Functional Basis of Sinus Bradycardia in Congenital Heart Block
Congenital heart block (CHB) is a conduction abnormality characterized by complete atrioventricular (AV) block. CHB affects fetuses and/or newborn of mothers with autoantibodies reactive with ribonucleoproteins 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro. We recently established animal models of CHB and reported, for the first time, significant sinus bradycardia preceding AV block. This unexpected observation implies that the spectrum of conduction abnormalities extends beyond the AV node to also affect the SA node. To test this hypothesis, we investigated the functional basis of this sinus bradycardia by characterizing the effects of antibodies from mothers with CHB children (positive IgG) on ionic currents that are known to significantly contribute to spontaneous pacing in SA node cells. We recorded L- (ICa.L) and T- (ICa.T) type Ca2+, delayed rectifier K+ (IK), hyperpolarization-activated (If) currents, and action potentials (APs) from young rabbit SA node cells. We demonstrated that positive IgG significantly inhibited both ICa.T and ICa.L and induced sinus bradycardia but did not affect If and IK. Normal IgG from mothers with healthy children did not affect all the currents studied and APs. These results establish that IgG from mothers with CHB children causes substantial inhibition of ICa.T and ICa.L, two important pacemaker currents in rabbit SA node cells and point to both ICa.T and ICa.L as major players in the ionic mechanism by which maternal antibodies induce sinus bradycardia in CHB. These novel findings have important clinical significance and suggest that sinus bradycardia may be a potential marker in the detection and prevention of CHB. The full text of this article is available online at http://circres.ahajournals.org
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 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro.1 CHB is presumed to be due to the transplacental passage of these IgG autoantibodies from the mother into the fetal circulation.2 In addition to various degree of atrioventricular (AV) block, other neonatal abnormalities affecting the skin, liver, and blood elements are also associated with anti-SSA/Ro and -SSB/La antibodies in the maternal and fetal circulation and are grouped under the heading of neonatal lupus syndromes.3 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.4
We have recently reported that perfusion of Langendorff perfused rabbit hearts with IgG from mothers with CHB children (positive IgG) caused sinus bradycardia preceding AV block using surface ECG and optical action potentials.5 A significant and unexpected sinus bradycardia was also observed in the animal models of CHB developed by either passive transfer of positive IgG into pregnant mice6 or by active immunization of female mice7 or rabbits8 with SSA/Ro antigen. This high incidence of sinus bradycardia both in vitro and in vivo suggests the possible involvement of the sinoatrial (SA) node. Indeed, Brucato et al9 found sinus bradycardia in infants born to mothers seropositive to SSA/Ro antibodies. Based on these animal and clinical data, we hypothesized that positive IgG may affect SA node ion currents underlying the pacemaker, thus providing a functional explanation for this sinus bradycardia.
Pacemaker activity in SA node cells is known to be due to a complex interplay of various ionic currents.10 Among these currents, L-type Ca2+ current, ICa.L, plays a significant role in both the diastolic depolarization and upstroke phase. Delayed rectifier K+ current, IK, is also important in diastolic depolarization. Hyperpolarization-activated inward current, If, and T-type Ca2+ current, ICa.T, are operative in early and late phases of the diastolic depolarization, respectively. In addition, changes in the time-independent currents can also affect the electrical activity in SA node.10 In the present study, we focused on the major time-dependent pacemaker currents to understand the negative chronotropic mechanism of autoantibodies from mothers with CHB children.
Materials and Methods
All experiments were performed in accordance with animal studies subcommittee regulations at VA New York Harbor Healthcare System. The isolation of single SA node cells was performed by the chopping method11 with a slight modification. New Zealand young rabbits weighing 0.9 to 1.5 kg were anesthetized with intravenous injection of pentobarbital sodium (40 mg/kg). The heart was rapidly excised and immersed in a normal Tyrode’s solution containing the following (in mmol/L): 140 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, and 5 HEPES (pH 7.4). The SA node region was excised from the heart and strips about 0.5 to 1 mm wide were cut perpendicularly to the crista terminalis border. The strips were first incubated in an oxygenated Ca2+-free Tyrode’s solution for 15 minutes, then in the Ca2+-free Tyrode’s solution containing elastase (0.2 mg/mL, Boehringer Mannheim) and collagenase (85 U/mL, Worthington) for 45 to 60 minutes at 37°C. The cells were then dispersed by gentle trituration and were stored at 4°C in a KB solution containing the following (in mmol/L): K glutamate 70, KCl 30, KH2PO4 10, MgCl2 1, taurine 20, glucose 10, and HEPES 10. The cells used in this study were visually identified as long spindle-shaped cells and showed faint striation and prominent centrally located nuclei.
At the beginning of each experiment, all cells were first superfused with normal Tyrode’s solution and then switched to the appropriate solution for each current to be studied. Unless otherwise indicated, the standard pipette solution contained (in mmol/L): 140 KCl, 0.5 MgCl2, 10 HEPES, 10 EGTA, 0.1 Na2GTP and 5 Na2ATP (pH 7.2).
For both L-type (ICa.L) and T-type (ICa.T) Ca2+ currents recording, K+ currents were blocked with intracellular and extracellular Cs2+ and 4-aminopyridine (4-AP).7 ICa.T was recorded in a Na-free and Tris Tyrode’s solution with 20 μmol/L tetrodotoxin plus 1 μmol/L nifedipine. The composition of external solutions for ICa.L contained (in mmol/L): NaCl 132, CsCl 5.4, CaC12 1.8, MgCl2 1.8, NaH2PO4 0.6, HEPES 10, dextrose 5 (pH 7.4), and for ICa.T, contained (in mmol/L): 140 Tris-Cl, 5.4 KCl, 1.0 MgCl2, 2.5 CaCl2, 10 glucose, 5 HEPES (pH 7.4). The internal solution for both ICa.L and ICa.T recordings was the same and contained (in mmol/L): CsCl 139.8, K2EGTA 10, MgCl2 2, CaC12 0.062, Na2-creatine phosphate 5, HEPES 10, Na2ATP 3.1, Na2GTP 0.42 (pH 7.2). The normal Tyrode’s solution was used for IK in the presence of 10 μmol/L nifedipine and 5 mmol/L 4-AP, and for If in the presence of 1 mmol/L BaCl2. The internal solution for IK contained (in mmol/L): 150 KOH, 30 HCl, 10 NaCl, 2 CaCl2, 5 EGTA, 5 MgCl2, 0.1 Na2-GTP, and 5 HEPES (pH 7.2 with aspartic acid). The standard pipette solution as mentioned above was used as the internal solution for If.
Purification of IgG has been performed as previously described.5,7,8 Briefly, immunoglobulin fractions containing IgG were purified from serum by protein A-Sepharose columns and confirmed to be pure by electrophoresis. IgG were obtained from three mothers whose children have CHB. These IgGs are referred to as positive IgG and contain antibodies against 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro, as tested by ELISA and immunoblot. Negative IgG (normal IgG) was purified from sera of three healthy mothers with healthy children, and tested negative for anti-SSA/Ro and anti-SSB/La antibodies by ELISA and immunoblot.
Membrane currents were recorded with amphotericin-perforated patch clamp techniques.12 Amphotericin B (6 mg) was dissolved in 100 μL dimethyl sulfoxide, from which 10 μL was added to a 3-mL pipette solution. The perforated patches were usually established within 10 minutes. The amphotericin-perforated patch recordings were used to reduce dilution of intracellular components, a possible cause of rundown of some membrane currents.
Data were sampled with an A/D converter (Digital 1200, Axon Instruments) and stored on the hard disk of a computer for subsequent analysis. A programmable horizontal puller (Model P-87, Sutter Instrument Company) was used to pull the electrodes. Borosilicate glass electrode (outer diameter, 1.5 mm) with resistances of 2 to 5 MΩ when filled were connected to a patch-clamp amplifier (Dagan 3900A, Dagan Corporation). Junction potentials were zeroed before the pipette touched the cell and always compensated. Pipette series resistance was compensated to minimize the duration of the capacitive transient on 10-mV depolarization from −80 mV.
ICa.L was activated by depolarization pulses from a holding potential of −40 mV. For some cells, a double pulse protocol consisting of a 150-ms prepulse from a holding potential of −80 to −40 mV followed by 300-ms depolarization to 10 mV was used to inactivate the fast Na+ current and ICa.T and minimize the rundown of ICa.L. ICa.T was elicited by depolarizing pluses from a holding potential of −80 mV. A 1000-ms depolarization pulse from a holding potential of −40 mV to the test potentials was used to record IK. If was elicited on hyperpolarizations from a holding potential of −40 to −110 mV.
Action Potential Recordings
Action potentials (APs) were recorded during stable spontaneous electrical activity (current clamp conditions) using the same set-up for current recordings described above. Cells were superfused with Tyrode’s solution containing the following (in mmol/L): NaCl 140.0, KCl 5.4, NaH2PO4 0.33, CaCl2 1.8, MgCl2 0.5, glucose 5.5, and HEPES 5.0; pH was adjusted to 7.4 with NaOH. The pipette solution contained the following (in mmol/L): aspartic acid 100.0, KCl 30.0, MgCl2 0.5, ATP- Na2 5.0, GTP-Na2 0.1, EGTA 11.0, and HEPES 10.0; CaCl2 5.0 (pH was adjusted to 7.2 with KOH). Experiments were performed at 35±0.5°C.
All results are presented as mean±SEM. Current density expressed as pA/pF was determined by dividing current amplitude with cell capacitance. Statistical significance was determined by a Student’s t test for paired data. A value of P≤0.05 was considered significant.
Effects of Positive IgG on ICa.L and ICa.T
To determine the relative role of ionic currents affected by maternal antibodies from mothers with CHB children in single SA node cells, first, the effect of positive IgG (10, 50, 100, and 200 μg/mL) on peak ICa.L was examined. The dose-response curve of positive IgG on ICa.L yielded an IC50 of 59.4 μg/mL (Figure 1A). Figure 1B shows the time course of a representative current recording elicited by 300 ms depolarizing pulses to 10 mV from a holding potential of −40 mV (activating ICa.L preferentially over ICa.T) in a K+ free, Cs+-containing solution in the presence of 5 mmol/L 4-AP before, during application of, and after washout of positive IgG. Application of positive IgG (100 μg/mL) reduced the peak of ICa.L from 191 to 112 pA (41.4%). The inhibition was partly reversed on washout of IgG (160.0 pA, 83.7% recovery). In the contrary, negative IgG (100 μg/mL) did not significantly alter the time course of ICa.L. Figure 1C shows current density-voltage (I-V) relations of ICa.L during control and positive IgG (100 μg/mL). Positive IgG, but not negative IgG, significantly reduced ICa.L at voltages between 0 and +30 mV (Figure 1C; 46.2%±5.6% at 10 mV, n=5; P<0.01) without a significant shift in the steady state activation curve (Figure 1D; V1/2=3.9±0.8 mV in control versus V1/2=5.4±0.9 mV in positive IgG group, n=5; P=NS).
Next, the effect of positive IgG on ICa.T was investigated. Figure 2A shows that the dose-response curve of positive IgG on ICa.T yielded an IC50 of 56.4 μg/mL. The time course of ICa.T before and during application of positive IgG (100 μg/mL) from representative cells is shown in Figure 2B. The inset of Figure 2B shows a current recording in response to a 200-ms depolarization test pulse to −40 mV (activating ICa.T preferentially over ICa.L) from a holding potential of −80 mV recorded in a Na-free and Tris Tyrode’s solution in the presence of nifedipine (1 μmol/L) and tetrodotoxin (20 μmol/L). On depolarization, a peak inward current was elicited, mainly ICa.T. Similar to ICa.L, ICa.T was significantly reduced at 100 μg/mL positive IgG (109 to 75 pA, 31.2%). ICa.T elicited at −40 mV from a holding potential of −80 mV was totally abolished by 40 μmol/L Ni2+ (n=3, data not shown). Negative IgG (100 μg/mL) did not affect ICa.T at all voltages tested. Figure 2C summarizes the effect of positive IgG on I-V relations of ICa.T. The average inhibition of ICa.T by positive IgG was 31.4±5.2% at −40 mV and 44.1%±6.1% at −20 mV (n=5, P<0.01).
Effects of Positive IgG on IK and If
In the experiment shown in Figure 3, the effect of positive IgG on IK was examined in normal Tyrode’s solution with 10 μmol/L nifedipine and 5 mmol/L 4-AP. Figure 3A shows the original superimposed traces recorded at 0 mV from a holding potential of −40 mV under control condition, and after superfusion with positive IgG. The current amplitudes measured near the end of depolarizing pulses and tail currents on repolarization to −40 mV were not affected by 100 μg/mL positive IgG. Even a relatively high concentration of positive IgG (500 μg/mL) did not have significant effect on IK (data not shown). Figure 3B shows I-V relations (current measured at the end of pulse) in response to 1-second pulses to various potentials from −40 mV under control conditions, E-4031–sensitive current (subtracting the current after E-4031 from control) and after positive IgG. In the control, depolarization of the membrane to the test potentials between −40 to 60 mV activated a time-dependent outward current, IK. In the presence of 5 μmol/L E-4031, IK was almost completely blocked and no obvious time-dependent outward current was noted. E-4031–sensitive current density-voltage relations show inward rectification indicating that the rapid component of IK is predominant in our single SA node cells. Positive IgG had no effect on IK. Similar findings were obtained in five other cells (step current at 0 mV: 6.0±0.5 pA/pF for control versus 6.2±0.6 pA/pF for positive IgG group, n=6; P=NS).
The effect of positive IgG on hyperpolarization-activated current, If, is shown in Figure 4. If was elicited on hyperpolarization from a holding potential of −40 mV to the test potentials between −40 to −110 mV every 5 seconds in the normal Tyrode’s solution with the presence of 1 mmol/L BaCl2. Application of positive IgG (100 μg/mL) did not significantly change the amplitude of If (Figure 4A). I-V relations for If is shown in Figure 4B (10.4±1.1 pA/pF for control versus 10.0±1.2 pA/pF for positive IgG, n=5, P=NS). Similar to IK, 500 μg/mL IgG did not significantly affect the amplitude of If (data not shown).
Effects of Positive IgG on SA Node Action Potential (AP) Rate
The effects of positive IgG (100 μg/mL) were tested in spontaneously beating (162.5±11.6 bpm, n=5) SA node myocytes. A typical recording is shown in Figure 5. Figure 5A shows control APs at a firing normal sinus rhythm of 155 bpm in Tyrode’s solution. After 1-minute superfusion of the SA node myocytes with positive IgG, there was sinus bradycardia at irregular firing intervals (about 78 bpm, Figure 5B). After 2 minutes of superfusion with positive IgG, further bradycardia (about 66 bpm) was observed (Figure 5C). After 10 minutes superfusion with Tyrode’s solution, only partial recovery was seen (Figure 5D). During positive IgG application, the AP amplitude was reduced (from 84.3±7.9 to 71.5±12.6 mV; P<0.05, n=5), the slope of phase 4 was also reduced (from 62.8±4.2 to 41.9±6.9 mV/sec; P<0.05, n=5) without significant change in the maximum diastolic potential (MDP; control −63.8±2.6 mV versus −58.0±7.0 mV; P=NS, n=5). In contrast, superfusion of SA node myocytes (n=3) with negative IgG did not alter the spontaneous AP rate, AP amplitude, phase 4 slope, or MDP (AP rate, 164.3±10.5 versus 162.5±12.5 bpm; AP amplitude, 85.5±8.6 versus 86.3±9.6 mV; phase 4 slope, 61.5±5.5 to 62.4±9.6 mV/sec; P=NS, n=5; MDP, −62.5±4.5 versus −61.8±6.6 mV, respectively).
In the present study, we found that maternal antibodies from mothers of children with CHB decreased ICa.L and ICa.T, two important currents to spontaneous cardiac pacing, without altering IK and If in rabbit SA node cells. In addition, positive IgG caused sinus bradycardia in single SA node myocytes. These effects were not seen with normal IgG from healthy mothers with healthy children. This is the first study that provides a functional basis for sinus bradycardia associated with CHB, and points to the important role of both ICa.T and ICa.L in this sinus bradycardia.
Bradycardia and SA Node Involvement
Because abnormalities of the AV node are the hallmark of autoantibody-associated CHB, the AV node rather than the SA node was the main focus of previous publications5–8 even during routine clinical diagnosis of CHB.9 Although Garcia et al,13 using isolated rabbit heart, and our group, using Langendorff perfused isolated rabbit5 and human fetal hearts,7 have also observed significant sinus bradycardia in their models, this bradycardia was not emphasized and its electrophysiological basis have not been investigated. Unexpectedly, we also observed high incidence of sinus bradycardia in an experimental mouse model of CHB developed by passive transfer of human autoantibodies into pregnant mice6 and in by directly immunization of female mice or rabbits with SSA/Ro antigen,7,8 suggesting a possible involvement of SA node. This observation is further supported by clinical data by Brucato et al,9 demonstrating sinus bradycardia in children born to mothers seropositive to SSA/Ro antibodies. This novel finding is of clinical importance because it is only recently that clinicians caring for infants with CHB have begun focusing their attention on sinus bradycardia in addition to AV node conduction abnormalities. Indeed, human fetal autopsies14,15 showed calcification of the SA node, further suggesting that the SA node may be affected. Because circulating maternal autoantibodies are directed against intracellular autoantigens, hypotheses have been proposed that intracellular SSA/Ro and SSA/La proteins are being trafficked to the cell surface during development by the induction of stress proteins, hormonal influences, viral infection, or apoptosis.16–19 The mechanisms by which these events alter SA node pacemaker activity remain unclear.
Effect of Positive IgG on the Membrane Currents and Action Potential in SA Node Cells
In general, the total IgG levels of CHB patients are higher than that from healthy individuals. The level of IgG in CHB cord sera at the time of delivery varied from 500 to 1500 mg/dL,20 which corresponds to 5 to 15 mg/mL. To study the role of antibodies from mothers with CHB children on currents involved in spontaneous pacing, we used the concentration of IgG (80 to 100 μg/mL), which we have previously shown to inhibit Ca channels in single ventricular myocytes7,21 and determined dose-response curves as shown in Figures 1A and 2⇑A. Higher concentrations of 800 to 1200 μg/mL were required to induce compete AV block in whole human fetal and rabbit heart perfused in a Langendorff fashion. Because the serum specimens from the infants were obtained after birth, ie, weeks later after CHB manifestation in the fetus, the exact concentration of IgG at that time is not known.
Single SA node cells were voltage-clamped to assess the effect of positive IgG on the four major time-dependent ionic currents involved in diastolic depolarization, IK, ICa.L, ICa.T, and If. Our data show that both ICa.L and ICa.T are reduced significantly by positive IgG in the single rabbit SA node cells. This is consistent with the observed inhibition of phase 4 diastolic depolarization in SA node cells, suggesting that the negative chronotropic effect of positive IgG is derived, at least in part, from reduction of both ICa.L and ICa.T.
In the present study, we showed ICa.L inhibition by positive IgG, suggesting that the L-type Ca2+ channel is a target for maternal antibodies in SA node cells. The consequences of this inhibition may account for the sinus bradycardia. Because we show that positive IgG reduces ICa.L without shifting I-V relations or activation curve (voltage dependence was unchanged), the underlying mechanism for positive IgG-induced reduction of ICa.L may not be due to changes in channel gating. Indeed, we have previously demonstrated that positive IgG inhibited ventricular ICaL by reducing open times and increasing closed times at the single channel level in both human fetal7 and rat21 heart. We proposed that these data may, in part, explain the basis of the whole-cell ICa.L inhibition by positive IgG in the present study. However, the exact mechanism by which positive IgG affects ICa.L gating in the rabbit SA node is yet to be determined.
T-type Ca2+ channels are usually present in the SA cells and Purkinje fibers of the heart. The physiological role of the T-type Ca2+ channel is not completely understood, and believed to be involved in the pacemaker activity. Indeed, in vivo studies have shown that sinus bradycardia can be induced in conscious rats22 and in anesthetized dogs23 by mibefradil alone, a selective T-type Ca current blocker. Similar dose-dependent decrease in heart rate has been also reported in human.24 Therefore, both ICa.L and ICa.T in SA node cells may contribute to the negative chronotropic effect of maternal antibodies to Ro/La.
It is noteworthy that normal IgG lacking anti-Ro/SSA and anti-La/SSB antibodies did not affect both Ca2+ channels (L- and T-types), indicating that it is unlikely that other unidentified components of IgG may contribute to the effects. However, because we did not use affinity-purified antibodies in this study, we cannot completely rule out the contribution of unidentified components of IgG to our observations.
We have previously shown that positive IgG did not alter the transient outward K current, Ito, the inward rectifier K current, IK, and the fast Na current, INa.21 Because time-dependent currents are absent after the administration of E-4031 plus nifedipine, the instantaneous I-V relation represents a background current. This background current could be a mix of some time-independent current that have been suggested to be involved in SA node pacemaking activity, ie, Na+-K+ pump, Na+-Ca2+ exchanger. However, we did not see any effect on the net current after applying positive IgG, indicating that this time-independent background current may not be involved in bradycardia associated with CHB. Altogether, positive IgG seems to selectively affect Ca2+ channels (L- and T-types) but not other currents such as If, IK, Ito, and INa suggesting specificity to Ca2+ channels.
In rabbit SA node cells, ICa.L and ICa.T are important currents in late phase of diastolic depolarization.10 In the present study, we found that both ICa.L and ICa.T were reduced by positive IgG in SA node cells. Our observations provide direct evidence for the ionic mechanism of the negative chronotropic action of maternal antibodies to SSA/Ro and SSB/La proteins associated with CHB. The findings raise the possibility that sinus bradycardia which often precedes AV block may indicate the potential for AV conduction abnormalities. The findings also provide new insights to the pathogenesis of CHB and potentially to the therapeutic management of a disease considered irreversible and for which currently available therapies are refractory.
This study was supported by an NIH grant (HL-55401) and VA Medical Research Funds (Merit Grant Award and REAP grant) to M.B. IgGs were kindly provided by Dr Jill Buyon through the Research Registry for Neonatal Lupus (AR-4220). We would like to thank the animal laboratory staff for their assistance.
Original received December 15, 2003; resubmission received January 28, 2004; accepted February 2, 2004.
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