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Molecular and Ionic Basis of Congenital Complete Heart Block
Wolpers HG, Burchert W, van den Hoff J, et al.: 1997. Assessment of myocardial viability by use of 11C-acetate and positron emission tomography. Threshold criteria of reversible dysfunction. Circulation 95:1417–1424.
Zhang J, Ishibashi Y, Zhang Y, et al.: 1997. Myocardial bioenergetics during acute hibernation. Am J Physiol 273:H1452–H1463. PII S1050-1738(00)00058-X
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Molecular and Ionic Basis of Congenital Complete Heart Block Mohamed Boutjdir*
Congenital heart block (CHB), detected at or before birth in a structurally normal heart, is strongly associated with autoantibodies reactive with the intracellular soluble ribonucleoproteins 48kD SSB/La, 52kD SSA/Ro, and 60kD SSA/Ro. CHB is presumed to be due to the transplacental passage of autoantibodies from the mother into the fetal circulation. Varying degrees of heart block have been reported. Although second degree block has, on rare occasion, reverted to normal sinus rhythm, complete atrio-ventricular (AV) block is irreversible. CHB carries substantial mortality and morbidity, with .60% of affected children requiring lifelong pacemakers. The recurrence rate exceeds, by at least twofold, that of the first birth and is likely to influence the decision to have more children. Curiously, the mother’s heart is almost never affected (with complete heart block) despite exposure to identical circulating autoantibodies. As part of our continuing effort to understand the complex factors contributing to the pathogenesis of CHB, we have established an animal model of CHB by immunizing female mice with recombinant proteins/antigens, reproduced the human complete AV block in an isolated Langendorff perfused fetal heart, and correlated these findings with L-type Ca channel inhibition by maternal antibodies from mothers of children with CHB. In addition, we established a passive animal model by directly injecting maternal antibodies into pregnant mice and reported significant sinus bradycardia, indicating that the spectrum of conduction abnormalities may extend beyond the AV node. All together, the data provided strong evidence supporting an etiologic role of antibody/Ca channel involvement in the pathogenesis of CHB. However, other yet unknown factors seem necessary to explain the full expression of CHB. (Trends Cardiovasc Med 2000;10:114–122). © 2001, Elsevier Science Inc.
Mohamed Boutjdir is at the Molecular and Cellular Cardiology Program, New York Harbor Healthcare System and SUNY Health Science Center, Brooklyn, New York, USA. * Address correspondence to: Dr. Mohamed Boutjdir, Research and Development Office (151), New York Harbor Healthcare System, 800 Poly Place, Brooklyn, NY 11209. Tel.: 718630-3645; fax: 718-630-3796; e-mail: [email protected] © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
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Isolated congenital heart block (CHB) was first recognized in 1901 by Morquio and later by Plant using auscultation and electrocardiography (Morquio 1901, Plant and Steven 1945). The association between isolated CHB and maternal connective tissue disease was described by Hull and others in the late 1960s and 1970s (Chameides et al. 1977, Hull et al. 1966, McCue et al. 1977). This relationship has since become well established and ascribed to the presence of SSA/Ro-SSB/La antibodies in maternal sera. SSA/Ro-SSB/La antibodies (maternal antibodies and maternal IgG are interchangeably used throughout) produce conduction abnormalities in the otherwise normally developing fetus (with no evidence of major cardiac structural abnormalities), yet third-degree AV block has not been documented in the mother (O’Neill et al. 1993). Because of the rarity and complex etiology of CHB, the true incidence is not well established. In general 1 in 15,000– 20,000 live births is often reported although this was based on information collected prior to the 1960s with a limited number of affected children (Michaelsson and Engle 1972). More recent studies (Buyon et al. 1998, Siren et al. 1998) using data collected during the latter decades, seem to indicate that the incidence of CHB is higher than previously reported (about 1 in 11,000). It was suggested that this is likely due to more effective detection and improved diagnostics of CHB during pregnancy (Siren et al. 1998). In the lupus population with anti-SSA/Ro and anti-SSB/La antibodies, the incidence rises to 3–5 in 100 (Buyon 1999). Patients at greatest risk are infants of mothers with a prior history of a CHB delivery, with a recurrence rate of 16–20%. CHB presents with first-, second-, or third-degree AV block. Third-degree AV block is most common and once manifest is always permanent. Mortality approaches 30%, usually within the first three months of life (Buyon 1999). Current therapies include dexamethasone, plasmapheresis, sympathomimetics, and in utero cardiac pacing. Unfortunately none have significantly altered mortality justifying the need for more aggressive support for both basic and clinical investigations. • Intracellular Autoantigens to Maternal Antibodies The candidate antigens and their cognate antibodies have been extensively characTCM Vol. 10, No. 3, 2000
terized at the molecular level. 60 kD SSA/Ro contains a putative zinc finger and an RNA-binding protein consensus motif (Ben-Chetrit et al. 1989), both of which could account for its direct interaction with small cytoplasmic hY-RNAs. Many sera which recognize 60 kD SSA/ Ro protein also react with another protein of 52 kD (SSA/Ro), 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 (BenChetrit el al. 1988). Anti-SSB/La antibodies recognize a 48-kD polypeptide that does not share antigenic determinants with either 52 kD or 60 kD SSA/Ro (Chan et al. 1986). 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 (Gottlieb and Steitz 1989). An alternative mRNA transcript of 52 kD SSA/Ro, derived from the splicing of exon 4, was identified and encodes a smaller protein, 52b of 45 kD (Buyon et al. 1997). Interestingly, the expression of 52b mRNA is maximal at 14–16 weeks gestation, which coincides with the time of cardiac ontogeny when placental transfer of maternal antibodies into the fetal circulation becomes effective (Buyon 1999). • Other Potential Antigenic Targets to Maternal Antibodies The possibility that maternal autoantibodies may cross-react with some antigenic targets other than intracellular Ro/La autoantigen has been explored. Antibodies from mothers, whose children had CHB, were reported to cross-react with laminin B-1 and to human cardiac myosin heavy chain (Horsfall and Rose 1992). Computer-based analysis of the amino acid sequence from the SWISSPROT database between the 52 kD SSA/ Ro, 60 kD SSA/Ro, 48 kD SSB/La proteins and the a1-subunit of L-type human cardiac sarcolemmal Ca channels revealed that most of the homology is with 52 kD SSA/Ro with very minor homology to 60 kD SSA/Ro and 48 kD SSB/La. When the amino acid sequence was compared in a linear representation, the maximum number of homologous amino acids between the 52 kD SSA/Ro and a1-subunit of L-type Ca channel did not exceed four amino acids. However, when TCM Vol. 10, No. 3, 2000
a three-dimensional representation of the a1-subunit structure was used, numerous homologous epitopes became adjacent in the extracellular loops. Epitopic regions on the a1-subunit could then be identified; one of which corresponds primarily with the pore-forming region between segments S5 and S6 of domain VI. This indicates potential direct interaction sites of maternal antibodies with sarcolemmal L-type cardiac Ca channel a1-subunit. • Proposed Pathophysiological Mechanisms of CHB Molecular and Biochemical Evidence Because the candidate antigens are intracellular and there is no convincing evidence that maternal antibodies can cross the sarcolemma of a normal cell, effort has been directed toward mechanisms that may cause the translocation of these antigens to the cell surface membrane. The conventional wisdom is that these antigens must be accessible to maternal antibodies to explain the pathological events, mainly the inflammatory process that may lead to CHB. Substantial experimental evidence has been proposed to account for the accessibility of antigens to maternal antibodies. This includes: 1) viral infection of cells to induce the antigen’s translocation to the cell surface (Baboonian et al. 1989); 2) IFNg treatment of epithelial T24 cells resulting in surface expression of SSB/La; 3) the expression of SSA/Ro and SSB/La antigens on the keratinocyte cell surface by ultraviolet light (LeFeber et al. 1984); 4) 17 b-estradiol enhanced binding of anti-SSA/Ro and anti-SSB/La antibodies to keratinocytes (Furukawa et al. 1988); 5) autopsy studies showing that maternal IgG was associated with specificity for the SSB/La antigen on the surface of fetal myocardial fibers (Horsfall et al. 1991); and 6) unknown inductive events in utero which may lead to apoptotic cell death, thus exposing the antigens (Miranda et al. 1998a). While autoantigen translocation to cell surface could be demonstrated, it is not yet established what are the resulting cellular events and signaling pathways that could account for the tissue injury. Electrophysiological Evidence While the molecular aspects of CHB have been extensively characterized [see Buyon
(1999) for review] the basic electrophysiological mechanisms of CHB are emerging. First, Alexander et al. (1989) demonstrated that IgG fraction of anti-SSA/ Ro SSB/La-positive maternal sera shortened neonatal rabbit cardiac action potential repolarization. Garcia et al. (1994), showed that electrocardiographic conduction disorders associated with neonatal lupus could be reproduced in an isolated adult rabbit heart. Further, they showed that anti-SSA/Ro positive sera inhibited L-type Ca current, ICa-L in isolated ventricular myocytes. Subsequently we established an active, antigen-specific (Boutjdir et al. 1997, Miranda et al. 1998b), and a passive (Mazel et al. 1999) animal model of the human CHB, showed that maternal autoantibodies can induce complete AV block in Langendorff perfused beating heart and in isolated atrioventricular multicellular preparation (Boutjdir et al. 1997, 1998). In addition, these findings correlated well with the inhibition of ICa-L [responsible for electrogenesis at the AV node (Zipes and Mendez 1973)] by the antibodies in isolated cardiac myocytes (Boutjdir et al. 1997, 1998). Below is a brief summary of the above work related to CHB from our laboratory. For clarity and easy flow of thought, characterization of CHB is presented starting from the whole animal, isolated beating heart, multicellular AV nodal preparation and finally single myocyte. Characterization of CHB at the whole animal level. It is a widely held view that CHB is caused by the transplacental transfer of maternal autoantibodies (anti-SSA/ Ro and/or anti-SSB/La) into the fetal circulation. We tested this hypothesis in a passive model in an experimental mouse (BALB/c) by passive transfer of human autoantibodies into pregnant mice (Mazel et al. 1999). Timed pregnant mice were injected with a single intravenous bolus of purified IgG containing human antiSSA/Ro and anti-SSB/La antibodies from mothers of children with CHB. Since the timing of human fetal injury is not random, but instead, occurs during a welldefined period between 15–24 weeks of gestation (Buyon et al. 1995), three groups of mice were used: 8, 11, and 16 days gestation. Within each group, we tested 10, 25, 50, and 100 mg of IgG. At delivery, ECGs were recorded and analyzed for conduction abnormalities. The results are
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Table 1. Summary of ECG parameters in the passive model of CHB
Control Passive 8d 11d 16d
Pups (n)
HR (bpm)
PR (ms)
QRS (ms)
Brady.
I8
II8 & III8
52 16 10 30
526.5 6 8.1 289.3 6 14.6* 233.9 6 19.0* 303.9 6 12.0*
40.4 6 1.1 62.9 6 2.3* 74 6 4.5* 57.8 6 2.3*
22.2 6 1.21 24 6 0.92 24.5 6 1.25 22.8 6 0.52
0 44% 70% 35%
0 88% 90% 45%
0 0 0 0
* p , 0.05 compared to control. 8d, 11d, and 16d indicate the 8th day, 11th day, and 16th day of gestation, respectively.
summarized in Table 1. Marked sinus bradycardia and PR interval prolongation were observed in 8-, 11-, and 16-day gestational groups when compared to controls. Interestingly no complete AV block was seen in this passive model. Antibody levels measured by ELISA in both mothers and their offspring confirmed the transplacental transfer of the human antibodies to the pups. The high incidence of sinus bradycardia suggests possible SA node involvement. These novel findings brought clinical attention not only to AV nodal disorders but also to SA nodal conduction abnormalities. In this regard, Menon et al. (1998) and Brucato et al. (2000) recently reported similar sinus bradycardia in newborns born to mothers seropositive to anti-SSA/Ro antibodies, suggesting that the spectrum of conduction abnormalities associated with maternal autoantibodies extend beyond the AV node and raise the question of whether screening of infants for sinus bradycardia should be part of standard diagnosis in mothers seropositive to anti-Ro/La antibodies. In the active model, the hypothesis being tested was whether the administration of a specific immunogen, not human antibody as in the passive model, to pregnant mice would result in conduction disorders in the pups (Boutjdir et al.
1997, Miranda et al. 1998b). An antigenspecific model was developed using female BALB/c mice immunized with human recombinant 48 kD SSB/La, 60 kD SSA/Ro, 52 kD SSA/Ro (52a), and 52b as well as with murine recombinant 52 kD SSA/Ro. The immunization resulted in high titer responses to the respective antigens as established by ELISA, immunoblotting, and immunoprecipitation. The results are summarized in Table 2. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48 kD SSB/La, 20% of 54 pups born to 60 kD SSA/Ro-immunized mothers, 6% of 56 pups born to 52 kD SSA/Ro-a immunized mothers, 7% of 86 pups born to 52b-immunized mothers and 9% of 22 pups born to mothers immunized with murine 52 kD SSA/Ro. Higher degrees of AV block were only identified in offspring of 52a- or 52b-immunized mice. In the 52a group, one pup had complete AV block and another had second-degree block (Wenckebach type); in the 52b group, five pups developed complete AV block. Maternal antibodies to the primary immunogens were detected in the pups. None of control pups had any conduction abnormalities. This antigenspecific animal model provides strong evidence for a pathogenic role of anti-SSA/ Ro-SSB/La antibodies, particularly 52Ro,
in the development of CHB. The range and frequency of conduction defects suggest that additional factors may promote disease expression. Characterization of CHB at the isolated heart level. Having demonstrated the reproducibility of the clinical CHB in an animal model, we asked the question whether direct perfusion of an isolated beating rat heart with maternal IgG would also result in electrocardiographic conduction abnormalities similar to those seen in affected infants (Boutjdir et al. 1997, 1998). The effects of maternal IgG containing anti-SSA/Ro-SSB/La antibodies on the ECG recording of an isolated rat heart perfused by the Langendorff technique were assessed. Recordings were done using a conventional ECG machine in lead I. After 5 min of perfusion with maternal IgG (800 mg/ml), there was bradycardia associated with 2:1 second-degree AV block that degenerated into complete AV block at about 15 min of perfusion. The QRS complex is absent but the P waves were clearly seen. After 25 min of reperfusion with Tyrode’s solution, only partial recovery was seen. In contrast, perfusion of the heart with normal IgG from healthy mothers with healthy children did not alter ECG parameters. Identical results were obtained
Table 2. Active model of CHB
Immunogen 48-kD La 52a Ro 52b Ro 60-kD Ro Murine 52-kD Ro b-gal (control) Vector (control)
Degree of AV block
Immunized mothers (n)
Fertile mothers (n)
Pups (n)
I8 (n)
II8 (n)
III8 (n)
10 8 5 7 5 3 4
3 5 5 6 4 2 4
27 56 86 54 22 21 43
2 3 5 10 2 0 0
0 1 0 0 0 0 0
0 1 5 0 0 0 0
From Miranda et al. 1998b.
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with maternal IgG for approximately 10 min resulted in 2:1 AV block indicated by the arrows. Longer IgG superfusion (10–15 min) resulted in almost complete inhibition of the AV-node action potential (trace at the bottom of Panel B). Similarly, conduction abnormalities were not seen with normal IgG. These results are consistent with the ECG findings from the whole heart and could account for the conduction abnormalities seen in vitro.
Figure 1. Effects of maternal antibodies on an isolated multicellular rat AV nodal preparation. (A) Simultaneous control action potentials from the crista-terminalis (upper tracing) and the AV node area (lower tracing). (B) Superfusion of the preparation with maternal IgG for about 10 min resulted in 2:1 AV block shown by the arrows. Longer IgG superfusion (15 min) resulted in complete inhibition of the AV-node action potential (lower tracing).
using human fetal heart (Boutjdir et al. 1997). Induction of AV block in the whole heart by maternal IgG led us next to hypothesize that conduction abnormalities could be reproduced in an AV nodal preparation (Boutjdir et al. 1998). Briefly, the technique consists of cutting the ventricles 5–7 mm below the AV groove. A round-edged steel cannula was inserted from the right ventricle through the superior vena cava. An incision was then performed along the cannula. The preparation was opened face up and pinned in the tissue bath to expose the sinus node, the crista terminalis, the AV nodal area and part of the ventricle. The AV nodal area was recognized by its location between the tricuspid valve and the coronary sinus. The effects of maternal IgG (80 mg/ml) were then tested in spontaneously beating AV nodal preparations using multiple microelectrode techniques. Figure 1 shows a representative recording in which simultaneous action potentials from the crista terminalis (upper tracing of Panel A) and AV nodal area (lower tracing of Panel A) were obtained without the addition of IgG. Note the typical triangular shape of TCM Vol. 10, No. 3, 2000
the action potential from the crista terminalis and the typical slow action potential with a phase 4 from the AV-node area. Superfusion of the preparation
Characterization of CHB at the isolated cell level. The observed induction of AV block by maternal antibodies in the Langendorff perfused heart and multicellular AV nodal preparation strongly suggest the involvement of L-type Ca channels, since AV nodal electrogenesis is mainly dependent on ICa-L (Zipes and Mendez 1974). This hypothesis was tested by characterizing the effects of IgG fractions and affinity purified anti-52 kD SSA/Ro antibodies on whole cell ICa-L in the AV node and ventricular myocytes (Boutjdir et al. 1997). IgG from mothers whose children had CHB but not from control mothers, inhibited peak ICa-L in myocytes from both the AV node (Figure 2) and the ventricle (Figure 3A). The range of inhibition at 0 mV was about 50–59%. Washout of IgG resulted in only partial recovery of ICa-L. Similarly, affinity puri-
Figure 2. Effects of maternal antibodies on whole cell L-type Ca current, ICa-L in rabbit AV node myocyte. ICa-L was recorded at holding potential of 240 mV to depolarizing potentials ranging from 230 to 60 mV in increments of 10 mV. The figure shows an I-V relation of ICa-L and selected current tracings at 0 mV (inset) before and after addition of IgG (100 mg/ml). IgG inhibited ICa-L by 51%.
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Figure 3. Effect of IgG and affinity purified anti-52 kD SSA/Ro antibodies on whole cell L-type Ca current, I Ca-L in human fetal ventricular myocytes. (A) Series of time- and voltage-dependent ICa-L tracings recorded from a cell of an 18-week heart at voltages ranging between 230 mV and 160 mV with a 10-mV increment, during control and during steady state effect of 80 mg/ml of IgG from a mother whose child has CHB. (B) Shows the lack of IgG effect from a control healthy mother with healthy children on ICa-L in a cell from a 22-week heart. However, affinity purified anti-52 kD SSA/Ro antibody (Ro52 Ab) inhibited peak ICa-L by 46% in another cell from a 22-week heart.
fied anti-52 kD SSA/Ro antibodies from mothers whose children had CHB inhibited peak ICa-L by 56% at 0 mV (Figure 3B). Inhibition of ICa-L by the autoantibodies in the isolated myocytes further supported the contribution of Ca channels to the conduction abnormalities observed in the whole heart. To gain insight into the biophysical properties, by which the autoantibodies inhibited whole cell ICa-L, we next investigated the effects of maternal antibodies on the L-type Ca channel kinetics at the single channel level. We recorded barium currents through Ca channels as described (Boutjdir et al. 1997, 1998). Bath application of 80 mg/ml affinity purified anti-52 kD SSA/Ro antibody from mothers with CHB children produced a significant decrease in the Ca channel activity and the ensemble average current (Figure 4A). The ensemble average currents were inhibited by 43% without any significant change in the channel conductance (Figure 4C). Analysis of single channel kinetics indicated that this inhibition was the result of shorter open times and longer closed times. The open probability (Figure 4B) calculated from all current sweeps averaged 0.22 6 0.05 for control and 0.11 6 0.02 for anti-52 kD SSA/Ro antibody. This may, in part,
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also explain the basis of the whole cell ICa-L inhibition by the autoantibody. The inhibitory effect of the affinity purified anti-52 kD SSA/Ro antibody and IgG fractions 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, such as receptors that negatively modulates L-type Ca channels, cannot be ruled out. To test whether maternal IgG functionally and biochemically interact directly with L-type Ca channel proteins, we expressed the pore forming a1C subunit of L-type Ca channel encoding rabbit cardiac DHP-receptor in Xenopus oocytes (Xiao et al. 2001a). Whole-cell Ba (40 mM) currents, IBa were recorded in Cl- and Ca-free bath solutions using double-electrode voltage clamp technique. Maternal IgG containing anti-SSA/Ro and anti-SSB/La antibodies (350 mg/ml) consistently inhibited a1C-IBa by 50.6 6 4.7% (p < 0.05). To unambiguously demonstrate a direct interaction of maternal IgG with Ca channels a1C protein, we immunoprecipitated a1C subunit from
membranes of oocytes injected with a1C subunit cRNA. The a1C subunit was detected as a band migrating about 200kD by Card I (antibody against a1C subunit) and maternal IgG but not by control IgG indicating that maternal IgG directly crossreact with L-type Ca channel pore forming a1C subunit. In conclusion, these data are consistent with those obtained in native cardiac myocytes and unequivocally indicate a direct interaction of maternal IgG with the pore-forming a1C subunit of cardiac Ca channels. • Ca Channel Blockade Hypothesis The electrophysiological data presented above raise two questions: 1) How do maternal antibodies blockade of L-type Ca channel in utero lead to CHB? and 2) Why is CHB irreversible in affected infants while in vitro experiments demonstrated partial reversibility of the AV block and L-type Ca current inhibition? Figure 5 is a schematic representation whereby L-type Ca channel blockade may lead to the development of CHB. Two distinct consequences of L-type Ca channel blockade by antibodies can be identified: chronic (minutes) and acute (weeks) effects. Fetal heart Ca channels are chronically exposed to maternal TCM Vol. 10, No. 3, 2000
Figure 4. Effect of affinity purified anti-52 kD SSA/Ro antibodies on single Ca channel unitary currents in human fetal ventricular myocytes. (A) Unitary barium sweeps during control (left) and after addition of 80 mg/ml of affinity purified anti-52 kD SSA/Ro antibodies (right) to the external solution of a myocyte from an 18-week fetal heart. Pipette solution contained 1 mM (2) Bay K 8644. The antibody produced a pronounced decrease in the channel activity, the ensemble averaged current and the average open state probability, Po (B). (C) Voltage dependence of the unitary currents. The slope conductance of control is 20.3 pS and that of affinity purified anti-52 kD SSA/Ro antibodies (Ro52 Ab) is 20.1 pS.
antibodies during pregnancy (starting at about the 12th week of gestation when significant amounts of IgGs are detected). Our hypothesis is that this chronic exposure of L-type Ca channels to maternal IgG could lead to internalization, degradation and eventually cell death since TCM Vol. 10, No. 3, 2000
L-type Ca channels have been reported to play a vital role in fetal excitationcontraction coupling (Fisher 1995). This hypothesis is supported by the data demonstrating that functional ICa-L density in hearts of 52kD-SSA/Roimmunized pups was reduced by 31%. In
addition, the amount of L-type Ca channel proteins as assessed by ELISA and Western blot were significantly lower (20%–30%) in pup hearts from immunized mothers (Xiao et al. 2001b). This is consistent with the findings that crosslinking of adjacent ion channels by the
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Figure 5. Schematic representation of maternal antibodies interaction with cardiac Ca channels: The Ca channel blockade hypothesis. (see text for details).
two Fab arms of IgG increases the rate of normal internalization of the target protein/antibody complex and thereby decreases the channel density on the cell surface (Lennon et al. 1995). Cell death could lead to exposure of autoantigens to antibodies, thus triggering an inflammatory process. Alternatively, cell death, per se, could trigger inflammation subsequent to leukocyte influx resulting in damage of the surrounding healthy fetal myocytes. Furthermore, unlike the affected keratinocytes (skin rash which is reversible), cardiac myocytes do not undergo mitosis with a sufficient rate after birth to replace the damaged myocytes, thus providing a plausible explanation for the irreversibility of CHB. In this regard, whereas it is true that the noncardiac manifestations (skin rash for example) of CHB resolve at about 6 months after birth coinciding with the disappearance of antibodies from infant’s circulation, complete AV block remains and is irreversible. This chronic effect must be clearly distinguished from the acute effect seen in experiments using Langendorff perfused hearts or isolated myo-
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cytes, which show that maternal IgG induced complete AV block and inhibited L-type Ca current in a partially reversible manner, respectively. Of particular interest is that some of deaths reported in children with CHB seem to be related to heart failure even in those with pacemakers, likely because of maternal antibody’s inhibition of ventricular L-type Ca channels responsible for generating the contractile force. Chronic ventricular L-type Ca channel blockade with maternal antibodies may provide a functional basis for heart failure and electromechanical dissociation reported clinically in infants with a pacemaker. In support of this hypothesis is the finding that ICa-L density is down-regulated in isolated human atrial myocytes after cessation of chronic treatment with Ca channel antagonists (Le Grand et al. 1991). The reduced ICa-L density could be attributed to a decrease in the number of functional Ca channels and/or number of DHP receptor binding sites, probably due to internalization and degradation of Ca channels during chronic exposure in utero to maternal antibodies (Figure 5). Alterna-
tively, L-type Ca channel inhibition could also occur indirectly via sarcolemmal receptors such as muscarinic (Bacman et al. 1994) and 5-HT4 serotonergic receptors (Eftekhari et al. 2000). In one hand, Bacman et al. reported that sera and purified IgGs from mothers of infants with CHB and sera of children with CHB react with b-adrenergic and cholinergic receptors of neonatal rat heart. On the other hand, Eftikhari et al. have recently affinity purified anti-G21V autoantibodies from Lupus patients and showed that they recognize both the 5-HT4 receptor and the rSSA/Ro protein in a specific manner indicating the existence of cross-reactive epitopes. G21V is a peptide derived from the human 5-HT4 serotoninergic receptor. Using the patch clamp technique, they further showed the ability of anti-G12V antibody to antagonize serotonin-induced ICaL activation in adult human atrial myocytes. They proposed that blockade of 5HT4 receptors could lead to reduction of calcium channel activation and even possibly the pacemaker “If” current channel leading to conduction abnormalities. Therefore, maternal antibodies can either directly (by interacting with calcium channel protein) or indirectly (through receptors that modulate channels) alter ion channel function. • Vulnerability of Fetal Heart vs. Mother’s Heart Although antibodies to components of the SSA/Ro-SSB/La ribonucleoproteins complex are essential to the development of CHB in offspring, it is intriguing that there are almost no reports of thirddegree AV block in mothers despite exposure to the identical circulating antibodies. However, two independent studies (Behan et al. 1989, Dorner et al. 1992) reported simultaneous existence of firstdegree AV block in the same anti-SSA/ Ro positive mothers whose children have CHB. One attractive hypothesis would be that ICa-L density is higher in the mother than the fetal heart as demonstrated in other mammalian hearts (Huynh et al. 1992). The implication is that while both the mother and the fetal heart are exposed to the same levels of antibodies, only first-degree AV block could be induced in the mothers likely because of higher L-type Ca channel density and that the same levels of antibodies caused complete AV block in the fetal heart (lower TCM Vol. 10, No. 3, 2000
density of L-type Ca channels). Furthermore, the clinical observation that Ltype Ca channel blockers are not the first choice of therapy for pregnant women (except for severe hypertension) is likely due to the vulnerability of the fetal heart to Ca channel blockers (Brillantes et al. 1994). It has been suggested that during fetal and neonatal stages, when expression of Ca channels involved in EC-coupling is low, small doses of channel blockers could have adverse effects on the myocardium (Brillantes et al. 1994). The Ca channel blocking effect could result in diminished cardiac contractility and performance. Other hypotheses to explain the maternal/fetal discordance vis à vis complete AV block have been proposed and recently reviewed by Buyon (1999). These include: 1) the fact that fetal heart contains a greater quantity of SSA/Ro/ mg protein than an adult heart, 2) the possibility that complement regulatory molecules may be diminished on the surface of fetal cells, and 3) the existence of maternal protective hormonal factors. • Summary and Future Directions The establishment of animal model for CHB and the induction of complete AV block in isolated Langendorff perfused fetal hearts and the well correlated inhibition of L-type Ca channels at the AV node and ventricle by maternal antibodies from mothers of children with CHB provide strong evidence supporting an etiologic role of antibody involvement in the pathogenesis of CHB. In addition, the newly reported sinus bradycardia in these animal models indicates that the spectrum of conduction abnormalities extend beyond the AV node. The Ca channel blockade hypothesis seems to account for both the chronic and acute effects of maternal antibodies. AV nodal L-type Ca channel blockade could explain the in vivo AV block and ventricular L-type Ca channel blockade, the heart failure reported in infants with CHB. With this in mind, the Ca channel blockade hypothesis does not explain all aspects of CHB, suggesting that other factors and avenues should be explored. Several unresolved and challenging questions remain and need to be addressed in the future. For example: 1) the low prevalence of CHB in mothers TCM Vol. 10, No. 3, 2000
seropositive to anti-SSA/Ro-SSB/La antibodies (i.e., why is CHB not expressed in all infants of mothers with anti-Ro/La antibodies); 2) why there is discordance in twins both being exposed to maternal antibodies and yet only one is affected; 3) the unique vulnerability of the fetal heart to maternal antibodies; 4) does sinus bradycardia represent an early event in the expression of the disease?; 5) what causes heart failure in CHB infants?; and 6) is it possible that maternal antibodies act as agonists/ antagonists on cell surface receptors to regulate L-type Ca channels? Finally, it is appealing to think that maternal antibodies (anti-Ro/La) are required for the development of CHB but are somehow prevented by yet unknown factor(s) from causing injuries in the healthy heart of the majority of infants born to mothers carrying these antibodies.
References Alexander EL, Buyon JP, Lane J, et al.: 1989. Anti-SSA/Ro SSB/La antibodies bind to neonatal rabbit cardiac cells and preferentially inhibit in vitro cardiac repolarization. J Autoimmun 2:463–469. Baboonian C, Venables PJW, Booth J, et al.: 1989. Virus infection induces redistribution and membrane localization of the nuclear antigen La (SSB): a possible mechanism for autoimmunity. Clin Exp Immunol 78:454–459. Bacman S, Sterin-Borda L, Camusso JJ, et al.: 1994. Circulating antibodies against neurotransmitter receptor activities in children with congenital heart block and their mothers. FASEB J 8:1170–1176. Behan WMH, Behan PO, Reid JM, et al.: 1989. Family studies of congenital heart block associated with Ro antibody. Br Heart J 62:320–324. Ben-Chetrit E, Gandy BJ, Tan EM, et al.: 1989. Isolation and characterization of a cDNA clone encoding the 60-kD component of the human SSA/Ro ribonucleoprotein autoantigen. J Clin Invest 83:1284–1292. Ben-Chetrit E, Chan EKL, Sullivan KF, et al.: 1988. A 52-kD protein is a novel component of the SSA/Ro antigenic particles. J Exp Med 167:1560–1571. Boutjdir M, Chen L, Zhang ZH, et al.: 1997. Arrhythmogenicity of IgG and anti-52-kD SSA/Ro affinity-purified antibodies from mothers of children with congenital heart block. Circ Res 80:354–362. Boutjdir M, Chen L, Zhang ZH, et al.: 1998. Serum and immunoglobulin G from the
mother of a child with congenital heart block induce conduction abnormalities and inhibit L-type calcium channels in a rat heart model. Pediatr Res 44:11–19. Brillantes AM, Bezprozvannaya S, Marks AR: 1994. Developmental and tissue-specific regulation of rabbit skeletal and cardiac muscle calcium channels involved in excitationcontraction coupling. Circ Res 75:503–510. Brucato A, Cimaz A, Catelli L, et al.: 2000. Anti-Ro associated sinus bradycardia in newborns. Circulation, 102:88–89. Buyon JP: 1999. Neonatal lupus syndromes in systemic lupus erythematousus. In Lahita R, ed. Systemic Lupus Erythematosus. New York, Churchill Livingstone, pp 337– 359. Buyon JP, Hiebert R, Copel J, et al.: 1998. Autoimmune-associated congenital heart block: demographics, mortality, morbidity and recurrence rates obtained from a national neonatal lupus registry. J Am Coll Cardiol 31:1658–1666. Buyon JP, Tseng CE, Di Donato F, et al.: 1997. Cardiac expression of 52b, an alternative transcript of the congenital heart blockassociated 52-kD SSA/Ro autoantigen, is maximal during fetal development. Arthritis Rheum 40:655–660. Buyon JP, Waltuck J, Kleinman C, et al.: 1995. In utero identification and therapy of CHB. Lupus 4:116–121. Chameides L, Truex RC, Vetter V, et al.: 1977. Association of maternal lupus erythematosus with congenital complete heart block. N Engl J Med 297:1204–1207. Chan EKL, Francoeur AM, Tan EM: 1986. Epitopes, structural domains and asymmetry of amino acid residues in SSA-B/La nuclear protein. J Immunol 136:3744–3749. Dorner T, Hiepe F, Goldner B, et al.: 1992. Investigations into Ro-specific antibodyassociated congenital cardiac conduction defects. Clin Invest 70:492–496. Eftekhari P, Salle L, Lezoualc’h F, et al.: 2000. Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block. Eur J Immunol 30:2782–2790. Fisher DJ: 1995. Recent insights into the regulation of cardiac Ca flux during perinatal development and in cardiac failure. Curr Opin Cardiol 10:44–51. Furukawa F, Lyons MB, Lee LA, et al.: 1988. Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. J Immunol 141:1480–1488. Garcia S, Nascimento JHM, Bonfa E, et al.: 1994. Cellular mechanism of the conduction abnormalities induced by serum from anti-Ro/SSA-positive patients in rabbit hearts. J Clin Invest 93:718–724.
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Gottlieb E, Steitz JA: 1989. Function of mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J 8:851–861. Horsfall AC, Rose LM: 1992. Cross-reactive maternal autoantibodies and congenital heart block. J Autoimmun 5:479–493. Horsfall AC, Venables PJW, Taylor PV, et al.: 1991. Ro and La antigens and maternal autoantibody idiotype on the surface of myocardial fibers in congenital heart block. J Autoimmun 4:165–176. Hull D, Binns BAO, Joyce D: 1966. Congenital heart block and widespread fibrosis due to maternal erythematosus. Arch Dis Child 41:688–690. Huynh TV, Chen F, Wetzel GT, et al.: 1992. Developmental changes in membrane Ca and K currents in fetal, neonatal, and adult rabbit ventricular myocytes. Circ Res 70:508–515. Le Grand B, Hatem S, Deroubaix E, et al.: 1991. Calcium current depression in isolated human atrial myocytes after cessation of chronic treatment with calcium antagonists. Circ Res 69:292–300. Lennon VA, Kryzer TJ, Griesmann GE, et al.: 1995. Calcium channel antibodies in the Lambert-Eaton syndromes and other paraneoplastic syndromes. N Engl J Med 332:1467–74. LeFeber WP, Norris DA, Ryan SB, et al.: 1984. Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes. J Clin Invest 74:1545–1551. Mazel JA, El-Sherif N, Buyon JP, et al.: 1999. Conduction abnormalities in a murine model for the clinical syndrome of congenital heart block. Circulation 99:1914–1918. McCue CM, Mantakas ME, Tingelstad JB, et al.: 1977. Congenital heart block in newborns of mothers with connective tissue disease. Circulation 56:82–90. Menon A, Silverman ED, Gow RM, et al.: 1998. Chronotropic competence of the sinus node in congenital complete heart block. Am J Cardiol 82:1119–1121. Michaelsson M, Engle MA: 1972. Congenital complete heart block: an international study of the natural history. Cardiovasc Clin 4:85–101. Miranda E, Boutjdir M, Tseng CE, et al.: 1998b. Induction of antibodies reactive with SSA/Ro-SSB/La and development of congenital heart block in a murine model. J Rheum 161:5886–5892. Miranda ME, Tseng CE, Rashbaum W, et al.: 1998a. Accessibility of SSA/Ro and SSB/La antigens to maternal autoantibodies in apoptotic human fetal cardiac myocytes. J Immunol 161:5061–5069.
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Morquio L: 1901. Sur une maladie infantile et familiale characterisee par des modifications permanentes du pouls des attaques syncopales et eptileptiformes et la mort subite. Arch Med Inf 4:467–469.
Xiao GQ, Hu K, Boutjdir M: 2001a. Direct inhibition of expressed cardiac L- and Ttype calcium channels by IgG from mothers whose children have congenital heart block. Circulation, in press.
O’Neill TW, Mohmoud A, Tooke A, et al.: 1993. Is there a relationship between subclinical myocardial abnormalities, conduction defects, and Ro/La antibodies in adults with systemic lupus erythematosus? Clin Exp Rheumato 11:409.
Xiao GQ, Qu Y, Hu K, Boutjdir M: 2001b. Downregulation of L-type calcium channel in pups born to 52KD SSA/Ro immunized rabbits. Manuscript submitted for publication.
Plant RK, Steven RA: 1945. Complete atrioventricular block in the fetus. Am Heart J 102:117–122. Siren MK, Julkunen H, Kaaja R: 1998. The increasing incidence of isolated congenital heart block in Finland. J Rheumatol 25:1862–1864.
Zipes DP, Mendez C: 1973. Action of manganese ions and tetrodotoxin on atrioventricular nodal transmembrane potentials in isolated rabbit hearts. Circ Res 32:447– 454.
PII S1050-1738(00)00059-1
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Invasive Cardiac Electrophysiology in the Mouse: Techniques and Applications Samir Saba, Paul J. Wang, and N.A. Mark Estes III*
As the genetic nature of a wide spectrum of cardiovascular diseases is being elucidated, it is increasingly important to understand the functional role of specific genes on cardiac arrhythmia and conduction disturbances. The progress made in molecular genetics has allowed the creation of mice with targeted gene overexpression or elimination. These animals are valuable tools for researchers who have adapted their clinical and technical skills to the mouse, in order to extract information on the phenotypic consequences of the specific genetic disruption. In this review, we summarize the progress made in the field of invasive murine electrophysiology, focusing on the recent technical advances in in vivo electrophysiologic testing and its application to various genetically engineered mouse models. The authors’ views on the future needs and trends in the field are also presented. (Trends Cardiovasc Med 2000;10:122–132). © 2001 Elsevier Science Inc.
Samir Saba, Paul J. Wang, and N.A. Mark Estes III are at the New England Cardiac Arrhythmia Center, Tufts University–New England Medical Center, Boston, Massachusetts, USA. * Address correspondence to: N.A. Mark Estes III, MD, New England Cardiac Arrhythmia Center, New England Medical Center, 750 Washington Street, Boston, MA 02111. Tel.: 617-636 6136; fax: 617-636 4586; e-mail: [email protected] © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
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Zhang J, Ishibashi Y, Zhang Y, et al.: 1997. Myocardial bioenergetics during acute hibernation. Am J Physiol 273:H1452–H1463. PII S1050-1738(00)00058-X
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Molecular and Ionic Basis of Congenital Complete Heart Block Mohamed Boutjdir*
Congenital heart block (CHB), detected at or before birth in a structurally normal heart, is strongly associated with autoantibodies reactive with the intracellular soluble ribonucleoproteins 48kD SSB/La, 52kD SSA/Ro, and 60kD SSA/Ro. CHB is presumed to be due to the transplacental passage of autoantibodies from the mother into the fetal circulation. Varying degrees of heart block have been reported. Although second degree block has, on rare occasion, reverted to normal sinus rhythm, complete atrio-ventricular (AV) block is irreversible. CHB carries substantial mortality and morbidity, with .60% of affected children requiring lifelong pacemakers. The recurrence rate exceeds, by at least twofold, that of the first birth and is likely to influence the decision to have more children. Curiously, the mother’s heart is almost never affected (with complete heart block) despite exposure to identical circulating autoantibodies. As part of our continuing effort to understand the complex factors contributing to the pathogenesis of CHB, we have established an animal model of CHB by immunizing female mice with recombinant proteins/antigens, reproduced the human complete AV block in an isolated Langendorff perfused fetal heart, and correlated these findings with L-type Ca channel inhibition by maternal antibodies from mothers of children with CHB. In addition, we established a passive animal model by directly injecting maternal antibodies into pregnant mice and reported significant sinus bradycardia, indicating that the spectrum of conduction abnormalities may extend beyond the AV node. All together, the data provided strong evidence supporting an etiologic role of antibody/Ca channel involvement in the pathogenesis of CHB. However, other yet unknown factors seem necessary to explain the full expression of CHB. (Trends Cardiovasc Med 2000;10:114–122). © 2001, Elsevier Science Inc.
Mohamed Boutjdir is at the Molecular and Cellular Cardiology Program, New York Harbor Healthcare System and SUNY Health Science Center, Brooklyn, New York, USA. * Address correspondence to: Dr. Mohamed Boutjdir, Research and Development Office (151), New York Harbor Healthcare System, 800 Poly Place, Brooklyn, NY 11209. Tel.: 718630-3645; fax: 718-630-3796; e-mail: [email protected] © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
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Isolated congenital heart block (CHB) was first recognized in 1901 by Morquio and later by Plant using auscultation and electrocardiography (Morquio 1901, Plant and Steven 1945). The association between isolated CHB and maternal connective tissue disease was described by Hull and others in the late 1960s and 1970s (Chameides et al. 1977, Hull et al. 1966, McCue et al. 1977). This relationship has since become well established and ascribed to the presence of SSA/Ro-SSB/La antibodies in maternal sera. SSA/Ro-SSB/La antibodies (maternal antibodies and maternal IgG are interchangeably used throughout) produce conduction abnormalities in the otherwise normally developing fetus (with no evidence of major cardiac structural abnormalities), yet third-degree AV block has not been documented in the mother (O’Neill et al. 1993). Because of the rarity and complex etiology of CHB, the true incidence is not well established. In general 1 in 15,000– 20,000 live births is often reported although this was based on information collected prior to the 1960s with a limited number of affected children (Michaelsson and Engle 1972). More recent studies (Buyon et al. 1998, Siren et al. 1998) using data collected during the latter decades, seem to indicate that the incidence of CHB is higher than previously reported (about 1 in 11,000). It was suggested that this is likely due to more effective detection and improved diagnostics of CHB during pregnancy (Siren et al. 1998). In the lupus population with anti-SSA/Ro and anti-SSB/La antibodies, the incidence rises to 3–5 in 100 (Buyon 1999). Patients at greatest risk are infants of mothers with a prior history of a CHB delivery, with a recurrence rate of 16–20%. CHB presents with first-, second-, or third-degree AV block. Third-degree AV block is most common and once manifest is always permanent. Mortality approaches 30%, usually within the first three months of life (Buyon 1999). Current therapies include dexamethasone, plasmapheresis, sympathomimetics, and in utero cardiac pacing. Unfortunately none have significantly altered mortality justifying the need for more aggressive support for both basic and clinical investigations. • Intracellular Autoantigens to Maternal Antibodies The candidate antigens and their cognate antibodies have been extensively characTCM Vol. 10, No. 3, 2000
terized at the molecular level. 60 kD SSA/Ro contains a putative zinc finger and an RNA-binding protein consensus motif (Ben-Chetrit et al. 1989), both of which could account for its direct interaction with small cytoplasmic hY-RNAs. Many sera which recognize 60 kD SSA/ Ro protein also react with another protein of 52 kD (SSA/Ro), 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 (BenChetrit el al. 1988). Anti-SSB/La antibodies recognize a 48-kD polypeptide that does not share antigenic determinants with either 52 kD or 60 kD SSA/Ro (Chan et al. 1986). 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 (Gottlieb and Steitz 1989). An alternative mRNA transcript of 52 kD SSA/Ro, derived from the splicing of exon 4, was identified and encodes a smaller protein, 52b of 45 kD (Buyon et al. 1997). Interestingly, the expression of 52b mRNA is maximal at 14–16 weeks gestation, which coincides with the time of cardiac ontogeny when placental transfer of maternal antibodies into the fetal circulation becomes effective (Buyon 1999). • Other Potential Antigenic Targets to Maternal Antibodies The possibility that maternal autoantibodies may cross-react with some antigenic targets other than intracellular Ro/La autoantigen has been explored. Antibodies from mothers, whose children had CHB, were reported to cross-react with laminin B-1 and to human cardiac myosin heavy chain (Horsfall and Rose 1992). Computer-based analysis of the amino acid sequence from the SWISSPROT database between the 52 kD SSA/ Ro, 60 kD SSA/Ro, 48 kD SSB/La proteins and the a1-subunit of L-type human cardiac sarcolemmal Ca channels revealed that most of the homology is with 52 kD SSA/Ro with very minor homology to 60 kD SSA/Ro and 48 kD SSB/La. When the amino acid sequence was compared in a linear representation, the maximum number of homologous amino acids between the 52 kD SSA/Ro and a1-subunit of L-type Ca channel did not exceed four amino acids. However, when TCM Vol. 10, No. 3, 2000
a three-dimensional representation of the a1-subunit structure was used, numerous homologous epitopes became adjacent in the extracellular loops. Epitopic regions on the a1-subunit could then be identified; one of which corresponds primarily with the pore-forming region between segments S5 and S6 of domain VI. This indicates potential direct interaction sites of maternal antibodies with sarcolemmal L-type cardiac Ca channel a1-subunit. • Proposed Pathophysiological Mechanisms of CHB Molecular and Biochemical Evidence Because the candidate antigens are intracellular and there is no convincing evidence that maternal antibodies can cross the sarcolemma of a normal cell, effort has been directed toward mechanisms that may cause the translocation of these antigens to the cell surface membrane. The conventional wisdom is that these antigens must be accessible to maternal antibodies to explain the pathological events, mainly the inflammatory process that may lead to CHB. Substantial experimental evidence has been proposed to account for the accessibility of antigens to maternal antibodies. This includes: 1) viral infection of cells to induce the antigen’s translocation to the cell surface (Baboonian et al. 1989); 2) IFNg treatment of epithelial T24 cells resulting in surface expression of SSB/La; 3) the expression of SSA/Ro and SSB/La antigens on the keratinocyte cell surface by ultraviolet light (LeFeber et al. 1984); 4) 17 b-estradiol enhanced binding of anti-SSA/Ro and anti-SSB/La antibodies to keratinocytes (Furukawa et al. 1988); 5) autopsy studies showing that maternal IgG was associated with specificity for the SSB/La antigen on the surface of fetal myocardial fibers (Horsfall et al. 1991); and 6) unknown inductive events in utero which may lead to apoptotic cell death, thus exposing the antigens (Miranda et al. 1998a). While autoantigen translocation to cell surface could be demonstrated, it is not yet established what are the resulting cellular events and signaling pathways that could account for the tissue injury. Electrophysiological Evidence While the molecular aspects of CHB have been extensively characterized [see Buyon
(1999) for review] the basic electrophysiological mechanisms of CHB are emerging. First, Alexander et al. (1989) demonstrated that IgG fraction of anti-SSA/ Ro SSB/La-positive maternal sera shortened neonatal rabbit cardiac action potential repolarization. Garcia et al. (1994), showed that electrocardiographic conduction disorders associated with neonatal lupus could be reproduced in an isolated adult rabbit heart. Further, they showed that anti-SSA/Ro positive sera inhibited L-type Ca current, ICa-L in isolated ventricular myocytes. Subsequently we established an active, antigen-specific (Boutjdir et al. 1997, Miranda et al. 1998b), and a passive (Mazel et al. 1999) animal model of the human CHB, showed that maternal autoantibodies can induce complete AV block in Langendorff perfused beating heart and in isolated atrioventricular multicellular preparation (Boutjdir et al. 1997, 1998). In addition, these findings correlated well with the inhibition of ICa-L [responsible for electrogenesis at the AV node (Zipes and Mendez 1973)] by the antibodies in isolated cardiac myocytes (Boutjdir et al. 1997, 1998). Below is a brief summary of the above work related to CHB from our laboratory. For clarity and easy flow of thought, characterization of CHB is presented starting from the whole animal, isolated beating heart, multicellular AV nodal preparation and finally single myocyte. Characterization of CHB at the whole animal level. It is a widely held view that CHB is caused by the transplacental transfer of maternal autoantibodies (anti-SSA/ Ro and/or anti-SSB/La) into the fetal circulation. We tested this hypothesis in a passive model in an experimental mouse (BALB/c) by passive transfer of human autoantibodies into pregnant mice (Mazel et al. 1999). Timed pregnant mice were injected with a single intravenous bolus of purified IgG containing human antiSSA/Ro and anti-SSB/La antibodies from mothers of children with CHB. Since the timing of human fetal injury is not random, but instead, occurs during a welldefined period between 15–24 weeks of gestation (Buyon et al. 1995), three groups of mice were used: 8, 11, and 16 days gestation. Within each group, we tested 10, 25, 50, and 100 mg of IgG. At delivery, ECGs were recorded and analyzed for conduction abnormalities. The results are
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Table 1. Summary of ECG parameters in the passive model of CHB
Control Passive 8d 11d 16d
Pups (n)
HR (bpm)
PR (ms)
QRS (ms)
Brady.
I8
II8 & III8
52 16 10 30
526.5 6 8.1 289.3 6 14.6* 233.9 6 19.0* 303.9 6 12.0*
40.4 6 1.1 62.9 6 2.3* 74 6 4.5* 57.8 6 2.3*
22.2 6 1.21 24 6 0.92 24.5 6 1.25 22.8 6 0.52
0 44% 70% 35%
0 88% 90% 45%
0 0 0 0
* p , 0.05 compared to control. 8d, 11d, and 16d indicate the 8th day, 11th day, and 16th day of gestation, respectively.
summarized in Table 1. Marked sinus bradycardia and PR interval prolongation were observed in 8-, 11-, and 16-day gestational groups when compared to controls. Interestingly no complete AV block was seen in this passive model. Antibody levels measured by ELISA in both mothers and their offspring confirmed the transplacental transfer of the human antibodies to the pups. The high incidence of sinus bradycardia suggests possible SA node involvement. These novel findings brought clinical attention not only to AV nodal disorders but also to SA nodal conduction abnormalities. In this regard, Menon et al. (1998) and Brucato et al. (2000) recently reported similar sinus bradycardia in newborns born to mothers seropositive to anti-SSA/Ro antibodies, suggesting that the spectrum of conduction abnormalities associated with maternal autoantibodies extend beyond the AV node and raise the question of whether screening of infants for sinus bradycardia should be part of standard diagnosis in mothers seropositive to anti-Ro/La antibodies. In the active model, the hypothesis being tested was whether the administration of a specific immunogen, not human antibody as in the passive model, to pregnant mice would result in conduction disorders in the pups (Boutjdir et al.
1997, Miranda et al. 1998b). An antigenspecific model was developed using female BALB/c mice immunized with human recombinant 48 kD SSB/La, 60 kD SSA/Ro, 52 kD SSA/Ro (52a), and 52b as well as with murine recombinant 52 kD SSA/Ro. The immunization resulted in high titer responses to the respective antigens as established by ELISA, immunoblotting, and immunoprecipitation. The results are summarized in Table 2. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48 kD SSB/La, 20% of 54 pups born to 60 kD SSA/Ro-immunized mothers, 6% of 56 pups born to 52 kD SSA/Ro-a immunized mothers, 7% of 86 pups born to 52b-immunized mothers and 9% of 22 pups born to mothers immunized with murine 52 kD SSA/Ro. Higher degrees of AV block were only identified in offspring of 52a- or 52b-immunized mice. In the 52a group, one pup had complete AV block and another had second-degree block (Wenckebach type); in the 52b group, five pups developed complete AV block. Maternal antibodies to the primary immunogens were detected in the pups. None of control pups had any conduction abnormalities. This antigenspecific animal model provides strong evidence for a pathogenic role of anti-SSA/ Ro-SSB/La antibodies, particularly 52Ro,
in the development of CHB. The range and frequency of conduction defects suggest that additional factors may promote disease expression. Characterization of CHB at the isolated heart level. Having demonstrated the reproducibility of the clinical CHB in an animal model, we asked the question whether direct perfusion of an isolated beating rat heart with maternal IgG would also result in electrocardiographic conduction abnormalities similar to those seen in affected infants (Boutjdir et al. 1997, 1998). The effects of maternal IgG containing anti-SSA/Ro-SSB/La antibodies on the ECG recording of an isolated rat heart perfused by the Langendorff technique were assessed. Recordings were done using a conventional ECG machine in lead I. After 5 min of perfusion with maternal IgG (800 mg/ml), there was bradycardia associated with 2:1 second-degree AV block that degenerated into complete AV block at about 15 min of perfusion. The QRS complex is absent but the P waves were clearly seen. After 25 min of reperfusion with Tyrode’s solution, only partial recovery was seen. In contrast, perfusion of the heart with normal IgG from healthy mothers with healthy children did not alter ECG parameters. Identical results were obtained
Table 2. Active model of CHB
Immunogen 48-kD La 52a Ro 52b Ro 60-kD Ro Murine 52-kD Ro b-gal (control) Vector (control)
Degree of AV block
Immunized mothers (n)
Fertile mothers (n)
Pups (n)
I8 (n)
II8 (n)
III8 (n)
10 8 5 7 5 3 4
3 5 5 6 4 2 4
27 56 86 54 22 21 43
2 3 5 10 2 0 0
0 1 0 0 0 0 0
0 1 5 0 0 0 0
From Miranda et al. 1998b.
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with maternal IgG for approximately 10 min resulted in 2:1 AV block indicated by the arrows. Longer IgG superfusion (10–15 min) resulted in almost complete inhibition of the AV-node action potential (trace at the bottom of Panel B). Similarly, conduction abnormalities were not seen with normal IgG. These results are consistent with the ECG findings from the whole heart and could account for the conduction abnormalities seen in vitro.
Figure 1. Effects of maternal antibodies on an isolated multicellular rat AV nodal preparation. (A) Simultaneous control action potentials from the crista-terminalis (upper tracing) and the AV node area (lower tracing). (B) Superfusion of the preparation with maternal IgG for about 10 min resulted in 2:1 AV block shown by the arrows. Longer IgG superfusion (15 min) resulted in complete inhibition of the AV-node action potential (lower tracing).
using human fetal heart (Boutjdir et al. 1997). Induction of AV block in the whole heart by maternal IgG led us next to hypothesize that conduction abnormalities could be reproduced in an AV nodal preparation (Boutjdir et al. 1998). Briefly, the technique consists of cutting the ventricles 5–7 mm below the AV groove. A round-edged steel cannula was inserted from the right ventricle through the superior vena cava. An incision was then performed along the cannula. The preparation was opened face up and pinned in the tissue bath to expose the sinus node, the crista terminalis, the AV nodal area and part of the ventricle. The AV nodal area was recognized by its location between the tricuspid valve and the coronary sinus. The effects of maternal IgG (80 mg/ml) were then tested in spontaneously beating AV nodal preparations using multiple microelectrode techniques. Figure 1 shows a representative recording in which simultaneous action potentials from the crista terminalis (upper tracing of Panel A) and AV nodal area (lower tracing of Panel A) were obtained without the addition of IgG. Note the typical triangular shape of TCM Vol. 10, No. 3, 2000
the action potential from the crista terminalis and the typical slow action potential with a phase 4 from the AV-node area. Superfusion of the preparation
Characterization of CHB at the isolated cell level. The observed induction of AV block by maternal antibodies in the Langendorff perfused heart and multicellular AV nodal preparation strongly suggest the involvement of L-type Ca channels, since AV nodal electrogenesis is mainly dependent on ICa-L (Zipes and Mendez 1974). This hypothesis was tested by characterizing the effects of IgG fractions and affinity purified anti-52 kD SSA/Ro antibodies on whole cell ICa-L in the AV node and ventricular myocytes (Boutjdir et al. 1997). IgG from mothers whose children had CHB but not from control mothers, inhibited peak ICa-L in myocytes from both the AV node (Figure 2) and the ventricle (Figure 3A). The range of inhibition at 0 mV was about 50–59%. Washout of IgG resulted in only partial recovery of ICa-L. Similarly, affinity puri-
Figure 2. Effects of maternal antibodies on whole cell L-type Ca current, ICa-L in rabbit AV node myocyte. ICa-L was recorded at holding potential of 240 mV to depolarizing potentials ranging from 230 to 60 mV in increments of 10 mV. The figure shows an I-V relation of ICa-L and selected current tracings at 0 mV (inset) before and after addition of IgG (100 mg/ml). IgG inhibited ICa-L by 51%.
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Figure 3. Effect of IgG and affinity purified anti-52 kD SSA/Ro antibodies on whole cell L-type Ca current, I Ca-L in human fetal ventricular myocytes. (A) Series of time- and voltage-dependent ICa-L tracings recorded from a cell of an 18-week heart at voltages ranging between 230 mV and 160 mV with a 10-mV increment, during control and during steady state effect of 80 mg/ml of IgG from a mother whose child has CHB. (B) Shows the lack of IgG effect from a control healthy mother with healthy children on ICa-L in a cell from a 22-week heart. However, affinity purified anti-52 kD SSA/Ro antibody (Ro52 Ab) inhibited peak ICa-L by 46% in another cell from a 22-week heart.
fied anti-52 kD SSA/Ro antibodies from mothers whose children had CHB inhibited peak ICa-L by 56% at 0 mV (Figure 3B). Inhibition of ICa-L by the autoantibodies in the isolated myocytes further supported the contribution of Ca channels to the conduction abnormalities observed in the whole heart. To gain insight into the biophysical properties, by which the autoantibodies inhibited whole cell ICa-L, we next investigated the effects of maternal antibodies on the L-type Ca channel kinetics at the single channel level. We recorded barium currents through Ca channels as described (Boutjdir et al. 1997, 1998). Bath application of 80 mg/ml affinity purified anti-52 kD SSA/Ro antibody from mothers with CHB children produced a significant decrease in the Ca channel activity and the ensemble average current (Figure 4A). The ensemble average currents were inhibited by 43% without any significant change in the channel conductance (Figure 4C). Analysis of single channel kinetics indicated that this inhibition was the result of shorter open times and longer closed times. The open probability (Figure 4B) calculated from all current sweeps averaged 0.22 6 0.05 for control and 0.11 6 0.02 for anti-52 kD SSA/Ro antibody. This may, in part,
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also explain the basis of the whole cell ICa-L inhibition by the autoantibody. The inhibitory effect of the affinity purified anti-52 kD SSA/Ro antibody and IgG fractions 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, such as receptors that negatively modulates L-type Ca channels, cannot be ruled out. To test whether maternal IgG functionally and biochemically interact directly with L-type Ca channel proteins, we expressed the pore forming a1C subunit of L-type Ca channel encoding rabbit cardiac DHP-receptor in Xenopus oocytes (Xiao et al. 2001a). Whole-cell Ba (40 mM) currents, IBa were recorded in Cl- and Ca-free bath solutions using double-electrode voltage clamp technique. Maternal IgG containing anti-SSA/Ro and anti-SSB/La antibodies (350 mg/ml) consistently inhibited a1C-IBa by 50.6 6 4.7% (p < 0.05). To unambiguously demonstrate a direct interaction of maternal IgG with Ca channels a1C protein, we immunoprecipitated a1C subunit from
membranes of oocytes injected with a1C subunit cRNA. The a1C subunit was detected as a band migrating about 200kD by Card I (antibody against a1C subunit) and maternal IgG but not by control IgG indicating that maternal IgG directly crossreact with L-type Ca channel pore forming a1C subunit. In conclusion, these data are consistent with those obtained in native cardiac myocytes and unequivocally indicate a direct interaction of maternal IgG with the pore-forming a1C subunit of cardiac Ca channels. • Ca Channel Blockade Hypothesis The electrophysiological data presented above raise two questions: 1) How do maternal antibodies blockade of L-type Ca channel in utero lead to CHB? and 2) Why is CHB irreversible in affected infants while in vitro experiments demonstrated partial reversibility of the AV block and L-type Ca current inhibition? Figure 5 is a schematic representation whereby L-type Ca channel blockade may lead to the development of CHB. Two distinct consequences of L-type Ca channel blockade by antibodies can be identified: chronic (minutes) and acute (weeks) effects. Fetal heart Ca channels are chronically exposed to maternal TCM Vol. 10, No. 3, 2000
Figure 4. Effect of affinity purified anti-52 kD SSA/Ro antibodies on single Ca channel unitary currents in human fetal ventricular myocytes. (A) Unitary barium sweeps during control (left) and after addition of 80 mg/ml of affinity purified anti-52 kD SSA/Ro antibodies (right) to the external solution of a myocyte from an 18-week fetal heart. Pipette solution contained 1 mM (2) Bay K 8644. The antibody produced a pronounced decrease in the channel activity, the ensemble averaged current and the average open state probability, Po (B). (C) Voltage dependence of the unitary currents. The slope conductance of control is 20.3 pS and that of affinity purified anti-52 kD SSA/Ro antibodies (Ro52 Ab) is 20.1 pS.
antibodies during pregnancy (starting at about the 12th week of gestation when significant amounts of IgGs are detected). Our hypothesis is that this chronic exposure of L-type Ca channels to maternal IgG could lead to internalization, degradation and eventually cell death since TCM Vol. 10, No. 3, 2000
L-type Ca channels have been reported to play a vital role in fetal excitationcontraction coupling (Fisher 1995). This hypothesis is supported by the data demonstrating that functional ICa-L density in hearts of 52kD-SSA/Roimmunized pups was reduced by 31%. In
addition, the amount of L-type Ca channel proteins as assessed by ELISA and Western blot were significantly lower (20%–30%) in pup hearts from immunized mothers (Xiao et al. 2001b). This is consistent with the findings that crosslinking of adjacent ion channels by the
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Figure 5. Schematic representation of maternal antibodies interaction with cardiac Ca channels: The Ca channel blockade hypothesis. (see text for details).
two Fab arms of IgG increases the rate of normal internalization of the target protein/antibody complex and thereby decreases the channel density on the cell surface (Lennon et al. 1995). Cell death could lead to exposure of autoantigens to antibodies, thus triggering an inflammatory process. Alternatively, cell death, per se, could trigger inflammation subsequent to leukocyte influx resulting in damage of the surrounding healthy fetal myocytes. Furthermore, unlike the affected keratinocytes (skin rash which is reversible), cardiac myocytes do not undergo mitosis with a sufficient rate after birth to replace the damaged myocytes, thus providing a plausible explanation for the irreversibility of CHB. In this regard, whereas it is true that the noncardiac manifestations (skin rash for example) of CHB resolve at about 6 months after birth coinciding with the disappearance of antibodies from infant’s circulation, complete AV block remains and is irreversible. This chronic effect must be clearly distinguished from the acute effect seen in experiments using Langendorff perfused hearts or isolated myo-
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cytes, which show that maternal IgG induced complete AV block and inhibited L-type Ca current in a partially reversible manner, respectively. Of particular interest is that some of deaths reported in children with CHB seem to be related to heart failure even in those with pacemakers, likely because of maternal antibody’s inhibition of ventricular L-type Ca channels responsible for generating the contractile force. Chronic ventricular L-type Ca channel blockade with maternal antibodies may provide a functional basis for heart failure and electromechanical dissociation reported clinically in infants with a pacemaker. In support of this hypothesis is the finding that ICa-L density is down-regulated in isolated human atrial myocytes after cessation of chronic treatment with Ca channel antagonists (Le Grand et al. 1991). The reduced ICa-L density could be attributed to a decrease in the number of functional Ca channels and/or number of DHP receptor binding sites, probably due to internalization and degradation of Ca channels during chronic exposure in utero to maternal antibodies (Figure 5). Alterna-
tively, L-type Ca channel inhibition could also occur indirectly via sarcolemmal receptors such as muscarinic (Bacman et al. 1994) and 5-HT4 serotonergic receptors (Eftekhari et al. 2000). In one hand, Bacman et al. reported that sera and purified IgGs from mothers of infants with CHB and sera of children with CHB react with b-adrenergic and cholinergic receptors of neonatal rat heart. On the other hand, Eftikhari et al. have recently affinity purified anti-G21V autoantibodies from Lupus patients and showed that they recognize both the 5-HT4 receptor and the rSSA/Ro protein in a specific manner indicating the existence of cross-reactive epitopes. G21V is a peptide derived from the human 5-HT4 serotoninergic receptor. Using the patch clamp technique, they further showed the ability of anti-G12V antibody to antagonize serotonin-induced ICaL activation in adult human atrial myocytes. They proposed that blockade of 5HT4 receptors could lead to reduction of calcium channel activation and even possibly the pacemaker “If” current channel leading to conduction abnormalities. Therefore, maternal antibodies can either directly (by interacting with calcium channel protein) or indirectly (through receptors that modulate channels) alter ion channel function. • Vulnerability of Fetal Heart vs. Mother’s Heart Although antibodies to components of the SSA/Ro-SSB/La ribonucleoproteins complex are essential to the development of CHB in offspring, it is intriguing that there are almost no reports of thirddegree AV block in mothers despite exposure to the identical circulating antibodies. However, two independent studies (Behan et al. 1989, Dorner et al. 1992) reported simultaneous existence of firstdegree AV block in the same anti-SSA/ Ro positive mothers whose children have CHB. One attractive hypothesis would be that ICa-L density is higher in the mother than the fetal heart as demonstrated in other mammalian hearts (Huynh et al. 1992). The implication is that while both the mother and the fetal heart are exposed to the same levels of antibodies, only first-degree AV block could be induced in the mothers likely because of higher L-type Ca channel density and that the same levels of antibodies caused complete AV block in the fetal heart (lower TCM Vol. 10, No. 3, 2000
density of L-type Ca channels). Furthermore, the clinical observation that Ltype Ca channel blockers are not the first choice of therapy for pregnant women (except for severe hypertension) is likely due to the vulnerability of the fetal heart to Ca channel blockers (Brillantes et al. 1994). It has been suggested that during fetal and neonatal stages, when expression of Ca channels involved in EC-coupling is low, small doses of channel blockers could have adverse effects on the myocardium (Brillantes et al. 1994). The Ca channel blocking effect could result in diminished cardiac contractility and performance. Other hypotheses to explain the maternal/fetal discordance vis à vis complete AV block have been proposed and recently reviewed by Buyon (1999). These include: 1) the fact that fetal heart contains a greater quantity of SSA/Ro/ mg protein than an adult heart, 2) the possibility that complement regulatory molecules may be diminished on the surface of fetal cells, and 3) the existence of maternal protective hormonal factors. • Summary and Future Directions The establishment of animal model for CHB and the induction of complete AV block in isolated Langendorff perfused fetal hearts and the well correlated inhibition of L-type Ca channels at the AV node and ventricle by maternal antibodies from mothers of children with CHB provide strong evidence supporting an etiologic role of antibody involvement in the pathogenesis of CHB. In addition, the newly reported sinus bradycardia in these animal models indicates that the spectrum of conduction abnormalities extend beyond the AV node. The Ca channel blockade hypothesis seems to account for both the chronic and acute effects of maternal antibodies. AV nodal L-type Ca channel blockade could explain the in vivo AV block and ventricular L-type Ca channel blockade, the heart failure reported in infants with CHB. With this in mind, the Ca channel blockade hypothesis does not explain all aspects of CHB, suggesting that other factors and avenues should be explored. Several unresolved and challenging questions remain and need to be addressed in the future. For example: 1) the low prevalence of CHB in mothers TCM Vol. 10, No. 3, 2000
seropositive to anti-SSA/Ro-SSB/La antibodies (i.e., why is CHB not expressed in all infants of mothers with anti-Ro/La antibodies); 2) why there is discordance in twins both being exposed to maternal antibodies and yet only one is affected; 3) the unique vulnerability of the fetal heart to maternal antibodies; 4) does sinus bradycardia represent an early event in the expression of the disease?; 5) what causes heart failure in CHB infants?; and 6) is it possible that maternal antibodies act as agonists/ antagonists on cell surface receptors to regulate L-type Ca channels? Finally, it is appealing to think that maternal antibodies (anti-Ro/La) are required for the development of CHB but are somehow prevented by yet unknown factor(s) from causing injuries in the healthy heart of the majority of infants born to mothers carrying these antibodies.
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PII S1050-1738(00)00059-1
TCM
Invasive Cardiac Electrophysiology in the Mouse: Techniques and Applications Samir Saba, Paul J. Wang, and N.A. Mark Estes III*
As the genetic nature of a wide spectrum of cardiovascular diseases is being elucidated, it is increasingly important to understand the functional role of specific genes on cardiac arrhythmia and conduction disturbances. The progress made in molecular genetics has allowed the creation of mice with targeted gene overexpression or elimination. These animals are valuable tools for researchers who have adapted their clinical and technical skills to the mouse, in order to extract information on the phenotypic consequences of the specific genetic disruption. In this review, we summarize the progress made in the field of invasive murine electrophysiology, focusing on the recent technical advances in in vivo electrophysiologic testing and its application to various genetically engineered mouse models. The authors’ views on the future needs and trends in the field are also presented. (Trends Cardiovasc Med 2000;10:122–132). © 2001 Elsevier Science Inc.
Samir Saba, Paul J. Wang, and N.A. Mark Estes III are at the New England Cardiac Arrhythmia Center, Tufts University–New England Medical Center, Boston, Massachusetts, USA. * Address correspondence to: N.A. Mark Estes III, MD, New England Cardiac Arrhythmia Center, New England Medical Center, 750 Washington Street, Boston, MA 02111. Tel.: 617-636 6136; fax: 617-636 4586; e-mail: [email protected] © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
TCM Vol. 10, No. 3, 2000