 s. The digitized
data were monitored and then transferred to the computer
hard drive after each epoch. At the end of each experiment,
data were archived on a recordable CD-ROM drive (model
HP8100, Hewlett-Packard; Palo Alto, CA).
Stimulation and electrocardiogram. Stimulation electrodes were placed at the right atrial appendage near the
superior vena cava. The bipolar stimulation electrodes were
composed of Teflon-coated silver wires (75-␮m diameter) with
an exposed length of 500 ␮m. The hooked ends were inserted
into the myocardium with an interpolar distance of ⬍0.5 mm.
Stimulation was applied from a constant current source at
two times diastolic threshold.
An electrocardiogram (ECG) was recorded from two electrodes located in positioning plungers at opposite sides of the
heart. The ECG was filtered at a band pass of 0.05–250 Hz
and monitored continuously on a digital oscilloscope (model
2522A, BK Precision; Chicago, IL). The same ECG was also
recorded simultaneously with the optical signals.
IgG purification. Purification of IgG was performed as
previously described (3, 4, 17, 20). Briefly, immunoglobulin
fractions containing IgG were purified from serum by protein
A-sepharose columns and confirmed pure by electrophoresis.
Throughout this study, we tested IgG from mothers (n ⫽ 3)
whose children have CHB and contain antibodies against
48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro, as
tested by enzyme-linked immunosorbent assay (ELISA) and
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OPTICAL MAPPING AND CONDUCTION ABNORMALITIES
Table 2. dF/dt and APD90 values before and after
positive autoantibodies
Atrium
Control
IgG
%Change
Ventricle
n
APD90
dF/dt
APD90
dF/dt
4
4
78 ⫾ 0.3
75 ⫾ 0.5*
⫺5
1.34 ⫾ 0.07
1.30 ⫾ 0.04†
⫺4.1
128 ⫾ 0.4
151 ⫾ 2.0*
15
0.94 ⫾ 0.03
0.93 ⫾ 0.07†
⫺0.7
Values are means ⫾ SE; n, no. of hearts. APD90, 90% recovery of
normalized optical action potential duration; dF/dt, rate of change of
fluorescence. All IgG measurements were made 15 min after start of
perfusion. Student’s t-test for paired data. * P ⬍ 0.05 compared with
control. † Not statistically significant.
Fig. 2. Electrocardiogram (ECG) recordings from Langendorff perfused rabbit heart before and after positive immunoglobulin (IgG)
(800 ␮g/ml). A: control; B: slowing of sinus rate after positive IgG;
C: second-degree heart block in the same heart with a 2:1 pattern,
which later evolved into 4:1 AV block (D). After washout, sinus
bradycardia persisted (E). Arrows, P waves.
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partial recovery was observed when the hearts were
perfused with positive IgG-free modified Tyrode solution for up to 45 min. The summary of ECG measurements is shown in Table 1. Table 2 compares dF/dt and
APD90 before and after positive IgG for four hearts. In
these experiments, the same six sites were selected
and the data were pooled (n ⫽ 24). The right atrial sites
were chosen from the free right atrial wall, distant
from the vicinity of the SA. There was a modest increase of 5% in APD90 in the right atrium and an
increase of 15% in the imaged portion of the right
ventricle. Both effects were statistically significant. On
the other hand, there was no statistically significant
effect of positive IgG on dF/dt in both the right atrium
and ventricle.
Figure 2 shows ECG recordings from a typical experiment that produced CHB in the presence of positive
IgG (800 ␮g/ml). The control ECG shows normal sinus
rhythm with a P wave, followed by a QRS complex at a
spontaneous heart rate of 160 beats/min (Fig. 2A). The
P-R interval was 67 ms. Fifteen minutes after infusion
of positive IgG, the heart rate slowed to 113 beats/min
and the P-R interval increased to 180 ms (Fig. 2, trace
B). Note that the QRS complex duration was essentially unaffected by positive IgG infusion. Fig. 2, traces
C and D, shows 2° AV block developed 19 min after
start of positive IgG infusion and 2° AV block with a 4:1
pattern at 24 min, respectively. Figure 2E shows partial recovery of heart block 45 min after perfusion with
positive IgG-free Tyrode solution. However, sinus bradycardia and 1° AV block at a heart rate of 138 beats/
min were maintained.
Figure 3 shows atrial and ventricular activation
maps, along with selected optical APs during fixed
atrial pacing (A1⫺A1 ⫽ 250 ms) in the presence of
positive IgG (800 ␮g/ml). Optical AP were mapped
using a photodiode array (124 sites) and di-4-ANEPPS.
Stimulation was applied in the sinus node region (see
asterisk in Fig. 3). The map projections shown in the
top panels are right-sided projections of the epicardial
heart surface in control (Fig. 3, left) and in the presence
of positive IgG (Fig. 3, right). A rough schematic of the
right atrium is outlined in the lower portion of the
maps with anatomic landmarks indicated. Optical AP
were acquired to simultaneously record from the right
atrium and ventricle. In the activation maps, each
shaded region correspond to an isochronal area acti-
vated each successive millisecond. The arrows indicate
the direction of activation wave front propagation. In
the control right atrial map, total activation time was 4
ms; total activation time for the imaged portion of the
right ventricle was 5 ms. Complete heart block was
obtained after positive IgG infusion (Fig. 3, right). In
the right atrial activation map, there was considerable
delay at the SA junction, as indicated by the crowded
isochrones. Whereas right atrial activation time was 9
ms, most of the delay was at the SA junction. The
majority of the remaining atrium was activated within
3 ms, as in control. The right ventricular activation
time for the ventricular escape beat was 4 ms. The
ECG and a selected atrial and ventricular optical action potential during control (left) and positive IgG
infusion (right) are shown at the lower portion of Fig. 3.
The role of atrial conduction disturbances during
spontaneous sinus rhythm in control and after positive
IgG perfusion (1,200 ␮g/ml) is shown in Fig. 4. The
topology of these maps was similar for all six hearts
that developed 3° AV block. The control right atrial
map (Fig. 4, left) shows that the right atrium was
activated within 5 ms. The spontaneous atrial rate was
180 beats/min with 1:1 AV conduction. The atrial isochrones were evenly spaced, indicating that there were
no conduction disturbances in the right atrium. The
right ventricle also activated within 5 ms. The activation pattern was from apex to base, indicating normal
sinus activation of the ventricles. Perfusion of positive
IgG resulted in sinus bradycardia, followed by complete AV block corresponding to an atrial rate of 144
OPTICAL MAPPING AND CONDUCTION ABNORMALITIES
H1893
beats/min. Examination of the atrial activation map
(Fig. 4, right) shows that sinus bradycardia was associated with marked conduction delay at the SA junction. The activation isochrones were crowded in the SA
region, similar to the paced example shown in the Fig.
3. Total right atrial activation time increased to 7 ms.
The right ventricle activated within 6 ms and was
similar in activation sequence and timing to the control
map. In this and all other experiments, sinus bradycardia was associated with only minor changes in overall atrial and ventricular activation patterns.
To understand the electrophysiological effects of positive IgG better, optical APs were obtained at a higher
magnification in the region of the sinus node and SA
junction. The results from a representative experiment, in which complete AV dissociation was obtained
in the presence of positive IgG (800 ␮g/ml), are shown
in Fig. 5. Optical APs are from one column of the grid.
Figure 5, traces A-D, shows that most of the conduction
occurred in the sinus node to SA junction region. In
this zone, there was a reduced upstroke in optical AP.
Optical AP from the free right atrial wall before and
after positive IgG were similar, indicating that positive
IgG had little effect on the rest of the atrium. Although
there was a modest increase (15%) in APD90, there was
no statistically significant effect on dF/dt at this con-
Fig. 4. Optical maps of atrial and ventricular activation along with selected optical
AP during spontaneous sinus rhythm in
control and after positive IgG (1,200 ␮g/
ml) perfusion. Left: control RA and ventricular activation maps. No conduction
disturbances were evident in the right
atrium in control. RV had a normal sinus
activation pattern. Right: positive IgG
caused third-degree heart block with a
ventricular escape rhythm. Sinus bradycardia was associated with marked conduction delay at the sinoatrial junction as
indicated by the crowded isochrones in the
sinoatrial region. Maps and abbreviations
are the same as in Fig. 3.
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Fig. 3. Optical maps of atrial and ventricular
activation along with selected optical action
potentials (AP) during fixed atrial pacing at a
rate of 240 beats/min. Stimulation (*) was
applied in the sinus node region. Rough schematic of RA is outlined in the lower portion of
the maps with anatomic landmarks indicated.
Activation maps are right-sided projections of
epicardium in control (left) and in the presence of 800 ␮g/ml positive IgG (right). Each
isochronal zone (shaded region) corresponds
to an isochronal time of 1 ms. Color code key
for atrial and ventricular isochrone intervals
is shown in between the 2 maps.
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OPTICAL MAPPING AND CONDUCTION ABNORMALITIES
centration of positive IgG, indicating that IgG had
little effect on the conduction in the imaged portion of
the right ventricle.
DISCUSSION
The primary finding of this study is that positive
maternal IgG causes bradycardia in the isolated Langendorff, perfused young rabbit heart. Whereas complete heart block is a hallmark of this potentially fatal
congenital disorder, bradycardia was a consistent finding in this study. Along with slowing of the intrinsic
sinus rate, optical mapping revealed marked conduction delays for propagation of the sinus impulse to the
atrium. Comparison of the activation maps and dF/dt
measurements indicates that there was no effect on
conduction in other parts of the right atrium and the
mapped portion of right ventricle. The fact that conduction is affected at the SA junction is not unlike the
documented concept that these IgG target specific cardiac conduction systems in the heart, such as the AV
node. The reason that sinus node and SA dysfunction
have been overlooked until recently may be related to
the severity of the disease during high-level AV nodal
block and the limited number of electrophysiological
studies in this relatively rare congenital disorder.
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Fig. 5. Optical AP obtained at a magnification of 0.4 mm/pixel along
one column of the grid in the presence of positive IgG (800 ␮g/ml)
going from A (sinus node region) to L (free RV wall).
Whereas optical mapping of hearts with CHB need to
be investigated in detail at many levels, our attention
to atrial and SA conduction disorders in the present
study was precipitated by the recent and somewhat
unexpected findings from our laboratory, that sinus
bradycardia was more common in animal models of
CHB than previously realized (4, 17). These findings
have been extended to the clinical settings, where
sinus node dysfunction in infants of mothers with
anti-Ro antibodies has been documented (5, 19). It is
tempting to speculate that because of the significant
mortality and morbidity associated with AV node dysfunction, the role of sinus node dysfunction may well
have been overlooked for years.
There are two implications to the findings of this
study. First, sinus bradycardia may have long been an
early electrophysiological consequence in the CHB syndrome. Our results, along with the observations of
others (5, 19), hopefully will draw more attention of
health care providers to the potential critical role of
sinus bradycardia for the early diagnosis of CHB. Second, because it appears that maternal IgG target specific conduction systems in the heart, there is a continued need for basic scientific investigation into the
histopathogical mechanism by which these antibodies
interact with systems responsible for the initiation of
the cardiac impulse in the SA node and its propagation
to the atria and ventricles.
Proposed mechanisms of tissue injury. Whereas the
molecular and biochemical mechanisms of CHB have
been explored to a certain extent (see Ref. 7 for review),
the electrophysiological mechanisms of CHB are just
emerging (3, 4, 12, 17, 20). Work from our laboratory
was based on the initial observation of Garcia et al.
(12), who showed that the use of a single-surface ECG
that conduction disorders associated with neonatal lupus could be reproduced in an isolated adult rabbit
heart. They further showed that anti-SSA/Ro positive
sera induced a reduction in ICa,L. Subsequently, and
unlike Garcia et al. (12), we used a large panel of sera
only from mothers whose children have CHB and
tested their electrophysiological effects on human fetal
heart (4). We provided evidence that IgG-enriched fractions and anti-52-kDa SSA/Ro antibodies affinity purified from sera of mothers whose children have CHB
induce complete AV block in the fetal heart perfused by
the Langendorff technique and inhibit ICa,L at the
whole cell and single channel level (3, 4). In addition,
immunization of female BALB/C mice with recombinant proteins generated high-titer antibodies, which
crossed the placenta during pregnancy and were associated with varying degrees of AV conduction abnormalities, including complete AV block, in the pups (4,
17, 20). Because conduction in the SA and AV node is
essentially dependent on Ca electrogenesis, sinus bradycardia, and AV block would be expected to result
from interventions that reduce Ca2⫹ currents. The
possibility that these IgG interact with other currents
in the SA or AV node cannot be ruled out. We propose
that chronic exposure of Ca2⫹ channels to maternal
antibodies during pregnancy could lead to Ca2⫹ chan-
OPTICAL MAPPING AND CONDUCTION ABNORMALITIES
The authors thank Joyce Ince in the Animal Care Facility for
technical assistance. IgG was obtained from the Research Registry
for Neonatal Lupus.
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-55401 (to M. Boutjdir) and Veterans Administration Medical Research Funds as Merit Grant Awards (to M.
Boutjdir and to M. Restivo).
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52-kD protein is a novel component of the SS-A/Ro antigenic
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nel internalization, degradation, cell death, and eventually fibrosis and calcification, as has been reported
from the autopsies of affected children (14). Because
cardiac myocytes do not undergo any significant mitosis after birth, affected cells are not replaced, and thus
this may explain the irreversibility of the 3° AV block
in human infants. The partial recovery of AV block
reported in this study is likely due to the acute exposure of the antibodies, unlike chronic exposure during the pregnancy term. Altogether, these findings
strongly suggest that maternal IgG are causally related to a number of electrophysiological abnormalities
in the development of CHB. However, it should be
emphasized that not all mothers with anti-SSA/RoSSB/La antibodies have affected children. This discordance may be related to undocumented electrophysiological effects or subclinical aspects of autoimmunerelated CHB indicating that additional factors are
required to promote disease expression.
In conclusion, the use of optical mapping techniques
allows for the correlation of conduction disorders to
specific anatomic structures in the heart because recordings could be mapped from multiple sites. We
showed that CHB was associated with marked sinus
bradycardia and conduction delays at the SA junction.
These findings provide potential new insights into understanding the pathogenesis of CHB and raise the
question of whether screening of infants from affected
mothers for sinus bradycardia should be part of standard diagnosis.
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