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Molecular and Cellular Cardiology Program, New York Harbor HealthCare System and SUNY Health Science Center, Brooklyn, New York 11209, USA
1Correspondence: Research and Development Office (151), New York Harbor Health Care System, 800 Poly Pl., Brooklyn, NY 11209, USA. E-mail: mohamed.boutjdir{at}med.va.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1C and ß2a
subunits of L-type Ca channel. Pups born to Ro52 immunized mothers
exhibited down-regulation of L-type calcium channels in heart. The data
provide new insight into the pathogenesis of congenital heart
block.—Xiao, C.-Q., Qu, Y., Hu, K., Boutjdir, M. Down-regulation of
L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits.
Key Words: autoantibody • AV block • immunization • cardiac myocyte
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. CHB is considered a model of passively
acquired autoimmune disease in which the mother generates anti-SSA/Ro
and anti-SSB/La antibodies that cross the placenta and presumably
injure the heart of the developing fetus (7)
.
CHB detected in utero is strongly associated with autoantibodies
reactive with the intracellular ribonucleoproteins SSA/Ro and SSB/La
(24)
. Anti-SSA/Ro antibodies recognize two proteins: a 60
kDa protein and a 52 kDa protein. An additional 75 kDa phosphoprotein
was recently reported to be associated with 60 kDa SSA/Ro
(25)
. The 60 kDa SSA/Ro protein contains an RNA binding
protein consensus motif (9)
. The 52 kDa SSA/Ro protein has
three distinct domains: two zinc fingers in the amino-terminal, a
central leucine zipper, and a carboxyl-terminal rfp-like domain
(8)
. SSB/La is a 48 kDa protein thought to facilitate the
maturation of RNA polymerase III transcripts (12)
. The
exact function of these autoantigens is yet to be defined. CHB carries
high mortality (
30%) and > 60% of affected children require
life-long pacemakers (24)
. ECG abnormalities manifest as
first, second, or third degree AV block and more recently as sinus
bradycardia (5)
. In addition to SA and AV node
dysfunction, heart failure (7)
has been reported in some
infants born to these mothers, invoking impairment of cardiac function.
We recently established a murine model for CHB where pups born to
mothers immunized with SSA/Ro recombinant proteins developed conduction
abnormalities (3
, 21)
identical to those seen clinically
in affected infants. We also showed that antibodies from mothers whose
children have CHB induced bradycardia and AV block in a
Langendorff-perfused fetal heart (3)
and correlated these
findings with inhibition of ICaL recorded from
isolated native cardiac myocytes (3
, 4)
. More recently,
direct interaction of maternal antibodies with the
1C subunit of cardiac Ca channels expressed in
Xenopus oocytes has been demonstrated (26)
. In
the present study, we immunized female rabbits with human recombinant
Ro52 protein and mated them after the establishment of primary immune
response. ECG, biochemical, and electrophysiological techniques were
used to assess the conduction abnormalities and the change of cardiac
L-type calcium channel density and proteins in pups born to these
mothers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Purified Ro52 protein was
verified by ELISA and Western blot.
Immunization of rabbits
All animal procedures conform to the principles embodied in the
Declaration of Helsinki. All experimental protocols were approved by
the Animal Studies Subcommittee and all procedures related to animal
use comply with the ‘Guiding Principles for Research Involving Animals
and Human Beings.’ The rabbit model was chosen over the mice model for
several reasons. The dimensions of 1-day-old rabbit pup aorta and heart
(size 
14x6x5 mm) are well suited for the Langendorff technique to
obtain single myocyte; this is not possible with mouse pup whose whole
heart size is 3 x 2 x 1.5 mm. Only eight hearts from
rabbit pups were needed to obtain the needed 10 g of tissue for
partial purification of L-type Ca channel proteins.
Seven white 8- to 9-wk-old female New Zealand rabbits (Hare-Marland, Hewitt, NJ) were immunized with human recombinant Ro52 protein. For initial immunization, 100 µg of the protein was injected intraperitoneally in complete Freud’s adjuvant (CFA) and 50 µg of the same recombinant in CFA given subcutaneously (s.c.) to the right and left scapular region. The first two boosts were given at 10 day intervals using 50 µg of recombinant protein in incomplete Freund’s adjuvant injected s.c. Subsequent boost injections were continued with a similar regularity until primary responses were detected in all rabbits. Blood tests for response to Ro52 were given every 10 days after the immunization and boosters. The rabbits were then mated after substantial immune response to Ro52 was established.
Enzyme-linked immunosorbent assay (ELISA) and immunoblots
Ro52 recombinant protein (0.1–0.2 µg) was coated overnight in
200 µl of 20 mM pH=9.0 Tris-HCl buffer in a 96-well plate (Corning
Lab. Science Co., Palo Alto, CA) at 4°C. Antigen was removed and
unreacted sites were blocked with 3%-BSA in 1x PBS-T at room
temperature for 2 h. Plates were washed completely with PBS-T.
Diluted serum (200 µl 1:800 in PBS) was added to each well and
incubated for 2–3 h at room temperature. Plates were then washed with
PBS-T. Anti-rabbit IgG (180 µl of 1:20000) conjugated with alkaline
phosphatase was added to each well and incubated for 1 h at
37°C. The plates were washed again. The p-nitrophenyl
phosphate alkaline phosphatase (200 µl) substrate (Sigma, St. Louis,
MO) was added to each well and incubated for 30 min at room
temperature. The plates were read at 405 nm for OD values by strip
reader (Biotek Instruments Inc, Winooski, VT).
Electrocardiographic recordings
On the day of delivery, ECG recording (leads I, II, and III)
were performed on each pup as described previously (19
, 21)
. Pups were placed on a warm pad and subjected to minimal
inhalation anesthetic metofane for stability of recordings. Paper speed
settings were adjusted to 25, 50, and 100 ms with filter settings at 40
or 100 Hz. Voltage amplification was 20 mV. Tracings were analyzed for
heart rate, QRS duration, and conduction abnormalities including first,
second, and third degree heart block. Bradycardia was defined as 40%
less than controls (3)
. PR prolongation was defined as
greater than 70 ms corresponding to the mean ± 2 SD
(mean control PR=55.0 and SD=6.5 ms)
(21)
.
IgG purification
Purification of IgG from rabbits was performed as described
previously (4)
. Immunoglobulin fractions containing IgG
were purified from serum by protein A-Sepharose columns, confirmed to
be pure by electrophoresis, and dialyzed in Tyrode’s solution for
electrophysiological studies. A concentration of serum and IgG of 400
µg/ml) was used in the oocyte experiments. This selected dose was
based on our previous observations in which maximum inhibition of
IBa in oocyte was obtained (26)
.
Isolation of cardiac myocytes
Cardiac myocytes were obtained from the heart of 1-day-old
newborn rabbit by the Langendorff technique as described previously
(3
, 4)
. The heart was perfused with an oxygenated (100%
O2) Ca-free Tyrode’s solution containing (in mM)
NaCl 117, KCI 5.7, NaHC03 4.4,
NaH2P04 1.5,
MgCl2 1.7, HEPES 20, glucose 11, creatine 10, and
taurine at 37°C for 5 to 10 min and then perfused with the same
solution containing collagenase type B (1.0 mg/ml, Boehringer Mannheim
Corporation, Indianapolis, IN) for 10 to 15 min. The softened
ventricular tissues were dissociated by triturating. Cells were
suspended in Tyrode’s solution with 1 mM CaC12
and 0.5% BSA (pH 7.4). After incubation for 30 min, noncontracting
cells with clear striations were used for whole-cell patch-clamp
studies.
Whole-cell recording of L-type Ca current, ICaL
To record whole-cell ICa.L, all
K+ currents were blocked with intracellular and
extracellular Cs+ and 4-aminopyridine (3
, 4)
. The fast Na current was blocked by a prepulse to -40 mV
from a holding potential of -80 mV in the presence of 50 nM TTX. Cells
were depolarized every 10 s from a holding potential of -80 mV to
a prepulse level of -40 mV for 100 ms and subsequently to a test pulse
of 10 mV for 300 ms. Pipette series resistance was compensated to
minimize the duration of the capacitative transient on 10 mV
hyperpolarizations from -80 mV. Junction potentials were zeroed before
the pipette touched the cell and always compensated.
The capacitative traces were fitted by a single exponential equation.
The membrane capacitance (Cm) was calculated
according to the equation: Cm =
c·Io/
Em,
where c is the time constant for cell membrane charge,
Io is the maximum capacitative current and
Em is the clamp voltage. We expressed all the
current data as density (pA/pF) by normalizing the peak
ICaL to cell capacitance. The composition of
external solutions is (in mM): NaCl 132, CsCl 5.4,
CaC12 1.8, MgCl2 1.8,
NaH2PO4 0.6,
4-aminopyridine 5, HEPES 10, dextrose 5 (pH=7.4). Patch electrodes were
filled with internal solution containing (in mM): CsCl 139.8,
K2EGTA 0.1–10, MgCl2 2,
CaC12 0.062, Na2-creatine
phosphate 5, HEPES 10, Na2ATP 3.1,
Na2GTP 0.42 adjusted to pH 7.1 with KOH.
Partial purification and immunoprecipitation of L-type Ca channel
1C subunit
Partial purification of Ca channel was carried out according to
Rengasamy et al. (23)
, with some modification. Briefly,
10 g of ventricles from 1-day-old pup hearts were homogenized in
0.25 M sucrose with 10 mM histidine (pH 7.4). This and all subsequent
buffers included the following mixture of protease inhibitors: 0.2 mM
PMSF, 0.1 mM benzamidine, 1 µM pepstatin A, 0.1 µM aprotinin, 1
µM leupeptin, calpain inhibitors I and II, 0.008 mg/ml, EDTA 2 mM,
EGTA 0.1 mM. Large pieces of tissue were removed by low-speed
centrifugation. The supernatant was loaded onto a three-layer sucrose
cushion of 15, 32, and 40%, respectively, and spun at 28 K for 3 h in Beckman ultracentrifuge. A faint band at the 32%/40% sucrose
interface was recovered and centrifuged at 24 K for 30 min; the
resulting pellet was resuspended in 10 mM Tris, 0.1M NaCl, 1%
digitonin. This sample was shaken on ice for 1 h, spun at 26 K for
30 min, after which the supernatant was recovered. Anti-Ca channel
1C subunit antibody was added to the
supernatant, which was precleaned with protein A-Sepharose at 4°C
overnight; 30 µl of 50% Sepharose suspension beads were added for
every 1 ml of sample and incubated at 4°C for 4 h. The protein
A-Sepharose antibody/antigen complex was collected by centrifugation,
washed, and eluted in nonreducing SDS sample buffer by boiling for 5
min.
Western blot
A Western blot of the above samples was performed as described
previously (22)
; 25 µl/lane of the above eluted antigens
were subjected to SDS-PAGE under nonreducing conditions. Proteins were
transferred to PVDF membrane, blocked for 2 h in 5% nonfat milk
and 0.3% Tween-20, and incubated with anti-Ca channel
1C subunit antibody (1:1000, Alamone Labs,
Israel) overnight at 4°C. After a complete wash, 1:5000 diluted
peroxidase-conjugated secondary antibody was added and incubated for 1
h. The reaction was detected with enhanced chemiluminescence and
quantified using Sigmagel software.
ELISA for L-type Ca channel proteins
ELISA was carried out as stated above, except protein
A-anti-1C antibody immunoprecipitated
1C protein was used as coating protein; 5%
donkey serum in 1x PBS-T was used as blocking agent;
anti-1C subunit antibody (raised in rabbit,
Alamone Labs, Israel) was used as primary antibody; and anti-rabbit IgG
conjugated with alkaline phosphatase was used as secondary antibody.
Preparation of Xenopus oocytes and cRNA injection
Mature female Xenopus frogs purchased from Xenopus I
(Ann Arbor MI) were anesthetized with 1.5 mg/ml tricaine. Surgically
removed ovarian lobes were dissected and treated for 1.5 h with
1.5 mg/ml collagenase type IA dissolved in Ca-free ND96 medium (mM:
NaCl 96, KCl 2, MgCl2 2, HEPES 5, pH=7.4). Stage
IV and V oocytes were selected. Plasmid pRC-Ca.1 containing cDNA
encoding the full-length rabbit cardiac L-type
1C subunit and ß2a
subunits was generously provided by Dr. Edward Perez-Reyes from the
University of Virginia Medical Center. In vitro transcription was
carried out by using the mMSSAGE mMACHINE (Ambion Inc., Austin, TX).
Each oocyte was injected with 55 nl volume containing 10 ng
1C cRNA and 5 ng of
ß2a subunit cRNA. The injected oocytes were
stored at 18°C and currents were recorded from the 4th to the 7th
day.
L-type Ca current recording from oocytes
The composition of external recording solution is (mM):
Ba(OH)2 40, NaOH 50, KOH 2, HEPES 5, 4-AP 5, and
TEA 10, adjusted to pH 7.4 with methanesulfonic acid. The expressed
macroscopic currents were recorded with a two-electrode voltage clamp
technique using GeneCLAMP 500 amplifier (Axon Instrument, Foster City,
CA). The volume of the recording chamber was 0.3 ml and the rate of
perfusion was 0.3 ml/min. Electrodes were filled with 3 M KCl. For
IBa current-voltage (I-V) relations, oocytes were
depolarized from a holding potential of -80mV to test potentials
ranging from -50 to 60mV with increments of 10 mV.
Data analysis
Data acquired from oocytes and myocytes were analyzed off-line
with Pclamp 6 software (Axon Instrument). Microcal Origin v5.0
(Microcal Software, Northampton, MA) program was used to generate
figures and perform statistical analysis. All the data are presented as
mean ± SE. The OD reading value at OD=405 nm with
more than 2 SD of control was considered significant.
Paired t test and independent t test or ANOVA
were used when appropriate. A P value of <0.05 was
considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and reveal that both immunized mothers and their pups showed high
levels (P<0.01) of anti-Ro52 antibodies, confirming
transplacental passage of maternal antibodies to the pups. In contrast,
no detectable antibody levels were present in controls.
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ECG recording from the pups
Seven female rabbits were immunized and gave birth to 152 pups. Of
these, 31 pups (20.4%) were born dead, 1 pup (0.7%) had second degree
AV block with 2:1 patterns, 7 pups (4.6%) showed sinus bradycardia, 8
pups (5.3%) showed first degree AV block, and 5 pups (3.3%) had both
sinus bradycardia and AV block. The remaining 100 pups had normal sinus
rhythm and normal PR interval. For clarity, two groups are identified
within the immunized pups: group I includes pups with ECG abnormalities
and group II includes pups with normal ECG parameters. A summary of the
ECG parameters in pups from control and immunized groups is shown in
Table 2
. Figure 1
shows an example of a control ECG (Fig. 1A
), an ECG with
sinus bradycardia and first degree AV block (Fig. 1B
), and
an ECG with second degree AV block with 2:1 AV pattern (Fig. 1C
). The body weight of individual pups from immunized and
control groups was not significantly different (Table 2)
.
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L-type Ca current density is reduced in myocytes from pups born to
immunized mothers
Because L-type Ca channels are involved in the generation and
conduction of action potential in the sinus node and AV node and we
showed the direct interaction between these autoantibodies and L-type
calcium channels (26)
, we tested the hypothesis that
chronic exposure of these channels to autoantibodies might lead to
their down-regulation and loss of function. The patch-clamp technique
was used to evaluate L-type Ca channel density from control pups and
pups from the immunized group (groups I and II). The average
Cm was 19.2 ± 5.3 pF (n=56); the
results are shown in Fig. 2
. ICaL density was reduced by 31 ± 3.4%
(P=0.02, n=24) in myocytes from pups of the
immunized groups. Note that although pups from immunized group II had
normal ECG parameters, they show a reduction in
ICaL density similar to that of the immunized
group I, which had abnormal ECG. Selected
ICaLcurrent tracings from control and
from groups I and II are shown in Fig. 2B
.
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L-type Ca channel proteins are reduced in hearts from pups born to
immunized mothers
To further investigate whether this functional decrease in L-type
Ca current density is accompanied by a similar decrease in Ca channel
proteins, we partially purified and immunoprecipitated the
1C subunit of L-type Ca channel from cardiac
crude membrane preparations of pups born to immunized and control
mothers. Because the above patch-clamp results did not show a
significant difference in cardiac membrane L-type Ca channel density
reduction between group I and group II and the number of pup’s hearts
for isolating enough L-type Ca channel protein is limited, we did not
differentiate group I and group II when we assessed the L-type calcium
channel protein level. We mixed all hearts from immunized group and
isolated Ca channel protein from these hearts. Immunoblotting was
performed using a 4–12% Tris-glycine gel under nonreducing conditions
and proteins were probed with antibody against
1C subunit. Figure 3A
shows the Western blot results from three experiments that
demonstrate a 19 ± 1.6% (P=0.03, n=3)
decrease of Ca channel 1C subunit in pups from
immunized groups. The anti-1C antibody
recognized a band slightly above 200 kDa corresponding to L-type Ca
channel 1C subunit (likely the truncated
form). We also used ELISA to compare Ca channel proteins between the
groups. The results are shown in Fig. 3B
. Ca channel
proteins were reduced by 30 ± 2.6% in pups from immunized group
(P=0.02, n=6). Note that the down-regulation of
Ca channel proteins as assessed by ELISA (30%) is similar to the
functional decrease of current density (31%; see Fig. 2
) as assessed
by whole-cell recordings.
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Serum and IgG from immunized rabbits inhibited IBa
recorded from oocytes expressed with
1C/ß2a subunits of L-type Ca channels
The above reduced L-type Ca channel current density and proteins
in pups born to immunized rabbits could be due to the autoantibody’s
functional interaction with ICaL. To test whether
antibody from these immunized rabbit has such an effect, Ba currents
were recorded from oocytes expressed with
1C/ß2a subunits of the
L-type Ca channel, and the effects of sera and purified IgG from
immunized and control rabbits were analyzed. Figure 4A
shows that whole serum inhibited IBa
by 43.5 ± 2.5% (P<0.05, n=5). This
inhibition was not seen with serum from control rabbits (Fig. 4B
, n=5). In addition, 400 µg/ml of purified
IgG from immunized (Fig. 3C
, n=6), but not from
control rabbits (Fig. 4D
, n=5), inhibited
IBa by 46.4 ± 3.3 (P<0.05). The
results are summarized in Table 3
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1C/ß2a
subunits of L-type Ca channel. All together, these data provide strong
evidence supporting an etiologic role of autoantibody-mediated, calcium
channel down-regulation in the pathogenesis of CHB.
Even though we did not obtain any complete AV block in pups of
immunized rabbits, 20% of the pups died after birth in the present
study. It is plausible, but not certain, that these pups (or at least
some of them) might have died because of complete AV block. This is
supported by recent clinical data (6)
showing that 19% of
children born to mothers with anti-SSA/Ro and/or anti-SSB/La antibodies
die within 3 months of birth. The fact that not all pups born to
immunized rabbits had electrocardiographic abnormalities is also
consistent with the clinical findings where 5–6% of CHB infants
were born to lupus mothers with anti-SSA/Ro and/or anti-SSB/La
antibodies (6)
.
The finding that chronic interaction of autoantibodies with Ca channels
caused down-regulation of membrane Ca channel proteins and function is
of pathophysiological relevance. The implication is that inhibition and
subsequent down-regulation of L-type Ca channels can affect SA and AV
node function since these channels play a major role in the
electrogenesis of both the SA and AV node. Compared with adult cardiac
cells, L-type Ca channel density is lower (2
, 15)
and
sarcoplasmic reticulum is less abundant(14, 18) in fetal heart cells.
Down-regulation of membrane Ca channels by autoantibodies will impose
an additional burden on those marginally functioning Ca channels.
Here we propose that maternal autoantibodies are causally related to
various electrophysiologic abnormalities in the developing heart and
that chronic exposure of Ca channels to maternal antibodies during
pregnancy could lead to Ca channels internalization, degradation, cell
death, and eventually fibrosis and calcification, as has been reported
from the autopsies of affected children (13)
. However, the
fact that not all pups were affected indicates that additional
promoting factors may be necessary for disease expression. These
include genetic factors (1
, 16)
, viruses
(17)
, apoptosis (20)
, and hormonal factors
(10
, 11)
.
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ACKNOWLEDGMENTS |
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Received for publication January 31, 2001. Accepted for publication March 9, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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