(Circulation. 2001;103:1599.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Direct Inhibition of Expressed Cardiac L- and T-Type Calcium Channels by IgG From Mothers Whose Children Have Congenital Heart Block
From the Molecular and Cellular Cardiology Program, New York Harbor Healthcare System and SUNY Health Science Center, Brooklyn, NY.
Correspondence to Dr Mohamed Boutjdir, R&D Office (151), Molecular and Cellular Cardiology Program, New York Harbor Healthcare System, 800 Poly Place, Brooklyn, NY 11209. E-mail mohamed.boutjdir{at}med.va.gov
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Abstract |
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Background—Congenital heart block (CHB) is a disease that affects the offspring of mothers with autoimmune diseases. We recently reported that maternal sera containing antibodies against SSA/Ro and SSB/La ribonucleoproteins (positive IgG) inhibited L-type Ca current in isolated cardiac myocytes and induced sinus bradycardia in a murine model of CHB. The direct interaction of positive IgG with L-type Ca channel proteins and the possible inhibition of T-type Ca current that could account for the sinus bradycardia remain unknown.
Methods and Results—The
2-electrode voltage-clamp technique was used to record currents via
L-type
(IBa-
1C
or
IBa-1C+ß2a+2/
)
and T-type
(IBa-1H)
Ca channels, Na channels
(INa-hH1),
and K channels
(IKs-minK+KvLQT1)
expressed in Xenopus oocytes.
Positive IgG (350 µg/mL) inhibited
IBa-1C
by 50.6±4.7% (P<0.01) and
IBa-1C+ß2a+2/
by 50.9±4.2% (P<0.01);
IBa-1H
was reduced by 18.9±1.0%
(P<0.01).
Immunoblot data show cross-reactivity of positive IgG with
1C subunit. Pretreatment of oocytes with
atropine (1 µmol/L) or acetylcholine (10 µmol/L) did not affect the
inhibitory effect of IgG on
IBa-1C
and
IBa-1C+ß2a+2/
(P<0.05). Positive IgG had no
effect, however, on either
INa-hH1
or
IKs-minK+KvLQT1.
Conclusions—Positive
IgG inhibited expressed L-type
IBa and
cross-reacted with the 1C subunit in
Xenopus oocytes, providing
strong evidence that maternal antibodies interact directly with the
pore-forming 1-subunit of Ca channels. In
addition, we show for the first time that positive IgG also inhibited
T-type
IBa but
not
INa-hH1
or
IKs-minK+KvLQT1.
This could provide, in part, the ionic basis of sinus bradycardia
reported in animal models of CHB and clinically in
humans.
Key Words: antibodies • ion channels • electrophysiology
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Congenital heart block (CHB) is a disease that affects children of mothers who may have autoimmune disease or may be entirely asymptomatic.1 CHB is usually detected between 16 and 24 weeks of gestation in fetuses with otherwise normally developing hearts.1 CHB carries high mortality (
30%), and >60% of affected children require
pacemakers.2 Although varying
degrees of block have been noted and second-degree block has on rare
occasions reverted to normal sinus
rhythm,3 CHB is irreversible.
CHB detected in utero is strongly associated with autoantibodies
reactive to the intracellular ribonucleoproteins SSA/Ro and
SSB/La.4 Anti-SSA/Ro
antibodies recognize 2 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.5 The 60-kDa SSA/Ro
protein contains an RNA-binding protein consensus
motif.6 The 52-kDa SSA/Ro
protein has 3 distinct domains: 2 zinc fingers in the N-terminal, a
central leucine zipper, and a C-terminal rfp-like
domain.7 SSB/La is a 48-kDa
protein, which is thought to have the function of facilitating the
maturation of RNA polymerase III
transcripts.8 The exact
function of these autoantigens is yet to be defined. The association between CHB and autoantibodies against SSA/Ro and SSB/La proteins has been known for >3 decades. The mechanisms underlying this disease, however, are just emerging. Several cellular and immunological mechanisms have been proposed to explain the pathogenesis of this disease.9 Recently, the development of animal models of CHB10 11 and the use of electrophysiological techniques10 12 13 to study the cellular and ionic mechanisms of maternal antibodies in heart cells provided new directions and alternative approaches for the pathogenesis of CHB. Garcia et al12 demonstrated that the IgG fraction of SSA/Ro and SSB/La antibodies induced abnormal conduction and reduction of Ca currents in rabbit heart. Subsequently, our laboratory demonstrated the arrhythmogenic effect of maternal autoantibodies in Langendorff-perfused hearts10 13 and further correlated these effects with the inhibition of the L-type Ca channel in isolated cardiac myocytes.10 13 In addition and unexpectedly, we reported significant sinus bradycardia in mouse pups born to mothers injected with human maternal antibodies.11 These same maternal antibodies, however, did not affect the Na current (INa), the transient outward current (Ito), and the inward rectifier K current (IK1) in rat ventricular myocytes.13
The present study was designed to address the following 3 questions: (1) Does inhibition of L-type Ca channels by maternal antibodies occur by direct interaction of the antibody with the channel pore-forming subunit? (2) Does maternal antibody affect T-type Ca channels (because this channel could be involved in the pacemaker activity of the heart)? (3) Does maternal antibody affect other channels, such as INa and delayed rectifier IK channels. Xenopus oocytes were used to individually express these currents. This is most relevant for L-type and T-type Ca channels because of the unique advantage of separating T-type from L-type current, which is usually difficult to achieve in native cardiocytes.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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IgG Purification
Purification of IgG has been performed as previously described.13 Briefly, immunoglobulin fractions containing IgG were purified from serum by protein A–Sepharose columns and confirmed to be pure by electrophoresis. IgGs were obtained from 3 mothers whose children have CHB. These IgGs were referred to as positive IgG and contain IgG against 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro, as tested by ELISA and immunoblot.13 Negative IgG (control IgG) was purified from sera of 3 healthy mothers with healthy children and tested negative for anti-SSA/Ro and anti-SSB/La antibodies by ELISA and immunoblot.13 The dose-response relationship for the inhibition of IBa-
1C
by positive IgG showed that the concentration of positive IgG that
produced a half-maximal response (EC50) was
173.5 µg/mL and that maximal inhibition was 350 µg/mL. Thus, an IgG
concentration of 350 µg/mL was used throughout.
Preparation of
Xenopus Oocyte and cRNA
Injection
Mature female
Xenopus frogs, purchased from
Xenopus I (Ann Arbor, Mich), were anesthetized with 1.5 mg/mL
tricaine. Surgically removed ovarian lobes were dissected and
treated for 1.5 hours with 1.5 mg/mL collagenase type IA
dissolved in Ca-free ND96 medium (mmol/L: NaCl 96, KCl 2,
MgCl2 2, HEPES 5, pH 7.4). Stage IV and V
oocytes were selected. cRNAs encoding the full length of the
1C subunit of rabbit cardiac Ca channel, the
ß subunit, and the 2/ subunit (both from
rabbit skeletal muscle) were kindly provided by Dr Mohamed Chahine from
Laval Hospital Research Center, Quebec,
Canada.14 Plasmid
pCDNA3.1(+)-minK, pCDNA3.1(+)-SCN5A, and pCDNA3.1(+)-KvLQT1 were
generously given by Dr Robert S. Kass from Columbia University. Plasmid
pGEM-HEA containing cDNA encoding the T-type
1H Ca channel of human heart was generously
provided by Dr Edward
Perez-Reyes15 from the
University of Virginia Medical Center. Plasmids were first linearized
with restriction enzymes, and in vitro transcription was carried out
with the mMSSAGE mMACHINE (Ambion Inc). Each oocyte was injected with a
55-nL volume containing 20 ng 1C cRNA (alone
or with ß2a and
2/ subunit cRNAs) or 5 to 10 ng
1H cRNA or 0.5 to 1 ng minK cRNA+1 to 2 ng
KvLQT1 or 1 to 2 ng hH1 cRNA. The injected oocytes were stored at
18°C in Leibovitz’s L-15 medium (GIBCO BRL) supplemented with 50
U/mL penicillin/streptomycin. Currents were recorded from days 4 to
7.
Solutions and Drugs
The composition of the external recording
solution for L-type and T-type currents is (mmol/L)
Ba(OH)2 40, NaOH 50, KOH 2, HEPES 5,
4-aminopyridine 5, and
tetraethylammonium chloride 10,
adjusted to pH 7.4 with methanesulfonic acid. ND96 bath
solution was used for
INa-hH1
and
IKs-minK+KvLQT1
recordings.16 17
All chemicals were purchased from Sigma except where
indicated.
Oocyte Ca Current Recordings
The expressed currents were recorded with the
2-electrode voltage-clamp technique with a GeneCLAMP 500 amplifier
(Axon Instrument Inc). The volume of the recording chamber was
0.5 mL, and the rate of perfusion was 0.3 mL/min. Oocytes were impaled
with electrodes filled with 3 mol/L KCl in ND96 external solution.
For L- and T-type Ba current-voltage (I-V) relations, oocytes were
depolarized from a holding potential of -80 mV to test potentials
ranging from -50 to 60 mV for L-type
IBa and
-70 to 60 mV for T-type
IBa,
with increments of 10 mV. The time course of L-type and T-type
IBa was
recorded by a depolarization pulse to 20 mV and -30 mV from a
holding potential of -80 mV, respectively. For
IKs-minK+KvLQT1,16
pulses were applied in 10-mV increments from -60 mV holding potential
to 60 mV and for
INa-hH117
from a holding potential of -130 mV to test potentials ranging from
-100 to 30 mV with increments of 5 mV.
Immunoprecipitation and Western Blot
L-type Ca channel proteins were obtained from
membranes of oocytes injected with 1C cRNA
and purified as previously
described.18 Briefly,
membranes were homogenized in 10% sucrose, 15 mmol/L
NaCl, 5 mmol/L KCl, and 20 mmol/L HEPES, pH 7.5, supplemented
with proteinase inhibitor
cocktail.18 After
centrifugation, membrane fractions from 20% to 50%
sucrose gradient interface were collected and solubilized in 2.5 mL
buffer (75 mmol/L KCl, 75 mmol/L NaCl, 50 mmol/L Na
phosphate [pH 7.2], 2 mg/mL soybean lipids, and 1% Triton X-100) and
centrifuged for supernatant collection.
Antibodies (Card I) against the L-type
1C
subunit19 were generously
given by Dr Marlene Hosey from Northwestern University, Chicago,
Ill, and were used to immunoprecipitate L-type Ca channel proteins as
described.19 Briefly, Card I
was added to the supernatant, which was precleared with protein
A–sepharose and shaken at 4°C overnight. Twenty-five microliters of
50% protein A–Sepharose beads was added for every 1 mL of sample and
incubated for 4 hours. Protein A–Sepharose antibody/antigen complex
was collected by centrifugation, washed, and eluted in
reducing SDS sample buffer by boiling for 5 minutes. For Western blot
analysis, 35 µL/lane of the above immunoprecipitated proteins
was subjected to SDS-PAGE on 8% to 16% gradient gel. Proteins were
transferred to a PVDF membrane by electrophoresis. The blot was blocked
for 2 hours in 5% donkey serum and washed twice in 1xPBS-Tween. For
immunoreaction, the blot was incubated with positive IgG, negative IgG,
and Card I overnight at 4°C. Blots were completely washed with
PBS-Tween. Immunodetection was carried out with a 1:5000 diluted
peroxidase-conjugated rabbit anti-goat IgG (for Card I) or sheep
anti-human IgG (for positive and negative IgG) for 1 hour. Blots were
washed again, then incubated with enhanced chemiluminescence detection
reagent (Amersham Pharmacia Biotech) for 1 minute and exposed to x-ray
film. To ensure that 1C protein was
present in the negative IgG lane, blots were stripped by shaking in
strip solution (100 mmol/L 2-mercaptoethanol, 2% SDS,
62.5 mmol/L Tris-HCl) at 50°C for 30 minutes. The stripped blot
was then washed with PBS-Tween, blocked with donkey serum, and
immunoblotted with Card I as described
above.
Data Analysis
Data acquired were stored, then analyzed
offline with Pclamp 6 software (Axon Instrument Inc). All values are
measured as the difference between zero and the peak current. The
Microcal Origin v5.0 (Microcal Software Inc) program was used to
generate figures and perform statistical analysis. Data are
presented as mean±SEM. Student’s
t test for paired data and
independent t test or ANOVA was
used when appropriate. A value of
P<0.05 was considered
statistically significant.
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Results |
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L-Type IBa Was Inhibited by Positive IgG
Figure 1
shows the inhibitory effect of positive
IgG on expressed L-type
IBa in
Xenopus oocytes.
Figure 1A and 1B illustrates the I-V relations for
IBa-1C+ß2a+2/
and
IBa-1C
before and after the addition of positive IgG. Positive IgG (350
µg/mL) inhibited
IBa-1C+ß2a+2/
and
IBa-1C
by 50.9±4.2% (P<0.01, n=18)
and 50.6±4.7% (P<0.01,
n=12), respectively.
Figure 1C shows the time course inhibition of
IBa-1C+ß2a+2/
by positive IgG in 1 typical oocyte. Application of positive IgG (350
µg/mL) resulted in 51% inhibition of
IBa-1C+ß2a+2/.
The effects of positive IgG were only partially reversible (86%
recovery). Negative IgG, however, had no significant effect on
IBa-1C+ß2a+2/
(Figure 1D, P=0.37,
n=6). Because it has been suggested that maternal IgG can act as a
muscarinic receptor
agonist,20 which in turn can
regulate L-type Ca channels, we tested whether blockade (negative
control) or stimulation (positive control) of muscarinic receptor with
atropine or acetylcholine, respectively, will affect IgG inhibition of
L-type
IBa.
Figure 2 shows that application of atropine (1 µmol/L, A
and B) or acetylcholine (10 µmol/L, C) per se had no effect on
IBa.
Addition of positive IgG (350 µg/mL) in the presence of atropine
resulted in the usual inhibition of
IBa-1C+ß2a+2/
by 47.5±4.6% (P<0.05, n=7)
and
IBa-1C
by 47.4±3.5% (P<0.05, n=5).
Similarly, the effects of positive IgG on
IBa-1C+ß2a+2/
were not affected by the presence of acetylcholine (48.2±3.0%
inhibition, P<0.05, n=5). In
summary, positive IgG but not negative IgG inhibited both
IBa-1C+ß2a+2/
and
IBa-1C.
No significant difference in the percent decrease of L-type
IBa by
positive IgG was found between
IBa-1C
and
IBa-1C+ß2a+2/
and between the groups in the absence or presence of atropine or
acetylcholine. The average statistical data are summarized in the
Table.
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T-Type
IBa Was
Inhibited by Positive IgG
Figure 3 shows
IBa-1H
in the absence and presence of IgG. Panel A illustrates the
inhibitory effects of positive IgG on the I-V relations of
IBa-1H.
Panel B shows the time course inhibition of
IBa-1H
by positive IgG. Application of 350 µg/mL positive IgG resulted in
18.9±1.0% inhibition of
IBa-1H
at -30 mV (P<0.01, n=10).
The effects of positive IgG were not completely reversible (88%
recovery after washing). Negative IgG did not significantly affect
IBa-1H
(Figure 3C). The average statistical data are summarized in
the
Table
.
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Positive IgG Did Not Affect Na
(INa-hH1)
and K
(IKs-minK+KvLQT1)
Channels
To check whether positive IgG affected other currents,
we expressed Na current,
INa-hH1,
and K current,
IKs-minK+KvLQT1,
in oocytes.
Figure 4 shows the effect of positive IgG on
INa-hH1
(A) and
IKs-minK+KvLQT1
(B). Positive IgG failed to significantly alter
INa-hH1
(P=0.07, n=8) and
IKs-minK+KvLQT1
(P=0.06,
n=7).
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Positive IgG Cross-Reacted With L-Type Ca
Channel 1-Subunit
To unambiguously demonstrate a direct interaction of
positive IgG with Ca channel 1C protein, we
immunoprecipitated 1C subunit from membranes
of oocytes injected with 1C subunit cRNA. A
representative Western blot of 6 experiments is
shown in
Figure 5. Lane 1 shows Card I used as positive control, lane
2 positive IgG, lane 3 negative IgG, and lane 4 Card I after the blots
of lane 3 were stripped. The 1C subunit was
detected as a band migrating above 200 kDa by Card I, as previously
reported,22 and
positive IgG but not negative IgG. This provides evidence that positive
IgG directly cross-reacts with the 1C
subunit. Western blot experiments for T-type
1H were not performed because antibodies
against T-type Ca channel protein are not yet
available.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results presented here demonstrate that (1) positive IgG containing anti-SSA/Ro and anti-SSB/La antibodies from mothers whose children have CHB inhibited both L-type and T-type Ca channels but did not affect Na and K channels expressed in Xenopus oocytes; (2) the inhibitory effect of positive IgG was more marked (
51%)
on L-type Ca channels than T-type Ca channels (19%); (3) positive
IgG inhibited
IBa-1C
and
IBa-1C+ß 2a+2/ to a similar extent; (4) pretreatment of oocytes with
atropine or acetylcholine did not alter the inhibition of expressed
L-type
IBa by
positive IgG; (5) negative IgG did not affect either L- or T-type
IBa; and
(6) immunoblot data unequivocally showed direct interaction
between positive IgG and L-type Ca channel 1C
subunit.
Maternal Antibody Inhibition of Expressed
L-Type Ca Channels
The present findings that positive IgG, but not
negative IgG, functionally inhibits Ca channels expressed in
Xenopus oocytes are
consistent with previous data from our laboratory in cardiac
myocytes.10 13
Although we do not exclude the possibility that other
endogenous auxiliary subunits, such as ß-subunits, may be
functionally associated with the expressed
1-subunit, our Western blot experiments
unambiguously demonstrate that maternal autoantibodies directly
cross-react with the pore-forming 1-subunit
(Figure 5). The 1C subunit migrated
at >200 kDa, and its size was similar to that expressed in oocytes
reported by
others.22
Conversely, the inhibition of IBa was not affected by pretreatment with atropine (a muscarinic receptor blocker). Bacman et al20 reported that IgG present in the sera of patients with CHB and their mothers could bind and activate muscarinic cholinergic receptors of neonatal rat atrial preparations. This raises the possibility that the inhibitory effect of positive IgG on IBa may be, at least in part, due to the activation of muscarinic receptors. Using an oocyte expression system, we did not find any difference in the inhibition of the expressed IBa by positive IgG in the absence and presence of atropine, suggesting either that the inhibitory effect of positive IgG does not involve muscarinic receptors or, alternatively, that oocyte muscarinic receptors are not coupled with expressed Ca channels. The present oocyte experiments do not rule out the possible regulation of Ca channels by positive IgG through normally coupled sarcolemmal receptors in native cardiocytes.
Maternal Antibody Inhibition of Expressed
T-Type Ca Channels
Our data showed that maternal antibody blocks not
only L-type Ca channels but also T-type Ca channels. Because T-type Ca
channels have been implicated in the pacemaker activity in the
heart,23 these findings may
provide, at least in part, an ionic basis for the sinus bradycardia
reported in murine models of
CHB.10 11 This is
further supported by in vivo data in conscious
rats24 and in
anesthetized dogs25
demonstrating a decrease in heart rate by mibefradil. Similar
dose-dependent decreases in heart rate have been reported in
humans.26 These novel
findings are of clinical importance because it is only recently that
clinicians caring for infants with CHB have begun focusing their
attention on sinus bradycardia in addition to
atrioventricular (AV) node conduction abnormalities. In
this regard, Brucato et al27
confirmed the sinus bradycardia we reported in the murine
model11 in infants born to
mothers seropositive to SSA/Ro antibodies.
Maternal Antibody Did Not Affect Na and K
Channels
Positive IgG failed to affect expressed
INa-hH1
and
IKs-minK+KvLQT1
in oocytes. These findings are consistent with those obtained
in native cardiac myocytes13
showing lack of effect of positive IgG on fast
INa,
Ito, and
IK1.
Furthermore, the lack of effect on these channels suggests that
positive IgG preferentially interacts with Ca
channels.
Pathogenesis of CHB
Available autopsies from affected infants showed the
existence of myocarditis and fibrosis of the AV
node.28 29
Because circulating maternal autoantibodies are directed against
intracellular autoantigens, hypotheses have been proposed that
intracellular SSA/Ro and SSA/La proteins are being trafficked to the
cell surface during development by the induction of stress proteins,
hormonal influences, viral infection, or
apoptosis.30 31 32 33
The mechanisms by which these events alter AV conduction in fetal heart
remain unclear.
It is only recently that electrophysiological and functional data10 11 12 13 proposed alternative explanations for CHB pathogenesis. Active10 and passive11 animal models for CHB have been established. Immunized pregnant mice gave birth to pups with complete AV block and significant sinus bradycardia. Furthermore, positive IgG induced AV block and bradycardia in acutely perfused isolated hearts and inhibited L-type Ca current from isolated cardiac myocytes.10 12 13 These findings suggest that apparent pathological changes, such as inflammation, are not necessarily a primary event for this disease and that the autopsy evidence may represent an advanced stage of the maternal antibody blockade of Ca channels, which play a vital role in the excitation-contraction coupling of the developing heart.
Consequences of L- and T-Type Ca Channel
Blockade by Maternal Antibodies
L-type Ca channels are widespread in the
cardiovascular system and are crucial in action
potential propagation, conduction in the AV node, and
excitation-contraction coupling in the heart. Blockade of L-type Ca
channels by positive IgG coincides with the conduction block at the AV
node and with the clinical finding that infants with CHB often have
diminished ventricular function and heart
failure.29 34 The
function of the T-type Ca channel is less clearly defined, but it is
thought to be involved in pacemaker activity in the
heart.23
L-type Ca channel density is lower35 and sarcoplasmic reticulum is less abundant36 in fetal heart cells than in adult cardiac cells. Thus, blockade of Ca channels by autoantibodies will impose a further burden on those marginally functioning Ca channels. It is also possible that prolonged and chronic exposure of fetal cardiac Ca channels to maternal antibodies could result in downregulation of the channels by internalization, leading to cell death, further exposing the intracellular SSA/Ro and SSB/La antigens to the circulating autoantibodies and ultimately resulting in inflammation, fibrosis, and at later stages, calcification. Thus, it is possible that the pathogenic activity of these autoantibodies may be primarily through Ca channel blockade and that the SSA/Ro and SSB/La ribonucleoproteins contribute as a secondary mechanism. Taken together, the present findings and those from several previous reports10 12 13 make the direct interaction of positive IgG with Ca channels an attractive hypothesis that could account, at least in part, for the pathogenesis of CHB.
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Acknowledgments |
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This work was supported by National Heart, Lung, and Blood Institute grant HL-55401 and a VA Merit Grant Award (to Dr Boutjdir).
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Footnotes |
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Guest Editor for this article was Eduardo Marbán, MD, PhD, Circulation Research Editorial Office, Baltimore, Md.
Received June 26, 2000; revision received September 13, 2000; accepted October 4, 2000.
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