Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block

Abstract

Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200-239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells.

Figure 1.

Maternal anti-p200 antibody levels correlate with fetal AV time. (A) Schematic representation of Ro52, indicating functional domains with two zinc fingers, a RING finger and a B-box (gray boxes), a leucine zipper (black box; amino acids 211–232) within a coiled coil domain (white box), and a B30.2 domain (white box), as well as peptides p136–p200. Echocardiographic representation of fetal hearts illustrating the view for (B) mitral valve/aortic outflow (MAV-time) measurement and (C) superior vena cava/aorta (SAV-time) measurement with a Doppler registration from each projection. Circles mark the area from where the registrations are sampled. LV, left ventricle; RA, right atrium; RV, right ventricle; AO, ascending aorta; SVC, superior vena cava. (D–G) Maternal anti-p200 levels and the ratio of anti-p200/anti-p176 antibodies plotted against the highest AV time intervals measured in each fetus. Vertical lines represent the upper 95% reference limits at 24 wk of gestation. Horizontal lines are drawn to exemplify the potential of antibody analyses to identify fetuses with prolonged AV time intervals. Square and diamond dots denote the fetuses with AV block II and III, respectively. Clinical data of these patients have been previously presented (reference 3).

Figure 2.

Immunization of rats with p200 peptide leads to AV block in the offspring. Rats immunized with p200 developed an antibody response against p200 (A) and full-length Ro52 (B), whereas rats immunized with control peptide did not. (C) No epitope spreading to adjacent Ro52 peptides was observed in p200-immunized animals, although to some extent antibodies did bind to the highly overlapping p197 peptide. (D) The induced anti-p200 antibodies were mainly of IgG2a, IgG2b, and IgG2c subclasses in mothers as well as pups. (E) Immunized rats were mated and antibody titers and AV time were monitored in the pups by ECG performed on all pups within 12 h after birth. HR, heart rate. ECGs were sampled for 5 s four times per minute and the PR interval was calculated from an averaged complex (F). Arrows denote start and end points for measurements on the averaged ECG. (G) ECG from a total of 52 pups from p200-immunized rats was analyzed and 10 (19%) had a first-degree AV block (filled symbols). The horizontal line marks the mean PR interval of the 26 pups from control immunized animals (mean ± SD; 58.9 ± 3.2).

Figure 3.

An epitope within the predicted leucine zipper structure is recognized by similar antibodies from congenital heart block children, rat pups, and selected human anti-p200 monoclonals. (A) Ribbon structure representation of the predicted p200 secondary structure fold, including the leucine zipper motif. Leucines are represented in gray. (B) Schematic drawing of the region around the predicted leucine zipper and mutated peptides used in epitope mapping of anti-p200 antibodies. The amino acid sequence of p197 and p200 (leucines in gray and underlined) are shown, as well as indications of amino acid substitutions in the mutated peptides (pZIP in blue and in pOUT in red). The antigenic contribution from the COOH-terminal amino acid was investigated by an alanine scan of amino acids 233–239. S3A8 (C) and M4H1 (D) binding to the peptides was investigated. Sera from children with congenital heart block (E) and pups from p200-immunized animals with first-degree AV block (F) contained mainly S3A8 idiotype-like p200-specific antibodies. The level of p200 reactivity in each monoclonal or individual was set as 1 and the values for each monoclonal/individual normalized against their respective p200 reactivity is shown.

Figure 4.

S3A8 anti-p200 monoclonals and sera from p200- immunized rats, but not M4H1, p200 monoclonals, or vehicle bind cardiomyocytes and induce dysregulation of Ca^{2 +} homeostasis. (A) Primary cardiomyocytes prepared from neonatal rat hearts were stained with S3A8, M4H1, or PBS. (B) Primary cardiomyocyte cultures loaded with fluo-4 before the addition of monoclonal antibodies or serum. (C) Single cell tracing of [Ca²⁺]_i (calcium dye fluorescence intensity in arbitrary units) after the addition of respective antibody or vehicle. (D) The mean relative frequency of Ca²⁺ transients. (E) Change in baseline intracellular Ca²⁺ levels over time. ΔF% was calculated by dividing baseline fluorescence between transients by the original (time 0) baseline level. (D and E) A representation of results from 10 independent experiments for S3A8, 6 independent experiments for M4H1, 5 independent experiments for PBS, and 2 experiments with 2 different rat sera (1:10). Each rat serum is depicted individually (D and E). (F) Baseline fluorescence intensity after 20 min of substance application is depicted. A scale of 0–250 arbitrary units was used, where intensity of average pixel fluorescence signal is saturated when color turns blue (>250 arbitrary units).