Contribution of S100A4-expressing fibroblasts to anti-SSA/Ro-associated atrioventricular nodal calcification and soluble S100A4 as a biomarker of clinical severity.

Background: Fibrosis and dystrophic calcification disrupting conduction tissue architecture are histopathological lesions characterizing cardiac manifestations of neonatal lupus (cardiac-NL) associated with maternal anti-SSA/Ro antibodies. Objectives: Increased appreciation of heterogeneity in fibroblasts encourages re-examination of existing models with the consideration of multiple fibroblast subtypes (and their unique functional differences) in mind. This study addressed fibroblast heterogeneity by examining expression of α-Smooth Muscle Actin (myofibroblasts) and of S100 Calcium-Binding Protein A4 (S100A4). Methods: Using a previously established model of rheumatic scarring/fibrosis in vitro, supported by the evaluation of cord blood from cardiac-NL neonates and their healthy (anti-SSA/Ro-exposed) counterparts, and autopsy tissue from fetuses dying with cardiac-NL, the current study was initiated to more clearly define and distinguish the S100A4-positive fibroblast in the fetal cardiac environment. Results: S100A4 immunostaining was observed in 4 cardiac-NL hearts with positional identity in the conduction system at regions of dystrophic calcification but not fibrotic zones, the latter containing only myofibroblasts. In vitro, fibroblasts cultured with supernatants of macrophages transfected with hY3 (noncoding ssRNA) differentiated into myofibroblasts or S100A4+ fibroblasts. Myofibroblasts expressed collagen while S100A4+ fibroblasts expressed pro-angiogenic cytokines and proteases that degrade collagen. Cord blood levels of S100A4 in anti-SSA/Ro-exposed neonates tracked disease severity and, in discordant twins, distinguished affected from unaffected. Conclusions: These findings position the S100A4+ fibroblast alongside the canonical myofibroblast in the pathogenesis of cardiac-NL. Neonatal S100A4 levels support a novel biomarker of poor prognosis.

Segment Length in Cine Strain Analysis Predicts Cardiac Resynchronization Therapy Outcome Beyond Current Guidelines.

BACKGROUND: Patients with a class I recommendation for cardiac resynchronization therapy (CRT) are likely to benefit, but the effect of CRT in class II patients is more heterogeneous and additional selection parameters are needed in this group. The recently validated segment length in cine strain analysis of the septum (SLICE-ESSsep) measurement on cardiac magnetic resonance cine imaging predicts left ventricular functional recovery after CRT but its prognostic value is unknown. This study sought to evaluate the prognostic value of SLICE-ESSsep for clinical outcome after CRT. METHODS: Two hundred eighteen patients with a left bundle branch block or intraventricular conduction delay and a class I or class II indication for CRT who underwent preimplantation cardiovascular magnetic resonance examination were enrolled. SLICE-ESSsep was manually measured on standard cardiovascular magnetic resonance cine imaging. The primary combined end point was all-cause mortality, left ventricular assist device, or heart transplantation. Secondary end points were (1) appropriate implantable cardioverter defibrillator therapy and (2) heart failure hospitalization. RESULTS: Two-thirds (65%) of patients had a positive SLICE-ESSsep ≥0.9% (ie, systolic septal stretching). During a median follow-up of 3.8 years, 66 (30%) patients reached the primary end point. Patients with positive SLICE-ESSsep were at lower risk to reach the primary end point (hazard ratio 0.36; P<0.001) and heart failure hospitalization (hazard ratio 0.41; P=0.019), but not for implantable cardioverter defibrillator therapy (hazard ratio, 0.66; P=0.272). Clinical outcome of class II patients with a positive ESSsep was similar to those of class I patients (hazard ratio, 1.38 [95% CI, 0.66-2.88]; P=0.396). CONCLUSIONS: Strain assessment of the septum (SLICE-ESSsep) provides a prognostic measure for clinical outcome after CRT. Detection of a positive SLICE-ESSsep in patients with a class II indication predicts improved CRT outcome similar to those with a class I indication whereas SLICE-ESSsep negative patients have poor prognosis after CRT implantation.

Relapsing polychondritis associated with heart block.

OBJECTIVE: The present article aims at describing a rare case of an RP patient who evolved with heart block and was successfully treated with corticoid pulse therapy, without the need for pacemaker insertion. PATIENTS AND METHODS: A systematic research on relapsing polychondritis (RP) and heart block (HB) published in PubMed/MEDLINE, Web of Sciences, LILACS, and Scielo from 1966 to August 2020 was performed. RESULTS: It was found 10 studies on RP associated with HB, and we added a case. Most were male (7/10) with ages 30 to 66 years old. RP disease duration was 1 week-6 years. In most cases (7/10), the RP was active when the HB occurred. A complete HB was observed in 4/7, followed by type II degree block in 3/7, and one patient had a sinus node dysfunction. Most patients received glucocorticoids. A pacemaker was inserted in 4/9 cases. Good outcome was observed in 3/9 patients and mortality in 2/10. CONCLUSIONS: We report the first case of an RP patient who had a heart block and was successfully treated with methylprednisolone pulse therapy. The authors suggest that in these RP cases, an attempt with a glucocorticoid pulse therapy may be offered to treat the heart block and prevent the insertion of a pacemaker.

A missense mutation of ErbB2 produces a novel mouse model of stillbirth associated with a cardiac abnormality but lacking abnormalities of placental structure.

BACKGROUND: In humans, stillbirth describes the death of a fetus before birth after 28 weeks gestation, and accounts for approximately 2.6 million deaths worldwide annually. In high-income countries, up to half of stillbirths have an unknown cause and are described as "unexplained stillbirths"; this lack of understanding impairs efforts to prevent stillbirth. There are also few animal models of stillbirth, but those that have been described usually have significant placental abnormalities. This study describes a novel mutant murine model of fetal death with atrial conduction block due to an ErbB2 missense mutation which is not associated with abnormal placental morphology. METHODS: Phenotypic characterisation and histological analysis of the mutant mouse model was conducted. The mRNA distribution of the early cardiomyocyte marker Nkx2-5 was assessed via in situ hybridisation. Cardiac structure was quantified and cellular morphology evaluated by electron microscopy. Immunostaining was employed to quantify placental structure and cell characteristics on matched heterozygous and homozygous mutant placental samples. RESULTS: There were no structural abnormalities observed in hearts of mutant embryos. Comparable Nkx2-5 expression was observed in hearts of mutants and controls, suggesting normal cardiac specification. Additionally, there was no significant difference in the weight, placenta dimensions, giant cell characteristics, labyrinth tissue composition, levels of apoptosis, proliferation or vascularisation between placentas of homozygous mutant mice and controls. CONCLUSION: Embryonic lethality in the ErbB2 homozygous mutant mouse cannot be attributed to placental pathology. As such, we conclude the ErbB2M802R mutant is a model of stillbirth with a non-placental cause of death. The mechanism of the atrial block resulting from ErbB2 mutation and its role in embryonic death is still unclear. Studying this mutant mouse model could identify candidate genes involved in stillbirth associated with structural or functional cardiac defects.

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

AIMS: Investigating 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 RESULTS: Three 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. CONCLUSION: These 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.

Prenatal diagnosis and management of congenital complete heart block.

Congenital complete heart block (CCHB) is a life-threatening medical condition in the unborn fetus with insufficiently validated prenatal interventions. Maternal administration of medications aimed at decreasing the immune response in the fetus and beta-agonists intended to increase fetal cardiac output have shown only marginal benefits. Anti-inflammatory therapies cannot reverse CCHB, but may decrease myocarditis and improve heart function. Advances in prenatal diagnosis and use of strict surveillance protocols for delivery timing have demonstrated small improvements in morbidity and mortality. Ambulatory surveillance programs and wearable fetal heart rate monitors may afford early identification of evolving fetal heart block allowing for emergent treatment. There is also preliminary data suggesting a roll for prevention of CCHB with hydroxychloroquine, but the efficacy and safety is still being studied. To date, intrauterine fetal pacing has not been successful due to the high-risk invasive placement techniques and potential problems with lead dislodgement. The development of a fully implantable micropacemaker via a minimally invasive approach has the potential to pace fetal patients with CCHB and thus delay delivery and allow fetal hydrops to resolve. The challenge remains to establish accepted prenatal interventions capable of successfully managing CCHB in utero until postnatal pacemaker placement is successfully achieved.

Transient receptor potential melastatin 4 cation channel in pediatric heart block.

OBJECTIVE: Progressive cardiac conduction disease (PCCD) is a common pediatric heart conduction disorder. It is an autosomal inheritance of rare mutations, which leads to familial cases of PCCD. In these cases, the His-Purkinje system's conductive capacity is progressively deranged, involving either right or left bundle branch block. Also, QRS complexes display widening is an important characteristic that culminates in complete AV block, syncope, and sudden death. Mutations in TRPM4 gene that encodes for transient receptor potential melastatin 4 have recently been reported to cause familial cases of PCCD and heart block. TRPM4 conducts a Ca2+-activated non-selective monovalent cationic current leading to a negative plasma membrane potential. TRPM4 channels let Na+ ion influx, causing membrane depolarization, whereas, at positive membrane potentials, TRPM4 channels repolarize the membrane by facilitating K+ ion efflux from the cell. TRPM4 protein contains many regulatory motifs that confer voltage dependence, ATP/ADP sensitivity, and Ca2+ responsiveness. Mutational studies revealed the significance of the two-calmodulin binding sites at the N-terminus of for Ca2+ dependent activation of this channel. Mutations that reduce deSUMOylation increase the steady-state levels of active TRPM4 channels on the membrane without alteration of its sensitivity to Ca2+ or ATP or its voltage dependence of activation. Increased TRPM4 function interferes with cardiac conduction and eventually contributes to heart block. Both gain and loss of function mutations of TRPM4 are implicated in the cardiac block. Currently, the major therapeutic management of cardiac block due to TRPM4 mutations is implantation of a pacemaker to reinstate normal current propagation through AV node.

Cardiac fibroblast transcriptome analyses support a role for interferogenic, profibrotic, and inflammatory genes in anti-SSA/Ro-associated congenital heart block.

The signature lesion of SSA/Ro autoantibody-associated congenital heart block (CHB) is fibrosis and a macrophage infiltrate, supporting an experimental focus on cues influencing the fibroblast component. The transcriptomes of human fetal cardiac fibroblasts were analyzed using two complementary approaches. Cardiac injury conditions were simulated in vitro by incubating human fetal cardiac fibroblasts with supernatants from macrophages transfected with the SSA/Ro-associated noncoding Y ssRNA. The top 10 upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response [e.g., IFN-induced protein 44-like (IFI44L), of MX dynamin-like GTPase (MX)1, MX2, and radical S-adenosyl methionine domain containing 2 (Rsad2)]. Within the fibrotic pathway, transcript levels of endothelin-1 (EDN1), phosphodiesterase (PDE)4D, chemokine (C-X-C motif) ligand (CXCL)2, and CXCL3 were upregulated, while others, including adenomedullin, RAP guanine nucleotide exchange factor 3 (RAPGEF3), tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, and dual specificity phosphatase 1, were downregulated. Agnostic Database for Annotation, Visualization and Integrated Discovery analysis revealed a significant increase in inflammatory genes, including complement C3A receptor 1 (C3AR1), F2R-like thrombin/trypsin receptor 3, and neutrophil cytosolic factor 2. In addition, stimulated fibroblasts expressed high levels of phospho-MADS box transcription enhancer factor 2 [a substrate of MAPK5 (ERK5)], which was inhibited by BIX-02189, a specific inhibitor of ERK5. Translation to human disease leveraged an unprecedented opportunity to interrogate the transcriptome of fibroblasts freshly isolated and cell sorted without stimulation from a fetal heart with CHB and a matched healthy heart. Consistent with the in vitro data, five IFN response genes were among the top 10 most highly expressed transcripts in CHB fibroblasts. In addition, the expression of matrix-related genes reflected fibrosis. These data support the novel finding that cardiac injury in CHB may occur secondary to abnormal remodeling due in part to upregulation of type 1 IFN response genes.NEW & NOTEWORTHY Congenital heart block is a rare disease of the fetal heart associated with maternal anti-Ro autoantibodies which can result in death and for survivors, lifelong pacing. This study provides in vivo and in vitro transcriptome-support that injury may be mediated by an effect of Type I Interferon on fetal fibroblasts.

No histologic evidence of foetal cardiotoxicity following exposure to maternal hydroxychloroquine.

It is currently recommended that hydroxychloroquine (HCQ) be maintained during pregnancy in patients with systemic lupus erythematosus. Recent data suggest that this Toll-like receptor inhibitor may also reduce the recurrence rate of anti-SSA/Ro associated congenital heart block (CHB). This case report describes a unique situation in which a CHB-afflicted, HCQ-exposed pregnancy was electively terminated. The heart did not reveal any characteristic features of cardiotoxicity, providing further evidence supporting the safety of foetal exposure to HCQ.

Myxedema Coma Secondary to Central Hypothyroidism: A Rare but Real Cause of Altered Mental Status in Pediatrics.

BACKGROUND: Myxedema coma (MC), a medical emergency defined as severe hypothyroidism leading to altered mental status, is more common in older women with hypothyroidism. METHODS/RESULTS: A 7-year-old Caucasian male with chromosome 1q deletion presented with altered mental status preceded by milestone regression. His presenting labs results were: thyroid-stimulating hormone (TSH) 0.501 µIU/ml and free thyroxine (T4) <0.5 ng/dl. His morning cortisol level was 8.1 µg/dl with repeat testing, while TSH was 1.119 µIU/ml and free T4 was 0.5 ng/dl. Low-dose cosyntropin test showed baseline and peak cortisol levels of 1.9 and 16 µg/dl, respectively. Aside from altered mental status, heart block was present in addition to hypothermia and hypercarbia. Diffuse cerebral cortical and corpus callosum atrophy were seen on MRI. An intravenous (i.v.) stress dose of hydrocortisone was administered for 24 h prior to an i.v. loading dose of levothyroxine. His activity level subsequently returned to baseline within 48 h after treatment had been initiated. CONCLUSION: Though MC is rare, occurring mainly with noncompliance in primary hypothyroidism, it may occur at the diagnosis of secondary hypothyroidism. Based on features like hypothermia, hypoventilation, and cardiovascular instability occurring in the setting of central hypothyroidism, it should be suspected and managed urgently in order to avert the associated high mortality resulting from treatment delays.

The high incidence of anti-Ro/SSA and anti-p200 antibodies in female patients with connective tissue diseases confirms the importance of screening for congenital heart block-associated autoantibodies during pregnancy.

It is known that anti-Ro/SSA positivity leads to higher risk of miscarriage and fetal cardiac malformations. Particularly, anti-p200 antibodies against a finer specificity of the Ro/SSA antigen, have been associated with congenital heart block. The aim of the study was to assess the frequency of anti-p200 among female patients with different connective tissue diseases and, among these, the relevance of anti-p200 values in patients with cutaneous diseases compared to systemic diseases. Anti-p200 were investigated in 110 anti-Ro/SSA positive female sera, sent to our laboratory between 2008 and 2014 with suspect of connective disease, by using ELISA testing. Positivity was found in 40.9 % samples, 34 of them showed a strong positivity (values ≥ 1.0, cut off = 0.7). Patients with systemic diseases were anti-p200 positive in the 45.9 % of cases while patients with cutaneous diseases were positive in the 24.0 % of cases. Positivity for anti-p200 antibodies was revealed in 24.0 % of patients with discoid lupus erythematosus; 100 % of patients with dermatomyositis; 40.0 % of patients with mixed connective tissue disease; 25.0 % of patients with rheumatoid arthritis; 100 % of patients with Sjögren's syndrome; 33.3 % of patients with subacute cutaneous lupus erythematosus; 42.9 % of patients with systemic lupus erythematosus; 80.0 % of patients with systemic sclerosis. No significant difference in anti-p200 prevalence was found between systemic and cutaneous involvement, nevertheless, considering only positive sera, the antibody titer was higher in systemic diseases rather than in cutaneous diseases (2.6 ± 1.7 and 1.7 ± 1.9; p = 0.041). The authors think screenings for anti-Ro/SSA and anti-p200 antibodies should be included in the laboratory checklist for pregnancy.

Targeted laser ablation of the zebrafish larval heart induces models of heart block, valvular regurgitation, and outflow tract obstruction.

Mammalian models of cardiac disease have provided unique and important insights into human disease but have become increasingly challenging to produce. The zebrafish could provide inexpensive high-throughput models of cardiac injury and repair. We used a highly targeted laser, synchronized to fire at specific phases of the cardiac cycle, to induce regional injury to the ventricle, atrioventricular (AV) cushion, and bulbus arteriosus (BA). We assessed the impact of laser injury on hearts of zebrafish early larvae at 72 h postfertilization, to different regions, recording the effects on ejection fraction (EF), heart rate (HR), and blood flow at 2 and 24 h postinjury (hpi). Laser injury to the apex, midzone, and outflow regions of the ventricle resulted in reductions of the ventricle EF at 2 hpi with full recovery of function by 24 hpi. Laser injury to the ventricle, close to the AV cushion, was more likely to cause bradycardia and atrial-ventricular dysfunction, suggestive of an electrical conduction block. At 2 hpi, direct injury to the AV cushion resulted in marked regurgitation of blood from the ventricle to the atrium. Laser injury to the BA caused temporary outflow tract obstruction with cessation of ventricle contraction and circulation. Despite such damage, 80% of embryos showed complete recovery of the HR and function within 24 h of laser injury. Precision laser injury to key structures in the zebrafish developing heart provides a range of potentially useful models of hemodynamic overload, injury, and repair.

Arrhythmogenic role of the border between two areas of cardiac cell alignment.

The goal of this study is to develop experimental and computational models of the excitation transition between areas of cardiac tissue with different anatomical anisotropy. Alignment of seeded neonatal rat cardiomyocytes was achieved with the aid of guiding polymer (PMGI) nanofibers, and two areas with orthogonal alignment were placed into a contact. It was found that the excitation wave crossing border between the areas with different alignment direction experiences substantial perturbation, up to the complete conduction block. In addition to the experimental study, this effect was analyzed computationally using generic FitzHugh-Nagumo reaction-diffusion model. It was shown that the non-monotonous changes of the excitation wave velocity on this boundary may be explained by the source/sink mismatch. Thus, the border may play pro-arrhythmogenic role.

Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block.

Somatic reprogramming by reexpression of the embryonic transcription factor T-box 18 (TBX18) converts cardiomyocytes into pacemaker cells. We hypothesized that this could be a viable therapeutic avenue for pacemaker-dependent patients afflicted with device-related complications, and therefore tested whether adenoviral TBX18 gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. Biological pacemaker activity, originating from the intramyocardial injection site, was evident in TBX18-transduced animals starting at day 2 and persisted for the duration of the study (14 days) with minimal backup electronic pacemaker use. Relative to controls transduced with a reporter gene, TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity. Induced sinoatrial node cells could be identified by their distinctive morphology at the site of injection in TBX18-transduced animals, but not in controls. No local or systemic safety concerns arose. Thus, minimally invasive TBX18 gene transfer creates physiologically relevant pacemaker activity in complete heart block, providing evidence for therapeutic somatic reprogramming in a clinically relevant disease model.

Model of bipolar electrogram fractionation and conduction block associated with activation wavefront direction at infarct border zone lateral isthmus boundaries.

BACKGROUND: Improved understanding of the mechanisms underlying infarct border zone electrogram fractionation may be helpful to identify arrhythmogenic regions in the postinfarction heart. We describe the generation of electrogram fractionation from changes in activation wavefront curvature in experimental canine infarction. METHODS AND RESULTS: A model was developed to estimate the extracellular signal shape that would be generated by wavefront propagation parallel to versus perpendicular to the lateral boundary (LB) of the reentrant ventricular tachycardia (VT) isthmus or diastolic pathway. LBs are defined as locations where functional block forms during VT, and elsewhere they have been shown to coincide with sharp thin-to-thick transitions in infarct border zone thickness. To test the model, bipolar electrograms were acquired from infarct border zone sites in 10 canine heart experiments 3 to 5 days after experimental infarction. Activation maps were constructed during sinus rhythm and during VT. The characteristics of model-generated versus actual electrograms were compared. Quantitatively expressed VT fractionation (7.6±1.2 deflections; 16.3±8.9-ms intervals) was similar to model-generated values with wavefront propagation perpendicular to the LB (9.4±2.4 deflections; 14.4±5.2-ms intervals). Fractionation during sinus rhythm (5.9±1.8 deflections; 9.2±4.4-ms intervals) was similar to model-generated fractionation with wavefront propagation parallel to the LB (6.7±3.1 deflections; 7.1±3.8-ms intervals). VT and sinus rhythm fractionation sites were adjacent to LBs ≈80% of the time. CONCLUSIONS: The results suggest that in a subacute canine infarct model, the LBs are a source of activation wavefront discontinuity and electrogram fractionation, with the degree of fractionation being dependent on activation rate and wavefront orientation with respect to the LB.

Conduction block in novel cardiomyocyte electrical conduction line by photosensitization reaction.

We developed a novel cardiomyocyte electrical conduction line. We studied electrical conduction block by extra-cellular photosensitization reaction with this conduction line to study electrical blockade by the photosensitization reaction in vitro.

Congenital complete heart block: single tertiary centre experience.

BACKGROUND: Congenital complete heart block (CCHB) is an uncommon although important and potentially serious condition contributing to neonatal morbidity and mortality. AIMS: To study the characteristics and outcomes of infants born with CCHB at a single tertiary centre. METHODS: A retrospective review of all infants with CCHB over the last 20 years was carried out to determine the outcomes, and the indications and timing of pacemaker insertion. RESULTS: Fifteen live born infants (10 male, 5 female) with CCHB were identified. Their mean (and SD) gestation and birth weight were 37 (3.3) weeks and 3100 (448) grams respectively. Maternal systemic lupus erythematosus (SLE) antibodies were present in eight (53%) pregnancies and two infants had congenitally corrected transposition of the great arteries (cCTGA). The median heart rate/minute at birth was 60 (range 40-80). Thirteen (87%) patients to date required a pacemaker. The median age of insertion of a pacemaker device was six months (range 2 days-16 years). All patients were paced epicardially - six initially with a single chamber and five with a dual chamber pacemaker. At the time of generator change, dual chamber pacemakers were used. The median life of an implanted pacemaker was six years (3-10 years). Except for a patient with cCTGA who has undergone a double switch procedure, all the patients had good systemic ventricular function. There was one death in the group unrelated to CCHB. CONCLUSIONS: CCHB is a uncommon but potentially serious condition in infancy. While a significant number of infants need a pacemaker, the overall outcome of infants with CCHB in our experience is good.