Autoimmune-associated Congenital Heart Block Research Articles

Cell atlas of the foetal human heart and implications for autoimmune-mediated congenital heart block.

AimsInvestigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for normal functioning. We investigated foetal human heart single-cell transcriptomes from mid-gestational healthy and anti-SSA/Ro associated congenital heart block (CHB) samples.Methods and resultsThree healthy foetal human hearts (19th to 22nd week of gestation) and one foetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single-cell suspensions followed by 10× Genomics- and Illumina-based single-cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse sub-populations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types of the CHB heart compared with the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contribute to extracellular matrix deposition and subsequent fibrosis.ConclusionThese data provide an information-rich resource to further our understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease.

Autoimmune-associated Congenital Heart Block: A New Insight in Fetal Life.

Objective:Congenital heart block (CHB) is a rare but life-threatening disorder. More than half of CHB cases are associated with maternal autoimmune, which are termed as autoimmune-associated CHB. This review summarized the recent research findings in understanding autoimmune-associated CHB, discussed the current diagnostic approaches and management strategies, and summarized the problems and future directions for this disorder.Data Sources:We retrieved the articles published in English from the PubMed database up to January 2017, using the keywords including “Autoimmune-associated”, “Autoimmune-mediated”, and “Congenital heart block”.Study Selection:Articles about autoimmune-associated CHB were obtained and reviewed.Results:Observational studies consistently reported that transplacental maternal antibodies might recognize fetal or neonatal antigens in various tissues and result in immunological damages, but the molecular mechanisms underlying CHB pathogenesis still need illuminated. Multiple factors were involved in the process of atrioventricular block development and progression. While several susceptibility genes had been successfully defined, how these genes and their protein interact and impact each other remains to be explored. With currently available diagnostic tools, fetal ultrasound cardiography, and fetal magnetocardiography, most of CHB could be successfully diagnosed and comprehensively evaluated prenatally. The efficacy of current approaches for preventing the progression and recurrence of CHB and other autoimmune-mediated damages was still controversial.Conclusions:This review highlighted the relationships between autoimmune injuries and CHB and strengthened the importance of perinatal management and therapy for autoimmune-associated CHB.

Prevention and treatment in utero of autoimmune-associated congenital heart block.

Transplacental transfer of maternal anti-Ro and/or anti-La autoantibodies can result in fetal cardiac disease, including congenital heart block and cardiomyopathy, called cardiac neonatal lupus (NL). Thousands of women are faced with the risk of cardiac NL in their offspring, which is associated with significant morbidity and mortality. There are no known therapies to permanently reverse third-degree heart block in NL, although several treatments have shown some effectiveness in incomplete heart block and disease beyond the atrioventricular node. Fluorinated steroids taken during pregnancy have shown benefit in these situations, although adverse effects may be concerning. Published data are discordant on the efficacy of fluorinated steroids in the prevention of mortality in cardiac NL. β-agonists have been used to increase fetal heart rates in utero. The endurance of β-agonist effect and its impact on mortality are in question, but when used in combination with other therapies, they may provide benefit. No controlled experiments regarding the use of plasmapheresis in cardiac NL have been performed, despite its theoretical benefits. Intravenous immunoglobulin was not shown to prevent cardiac NL at a dose of 400 mg/kg, although it has shown effectiveness in the treatment of associated cardiomyopathy both in utero and after birth. Retrospective studies have shown that hydroxychloroquine may prevent the recurrence of cardiac NL in families with a previously affected child, and a prospective open-label trial is currently recruiting patients in order to fully evaluate this relationship.

Calreticulin negatively regulates the surface expression of Cav1.3 L-type calcium channel

BackgroundThe neuroendocrine Cav1.3 L-type Ca channels have been recently found in the Human fetal heart and shown to play a vital role in Ca entry from the sarcolemma into the cell and in Ca homeostasis. Calreticulin, a Ca binding endoplasmic reticulum (ER) resident protein, has been recently shown to translocate to the cell surface where its role and function are just emerging. Here, we demonstrated a novel mechanism of Cav1.3 and calreticulin interaction resulting in downregulation of Cav1.3 channel densities in native Human fetal cardiac cells and Human Embryonic Kidney cell lines (tsA201). Methods and resultsCell surface and cytoplasmic staining of calreticulin was demonstrated first in cultured human fetal cardiomyocytes (HFC), gestational age 18–24weeks, using confocal microscopy thereby establishing that calreticulin is present at the cell surface in HFC. Co-immunoprecipitation from HFC using anti-Cav1.3 Ca channel antibody, and probing with anti-calreticulin antibody revealed a 46kDa band corresponding to calreticulin suggesting that Cav1.3 Ca channel and calreticulin co-assemble in a macromolecular complex. Co-expression of Cav1.3 and calreticulin in tsA201 cells resulted in a decrease in surface expression of Cav1.3 Ca channels. These findings were consistent with the electrophysiological studies showing that co-transfection of Cav1.3 Ca channel and calreticulin resulted in 55% reduction of Cav1.3 Ca current densities recorded from tsA201 cells. ConclusionsThe results show the first evidence that calreticulin: (1) is localized outside the ER on the cell surface of HFC; (2) coimmunoprecipitates with Cav1.3 L-type Ca channel; (3) negatively regulates Cav1.3 surface expression thus resulting in decreased Cav1.3 Ca current densities. The data demonstrate a novel mechanism of modulation of Cav1.3 Ca channel by calreticulin, which may be involved in pathological settings such as autoimmune associated congenital heart block where Cav1.3 Ca channels are downregulated.

Role of the Urokinase Plasminogen Activator Receptor in Mediating Impaired Efferocytosis of Anti-SSA/Ro–Bound Apoptotic Cardiocytes

Rationale : Binding of maternal anti-Ro/La antibodies to cognate antigen expressed on apoptotic cardiocytes decreases clearance by healthy cardiocytes, which may contribute to the development of autoimmune associated congenital heart block and fatal cardiomyopathy. Objective : Given recent evidence implicating the urokinase plasminogen activator receptor (uPAR) as a “don't eat me” signal during efferocytosis, experiments addressed whether surface bound anti-Ro antibodies inhibit apoptotic cell removal via an effect on the expression/function of the urokinase-type plasminogen activator protease uPA/uPAR system. Methods and Results : As assessed by flow cytometry and confocal microscopy, uPAR colocalizes and interacts with Ro60 on the surface of apoptotic human fetal cardiocytes. Blocking of uPAR enhances phagocytosis of apoptotic cardiocytes by healthy cardiocytes and reverses the anti-Ro60–dependent impaired clearance of apoptotic cardiocytes. Binding of anti-Ro60 antibodies to apoptotic cardiocytes results in increased uPAR expression, as well as enhanced uPA activity. The binding of anti-Ro60 did not alter other surface molecules involved in cell recognition (calreticulin, CD31, or CD47). Conclusions : These data suggest that increased uPAR expression and uPA activity induced by anti-Ro60 binding to the apoptotic fetal cardiocyte provide a molecular basis by which these antibodies inhibit efferocytosis and ultimately lead to scar of the fetal conduction system and working myocardium.

Passively acquired anti‐SSA/Ro antibodies are required for congenital heart block following ovodonation but maternal genes are not

Anti-SSA/Ro antibodies are necessary but not sufficient to provoke autoimmune-associated congenital heart block (CHB). Genetic factors are likely contributory. Accordingly, HLA-related candidates and single-nucleotide polymorphisms in the promoter region of tumor necrosis factor α and codon 10 in transforming growth factor β1 (TGFβ1) were evaluated in a unique family: the surrogate mother (anti-SSA/Ro positive), the biologic father, and the CHB-affected child (product of ovodonation). There was an HLA mismatch between the affected child and the surrogate mother. However, both the biologic and the surrogate mothers shared DQ2 and the profibrosing leucine polymorphism at codon 10 of TGFβ. In conclusion, we observed that CHB can develop in a genetically unrelated child exposed in utero to anti-SSA/Ro antibodies. Testing for anti-SSA/Ro antibodies might be considered in women undergoing artificial fertilization. It is possible that there is no direct association of maternal genes beyond a contributory role in generating the autoantibody.

Ro60-Associated Single-Stranded RNA Links Inflammation with Fetal Cardiac Fibrosis via Ligation of TLRs: A Novel Pathway to Autoimmune-Associated Heart Block

Activation of TLR by ssRNA after FcgammaR-mediated phagocytosis of immune complexes (IC) may be relevant in autoimmune-associated congenital heart block (CHB) where the obligate factor is a maternal anti-SSA/Ro Ab and the fetal factors, protein/RNA on an apoptotic cardiocyte and infiltrating macrophages. This study addressed the hypothesis that Ro60-associated ssRNAs link macrophage activation to fibrosis via TLR engagement. Both macrophage transfection with noncoding ssRNA that bind Ro60 and an IC generated by incubation of Ro60-ssRNA with an IgG fraction from a CHB mother or affinity purified anti-Ro60 significantly increased TNF-alpha secretion, an effect not observed using control RNAs or normal IgG. Dependence on TLR was supported by the significant inhibition of TNF-alpha release by IRS661 and chloroquine. The requirement for FcgammaRIIIa-mediated delivery was provided by inhibition with an anti-CD16a Ab. Fibrosis markers were noticeably increased in fetal cardiac fibroblasts after incubation with supernatants generated from macrophages transfected with ssRNA or incubated with the IC. Supernatants generated from macrophages with ssRNA in the presence of IRS661 or chloroquine did not cause fibrosis. In a CHB heart, but not a healthy heart, TLR7 immunostaining was localized to a region near the atrioventricular groove at a site enriched in mononuclear cells and fibrosis. These data support a novel injury model in CHB, whereby endogenous ligand, Ro60-associated ssRNA, forges a nexus between TLR ligation and fibrosis instigated by binding of anti-Ro Abs to the target protein likely accessible via apoptosis.

Introduction: The first Merinoff Symposium, ‘Systemic Lupus: Bringing Science to the Patient’

Introduction: The first Merinoff Symposium, ‘Systemic Lupus: Bringing Science to the Patient’

Prospective Evaluation of Fetuses With Autoimmune-Associated Congenital Heart Block Followed in the PR Interval and Dexamethasone Evaluation (PRIDE) Study

We evaluated the efficacy of dexamethasone (DEX) in anti-SSA/Ro-exposed fetuses newly diagnosed with congenital heart block. Previous use of DEX has been anecdotal with varying reports of therapeutic benefit. This was a multicenter, open-label, nonrandomized study involving 30 pregnancies treated with DEX (22 with third-degree block, 6 with second-degree block, 2 with first-degree block) and 10 untreated (9 with third-degree block, 1 with first-degree block). Initial median ventricular rates, age at diagnosis, and degree of cardiac dysfunction were similar between groups. Six deaths occurred in the DEX group. There was no reversal of third-degree block with therapy or spontaneously. In fetuses treated with DEX, 1/6 with second-degree block progressed to third-degree block and 3 remained in second-degree block (postnatally 1 paced, 2 progressed to third degree); 2 reverted to normal sinus rhythm (NSR; postnatally 1 progressed to second degree). DEX reversed the 2 fetuses with first-degree block to NSR by 7 days with no regression at discontinuation. Absent DEX, the 1 with first-degree block detected at 38 weeks had NSR at birth (overall stability or improvement in 4 of 8 in the DEX group vs 1 of 1 in the non-DEX group). Median gestational birth age was 37 weeks in the DEX group versus 38 weeks in the non-DEX group (p = 0.019). Prematurity and small size for gestational age were restricted to the DEX group. Pacemaker use and growth parameters at birth and 1 year were similar between groups. In conclusion, these data confirm the irreversibility of third-degree block and progression of second- to third-degree block despite DEX. A potential benefit of DEX in reversing first- or second-degree block was supported in rare cases but should be weighed against potential steroid side effects such as growth restriction.

Autoimmune‐associated Congenital Heart Block: Treatment of the Mother With Immunoadsorption

Autoimmune-associated congenital heart block (CHB) is a rare complication of pregnancy in mothers with Anti-Ro/SSA antibodies (SSA-abs), resulting in fetal myocarditis, atrioventricular block, hydrops fetalis and/or intrauterine fetal death. As these antibodies are supposed to be directly involved in the pathogenesis of CHB, their removal should be associated with an improved clinical course. Extracorporeal immunoadsorption (IA) is the most efficient method to remove IgG-immunoglobulins like SSA-abs selectively. Two women with high titers of those auto-antibodies [mothers serum 615 and 612, respectively (normal range <3.0 IU/mL)] were treated with IA two to three times per gestation week in the outpatient department of the University of Rostock. In both patients, the mean removal of IgG (65 +/- 6%) to a target near 2.0 g/L after IA was successful. The SSA-abs were reduced from mean 328 +/- 138 and 247 +/- 105 pre IA to 88 +/- 124 and 98 +/- 42 post IA, respectively. One child received a pacemaker due to the persisting atrioventricular block grade III after birth. The second was unaffected. The removal of highly elevated SSA-antibodies by immunoadsorption is a possible treatment option in pregnant woman with high titers of those antibodies and/or a positive history of clinical complications. Further clinical studies are necessary.

Autoimmune-associated congenital heart block: dissecting the cascade from immunologic insult to relentless fibrosis.

Few diseases exemplify the integration of research from bench to bedside as well as neonatal lupus, often described as a model of passively acquired autoimmunity. The signature histologic lesion of autoimmune congenital heart block (CHB) is fibrosis of the conducting tissue and, in some cases, the surrounding myocardium. Although anti-SSA/Ro-SSB/La antibodies are detected in > 85% of mothers whose fetuses are identified with conduction abnormalities in a structurally normal heart, the risk for a woman with the candidate antibodies to have a child with CHB is 2%. The mechanism by which maternal anti-SSA/Ro-SSB/La antibodies initiate and finally eventuate in atrioventricular nodal scarring is not yet defined, but it is clear that the antibodies alone are insufficient to cause disease and fetal factors are likely contributory. Previous in vitro and in vivo studies suggest that the pathologic cascade is initiated via apoptosis, resulting in translocation of SSA/Ro-SSB/La antigens to the cell surface where they are bound by maternal autoantibodies. Subsequently, the Fc portion of the bound immunoglobulin engages Fcgamma receptors on tissue macrophages, resulting in release of TGF-beta at a threshold favoring a profibrotic milieu and irreversible scarring. This cascade also involves tissue-specific activation of TGF-beta, which promotes the modulation of fibroblasts into myofibroblasts, a scarring phenotype. Recent findings point to genetic polymorphisms that promote high production of TGF-beta as possible fetal risk factors for CHB. Further elucidation of maternal and fetal contributory factors should provide insight into the pathogenesis of CHB and the rarity of irreversible injury.

From antibody insult to fibrosis in neonatal lupus - the heart of the matter

Few diseases exemplify the integration of research from bench to bedside as well as neonatal lupus, often referred to as a model of passively acquired autoimmunity. In essence, this disease encompasses two patients, both the mother and her child. The signature histologic lesion of autoimmune-associated congenital heart block is fibrosis of the conducting tissue, and in some cases the surrounding myocardium. It is astounding how rapid and, in most cases, irreversible is the fibrotic response to injury. The mechanism by which maternal anti-SSA/Ro–SSB/La antibodies initiate and perpetuate inflammation, and eventuate in scarring of the atrioventricular node, is not yet defined. In vitro and in vivo studies suggest that one pathologic cascade leading to scarring may be initiated via apoptosis, resulting in translocation of SSA/Ro–SSB/La antigens and subsequent surface binding by maternal autoantibodies. These opsonized cardiocytes are phagocytosed by macrophages, which secrete factors that transdifferentiate fibroblasts into myofibroblasts, a scarring phenotype. Dissecting the individual components in this fibrotic pathway should provide insights into the rarity of irreversible injury and should form the basis of rational approaches to prevention and treatment.

Unitary current analysis of L-type Ca2+ channels in human fetal ventricular myocytes.

L-type calcium channels were studied in cell-attached patches from ventricular cell membranes of human fetal heart. Experiments were performed in the presence of 70 mM Ba2+ as the charge carrier at 22 degrees C to 24 degrees C. Unitary current sweeps were evoked by 300-msec depolarizing pulses to 0 mV from a holding potential of -50 mV at 0.5 Hz. Recorded currents were blocked by nisoldipine (1 microM) and stimulated by (-)Bay K 8644 (1 microM). During control, channel activity was seen in 13.9%+/-4.2% of the total 200 sweeps. Ensemble average current amplitude was 0.03+/-0.01 pA (n = 6) and average conductance was 20.4+/-0.2 pS (n = 5). Analysis of single channel kinetics showed open time and closed time histograms were best fit by one and two exponentials, respectively. Mean open time was tau(o) = 0.99+/-0.05 msec (n = 6). Mean closed time fast (tau(cf)) and slow (tau(cs)) component values were tau(cf) = 0.85+/-0.09 msec and tau(cs) = 8.0+/-0.94 msec (n = 6), respectively. With intrapipette (-)Bay K 8644 (1 microM), mean open time was best fit by two exponentials, tau(of) = 0.9+/-0.2 msec (n = 10) and tau(os) = 13.4+/-2.6 msec (n = 10); mean close time values were tau(cf) = 0.6+/-0.1 msec (n = 10) and tau(cs) = 9.8+/-1.9 msec (n = 10), respectively. With (-)Bay K 8644, channel activity was 66.5%+/-7.4%, the ensemble average current was 0.52+/-0.04 pA (n = 10) and the conductance 20.7+/-0.5 pS (n = 5). (1) the data establishes the characteristics of L-type Ca channels of human fetal hearts and their modulation by dihydropyridines; (2) the open time kinetics differ from those of avian embryonic and rat fetal hearts; and (3) the findings provide new and relevant information for understanding the physiologic behavior of unitary Ca2+ channels in the developing human heart and the baseline comparison for diseases that implicate Ca2+ channels in their etiology, such as autoimmune-associated congenital heart block.

Autoimmune-Associated Congenital Heart Block: Demographics, Mortality, Morbidity and Recurrence Rates Obtained From a National Neonatal Lupus Registry

Objectives. The present study describes the demographics, mortality, morbidity and recurrence rates of autoimmune-associated congenital heart block (CHB) using information from the Research Registry for Neonatal Lupus.Background. Isolated CHB detected at or before birth is strongly associated with maternal autoantibodies to 48-kD SSB/La, 52-kD SSA/Ro and 60-kD SSA/Ro ribonucleoproteins and is a permanent manifestation of the neonatal lupus syndromes (NLS). Available data are limited by the rarity of the disease.Results. The cohort includes 105 mothers whose sera contain anti-SSA/Ro or anti-SSB/La antibodies, or both, and their 113 infants diagnosed with CHB between 1970 and 1997 (56 boys, 57 girls). Of 87 pregnancies in which sufficient medical records were available, bradyarrhythmia confirmed to be CHB was initially detected before 30 weeks of gestation in 71 (82%) (median time 23 weeks). There were no cases in which major congenital cardiac anatomic defects were considered causal for the development of CHB; in 14 there were minor abnormalities. Twenty-two (19%) of the 113 children died, 16 (73%) within 3 months after birth. Cumulative probability of 3-year survival was 79%. Sixty-seven (63%) of 107 live-born children required pacemakers: 35 within 9 days of life, 15 within 1 year, and 17 after 1 year. Forty-nine of the mothers had subsequent pregnancies: 8 (16%) had another infant with CHB and 3 (6%) had a child with an isolated rash consistent with NLS.Conclusions. Data from this large series substantiate that autoantibody-associated CHB is not coincident with major structural abnormalities, is most often identified in the late second trimester, carries a substantial mortality in the neonatal period and frequently requires pacing. The recurrence rate of CHB is at least two- to three-fold higher than the rate for a mother with anti-SSA/Ro-SSB/La antibodies who never had an affected child, supporting close echocardiographic monitoring in all subsequent pregnancies, with heightened surveillance between 18 and 24 weeks of gestation.