© 1997 American Heart Association, Inc.
Articles |
Arrhythmogenicity of IgG and Anti–52-kD SSA/Ro Affinity-Purified Antibodies From Mothers of Children With Congenital Heart Block
the Division of Cardiology (M.B., L.C., Z.-H.Z., N.E.-S.), Veterans Administration Medical Center and State University of New York, Health Science Center, Brooklyn; the Department of Medicine (C.T., F.D., J.P.B.), Division of Rheumatology, New York University, and the Department of Rheumatic Diseases and Molecular Medicine (C.T., F.D., J.P.B.), Hospital for Joint Diseases, New York; and the Department of Obstetrics and Gynecology (W.R., A.M.), Beth Israel Medical Center, New York, NY.
Correspondence to Dr Mohamed Boutjdir, Cardiology Division (IIIA), VA Medical Center, 800 Poly Place, Brooklyn, NY 11209. E-mail boutjdir.mohamed@brooklyn.va.gov
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An important advance in the description and understanding of congenital heart block (CHB) came in the 1970s with the observation that mothers of affected infants frequently had autoimmune diseases and, in particular, that many maternal sera contained antibodies to SSA/Ro and SSB/La ribonucleoproteins. Although the molecular biology of the candidate antigens has been extensively defined, the arrhythmogenic and electrophysiological effects of their cognate antibodies on the human fetal heart are unknown. In the present study, we provide evidence that IgG-enriched fractions and anti–52-kD SSA/Ro antibodies affinity-purified from sera of mothers whose children have CHB induce complete atrioventricular (AV) block in the human fetal heart perfused by the Langendorff technique and inhibit L-type Ca2+ currents at the whole-cell and single-channel level. Immunization of female BALB/c mice with recombinant 52-kD SSA/Ro protein generated high-titer antibodies that crossed the placenta during pregnancy and were associated with varying degrees of AV conduction abnormalities, including complete AV block, in the pups. These findings strongly suggest that anti–52-kD SSA/Ro antibodies are causally related to the development of CHB.
Key Words: human fetal heart • patch-clamp technique • L-type Ca2+ current • atrioventricular block • immunization
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Over three decades ago, it was noted that mothers who gave birth to children with CHB often had autoimmune diseases.1 It is now well established that heart block detected before or at birth, in the absence of structural abnormalities, is strongly associated with maternal autoantibodies to SSA/Ro and/or SSB/La ribonucleoproteins, independent of whether the mother has systemic lupus erythematosus or Sjogren's syndrome or is totally asymptomatic.2 3 4 Autoimmune-associated CHB is most often detected between 16 and 24 weeks of gestation in an otherwise normally developing heart and is considered a consequence of transplacental passage of autoantibodies into the fetal circulation resulting in tissue injury.4 5 CHB carries a substantial mortality, approaching 30%, and morbidity, with >60% of affected children requiring lifelong pacemakers.4 To date, complete AV block is irreversible, although varying degrees of block have been noted, and second-degree block has on rare occasion reverted to normal sinus rhythm.6 Despite exposure to the identical circulating autoantibodies, third-degree AV block has not been reported in these mothers, but several cases of first-degree AV block have been described.7
The candidate antigens and their cognate antibodies have been extensively characterized at the molecular level. SSA/Ro (60 kD) contains a putative zinc finger and an RNA-binding protein consensus motif,8 both of which could account for its direct interaction with small cytoplasmic hY-RNAs (a class of low-molecular-weight molecules of 83 to 112 bases that are small uncapped cytoplasmic RNA).9 This protein may function as part of a novel quality control or discard pathway for 5S rRNA precursors in Xenopus oocytes.10 Many sera that recognize 60-kD SSA/Ro protein also react with another protein of 52 kD, comprising three distinct domains: an N-terminal region with three zinc fingers, a central region with a leucine zipper motif, and a C-terminal "rfp-like" domain.11 Anti-SSB/La antibodies recognize a 48-kD polypeptide that does not share antigenic determinants with either 52-kD or 60-kD SSA/Ro.12 SSB/La facilitates maturation of RNA polymerase III transcripts, directly binds a spectrum of RNAs, and associates at least transiently with 60-kD SSA/Ro.13 Antibodies to 60-kD SSA/Ro do not discriminate between mothers whose children have CHB or are unaffected, but at least by SDS immunoblot, we have demonstrated significantly higher responses against 52-kD than 60-kD SSA/Ro in mothers with affected versus unaffected children.3
The mechanism by which maternal autoantibodies directed to intracellular antigens (likely involved in transcriptional regulation) perturb cardiac function is not apparent, raising the possibility that they are "clinical markers" and not truly causal. In one study of fatal CHB, anti-SSA/Ro antibodies were eluted from the affected fetal heart.14 Only two publications, both in animal models, indirectly invoked potential arrhythmogenic effects of anti–SSA/Ro-SSB/La antibodies. Alexander et al15 reported that superfusion of newborn rabbit ventricular papillary muscles with IgG-enriched fractions from sera containing anti–SSA/Ro-SSB/La antibodies specifically reduced the plateau phase of the action potential. Garcia et al,16 using isolated adult rabbit hearts, showed that IgG fractions with anti–SSA/Ro-SSB/La antibodies induced conduction abnormalities and reduced Ca2+ currents. The possible contribution of the anti–52-kD SSA/Ro antibodies to the pathogenesis of CHB remains unknown. Moreover, antibodies isolated from mothers whose children have CHB have not been tested in the human fetal heart. In the present study, we provide evidence for the arrhythmogenicity of anti–52-kD SSA/Ro antibodies by using whole heart and isolated myocytes from human fetuses and for the pathogenicity of these antibodies by establishing an animal model of CHB.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Preparation of cDNA for Recombinant Proteins and Plasmid Construction
Based on previously reported sequences, primers containing internal restriction sites to facilitate in-frame directional cloning were designed to amplify DNA fragments encoding full-length 48-kD SSB/La and 52-kD and 60-kD SSA/Ro cDNA. These cDNA fragments were subcloned into pET-28a, -28b, or -28c (Novagen). Each construct was subcloned to allow expression of the 6XHis Tag for Ni2+ resin purification. DNA sequencing confirmed the identity of each subclone.
Expression and Purification of Recombinant Proteins
Escherichia coli (DH3) were transformed with each recombinant plasmid, and cultures were induced with isopropyl ß-D-thiogalactopyranoside. Stored cells were thawed and lysed in 5 mL buffer A (6 mol/L guanidine HCl, 0.1 mol/L sodium phosphate, and 0.01 mol/L Tris-HCl, pH 8.0). After sonication and centrifugation, the supernatant was collected and added to 8 mL of a 50% slurry of Ni-NTA resin (Qiagen). Recombinant proteins were incubated with the Ni-NTA resin, and the slurry was loaded into a 1.5-cm-diameter column. Columns were washed with buffer B (8 mol/L urea, 0.1 mol/L sodium phosphate, and 0.01 mol/L Tris-HCl, pH 8.0) until the A280 reached <0.01. This was followed by an additional wash with buffer C (8 mol/L urea, 0.1 mol/L sodium phosphate, and 0.01 mol/L Tris-HCl, pH 6.3) until the A280 again reached <0.01. The recombinant proteins were eluted with 20 mL buffer D (8 mol/L urea, 0.1 mol/L sodium phosphate, and 0.01 mol/L Tris-HCl, pH 5.9), 20 mL buffer E (8 mol/L urea, 0.1 mol/L sodium phosphate, and 0.01 mol/L Tris-HCl, pH 5.9), and 20 mL buffer F (6 mol/L guanidine HCl and 0.2 mol/L acetic acid), each collected in 3-mL fractions. Elution fractions were analyzed by SDS-PAGE. Samples containing recombinant protein were dialyzed in 0.5 mol/L NaCl and 0.1 mol/L NaHCO3, pH 8.3.
Purification of IgG Fractions
Immunoglobulin fractions containing IgG were purified from serum by protein A–Sepharose gel separation and confirmed to be pure by electrophoresis.
Affinity Purification of Antibodies
Antibodies against either 48-kD SSB/La or 52-kD or 60-kD SSA/Ro proteins were isolated from sera by affinity column chromatography, using the respective recombinant proteins coupled to CNBr-activated Sepharose 4B as antigens. Eluted antibodies were neutralized with 1 mol/L Tris and tested for specificity by ELISA, immunoblot, and immunoprecipitation (Fig 1
). Both IgG and affinity-purified antibodies were dialyzed against Tyrode's solution to obtain stock concentrations of 2 to 4 mg/mL as determined by the Pierce BCA protein assay according to the manufacturer's instructions.
|
ELISA
Recombinant SSA/Ro and SSB/La proteins were used as ELISA substrates. Typically, 60-kD and 52-kD SSA/Ro recombinant protein fractions were diluted 1000 and 16 000-fold, respectively, in PBS for coating 96-well microliter plates (
1 µg for the 60-kD protein and 0.1 µg for the 52-kD protein). Plates were incubated overnight at 4°C. The plates were then washed with PBS containing 0.05% Tween 20 (PBS-Tween) and blocked with 1% BSA in PBS-Tween, followed by incubation with patient or murine antisera at a 1:1000 dilution for 1 hour. Each serum was run in duplicate. After washing, F(ab')2 goat anti-human or mouse IgG alkaline phosphate conjugate was added for 1 hour. The plates were washed again and developed with p-nitrophenyl phosphate, disodium salt, in diethanolamine buffer. Results were expressed as the optical absorbance at 405 nm less reagent blank.
Immunoblot
The cell line MOLT4, derived from a patient with acute T-cell lymphoblastic leukemia, was used as the source of antigen as described previously.17
Immunoprecipitation
Immunoprecipitation of in vitro translation products was performed as described previously.18 Briefly, 10 µL of the patient's serum was mixed with 50 µL of 50% protein A–Sepharose, 100 µL of 10 mg/mL BSA, 200 µL reaction buffer (150 mmol/L NaCl, 4 mmol/L EDTA, 50 mmol/L Tris-HCl, pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS), and 1 to 10 µL labeled in vitro translation product. The reaction mixtures, in 1.5-mL Eppendorf tubes, were rotated at 4°C for 1 to 3 hours. The beads were then washed four times with chilled reaction buffer and then run on 15% high-ratio monomer/cross-linker acrylamide gels overnight at 7.5 mA until the dye front ran off. The gel was then stained and destained to visualize molecular weight markers and ensure uniformity of the immunoglobulin heavy and light chains precipitated in each reaction, dried with heat and vacuum, and placed with film at -70°C.
ECG Recordings
Twenty-five human fetal hearts at gestational ages of 15 to 24 weeks were obtained after elective termination of normal pregnancy by dilatation and evacuation. This was done in accordance with the guidelines of the Institutional Review Board and after obtaining consent from the mothers. After transport on iced PBS (<15 minutes), hearts were cannulated through the aorta for continuous perfusion of the coronary arteries with oxygenated (100% O2) Tyrode's solution at 37°C. Tyrode's solution contained (mmol/L) NaCl 117, KCl 5.7, CaCl2 1.8, NaHCO3 4.4, NaH2PO4 1.5, MgCl2 1.7, HEPES 20, glucose 11, creatine 10, and taurine 20. pH was adjusted to 7.4 with NaOH. The hearts were lowered into a double-jacketed beaker containing prewarmed Tyrode's solution. ECG tracings were obtained by conventional technique except for the use of silver wires (diameter, 0.3 mm each) at the recording end of the leads. One wire was inserted into the right atrium; one, in the left ventricle near the apex; and one, in Tyrode's solution (ground).
Cell Dissociation
Cardiac myocytes were obtained from human fetal hearts (aged 15 to 24 weeks) perfused by the Langendorff technique as previously described.19 20 Each heart was perfused with a HEPES-buffered solution containing (mmol/L) NaCl 117, KCl 5.7, NaHCO3 4.4, NH2PO4 1.5, MgCl2 1.7, HEPES 20, glucose 11, creatine 10, taurine 20, and 21 mU/mL insulin, and the pH was adjusted to 7.4 with NaOH. The perfusion solution was gassed with 100% O2 and prewarmed to 37°C. The heart was then perfused with fresh buffer mixed with 1 mg/mL collagenase type A or B (Boehringer Mannheim Corp) and 20 µmol/L Ca2+ for 5 to 10 minutes. The ventricles were then cut off and stirred to obtain cells. Cells were suspended in Petri dishes containing HEPES buffer with 1 mmol/L CaCl2 and 0.5% BSA (pH 7.4). Cells were transferred to a 0.5-mL recording chamber and superfused at a rate of 3 mL/min.
Solutions
For ICa recordings, external solution contained (mmol/L) NaCl 132, CsCl 5.4, CaCl2 1.8, MgCl2 1.8, NaH2PO4 0.6, 4-aminopyridine 5, HEPES 10, dextrose 5, and sodium pyruvate 5, pH 7.4. Patch electrodes (0.8 to 1.6 M
) were filled with control internal solution containing (mmol/L) CsCl 139.8, K2EGTA 10, MgCl2 4, CaCl2 0.062, Na2–creatine phosphate 5, HEPES 10, Na2ATP 3.1, and Na2GTP 0.42, adjusted to pH 7.1 with KOH.
Electrophysiology
The patch-clamp technique was used to record currents.21 Whole-cell ICa and unitary currents through Ca2+ channels were recorded as previously described.19 20 Briefly, K+ currents were blocked with intracellular and extracellular Cs+ and 4-aminopyridine. The fast Na+ and possible T-type Ca2+ currents were blocked by a prepulse to -40 mV from a holding potential of -80 mV in the presence of tetrodotoxin (50 µmol/L). Cells were depolarized every 10 seconds from a holding potential of -80 mV to a prepulse level of -40 mV for 100 milliseconds and subsequently to a test pulse of 0 mV for 300 milliseconds. Capacitive currents were elicited by a 10-mV depolarizing pulse from -80 mV and then compensated. The capacitive traces were fitted by a single-exponential equation, and Cm was calculated according to the following equation: Cm=
c·Io/
Em, where Cm is the membrane capacitance, c is the time constant for cell membrane charge, Io is the maximum capacitive current, and Em is the clamp voltage. The average Cm was 15.0±2.1 pF (mean±SEM, n=22). For single-channel recordings,20 the bath solution contained (mmol/L) potassium glutamate 120, KCl 25, MgCl2 2, ATP 1, EGTA 2, and HEPES 10, adjusted to pH 7.4 with KOH. The pipette solution contained (mmol/L) BaCl2 70, sucrose 110, and HEPES 10, adjusted to pH 7.4 with tetraethylammonium hydroxide. (-)Bay K 8644 (1 µmol/L) was added to the pipette solution. Unitary currents were evoked by a 300-millisecond step depolarization to 0 mV from a holding potential of -40 mV at 0.5 Hz. Control experiments (n=3) in the absence of antibodies showed no significant channel activity changes during time equivalent to drug exposure. Analysis of unitary currents was performed after digital subtraction of capacitive and leakage currents by averaging records without channel openings and subtracting the average from each record in the series. Measurements of open and closed times were made at 0 mV, and patches exhibiting more than one open-channel current level were not included in the analysis. The ensemble mean current was obtained by averaging all subtracted current records of the series. The open-state probability was determined by integrating over each sweep. Student's t test for paired data was used to compare control conditions with interventions. A value of P<.05 was considered statistically significant. All experiments were performed at 22°C to 24°C.
Immunization
Immunizations were carried out as previously described.22 Female mice (n=5) were initially immunized intraperitoneally and intracervically with 100 µg of recombinant 52-kD SSA/Ro protein in Freund's adjuvant solution and boosted twice with 50 µg of antigen in incomplete Freund's adjuvant at 10-day intervals. The age-matched vehicle group (n=5) was injected with only Freund's adjuvant solution at similar intervals, and another control group (n=4) did not receive any injections. Offspring were obtained by breeding female mice with syngeneic males.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Antibodies From Mothers Whose Children Have CHB Induce Conduction Defects in Beating Human Fetal Hearts
For evaluation in the isolated beating heart and myocytes, IgG fractions were purified from the serum of six women whose fetuses developed CHB in utero and four multiparous women with healthy children, by protein A/sepharose gel separation. The clinical and serological profiles of the maternal sera are provided in Table 1
. Affinity-purified antibodies were obtained by gel separation of CNBr-coupled histidine fusion proteins isolated by Ni2+ affinity chromatography. Reactivities of the affinity-purified antibodies are depicted in Fig 1
.
|
To assess the effect of IgG fractions and affinity-purified antibodies on conduction and heart rate, ECG recordings were obtained from whole human fetal hearts. Control ECGs were obtained after a stabilization period of 30 to 45 minutes. Fig 2A shows an example of a control ECG with a regular sinus rhythm at 130 bpm. Perfusion of the heart for 27 minutes with 800 µg/mL of purified anti–52-kD SSA/Ro antibody from mothers with affected children (Nos. 1, 2, and 4) resulted in bradycardia (80 bpm) associated with widening of the QRS complex (Fig 2B) that could represent bundle branch block or an intraventricular conduction defect. The average increase in the RR interval corresponded to 32.4±12.7% (n=8). At 33 minutes of perfusion, complete AV block was observed with the presence of only P waves (indicated by single-headed arrows) and missing QRS complexes (indicated by a double-headed arrow, Fig 2C). Reperfusion of the heart with antibody-free Tyrode's solution for 48 minutes resulted in partial and slow recovery (Fig 2D). Superfusion of hearts with IgG fractions (800 µg/mL) from mothers with affected children (Nos. 1, 3, and 4) also induced bradycardia and AV dissociation (not shown). The average increase in the RR interval was 38.4±12.7% (n=8). In contrast, IgG from control mothers (Nos. 7 through 10) did not have any measurable effect on AV conduction (n=6). An example of the effect of control IgG (800 µg/mL ) from mother No. 7 on ECG is shown in Fig 2F. A summary of the experiments sorted by patient's IgG and affinity-purified anti–52-kD SSA/Ro is shown in Table 1 (left).
|
Antibodies From Mothers Whose Children Have CHB Inhibit ICa at the Whole-Cell and Single-Channel Levels
The observed induction of AV block by affinity-purified anti–52-kD SSA/Ro antibodies and IgG fractions strongly suggested the involvement of Ca2+ channels, since AV nodal electrogenesis is largely dependent on ICa.25 26 This hypothesis was tested by characterizing the effects of IgG fractions and affinity-purified anti–52-kD SS/Ro antibodies on whole-cell ICa. Given the technical difficulties in isolating human fetal AV nodal myocytes, ventricular cells, which also contain Ca2+ channels, were used. ICa was routinely elicited by a 300-millisecond test pulse to 0 mV, preceded by a 100-millisecond prepulse to -40 mV from a holding potential of -80 mV every 10 seconds. Under our experimental conditions, the rundown of ICa was minimal (0.3±0.1 pA/min). Recorded currents were enhanced by Bay K 8644 (1 µmol/L), blocked by both 2 µmol/L nisoldipine and 5 mmol/L cobalt (Fig 3A).
|
IgG from mothers whose children had CHB (Nos. 1 and 4) but not from control mothers (Nos. 7, 9, and 10) inhibited peak ICa in a voltage-independent manner. The average inhibition at 0 mV was 58.8±18.6% (n=9, Table 2) at a rate of 11.8±3.5 pA/min. A representative effect of the IgG fraction (80 µg/mL) from a mother whose child had CHB (No. 1) on ICa tracings elicited by test pulses from -30 to 60 mV in a myocyte isolated from an 18-week-old heart is shown in Fig 3B. Washout of IgG resulted in partial recovery of ICa (31.5±6.2%, n=4). Similarly, affinity-purified anti–52-kD SSA/Ro antibodies from mothers whose children had CHB (Nos. 1, 2, and 4) inhibited peak ICa by 56.2±11.7% (n=10) at 0 mV (Fig 3C). The inhibition of ICa by both IgG and anti–52-kD SSA/Ro antibodies from mothers whose children had CHB had a rapid onset of action and slow and partial recovery. The summary of the effect of IgG and affinity-purified anti–52-kD antibodies on ICa is shown in Table 2.
|
Accordingly, inhibition of ICa by the autoantibodies in the isolated myocytes further supported the contribution of Ca2+ channels to the conduction abnormalities observed in the whole heart. The biophysical properties by which the autoantibodies inhibited whole-cell ICa were then investigated at the single-channel level.21 We recorded Ba2+ currents through Ca2+ channels as described previously.20 Depolarizing test pulses to 0 mV from a holding potential of -40 mV at 0.5 Hz elicited inward unitary currents (Fig 4A). These currents were also stimulated by (-)Bay K 8644 (1 µmol/L ), blocked by Ca2+ channel blockers nisoldipine (2 µmol/L) and cobalt (5 mmol/L) (not shown), and had a conductance of 20.3 pS. These characteristics substantiated that the channel activity recorded was attributable to L-type Ca2+ channels. Bath application of 80 µg/mL affinity-purified anti–52-kD SSA/Ro antibody from mothers 1 and 2 produced a significant decrease in the Ca2+ channel activity and the ensemble-averaged current (Fig 4A). The ensemble-averaged currents were inhibited by 43.3±12.5% (n=8) without any significant change in the channel conductance (20.9±0.8 pS during control and 20.5±0.9 pS with anti–52-kD SSA/Ro, n=4, P=NS). Channel conductance was obtained from the slope of the relationship between single-channel current amplitude and membrane potential during step depolarizations between -20 and 10 mV (Fig 4C). Comparable inhibition was obtained with IgG (80 µg/mL) from affected mothers 1 and 4 (48.6±15.4%, n=5), but no significant effect (2.5±2.0%, n=3) was observed with IgG (80 µg/mL) from control mothers 7, 9, and 10. Table 2 summarizes the percent inhibition of currents by the antibodies. Analysis of single-channel kinetics indicated that this inhibition was the result of shorter open times and longer closed times. The open probability (Fig 4B) calculated from all current sweeps averaged 0.22±0.05 for control and 0.11±0.02 for anti–52-kD SSA/Ro antibody (n=6, P<.02). This may, in part, also explain the basis of the whole-cell ICa inhibition by the autoantibody. The inhibitory effect of the affinity-purified anti–52-kD SSA/Ro antibody and IgG from mothers whose children have CHB was less pronounced in the cell-attached than the whole-cell recordings. This suggests the involvement of a diffusible cytosolic constituent in mediating part of the response to autoantibodies. However, a direct effect on the channel protein or a substrate in close proximity cannot be ruled out.
|
, mean±SEM; from 14 traces for each potential), and that of affinity-purified anti–52-kD SSA/Ro antibodies is 20.1 pS (
, mean±SEM; from 12 traces for each potential). Similar results were observed in three other patches.
Immunization of Female BALB/c Mice Induces AV Conduction Defects in the Pups
To correlate the arrhythmogenic effect of anti–52-kD SSA/Ro antibodies on the isolated human fetal heart with the in vivo genesis of CHB, we developed an antibody-specific animal model. This was accomplished by immunizing BALB/c inbred female mice with SSA/Ro 52-kD recombinant protein. Both control groups of pups (8 vehicle and 14 control) had normal heart rates of 500 to 580 bpm with sinus rhythm, a P-wave preceding each QRS complex, and a short PR interval (<30 milliseconds). In contrast, 9 (5 females and 4 males) of 20 pups from the immunized mothers had conduction system abnormalities (Table 3). Four pups had bradycardia defined as 
40% of the maximal normal heart rate. Three pups had PR prolongation defined as >50 milliseconds and wide QRS of 
50 milliseconds. Two pups had complete AV dissociation. Two pups died of unknown cause several hours after birth. Selected ECG tracings from different pups are shown in Fig 5.
|
|
To evaluate the effectiveness of the immunization, ELISAs were performed on sera from immunized and control mothers and their pups. The levels of anti–52-kD and anti–60-kD SSA/Ro and anti–48-kD SSB/La antibodies are summarized in Table 3. All immunized mothers and their corresponding pups showed high levels of anti–52-kD SSA/Ro antibodies, indicating the transplacental passage of the antibody into the fetal circulation. No anti–60-kD SSA/Ro or anti–48-kD SSB/La responses were detected. In the control groups, neither anti-SSA/Ro nor SSB/La antibodies were detected in the mothers or their pups. This antibody-specific animal model provides strong evidence for an etiopathological role of anti–52-kD SSA/Ro antibody in the development of CHB. The somewhat unexpectedly high frequency of expression of disease (45%) compared with women with systemic lupus erythematosus, whose risk of having a CHB baby is between 1% and 5%,27 28 could be attributed to the use of inbred mice that are identical in their genetic makeup.29
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The data presented herein establish that IgG fractions containing anti–SSA/Ro-SSB/La specificities and affinity-purified anti–52-kD SSA/Ro antibodies isolated from the sera of mothers whose children have CHB are arrhythmogenic in the human fetal heart. An effect on L-type Ca2+ channels is indirectly supported by the induction of AV block in the Langendorff-perfused heart and directly supported by the patch-clamp experiments in isolated myocytes. Furthermore, abnormalities in conduction at the AV node were reproduced in fetal mice born to mothers immunized with the 52-kD SSA/Ro recombinant protein.
The AV conduction defect induced by the autoantibodies in the beating human fetal heart coincides with the blockade of Ca2+ channel activity that is responsible for conduction at the AV node.25 26 Ca2+ channel density is lower30 31 and sarcoplasmic reticulum is less abundant32 33 in fetal compared with adult cardiac cells. This makes excitation-contraction coupling and other cytoplasmic Ca2+–regulated functions critically dependent on transsarcolemmal Ca2+ entry and possibly the Na+-Ca2+ exchanger. Moreover, there is developmental regulation in the expression of two alternatively spliced cardiac L-type Ca2+ channel 
1-subunit transcripts.34 Both isoforms are equally expressed in the fetal heart, but only one is predominantly expressed in the adult heart. Although each transcript encodes a functional Ca2+ channel, physiological comparisons to determine any functional consequences of the two isoforms have not been reported to date. These developmental differences could account for the apparently unique vulnerability of the fetal heart.
Prolonged exposure of fetal Ca2+ channels to the maternal anti–SSA/Ro-SSB/La antibodies may lead to internalization and degradation of the channel, cell death, and ultimately fibrosis. This hypothesis is supported by available autopsy reports that document fibrosis of the AV node.35 In utero echocardiographic evaluation of many fetuses with CHB demonstrates pleuropericardial effusions and decreased ventricular function. These findings are usually attributed to an antibody-induced inflammatory myocarditis36 37 ; however, inhibition of ventricular Ca2+ channels may also result in diminished contraction and congestive heart failure.
Several lines of evidence indicate that, in addition to anti-SSA/Ro antibodies, there is likely to be a fetal factor required for the development of heart block. Recurrent pregnancies complicated by CHB occur with a frequency <20%,4 38 39 and there is discordance in monozygotic twins.40 Expression of alternative Ca2+ channel isoforms may also contribute, in part, to the susceptibility of only some fetuses. Moreover, it has been demonstrated that an alternatively spliced form of the 52-kD SSA/Ro protein lacking the leucine zipper is preferentially expressed in fetal compared with adult hearts.41
Ca2+ channels have been identified as autoantigens in a number of diseases. Sera from patients with Lambert-Eaton myasthenic syndrome42 43 44 and amyotrophic lateral sclerosis45 contain IgG antibodies directed against Ca2+ channel proteins. Whether the maternal autoantibodies interact directly and/or indirectly with Ca2+ channel proteins is yet to be determined. The present data (1) provide important insights into the pathogenesis and etiology of CHB, (2) provide the platform for future investigations, and (3) could help in the development of new therapeutic strategies in a disease generally considered to be irreversible.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported in part by the National Institutes of Health, the American Heart Association, Veterans Administration Research Funds, and a Fellowship from the New York Chapter of the Arthritis Foundation.
|
Footnotes |
|---|
*Both authors contributed equally to this article.
Received July 17, 1996; accepted November 26, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. McCue CM, Mantakas ME, Tingelstad JB, Ruddy S. Congenital heart block in newborns of mothers with connective tissue disease. Circulation. 1977;56:82-90.
2. Chameides L, Truex RC, Vetter V, Rashkind WJ, Galioto FM, Noonan JA. Association of maternal systematic lupus erythematosus with congenital complete heart block. N Engl J Med. 1977;297:1204-1207.[Abstract]
3. Buyon JP, Winchester RJ, Slade SG, Arnett F, Copel J, Freidman D, Lockshin MD. Identification of mothers at risk for congenital heart block and other neonatal lupus syndromes in their children: comparison of ELISA and immunoblot to measure anti-SSA/Ro and anti-SSB/La antibodies. Arthritis Rheum. 1993;36:1263-1273.[Medline] [Order article via Infotrieve]
4.
Waltuck J, Buyon JP. Autoantibody associated complete heart block: outcome in mothers and children. Ann Intern Med. 1994;120:544-551.
5. Scott JS, Maddison PJ, Taylor PV, Esscher E, Scott O, Skinner RP. Connective tissue disease, antibodies to ribonucleoprotein and congenital heart block. N Engl J Med. 1983;309:209-212.[Abstract]
6. Copel J, Buyon J, Kleinman C. Successful in utero therapy of fetal heart block. Am J Obstet Gynecol. 1995;173:1384-1390.[Medline] [Order article via Infotrieve]
7. Dorner T, Hiepe F, Goldner B, Apostoloff E. Investigations into Ro-specific antibody-associated congenital cardiac conduction defects. Clin Invest. 1992;70:492-496.[Medline] [Order article via Infotrieve]
8. Ben-Chetrit E, Gandy BJ, Tan EM, Sullivan KF. Isolation and characterization of a cDNA clone encoding the 60-kD component of the human SS-A/Ro ribonucleoprotein auto antigen. J Clin Invest. 1989;83:1284-1292.
9.
Lerner MR, Boyle JA, Hardin JA, Steitz JA. Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus. Science. 1981;211:400-402.
10.
O'Brien CA, Wolin SL. A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors. Genes Dev. 1994;8:2891-2903.
11.
Ben-Chetrit E, Chan EKL, Sullivan KF, Tan EM. A 52-kD protein is a novel component of the SS-A/Ro antigenic particles. J Exp Med. 1988;167:1560-1571.
12. Chan EKL, Francoeur AM, Tan EM. Epitopes, structural domains and asymmetry of amino acid residues in SSA-B/La nuclear protein. J Immunol. 1986;136:3744-3749.[Abstract]
13. Gottlieb E, Steitz JA. Function of mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J. 1989;8:851-861.[Medline] [Order article via Infotrieve]
14. Reichlin M, Brucato A, Frank MB, Maddison PJ, McCubbin VR, Wolfson-Reichlin M, Lee L. Concentration of autoantibodies to native 60-kD Ro/SS-A and denatured 52-kD Ro/SS-A in eluates from the heart of a child who died with congenital complete heart block. Arthritis Rheum. 1994;37:1698-1703.[Medline] [Order article via Infotrieve]
15. Alexander E, Buyon JP, Provost TT, Guarnicri Y. Anti-Ro/SS-A antibodies in the pathophysiology of congenital heart block in neonatal lupus syndrome, an experimental model: in vitro electrophysiologic and immunocytochemical studies. Arthritis Rheum. 1992;35:176-189.[Medline] [Order article via Infotrieve]
16. Garcia S, Nascimento JHM, Bonfa E, Levey R, Oliveirn SF, Tavares AV, Campos deCarvalho AC. Cellular mechanism of the conduction abnormalities induced by serum from anti-Ro/SSA-positive patients in rabbit hearts. J Clin Invest. 1994;93:718-724.
17. Buyon JP, Slade SG, Chan EKL, Tan EM, Winchester R. Effective separation of the 52 kD SSA/Ro polypeptide from the 48 kD SSB/La polypeptide by altering condition of polyacrylamide gel electrophoresis. J Immunol Methods. 1990;129:207-210.[Medline] [Order article via Infotrieve]
18. Buyon JP, Slade SG, Reveille JD, Hamel JC, Chan EKL. Autoantibody responses to the `native' 52 kD SS-A/Ro protein in neonatal lupus syndromes, systemic lupus erythematosus and Sjogren's syndrome. J Immunol. 1994;152:3675-3684.[Abstract]
19.
Boutjdir M, Restivo M, Wei YS, El-Sherif N. 1- and ß-adrenergic interactions on L-type calcium current in cardiac myocytes. Pflugers Arch. 1992;421:397-399.[Medline]
[Order article via Infotrieve]
20.
Chen L, El-Sherif N, Boutjdir M. 1-Adrenergic activation inhibits ß-adrenergic–stimulated unitary Ca2+ currents in cardiac ventricular myocytes. Circ Res. 1996;79:184-193.
21. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth F. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.[Medline] [Order article via Infotrieve]
22.
Topfer F, Gordon T, McCluskey J. Intra- and intermolecular spreading of autoimmunity involving the nuclear self-antigens La (SS-B) and Ro (SS-A). Proc Natl Acad Sci U S A. 1995;92:875-879.
23. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Talal N, Winchester RJ. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271-1277.[Medline] [Order article via Infotrieve]
24. Boire G, Lopez-Longo FJ, Lapointe S, Menar HA. Sera from patients with autoimmune disease recognize conformational determinants on the 60 kD Ro/SSA protein. Arthritis Rheum. 1991;34:722-730.[Medline] [Order article via Infotrieve]
25.
Zipes DP, Mendez C. Action of manganese ions and tetrodotoxin on atrioventricular nodal transmembrane potentials in isolated rabbit hearts. Circ Res. 1973;32:447-454.
26.
Hancox JC, Levy AJ. L-type calcium current in rod- and spindle-shaped myocytes isolated from rabbit atrioventricular node. Am J Physiol. 1994;267:H1670-H1680.
27. Lee LA. Neonatal lupus erythematosus. J Invest Dermatol. 1993;100:9s-13s. Review.[Medline] [Order article via Infotrieve]
28. Ramsey-Goldman R, Hom D, Deng JS, Ziegler GC, Kahl LE, Steen V, La Porte RE, Medsger TA Jr. Anti SS-A antibodies and fetal outcome in maternal systematic lupus erythematosus. Arthritis Rheum. 1986;29:1269-1273.[Medline] [Order article via Infotrieve]
29. Kalush F, Rimon E, Keller A, Mozes E. Neonatal lupus erythematosis with cardiac involvement in offspring of mothers with experimental systemic lupus erythematosus. J Clin Immunol. 1994;14:314-322.[Medline] [Order article via Infotrieve]
30.
Huynh TV, Chen F, Wetzel GT, Friedman WF, Klitzner TS. Developmental changes in membrane Ca2+ and K+ currents in fetal, neonatal, and adult rabbit ventricular myocytes. Circ Res. 1992;70:508-515.
31. Boucek RJ Jr, Shelton M, Artman M, Mushlin PS, Starnes VA, Olsen RD. Comparative effects of verapamil, nifedipine and diltiazem on contractile function in the isolated fetal and adult rabbit heart. Pediatr Res. 1984;18:948-952.[Medline] [Order article via Infotrieve]
32. Hoerter J, Mazet F, Vassort G. Perinatal growth of the rabbit cardiac cell: possible implications for the mechanism of relaxation. J Mol Cell Cardiol. 1982;13:725-740.
33. Maylie JG. Excitation-contraction coupling in neonatal and adult myocardium of cat. Am J Physiol. 1982;242:H834-H843.
34.
Diebold RJ, Koch WJ, Ellinor PT, Wang JJ, Muthuchamy M, Wieczorek DF, Schwartz A. Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart. Proc Natl Acad Sci U S A. 1992;89:1497-1501.
35. Ho YS, Esscher E, Anderson RH, Michaelsson M. Anatomy of congenital complete heart block and relation to maternal anti-Ro antibodies. Am J Cardiol. 1986;58:291-294.[Medline] [Order article via Infotrieve]
36. Buyon J, Swersky S, Fox H, Bierman F, Winchester R. Intra uterine therapy of presumptive fetal myocarditis with heart block occurring in a mother with systemic lupus erythematosus and a predominance of La (SSB) antibodies. Arthritis Rheum. 1987;40:44-49.
37. Carreira PE, Gutierrez-Larraya F, Gomez-Reino JJ. Successful intrauterine therapy with dexamethasone for fetal myocarditis and heart block in a mother with systemic lupus erythematosus. J Rheumatol. 1993;20:1204-1207.[Medline] [Order article via Infotrieve]
38. McCune AB, Weston WL, Lee LA. Maternal and fetal outcome in neonatal lupus erythematosus. Ann Intern Med. 1987;106:518-523.
39. Julkunen H, Kaaja R, Wallgren E, Teramo K. Isolated congenital heart block in fetal and infant outcome and familial incidence of heart block. Obstet Gynecol. 1993;82:11-16.[Medline] [Order article via Infotrieve]
40. Buyon JP. Neonatal lupus syndrome. In: Lahita R, ed. Systemic Lupus Erythematosus. New York, NY: Churchill Livingstone; 1992:419-435.
41.
Chan EKL, DiDonato F, Hamel JC, Tseng CE, Buyon JP. 52 kD SSA/Ro: genomic structure and identification of alternatively spliced transcript encoding a novel leucine zipper-minus autoantigen. J Exp Med. 1995;182:983-992.
42.
Kim YI, Neher E. IgG from patients with Lambert-Eaton syndrome blocks voltage dependent calcium channels. Science. 1988;239:405-408.
43.
Peers C, Lang B, Newsom-Davis J, Wray WD. Selective action of myasthenic syndrome antibodies on calcium channel in a rodent neuroblastoma x glioma cell line. J Physiol (Lond). 1990;421:293-308.
44. Rosenfeld MR, Wong E, Dalmau J, Manley GF, Posner JB, Sher E, Furneaux HM. Cloning and characterization of a Lambert-Eaton myasthenic syndrome antigen. Ann Neurol. 1993;33:113-120.[Medline] [Order article via Infotrieve]
45.
Delbono O, Garcia J, Appel SH, Stephani E. IgG from amyotrophic lateral sclerosis affects tubular calcium channels of skeletal muscle. Am J Physiol. 1991;260:C1347-C1351.
This article has been cited by other articles:
 |
|
 |
|
  A.J.J.T. Rein, D. Mevorach, Z. Perles, S. Gavri, M. Nadjari, A. Nir, and U. Elchalal Early Diagnosis and Treatment of Atrioventricular Block in the Fetus Exposed to Maternal Anti-SSA/Ro-SSB/La Antibodies: A Prospective, Observational, Fetal Kinetocardiogram-Based Study Circulation, April 14, 2009; 119(14): 1867 - 1872. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.P. Buyon and R.M. Clancy Dying right to live longer: positing apoptosis as a link between maternal autoantibodies and congenital heart block Lupus, February 1, 2008; 17(2): 86 - 90. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Gerosa, R. Cimaz, M. Stramba-Badiale, K. Goulene, E. Meregalli, L. Trespidi, B. Acaia, R. Cattaneo, A. Tincani, M. Motta, et al. Electrocardiographic abnormalities in infants born from mothers with autoimmune diseases a multicentre prospective study Rheumatology, August 1, 2007; 46(8): 1285 - 1289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Multiple Annular Plaques in an Infant--Diagnosis Arch Dermatol, October 1, 2006; 142(10): 1351 - 1356. [Full Text] [PDF]
|
|
|
|
|
|
L Strandberg, S Salomonsson, K Bremme, S-E Sonesson, and M Wahren-Herlenius Ro52, Ro60 and La IgG autoantibody levels and Ro52 IgG subclass profiles longitudinally throughout pregnancy in congenital heart block risk pregnancies Lupus, June 1, 2006; 15(6): 346 - 353. [Abstract] [PDF]
|
|
|
|
|
|
Y. Qu, G. Baroudi, Y. Yue, and M. Boutjdir Novel Molecular Mechanism Involving {alpha}1D (Cav1.3) L-Type Calcium Channel in Autoimmune-Associated Sinus Bradycardia Circulation, June 14, 2005; 111(23): 3034 - 3041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Hu, Y. Qu, Y. Yue, and M. Boutjdir Functional Basis of Sinus Bradycardia in Congenital Heart Block Circ. Res., March 5, 2004; 94 (4): e32 - e38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A Brucato, A Jonzon, D Friedman, L D Allan, G Vignati, M Gasparini, J I Stein, S Montella, M Michaelsson, and J Buyon Proposal for a new definition of congenital complete atrioventricular block Lupus, June 1, 2003; 12(6): 427 - 435. [Abstract] [PDF]
|
|
|
|
|
|
E Rosenthal Spectrum of conduction abnormalities associated with maternal anti-Ro antibodies Lupus, March 1, 2002; 11(3): 135 - 136. [PDF]
|
|
|
|
|
|
A D Askanase, D M Friedman, J Copel, M R Dische, A Dubin, T J Starc, M C Katholi, and J P Buyon Spectrum and progression of conduction abnormalities in infants born to mothers with anti-SSA/Ro-SSB/La antibodies Lupus, March 1, 2002; 11(3): 145 - 151. [Abstract] [PDF]
|
|
|
|
 |
|
 J. P. Buyon, D. Nugent, E. Mellins, and C. Sandborg Maternal Immunologic Diseases and Neonatal Disorders NeoReviews, January 1, 2002; 3(1): e3 - 10. [Full Text] [PDF]
|
|
|
|
|
|
P. A. Gordon, M. A. Khamashta, G. R. V. Hughes, and E. Rosenthal A normal ECG at birth does not exclude significant congenital cardiac conduction disease associated with maternal anti-Ro antibodies Rheumatology, August 1, 2001; 40(8): 939 - 940. [Full Text] [PDF]
|
|
|
|
 |
|
 G.-Q. XIAO, Y. QU, K. HU, and M. BOUTJDIR Down-regulation of L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits FASEB J, July 1, 2001; 15(9): 1539 - 1545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Restivo, D. O. Kozhevnikov, and M. Boutjdir Optical mapping of activation patterns in an animal model of congenital heart block Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1889 - H1895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-Q. Xiao, K. Hu, and M. Boutjdir Direct Inhibition of Expressed Cardiac L- and T-Type Calcium Channels by IgG From Mothers Whose Children Have Congenital Heart Block Circulation, March 20, 2001; 103(11): 1599 - 1604. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Brucato, R. Cimaz, L. Catelli, P. Meroni, M. Boutjdir, N. El-Sherif, J. A. Mazel, and J. Buyon Anti-Ro-Associated Sinus Bradycardia in Newborns Response Circulation, September 12, 2000; 102 (11): e88 - e89. [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Qu, A. Ghatpande, N. El-Sherif, and M. Boutjdir Gene expression of Na+/Ca2+ exchanger during development in human heart Cardiovasc Res, March 1, 2000; 45(4): 866 - 873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Mazel, N. El-Sherif, J. Buyon, and M. Boutjdir Electrocardiographic Abnormalities in a Murine Model Injected With IgG From Mothers of Children With Congenital Heart Block Circulation, April 13, 1999; 99(14): 1914 - 1918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M-K Sire, H Julkunen, R Kaaja, P Kurki, and S Koskimies Role of HLA in congenital heart block: susceptibility alleles in mothers Lupus, January 1, 1999; 8(1): 52 - 59. [Abstract] [PDF]
|
|
|
|
 |
|
 M. E. Miranda-Carus, M. Boutjdir, C.-E Tseng, F. DiDonato, E. K. L. Chan, and J. P. Buyon Induction of Antibodies Reactive with SSA/Ro-SSB/La and Development of Congenital Heart Block in a Murine Model J. Immunol., December 1, 1998; 161(11): 5886 - 5892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Miranda, C.-E Tseng, W. Rashbaum, R. L. Ochs, C. A. Casiano, F. Di Donato, E. K. L. Chan, and J. P. Buyon Accessibility of SSA/Ro and SSB/La Antigens to Maternal Autoantibodies in Apoptotic Human Fetal Cardiac Myocytes J. Immunol., November 1, 1998; 161(9): 5061 - 5069. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E Rosenthal New insights into the pathogenesis of anti-Ro antibody associated congenital complete heart block Lupus, March 1, 1998; 7(3): 135 - 136. [PDF]
|
|
|
|
|
|
V S. Viana, S Garcia, J H. Nascimento, K B Elkon, N Brot, A C Campos de Carvalho, and E Bonfa Induction of in vitro heart block is not restricted to affinity purified anti-52 kDa Ro/SSA antibody from mothers of children with neonatal lupus Lupus, March 1, 1998; 7(3): 141 - 147. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||