Novel Molecular Mechanism Involving 1D (Cav1.3) L-Type Calcium Channel in Autoimmune-Associated Sinus Bradycardia
http://www.100md.com
《循环学杂志》
the Molecular and Cellular Cardiology Program, VA New York Harbor Healthcare System (Y.Q., G.B., Y.Y., M.B.)
SUNY Downstate Medical Center (Y.Q., G.B., M.B.), and NYU School of Medicine (M.B.), New York, NY.
Abstract
Background— Congenital heart block (CHB) is an autoimmune disease that affects fetuses/infants born to mothers with anti-Ro/La antibodies (positive IgG). Although the hallmark of CHB is complete atrioventricular block, sinus bradycardia has been reported recently in animal models of CHB. Interestingly, knockout of the neuroendocrine 1D Ca channel in mice results in significant sinus bradycardia and atrioventricular block, a phenotype reminiscent to that seen in CHB. Here, we tested the hypothesis that the 1D Ca channel is a novel target for positive IgG.
Methods and Results— Reverse transcription–polymerase chain reaction, confocal indirect immunostaining, and Western blot data established the expression of the 1D Ca channel in the human fetal heart. The effect of positive IgG on 1D Ca current (ICa-L) was characterized in heterologous expression systems (tsA201 cells and Xenopus oocytes) because of the unavailability of 1D-specific modulators. 1D ICa-L activated at negative potentials (between –60 and –50 mV). Positive IgG inhibited 1D ICa-L in both expression systems. This inhibition was rescued by a Ca channel activator, Bay K8644. No effect on 1D ICa-L was observed with negative IgG and denatured positive IgG. Western blot data showed that positive IgG binds directly to 1D Ca channel protein.
Conclusions— The data are the first to demonstrate (1) expression of the 1D Ca channel in human fetal heart, (2) inhibition of 1D ICa-L by positive IgG, and (3) direct cross-reactivity of positive IgG with the 1D Ca channel protein. Given that 1D ICa-L activates at voltages within the pacemaker’s diastolic depolarization, inhibition of 1D ICa-L in part may account for autoimmune-associated sinus bradycardia. In addition, Bay K8644 rescue of 1D ICa-L inhibition opens new directions in the development of pharmacotherapeutic approaches in the management of CHB.
Key Words: antibodies ; sinoatrial node ; heart block ; ion channels ; calcium
Introduction
Autoimmune-associated congenital heart block (CHB) is a passively acquired autoimmune disease. Fetal injury is presumed to occur as a consequence of transplacental maternal autoantibodies against the intracellular ribonucleoproteins 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro.1 CHB historically is characterized by irreversible various degrees of AV block, not accompanied by any cardiac anatomic abnormalities.1 Detection of CHB occurs after 16 weeks of gestation, when maternal antibodies effectively gain access to the fetal circulation.2,3 The incidence of CHB is variable, 1 in 11 000 in the general population, but rises to 5 in 100 in patients with lupus.2 CHB carries substantial morbidity and mortality approaching 30%, with >60% of affected children requiring lifelong pacemakers.2 The establishment of both active (by injection of Ro/La antigens)4 and passive (by injection of anti-Ro/La antibodies)5 mouse models of CHB provides the most compelling evidence that maternal anti-Ro/La antibodies cause CHB.
Because AV block has been the hallmark phenotype for CHB, the AV node, rather than the sinoatrial (SA) node, has been the main focus of previous publications4–6 and during clinical diagnosis of CHB.2 In this regard, sinus bradycardia unrelated to AV block was first reported in animal models of CHB.4,5 This was subsequently confirmed by Brucato et al7 and Menon et al,8 who reported significant sinus bradycardia in infants born to mothers seropositive to anti-Ro antibodies. The high incidence of sinus bradycardia in mouse models of CHB and in some affected infants indicates that the spectrum of ECG abnormalities in CHB extends beyond the AV node to affect the SA node. This previously underappreciated autoimmune-associated sinus bradycardia has drawn intense clinical and basic research attention because it may serve as a clinical marker for the detection of CHB, given that sinus bradycardia often precedes AV block.
In previous investigations of the mechanism of AV block in CHB, we demonstrated that positive IgG from mothers with CHB infants selectively inhibits 1C L-type (ICa-L) and T-type (ICa-T) Ca current, but did not affect the pacemaker current If, Ik, and INa.4,6,9–11 We proposed that inhibition of 1C ICa-L could account for the AV block seen in CHB, because impulse conduction in the AV node depends critically on ICa-L. 1C ICa-L activates at more positive (–40 and –30 mV) potentials, whereas SA node pacemaker depolarization occurs between –60 and –40 mV.12 Thus, the contribution of 1C ICa-L to diastolic depolarization of the SA node is generally considered to be minor. It is therefore logical to hypothesize that inhibition of 1C ICa-L by positive IgG will not be the only factor contributing to sinus bradycardia reported in CHB. In support of this hypothesis are recent reports showing that the neuroendocrine 1D Ca channel, which with the 1C Ca channel contributes to the formation of ICa-L, is also expressed in the heart, specifically in the SA node and atria but not in adult ventricles.13 1D ICa-L differs from 1C ICa-L in that 1D ICa-L activates at a more negative (–25mV) membrane potential. This unique biophysical property enables 1D ICa-L to play a more important role in spontaneous depolarization in the SA node. In fact, 1D Ca channel knockout mice exhibit profound sinus bradycardia.14–16 These observations offer convincing evidence to support an important role for 1D ICa-L in SA node pacemaking and evoke the possibility that the 1D Ca channel may be a novel target for positive IgG and thus contribute to the development of autoimmune-associated sinus bradycardia. To establish that the 1D Ca channel is a target for positive IgG, at least 2 criteria must be met: (1) the 1D Ca channel should be expressed in human fetal heart, and (2) 1D ICa-L should be inhibited by positive IgG. In the present study, we combined reverse transcription–polymerase chain reaction (RT-PCR), confocal indirect immunofluorescent staining, Western blot analysis, and electrophysiological techniques to address these criteria.
Methods
Antibodies and Reagents
Anti-Ro/La antibodies (positive IgG) were obtained from the National Institute of Arthritis and Musculoskeletal and Skin Diseases-supported National Research Registry for Neonatal Lupus (Dr Jill P. Buyon, NYU School of Medicine). In the present study, positive IgG was from 3 mothers whose children have CHB, as tested by ELISA and immunoblot.4–6,9–11 Negative IgG (control IgG) was from 3 healthy mothers with healthy children who tested negative for anti-Ro/La antibodies.4–6,9–11 The concentration of positive IgG used was 300 μg/mL for oocyte experiments and 100 μg/mL for tsA201 cells. This was based on data from our previous publications and the dose-response curve in the present study (Figure 5A).9,10 The anti-1D antibody is developed in rabbit with a synthetic peptide that corresponds to amino acids 809 to 825 of the 1D subunit of the rat L-type Ca channel (Sigma).
Human Fetal Heart Tissue
Human fetal hearts (15- to 20-week gestation) were obtained after elective termination of normal pregnancy from National Institutes of Health–sponsored tissue banks in Baltimore, Md, and Seattle, Wash. The use of human fetal heart tissue received an exemption from the VA New York Harbor Healthcare System Institutional Review Board.
Rabbit SA/AV Node Isolation
Rabbit SA and AV node tissues were prepared according to Denyer et al17 and Hancox et al,18 respectively. All animal protocols were approved by the Institutional Animal Care and Use Committee of VA New York Harbor Healthcare System. Young (6-week-old) New Zealand rabbits were anesthetized with intravenous injection of pentobarbital sodium (50 mg/kg). The heart was rapidly excised and immersed in a normal Tyrode solution containing (in mmol/L): 140 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, 5 HEPES (pH 7.4). The SA node, AV node, and atrial and ventricular tissue were snap-frozen in liquid nitrogen.
Reverse Transcription–Polymerase Chain Reaction
Total cellular RNA was isolated from human fetal and rabbit tissue, and reverse transcription was performed as described previously.19,20 The sense primer was 5'-TTAGTGACGCCTGGAACACG-3', and the antisense primer was 5'-CCTGTATCAGGAAAGTGG-3'. The primers were chosen from conserved regions among different species and were unique to the 1D Ca channel. The expected PCR amplification size was 1047 bp. Final PCR products were evaluated on ethidium bromide–stained 1% agarose gel. Sequencing of the PCR products was performed by Genemed.
Isolation of Human and Rat Fetal Cardiac Myocytes
Cardiac myocytes were obtained from Langendorff-perfused human fetal hearts as described previously.4 Hearts were perfused at 37°C with 100% O2 gassed Tyrode’s solution followed by Ca-free Tyrode’s solution with 0.5 mg/mL collagenase type B (Boehringer Mannheim). Cells were then dispersed in a Kraft-Brühe solution containing (in mmol/L): potassium glutamate 70, KCl 30, KH2PO4 10, MgCl2 1, taurine 20, glucose 10, HEPES 10. Isolated 17- to 19-day-old fetal Sprague-Dawley rat myocytes were obtained as described previously21 and cultured on coverslips in Dulbecco’s modified Eagle’s medium containing 10% calf serum (Gibco) overnight before being subjected to the immunostaining procedures.
Indirect Immunofluorescent Staining
Indirect immunostaining was performed on isolated human and rat fetal myocytes and on 1D/;2a/2-transfected tsA201 cells as described previously.19,20 Cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton. After they were blocked with 5% normal goat sera, the cells were incubated overnight at 4°C with anti-1D Ca channel antibody (1:200) and detected with FITC-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc). Secondary antibody alone and staining of nontransfected tsA201 cells with anti-1D antibody were included as negative controls. A confocal scanning laser microscope (MRC-600; Bio-Rad) was used for visualization.
Expression of 1D Ca Channel in tsA201 Cells
tsA201 cells were grown and transiently transfected with 10 μg of a mix of human 1D, rat ;2a, and 2 cDNAs (in pCMV6b vector, kindly provided by Drs J. Striessnig, Innsbruck, Austria, and S. Seino, Kobe, Japan) by the calcium phosphate method as described previously.22 Whole-cell voltage-clamp recording was performed with the Axopatch 200B (Axon Instruments) with pipette resistance of 1.5 to 3 M at 48 hours after transfection. The internal solution contained (in mmol/L): 135 CsCl, 4 MgCl2, 4 ATP, 10 HEPES, 10 EGTA, and 1 EDTA, adjusted to pH 7.2 with TEAOH. The bath solution contained (in mmol/L): 135 choline chlorine, 1 MgCl2, 2 CaCl2, and 10 HEPES, adjusted to pH 7.4 with TEAOH. Signals were sampled at 20 kHz and low-pass filtered at 2 kHz. Data were leak-subtracted online with a P/4 protocol and analyzed with pClamp version 8.0 (Axon Instruments). For 1D ICa-L current-voltage (I-V) relations, tsA201 cells were depolarized from a holding potential of –100 mV to test potentials between –80 and 60 mV with increments of 10 mV. For the time course, 1D ICa-L was recorded continuously at a test potential of –10 mV from a holding potential of –100 mV.
Expression and Recording of 1D Ca Current in Xenopus Oocytes
Stage IV and V oocytes were injected with 20 ng of 1D/;2a cRNA encoding the full length of human 1D (subcloned from pCMV6b to pCDNA vector) and rat ;2a subunits (in pBS bluescript SK vector, kindly provided by E. Perez-Reyes, University of Virginia, Charlottesville). Currents were recorded from the fourth to the seventh day after injection.9,10 The external recording solution contained (in mmol/L): Ba(OH)2 40, NaOH 50, KOH 2, HEPES 5, 4-AP 5, and TEA 10, pH 7.4. Barium was used instead of calcium to avoid the significant endogenous chloride current in Xenopus oocytes. Oocytes were impaled with electrodes filled with 3 mol/L KCl.
For IBa-L I-V relations, oocytes were depolarized from a holding potential of –80 mV to test potentials between –50 and 50 mV with increments of 10 mV. For the time course, IBa-L was recorded continuously at a test potential of –10 mV from a holding potential of –80 mV.
Western Blot
tsA201 cells were harvested at 48 hours after transfection with 1D/;2a/2 cDNA. Cells were lysed in a lysis buffer (in mmol/L: Tris/HCl 50, pH 7.4, NaCl 150, EDTA 5, 0.25% Triton X-100, 10% glycerol, NaF 1, Na3VO4 1, 10 μg/mL PMSF, aprotinin, leupeptin),23 and centrifuged at 40 000g for 30 minutes. The supernatant (150 μg) was resolved by 4% to 12% SDS/PAGE. Blots were probed with rabbit anti-1D antibody (Sigma, 1:250) at 4°C overnight and detected with a 1:5000 diluted peroxidase-conjugated anti-rabbit IgG. For the cross-reactivity experiments, after detection, the membrane blot was stripped, split into 2, and then reprobed with positive IgG (1:400) and negative IgG (1:400), respectively. Peroxidase-conjugated anti-human IgG was used for detection. Additional experiments with membrane proteins (150 μg) from human fetal heart and rat fetal heart prepared as described previously20 were performed to examine expression of 1D Ca channels in these tissues. Membrane protein from adult rat left ventricles was included as negative control.
Statistical Analysis
Statistical comparisons were evaluated with a Student t test. Data are presented as mean±SEM. A value of P<0.05 was considered significant.
Results
1D Ca Channel Is Expressed in Human Fetal Cardiac Myocytes
1D Ca Channel mRNA
CHB is detected in human fetal heart as early as 16 weeks of gestation; however, there are no available data about expression of 1D Ca channel in human fetal heart. We first performed RT-PCR to investigate the expression of 1D mRNA in human fetal hearts (15- to 20-weeks’ gestation). Because of the small size and limited access to human fetal hearts, young rabbit SA node tissue was used instead. With an 1D Ca channel–specific primer, 1D Ca channel mRNA was readily amplified from right atria, ventricles, and AV node of human fetal hearts (n=3; Figure 1A). This is in contrast to the young rabbit hearts (n=3), in which 1D Ca channel mRNA was not detected in the ventricles (Figure 1B) but was detected in SA node, atria, and AV node. The brain tissue was used as positive control. A 361-bp band corresponding to the S15 housekeeping gene was seen in all tissues (lower panels, Figures 1A and 1B), which confirmed accuracy in the RNA estimation and gel loading techniques.
1D Ca Channel Protein
To eliminate possible contamination from noncardiac tissue in the RT-PCR experiments above, indirect confocal immunostaining with anti-1D Ca channel antibody was performed in isolated human fetal cardiac myocytes (n=3 hearts). Figure 2 illustrates typical immunostaining experiments, with panels A, C, E, and G representing phase controls and panels B, D, F, and H the corresponding staining, respectively. 1D Ca channel protein was localized on the cell membrane of both human right atrial and ventricular myocytes (Figures 2B and 2D). No staining was seen with the secondary antibody alone (Figure 2F). Interestingly, nuclear staining was also observed (Figures 2B and 2D). Because the anti-1D Ca channel antibody was raised in rabbit against rat 1D Ca channel, we also performed immunostaining experiments using rat fetal ventricular myocytes to eliminate the possible species-related cross-reactivity of the antibody. As in the human fetal heart, both sarcolemmal and nuclear staining were observed in rat fetal myocytes, as shown in Figure 2H. We further tested the staining in both transfected and nontransfected tsA201 cells using the same anti-1D antibody. In 1D/;2a/2–transfected tsA201 cells, marked staining of 1D Ca channel was observed at the plasma membrane and intracellularly (Figures 3A, 3B, 3C, and 3D). No staining was observed under the same scanning settings with secondary antibody alone (Figures 3E and 3F) or in nontransfected tsA201 cells (Figures 3G and 3H). Human and rat fetal myocytes and transfected tsA201 cells all showed clear sarcolemmal staining. The significance of nuclear staining is not yet known. To complement the immunostaining experiments, Western blots were also performed to examine expression of the 1D Ca channel in fetal hearts. Figure 4 shows that the band corresponding to the 1D Ca channel was detected in both human and rat fetal hearts. This band was not seen in the negative control, in which the anti-1D antibody was preincubated with its antigen peptide for 1 hour at room temperature. Furthermore, no band was observed with proteins from adult rat ventricles, which indicates the specificity of the anti-1D Ca channel antibody. In summary, the confocal immunostaining and Western blot data establish for the first time that the 1D Ca channel is expressed in the human fetal heart.
1D ICa-L Was Inhibited by Positive IgG and Was Rescued by Bay K8644 in Mammalian tsA201 Cells
Presently, there are no pharmacological agents to separate 1D from 1C ICa-L in native cells. Thus, expression systems constitute an alternative for studying the effect of positive IgG on only the 1D Ca channel. Transfection of tsA201 cells with 1D plasmid alone failed to yield any functional channel. This is consistent with a previous report.24 Similarly, no detectable current was observed in tsA201 cells transfected with ;2a/2 subunits alone. Coexpression of 1D together with ;2a/2 subunits in tsA201 cells yielded functional 1D ICa-L that activated at approximately –60 to –50 mV and peaked at –10 mV with 2 mmol/L Ca used as a charge carrier (Figure 5C). We next tested whether positive IgG would inhibit ICa-L and whether this inhibition could be rescued by Bay K8644 (1 μmol/L), a dihydropyridine calcium channel activator. The dose-response curve of positive IgG on 1D ICa-L yielded an IC50 of 51.4 μg/mL (Figure 5A; n=5). Application of positive IgG (100 μg/mL) reduced the peak of 1D ICa-L (Figure 5B). Averaged data showed that inhibition of 1D ICa-L by positive IgG was 42.2±3% at –10 mV (Figure 5C). This inhibition of 1D ICa-L was rescued by 1 μmol/L Bay K8644 beyond the baseline level (Figure 6A; note that Bay K8644 was added to positive IgG). The specificity of the inhibition of 1D ICa-L by positive IgG was tested by the observation that neither denatured positive IgG (100 μg/mL; Figure 6B) nor the negative IgG (100 μg/mL) had any effect on 1D ICa-L.
1D ICa-L Was Also Inhibited by Positive IgG and Rescued by Bay K8644 in Xenopus Oocyte
To exclude that 1D ICa-L inhibition by positive IgG is not dependent on the expression system, the effect of positive IgG on 1D current was also characterized in Xenopus oocytes. Figure 7A shows I-V relations (panel A) and current traces (inset) of the expressed 1D IBa-L in Xenopus oocytes. 1D IBa-L activated at approximately –50 to –40 mV and peaked at –10 mV with 40 mmol/L barium as the charge carrier. Figure 7B shows the time course of one representative experiment in which 1D IBa-L recorded at –10 mV was inhibited by positive IgG (from 450 nA at control to 325 nA with positive IgG). This inhibition was also rescued by Bay K8644 beyond the baseline level. Averaged data showed that the inhibition of 1D IBa-L by positive IgG (300 μg/mL) was 33±10% at –10 mV; Bay K8644 (1 μmol/L) reversed the inhibition of 1D IBa-L by positive IgG and further increased the current to 12% above the baseline level (n=6, P<0.05; Figure 7C). Negative IgG did not have any effect on 1D IBa-L.
Positive IgG Cross-Reacts With 1D Ca Channel Protein
Discussion
The present data are the first to provide biochemical and functional evidence that the 1D Ca channel is expressed in human fetal heart and that positive IgG from mothers of children with CHB inhibits 1D ICa-L. Positive IgG from the same mothers used to demonstrate electrophysiological inhibition of 1D ICa-L also recognized the 1D subunit of the L-type Ca channel by Western blot. Together, the data in this study establish that the 1D Ca channel is a novel target for positive IgG, and consequently, 1D ICa-L inhibited by positive IgG may account in part for the autoimmune-associated sinus bradycardia seen in CHB.
The causal relationship of positive IgG to autoimmune-associated sinus bradycardia in CHB has been documented in animal models of CHB4,5,11 and in clinical settings7,8; however, the underlying molecular mechanism remains unknown. The candidate antigens SSA-Ro and SSB-La are intracellularly located, and there is no convincing evidence that maternal antibodies can cross the sarcolemma of a normal myocyte. Efforts have therefore been directed toward mechanisms that may cause the translocation of these antigens to the cell surface. Several experimental evidences have shown that viral infection,25 UV light treatment,26 and apoptotic death of cells could induce the translocation of Ro/La antigens to the cell surface27; however, it is not yet clear what the resulting cellular events and signaling pathways are that could account for the sinus bradycardia in CHB. Alternatively, another appealing hypothesis, which we termed the "Ca channel hypothesis," proposes that anti-Ro/La autoantibodies cross-react with sarcolemmal components, such as Ca channels, which play an important role in SA node pacemaking activity. This hypothesis is supported by previous observations that Ca channels have been the targets for autoantibodies in other autoimmune diseases.28–32 It has been reported that autoantibodies against the ADP/ATP carrier of the inner mitochondrial membrane from patients with myocarditis and dilated cardiomyopathy can cross-react with sarcolemmal Ca channel proteins and disturb Ca channel function.28 Similar cross-reactivity has been reported in other autoimmune disease, such as Lambert-Eaton myasthenic syndrome and lateral sclerosis.29–32 In addition, we have also previously shown that positive IgG cross-reacts with 1C Ca channel protein and inhibited 1C ICa-L.4,6,9–11 This hypothesis is further supported by the present data showing that positive IgG recognized the 1D Ca channel protein and functionally inhibited the expressed 1D ICa-L.
A number of ion currents have been implicated in SA node pacemaker activity. These include If, ICa-T, ICa-L, IK, and INa. We have previously shown that positive IgG specifically inhibited 1C ICa-L and, to a lesser extent, ICa-T but did not affect If, IK, or INa, which indicates specificity for the Ca channel famliy.4,9–11 However, the inhibition of 1C ICa-L by positive IgG cannot account for the reported sinus bradycardia in CHB, because the contribution of 1C ICa-L to diastolic depolarization of SA node is generally considered to be minor owing to its more positive activation threshold compared with the spontaneous pacemaker diastolic depolarization.12 The present data using human fetal heart, together with animal data from other studies, have demonstrated that the previously underappreciated 1D Ca channel appears to play unique and distinct roles in the heart.14–16 First, unlike the universally expressed 1C Ca channel in the heart, the 1D Ca channel is expressed only in adult SA node, atria, and AV node. Mangoni et al16 showed ICa-L density in SA node cells was decreased by 75% in 1D Ca channel knockout mice compared with wild-type mice, which indicates that the contribution of the 1D Ca channel to total ICa-L in the mouse SA node cell is significant. Second, we showed that 1D ICa-L was activated at between –60 and –50 mV in tsA201 cells, which falls within the range of pacemaker diastolic depolarization. The fact that the 1D Ca channel is expressed in SA node cells and activates at a low-voltage threshold range and that 1D Ca channel knockout mice exhibit profound sinus bradycardia14–16 indicates that the 1D Ca channel plays a critical role in maintenance of normal cardiac rhythm. Consequently, inhibition of SA nodal 1D ICa-L by positive IgG is expected to slow the heart rate. Indeed, positive IgG-superfused rat and rabbit hearts and pups from mice injected with positive IgG during pregnancy exhibited profound sinus bradycardia by ECG and optical action potential recordings.4,6,11 Furthermore, we recently showed that superfusion of spontaneously beating single rabbit SA node myocytes with positive IgG resulted in significant sinus bradycardia as measured by an increase in action potential cycle length.11
In the present study, we also unexpectedly observed expression of the 1D Ca channel in the fetal ventricle. This fetal ventricular expression of the 1D Ca channel is clinically relevant, because some deaths reported in children with CHB are related to heart failure.2,33,34 The inhibition by positive IgG of ventricular L-type Ca channels (both 1D and 1C), which are responsible for generating contractile force, will diminish the contraction status of the fetal heart, which depends significantly on sarcolemmal Ca entry. Thus, inhibition of 1D together with 1C ICa-L by positive IgG may account for both the sinus bradycardia and contractile impairment seen in CHB infants.
Despite the extensive research effort in CHB, there is no effective pharmacotherapy for CHB to date. If inhibition of ICa-L is critical in cardiac impulse generation and conduction found in CHB patients, then an increase in ICa-L should rescue or reverse cardiac electrical abnormalities seen in CHB. We demonstrated that application of a Ca channel activator, Bay K8644, reversed the 1D ICa-L inhibited by positive IgG beyond the baseline level. This finding is the first experimental investigation in CHB to correlate basic findings to potential therapeutic development of new strategies in the management of CHB. Although Bay K8644 may not be an ideal Ca channel agonist in patients because of its vascular effects, its ability to rescue IgG inhibition of 1D ICa-L points to the need to develop new therapeutic agents that will specifically target cardiac Ca channels.
In summary, the data demonstrate that the 1D Ca channel is expressed in human fetal heart, and 1D ICa-L is inhibited by positive IgG by direct binding to the channel protein. Given that the 1D Ca channel plays a critical role in SA node pacemaking and given our previous observation that 1C ICa-L and ICa-T were also inhibited by positive IgG,9,10 blockade of 1D, 1C ICa-L, and ICa-T in human fetal heart may contribute to the genesis of sinus bradycardia in CHB.
Acknowledgments
This work has been supported by National Heart, Lung, and Blood Institutes grant No. HL077494, the VA Merit Grant Award and REAP to Dr Boutjdir, National Institutes of Health F32 grant to Dr Qu, the Canadian Institutes of Health Research Award to Dr G. Baroudi, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases-supported National Research Registry for Neonatal Lupus.
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Smith RG, Hamilton S, Hofmann F, Schneider T, Nastainczyk W, Birnbaumer L, Stefani E, Appel SH. Serum antibodies to L-type calcium channels in patients with amyotrophic lateral sclerosis. N Engl J Med. 1992; 327: 1721–1728.
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Anandakumar C, Biswas A, Chew SS, Chia D, Wong YC, Ratnam SS. Direct fetal therapy for hydrops secondary to congenital atrioventricular heart block. Obstet Gynecol. 1996; 87: 835–837.(Novel Molecular Mechanism)
SUNY Downstate Medical Center (Y.Q., G.B., M.B.), and NYU School of Medicine (M.B.), New York, NY.
Abstract
Background— Congenital heart block (CHB) is an autoimmune disease that affects fetuses/infants born to mothers with anti-Ro/La antibodies (positive IgG). Although the hallmark of CHB is complete atrioventricular block, sinus bradycardia has been reported recently in animal models of CHB. Interestingly, knockout of the neuroendocrine 1D Ca channel in mice results in significant sinus bradycardia and atrioventricular block, a phenotype reminiscent to that seen in CHB. Here, we tested the hypothesis that the 1D Ca channel is a novel target for positive IgG.
Methods and Results— Reverse transcription–polymerase chain reaction, confocal indirect immunostaining, and Western blot data established the expression of the 1D Ca channel in the human fetal heart. The effect of positive IgG on 1D Ca current (ICa-L) was characterized in heterologous expression systems (tsA201 cells and Xenopus oocytes) because of the unavailability of 1D-specific modulators. 1D ICa-L activated at negative potentials (between –60 and –50 mV). Positive IgG inhibited 1D ICa-L in both expression systems. This inhibition was rescued by a Ca channel activator, Bay K8644. No effect on 1D ICa-L was observed with negative IgG and denatured positive IgG. Western blot data showed that positive IgG binds directly to 1D Ca channel protein.
Conclusions— The data are the first to demonstrate (1) expression of the 1D Ca channel in human fetal heart, (2) inhibition of 1D ICa-L by positive IgG, and (3) direct cross-reactivity of positive IgG with the 1D Ca channel protein. Given that 1D ICa-L activates at voltages within the pacemaker’s diastolic depolarization, inhibition of 1D ICa-L in part may account for autoimmune-associated sinus bradycardia. In addition, Bay K8644 rescue of 1D ICa-L inhibition opens new directions in the development of pharmacotherapeutic approaches in the management of CHB.
Key Words: antibodies ; sinoatrial node ; heart block ; ion channels ; calcium
Introduction
Autoimmune-associated congenital heart block (CHB) is a passively acquired autoimmune disease. Fetal injury is presumed to occur as a consequence of transplacental maternal autoantibodies against the intracellular ribonucleoproteins 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro.1 CHB historically is characterized by irreversible various degrees of AV block, not accompanied by any cardiac anatomic abnormalities.1 Detection of CHB occurs after 16 weeks of gestation, when maternal antibodies effectively gain access to the fetal circulation.2,3 The incidence of CHB is variable, 1 in 11 000 in the general population, but rises to 5 in 100 in patients with lupus.2 CHB carries substantial morbidity and mortality approaching 30%, with >60% of affected children requiring lifelong pacemakers.2 The establishment of both active (by injection of Ro/La antigens)4 and passive (by injection of anti-Ro/La antibodies)5 mouse models of CHB provides the most compelling evidence that maternal anti-Ro/La antibodies cause CHB.
Because AV block has been the hallmark phenotype for CHB, the AV node, rather than the sinoatrial (SA) node, has been the main focus of previous publications4–6 and during clinical diagnosis of CHB.2 In this regard, sinus bradycardia unrelated to AV block was first reported in animal models of CHB.4,5 This was subsequently confirmed by Brucato et al7 and Menon et al,8 who reported significant sinus bradycardia in infants born to mothers seropositive to anti-Ro antibodies. The high incidence of sinus bradycardia in mouse models of CHB and in some affected infants indicates that the spectrum of ECG abnormalities in CHB extends beyond the AV node to affect the SA node. This previously underappreciated autoimmune-associated sinus bradycardia has drawn intense clinical and basic research attention because it may serve as a clinical marker for the detection of CHB, given that sinus bradycardia often precedes AV block.
In previous investigations of the mechanism of AV block in CHB, we demonstrated that positive IgG from mothers with CHB infants selectively inhibits 1C L-type (ICa-L) and T-type (ICa-T) Ca current, but did not affect the pacemaker current If, Ik, and INa.4,6,9–11 We proposed that inhibition of 1C ICa-L could account for the AV block seen in CHB, because impulse conduction in the AV node depends critically on ICa-L. 1C ICa-L activates at more positive (–40 and –30 mV) potentials, whereas SA node pacemaker depolarization occurs between –60 and –40 mV.12 Thus, the contribution of 1C ICa-L to diastolic depolarization of the SA node is generally considered to be minor. It is therefore logical to hypothesize that inhibition of 1C ICa-L by positive IgG will not be the only factor contributing to sinus bradycardia reported in CHB. In support of this hypothesis are recent reports showing that the neuroendocrine 1D Ca channel, which with the 1C Ca channel contributes to the formation of ICa-L, is also expressed in the heart, specifically in the SA node and atria but not in adult ventricles.13 1D ICa-L differs from 1C ICa-L in that 1D ICa-L activates at a more negative (–25mV) membrane potential. This unique biophysical property enables 1D ICa-L to play a more important role in spontaneous depolarization in the SA node. In fact, 1D Ca channel knockout mice exhibit profound sinus bradycardia.14–16 These observations offer convincing evidence to support an important role for 1D ICa-L in SA node pacemaking and evoke the possibility that the 1D Ca channel may be a novel target for positive IgG and thus contribute to the development of autoimmune-associated sinus bradycardia. To establish that the 1D Ca channel is a target for positive IgG, at least 2 criteria must be met: (1) the 1D Ca channel should be expressed in human fetal heart, and (2) 1D ICa-L should be inhibited by positive IgG. In the present study, we combined reverse transcription–polymerase chain reaction (RT-PCR), confocal indirect immunofluorescent staining, Western blot analysis, and electrophysiological techniques to address these criteria.
Methods
Antibodies and Reagents
Anti-Ro/La antibodies (positive IgG) were obtained from the National Institute of Arthritis and Musculoskeletal and Skin Diseases-supported National Research Registry for Neonatal Lupus (Dr Jill P. Buyon, NYU School of Medicine). In the present study, positive IgG was from 3 mothers whose children have CHB, as tested by ELISA and immunoblot.4–6,9–11 Negative IgG (control IgG) was from 3 healthy mothers with healthy children who tested negative for anti-Ro/La antibodies.4–6,9–11 The concentration of positive IgG used was 300 μg/mL for oocyte experiments and 100 μg/mL for tsA201 cells. This was based on data from our previous publications and the dose-response curve in the present study (Figure 5A).9,10 The anti-1D antibody is developed in rabbit with a synthetic peptide that corresponds to amino acids 809 to 825 of the 1D subunit of the rat L-type Ca channel (Sigma).
Human Fetal Heart Tissue
Human fetal hearts (15- to 20-week gestation) were obtained after elective termination of normal pregnancy from National Institutes of Health–sponsored tissue banks in Baltimore, Md, and Seattle, Wash. The use of human fetal heart tissue received an exemption from the VA New York Harbor Healthcare System Institutional Review Board.
Rabbit SA/AV Node Isolation
Rabbit SA and AV node tissues were prepared according to Denyer et al17 and Hancox et al,18 respectively. All animal protocols were approved by the Institutional Animal Care and Use Committee of VA New York Harbor Healthcare System. Young (6-week-old) New Zealand rabbits were anesthetized with intravenous injection of pentobarbital sodium (50 mg/kg). The heart was rapidly excised and immersed in a normal Tyrode solution containing (in mmol/L): 140 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10 glucose, 5 HEPES (pH 7.4). The SA node, AV node, and atrial and ventricular tissue were snap-frozen in liquid nitrogen.
Reverse Transcription–Polymerase Chain Reaction
Total cellular RNA was isolated from human fetal and rabbit tissue, and reverse transcription was performed as described previously.19,20 The sense primer was 5'-TTAGTGACGCCTGGAACACG-3', and the antisense primer was 5'-CCTGTATCAGGAAAGTGG-3'. The primers were chosen from conserved regions among different species and were unique to the 1D Ca channel. The expected PCR amplification size was 1047 bp. Final PCR products were evaluated on ethidium bromide–stained 1% agarose gel. Sequencing of the PCR products was performed by Genemed.
Isolation of Human and Rat Fetal Cardiac Myocytes
Cardiac myocytes were obtained from Langendorff-perfused human fetal hearts as described previously.4 Hearts were perfused at 37°C with 100% O2 gassed Tyrode’s solution followed by Ca-free Tyrode’s solution with 0.5 mg/mL collagenase type B (Boehringer Mannheim). Cells were then dispersed in a Kraft-Brühe solution containing (in mmol/L): potassium glutamate 70, KCl 30, KH2PO4 10, MgCl2 1, taurine 20, glucose 10, HEPES 10. Isolated 17- to 19-day-old fetal Sprague-Dawley rat myocytes were obtained as described previously21 and cultured on coverslips in Dulbecco’s modified Eagle’s medium containing 10% calf serum (Gibco) overnight before being subjected to the immunostaining procedures.
Indirect Immunofluorescent Staining
Indirect immunostaining was performed on isolated human and rat fetal myocytes and on 1D/;2a/2-transfected tsA201 cells as described previously.19,20 Cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton. After they were blocked with 5% normal goat sera, the cells were incubated overnight at 4°C with anti-1D Ca channel antibody (1:200) and detected with FITC-conjugated anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, Inc). Secondary antibody alone and staining of nontransfected tsA201 cells with anti-1D antibody were included as negative controls. A confocal scanning laser microscope (MRC-600; Bio-Rad) was used for visualization.
Expression of 1D Ca Channel in tsA201 Cells
tsA201 cells were grown and transiently transfected with 10 μg of a mix of human 1D, rat ;2a, and 2 cDNAs (in pCMV6b vector, kindly provided by Drs J. Striessnig, Innsbruck, Austria, and S. Seino, Kobe, Japan) by the calcium phosphate method as described previously.22 Whole-cell voltage-clamp recording was performed with the Axopatch 200B (Axon Instruments) with pipette resistance of 1.5 to 3 M at 48 hours after transfection. The internal solution contained (in mmol/L): 135 CsCl, 4 MgCl2, 4 ATP, 10 HEPES, 10 EGTA, and 1 EDTA, adjusted to pH 7.2 with TEAOH. The bath solution contained (in mmol/L): 135 choline chlorine, 1 MgCl2, 2 CaCl2, and 10 HEPES, adjusted to pH 7.4 with TEAOH. Signals were sampled at 20 kHz and low-pass filtered at 2 kHz. Data were leak-subtracted online with a P/4 protocol and analyzed with pClamp version 8.0 (Axon Instruments). For 1D ICa-L current-voltage (I-V) relations, tsA201 cells were depolarized from a holding potential of –100 mV to test potentials between –80 and 60 mV with increments of 10 mV. For the time course, 1D ICa-L was recorded continuously at a test potential of –10 mV from a holding potential of –100 mV.
Expression and Recording of 1D Ca Current in Xenopus Oocytes
Stage IV and V oocytes were injected with 20 ng of 1D/;2a cRNA encoding the full length of human 1D (subcloned from pCMV6b to pCDNA vector) and rat ;2a subunits (in pBS bluescript SK vector, kindly provided by E. Perez-Reyes, University of Virginia, Charlottesville). Currents were recorded from the fourth to the seventh day after injection.9,10 The external recording solution contained (in mmol/L): Ba(OH)2 40, NaOH 50, KOH 2, HEPES 5, 4-AP 5, and TEA 10, pH 7.4. Barium was used instead of calcium to avoid the significant endogenous chloride current in Xenopus oocytes. Oocytes were impaled with electrodes filled with 3 mol/L KCl.
For IBa-L I-V relations, oocytes were depolarized from a holding potential of –80 mV to test potentials between –50 and 50 mV with increments of 10 mV. For the time course, IBa-L was recorded continuously at a test potential of –10 mV from a holding potential of –80 mV.
Western Blot
tsA201 cells were harvested at 48 hours after transfection with 1D/;2a/2 cDNA. Cells were lysed in a lysis buffer (in mmol/L: Tris/HCl 50, pH 7.4, NaCl 150, EDTA 5, 0.25% Triton X-100, 10% glycerol, NaF 1, Na3VO4 1, 10 μg/mL PMSF, aprotinin, leupeptin),23 and centrifuged at 40 000g for 30 minutes. The supernatant (150 μg) was resolved by 4% to 12% SDS/PAGE. Blots were probed with rabbit anti-1D antibody (Sigma, 1:250) at 4°C overnight and detected with a 1:5000 diluted peroxidase-conjugated anti-rabbit IgG. For the cross-reactivity experiments, after detection, the membrane blot was stripped, split into 2, and then reprobed with positive IgG (1:400) and negative IgG (1:400), respectively. Peroxidase-conjugated anti-human IgG was used for detection. Additional experiments with membrane proteins (150 μg) from human fetal heart and rat fetal heart prepared as described previously20 were performed to examine expression of 1D Ca channels in these tissues. Membrane protein from adult rat left ventricles was included as negative control.
Statistical Analysis
Statistical comparisons were evaluated with a Student t test. Data are presented as mean±SEM. A value of P<0.05 was considered significant.
Results
1D Ca Channel Is Expressed in Human Fetal Cardiac Myocytes
1D Ca Channel mRNA
CHB is detected in human fetal heart as early as 16 weeks of gestation; however, there are no available data about expression of 1D Ca channel in human fetal heart. We first performed RT-PCR to investigate the expression of 1D mRNA in human fetal hearts (15- to 20-weeks’ gestation). Because of the small size and limited access to human fetal hearts, young rabbit SA node tissue was used instead. With an 1D Ca channel–specific primer, 1D Ca channel mRNA was readily amplified from right atria, ventricles, and AV node of human fetal hearts (n=3; Figure 1A). This is in contrast to the young rabbit hearts (n=3), in which 1D Ca channel mRNA was not detected in the ventricles (Figure 1B) but was detected in SA node, atria, and AV node. The brain tissue was used as positive control. A 361-bp band corresponding to the S15 housekeeping gene was seen in all tissues (lower panels, Figures 1A and 1B), which confirmed accuracy in the RNA estimation and gel loading techniques.
1D Ca Channel Protein
To eliminate possible contamination from noncardiac tissue in the RT-PCR experiments above, indirect confocal immunostaining with anti-1D Ca channel antibody was performed in isolated human fetal cardiac myocytes (n=3 hearts). Figure 2 illustrates typical immunostaining experiments, with panels A, C, E, and G representing phase controls and panels B, D, F, and H the corresponding staining, respectively. 1D Ca channel protein was localized on the cell membrane of both human right atrial and ventricular myocytes (Figures 2B and 2D). No staining was seen with the secondary antibody alone (Figure 2F). Interestingly, nuclear staining was also observed (Figures 2B and 2D). Because the anti-1D Ca channel antibody was raised in rabbit against rat 1D Ca channel, we also performed immunostaining experiments using rat fetal ventricular myocytes to eliminate the possible species-related cross-reactivity of the antibody. As in the human fetal heart, both sarcolemmal and nuclear staining were observed in rat fetal myocytes, as shown in Figure 2H. We further tested the staining in both transfected and nontransfected tsA201 cells using the same anti-1D antibody. In 1D/;2a/2–transfected tsA201 cells, marked staining of 1D Ca channel was observed at the plasma membrane and intracellularly (Figures 3A, 3B, 3C, and 3D). No staining was observed under the same scanning settings with secondary antibody alone (Figures 3E and 3F) or in nontransfected tsA201 cells (Figures 3G and 3H). Human and rat fetal myocytes and transfected tsA201 cells all showed clear sarcolemmal staining. The significance of nuclear staining is not yet known. To complement the immunostaining experiments, Western blots were also performed to examine expression of the 1D Ca channel in fetal hearts. Figure 4 shows that the band corresponding to the 1D Ca channel was detected in both human and rat fetal hearts. This band was not seen in the negative control, in which the anti-1D antibody was preincubated with its antigen peptide for 1 hour at room temperature. Furthermore, no band was observed with proteins from adult rat ventricles, which indicates the specificity of the anti-1D Ca channel antibody. In summary, the confocal immunostaining and Western blot data establish for the first time that the 1D Ca channel is expressed in the human fetal heart.
1D ICa-L Was Inhibited by Positive IgG and Was Rescued by Bay K8644 in Mammalian tsA201 Cells
Presently, there are no pharmacological agents to separate 1D from 1C ICa-L in native cells. Thus, expression systems constitute an alternative for studying the effect of positive IgG on only the 1D Ca channel. Transfection of tsA201 cells with 1D plasmid alone failed to yield any functional channel. This is consistent with a previous report.24 Similarly, no detectable current was observed in tsA201 cells transfected with ;2a/2 subunits alone. Coexpression of 1D together with ;2a/2 subunits in tsA201 cells yielded functional 1D ICa-L that activated at approximately –60 to –50 mV and peaked at –10 mV with 2 mmol/L Ca used as a charge carrier (Figure 5C). We next tested whether positive IgG would inhibit ICa-L and whether this inhibition could be rescued by Bay K8644 (1 μmol/L), a dihydropyridine calcium channel activator. The dose-response curve of positive IgG on 1D ICa-L yielded an IC50 of 51.4 μg/mL (Figure 5A; n=5). Application of positive IgG (100 μg/mL) reduced the peak of 1D ICa-L (Figure 5B). Averaged data showed that inhibition of 1D ICa-L by positive IgG was 42.2±3% at –10 mV (Figure 5C). This inhibition of 1D ICa-L was rescued by 1 μmol/L Bay K8644 beyond the baseline level (Figure 6A; note that Bay K8644 was added to positive IgG). The specificity of the inhibition of 1D ICa-L by positive IgG was tested by the observation that neither denatured positive IgG (100 μg/mL; Figure 6B) nor the negative IgG (100 μg/mL) had any effect on 1D ICa-L.
1D ICa-L Was Also Inhibited by Positive IgG and Rescued by Bay K8644 in Xenopus Oocyte
To exclude that 1D ICa-L inhibition by positive IgG is not dependent on the expression system, the effect of positive IgG on 1D current was also characterized in Xenopus oocytes. Figure 7A shows I-V relations (panel A) and current traces (inset) of the expressed 1D IBa-L in Xenopus oocytes. 1D IBa-L activated at approximately –50 to –40 mV and peaked at –10 mV with 40 mmol/L barium as the charge carrier. Figure 7B shows the time course of one representative experiment in which 1D IBa-L recorded at –10 mV was inhibited by positive IgG (from 450 nA at control to 325 nA with positive IgG). This inhibition was also rescued by Bay K8644 beyond the baseline level. Averaged data showed that the inhibition of 1D IBa-L by positive IgG (300 μg/mL) was 33±10% at –10 mV; Bay K8644 (1 μmol/L) reversed the inhibition of 1D IBa-L by positive IgG and further increased the current to 12% above the baseline level (n=6, P<0.05; Figure 7C). Negative IgG did not have any effect on 1D IBa-L.
Positive IgG Cross-Reacts With 1D Ca Channel Protein
Discussion
The present data are the first to provide biochemical and functional evidence that the 1D Ca channel is expressed in human fetal heart and that positive IgG from mothers of children with CHB inhibits 1D ICa-L. Positive IgG from the same mothers used to demonstrate electrophysiological inhibition of 1D ICa-L also recognized the 1D subunit of the L-type Ca channel by Western blot. Together, the data in this study establish that the 1D Ca channel is a novel target for positive IgG, and consequently, 1D ICa-L inhibited by positive IgG may account in part for the autoimmune-associated sinus bradycardia seen in CHB.
The causal relationship of positive IgG to autoimmune-associated sinus bradycardia in CHB has been documented in animal models of CHB4,5,11 and in clinical settings7,8; however, the underlying molecular mechanism remains unknown. The candidate antigens SSA-Ro and SSB-La are intracellularly located, and there is no convincing evidence that maternal antibodies can cross the sarcolemma of a normal myocyte. Efforts have therefore been directed toward mechanisms that may cause the translocation of these antigens to the cell surface. Several experimental evidences have shown that viral infection,25 UV light treatment,26 and apoptotic death of cells could induce the translocation of Ro/La antigens to the cell surface27; however, it is not yet clear what the resulting cellular events and signaling pathways are that could account for the sinus bradycardia in CHB. Alternatively, another appealing hypothesis, which we termed the "Ca channel hypothesis," proposes that anti-Ro/La autoantibodies cross-react with sarcolemmal components, such as Ca channels, which play an important role in SA node pacemaking activity. This hypothesis is supported by previous observations that Ca channels have been the targets for autoantibodies in other autoimmune diseases.28–32 It has been reported that autoantibodies against the ADP/ATP carrier of the inner mitochondrial membrane from patients with myocarditis and dilated cardiomyopathy can cross-react with sarcolemmal Ca channel proteins and disturb Ca channel function.28 Similar cross-reactivity has been reported in other autoimmune disease, such as Lambert-Eaton myasthenic syndrome and lateral sclerosis.29–32 In addition, we have also previously shown that positive IgG cross-reacts with 1C Ca channel protein and inhibited 1C ICa-L.4,6,9–11 This hypothesis is further supported by the present data showing that positive IgG recognized the 1D Ca channel protein and functionally inhibited the expressed 1D ICa-L.
A number of ion currents have been implicated in SA node pacemaker activity. These include If, ICa-T, ICa-L, IK, and INa. We have previously shown that positive IgG specifically inhibited 1C ICa-L and, to a lesser extent, ICa-T but did not affect If, IK, or INa, which indicates specificity for the Ca channel famliy.4,9–11 However, the inhibition of 1C ICa-L by positive IgG cannot account for the reported sinus bradycardia in CHB, because the contribution of 1C ICa-L to diastolic depolarization of SA node is generally considered to be minor owing to its more positive activation threshold compared with the spontaneous pacemaker diastolic depolarization.12 The present data using human fetal heart, together with animal data from other studies, have demonstrated that the previously underappreciated 1D Ca channel appears to play unique and distinct roles in the heart.14–16 First, unlike the universally expressed 1C Ca channel in the heart, the 1D Ca channel is expressed only in adult SA node, atria, and AV node. Mangoni et al16 showed ICa-L density in SA node cells was decreased by 75% in 1D Ca channel knockout mice compared with wild-type mice, which indicates that the contribution of the 1D Ca channel to total ICa-L in the mouse SA node cell is significant. Second, we showed that 1D ICa-L was activated at between –60 and –50 mV in tsA201 cells, which falls within the range of pacemaker diastolic depolarization. The fact that the 1D Ca channel is expressed in SA node cells and activates at a low-voltage threshold range and that 1D Ca channel knockout mice exhibit profound sinus bradycardia14–16 indicates that the 1D Ca channel plays a critical role in maintenance of normal cardiac rhythm. Consequently, inhibition of SA nodal 1D ICa-L by positive IgG is expected to slow the heart rate. Indeed, positive IgG-superfused rat and rabbit hearts and pups from mice injected with positive IgG during pregnancy exhibited profound sinus bradycardia by ECG and optical action potential recordings.4,6,11 Furthermore, we recently showed that superfusion of spontaneously beating single rabbit SA node myocytes with positive IgG resulted in significant sinus bradycardia as measured by an increase in action potential cycle length.11
In the present study, we also unexpectedly observed expression of the 1D Ca channel in the fetal ventricle. This fetal ventricular expression of the 1D Ca channel is clinically relevant, because some deaths reported in children with CHB are related to heart failure.2,33,34 The inhibition by positive IgG of ventricular L-type Ca channels (both 1D and 1C), which are responsible for generating contractile force, will diminish the contraction status of the fetal heart, which depends significantly on sarcolemmal Ca entry. Thus, inhibition of 1D together with 1C ICa-L by positive IgG may account for both the sinus bradycardia and contractile impairment seen in CHB infants.
Despite the extensive research effort in CHB, there is no effective pharmacotherapy for CHB to date. If inhibition of ICa-L is critical in cardiac impulse generation and conduction found in CHB patients, then an increase in ICa-L should rescue or reverse cardiac electrical abnormalities seen in CHB. We demonstrated that application of a Ca channel activator, Bay K8644, reversed the 1D ICa-L inhibited by positive IgG beyond the baseline level. This finding is the first experimental investigation in CHB to correlate basic findings to potential therapeutic development of new strategies in the management of CHB. Although Bay K8644 may not be an ideal Ca channel agonist in patients because of its vascular effects, its ability to rescue IgG inhibition of 1D ICa-L points to the need to develop new therapeutic agents that will specifically target cardiac Ca channels.
In summary, the data demonstrate that the 1D Ca channel is expressed in human fetal heart, and 1D ICa-L is inhibited by positive IgG by direct binding to the channel protein. Given that the 1D Ca channel plays a critical role in SA node pacemaking and given our previous observation that 1C ICa-L and ICa-T were also inhibited by positive IgG,9,10 blockade of 1D, 1C ICa-L, and ICa-T in human fetal heart may contribute to the genesis of sinus bradycardia in CHB.
Acknowledgments
This work has been supported by National Heart, Lung, and Blood Institutes grant No. HL077494, the VA Merit Grant Award and REAP to Dr Boutjdir, National Institutes of Health F32 grant to Dr Qu, the Canadian Institutes of Health Research Award to Dr G. Baroudi, and the National Institute of Arthritis and Musculoskeletal and Skin Diseases-supported National Research Registry for Neonatal Lupus.
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