Loss of Nexilin function leads to a recessive lethal fetal cardiomyopathy characterized by cardiomegaly and endocardial fibroelastosis.

The Nexilin F-Actin Binding Protein (Nexilin) encoded by NEXN is a cardiac Z-disc protein important for cardiac function and development in humans, zebrafish, and mice. Heterozygote variants in the human NEXN gene have been reported to cause dilated and hypertrophic cardiomyopathy. Homozygous variants in NEXN cause a lethal form of human fetal cardiomyopathy, only described in two patients before. In a Swedish, four-generation, non-consanguineous family comprising 42 individuals, one female had three consecutive pregnancies with intrauterine fetal deaths caused by a lethal form of dilated cardiomyopathy. Whole-exome sequencing and variant analysis revealed that the affected fetuses were homozygous for a NEXN variant (NM_144573:c.1302del;p.(Ile435Serfs*3)). Moreover, autopsy and histology staining declared that they presented with cardiomegaly and endocardial fibroelastosis. Immunohistochemistry staining for Nexilin in the affected fetuses revealed reduced antibody staining and loss of striation in the heart, supporting loss of Nexilin function. Clinical examination of seven heterozygote carriers confirmed dilated cardiomyopathy (two individuals), other cardiac findings (three individuals), or no cardiac deviations (two individuals), indicating incomplete penetrance or age-dependent expression of dilated cardiomyopathy. RNA sequencing spanning the variant in cDNA blood of heterozygote individuals revealed nonsense-mediated mRNA decay of the mutated transcripts. In the current study, we present the first natural course of the recessively inherited lethal form of human fetal cardiomyopathy caused by loss of Nexilin function. The affected family had uneventful pregnancies until week 23-24, followed by fetal death at week 24-30, characterized by cardiomegaly and endocardial fibroelastosis.

Recessive ciliopathy mutations in primary endocardial fibroelastosis: a rare neonatal cardiomyopathy in a case of Alstrom syndrome.

Among neonatal cardiomyopathies, primary endocardial fibroelastosis (pEFE) remains a mysterious disease of the endomyocardium that is poorly genetically characterized, affecting 1/5000 live births and accounting for 25% of the entire pediatric dilated cardiomyopathy (DCM) with a devastating course and grave prognosis. To investigate the potential genetic contribution to pEFE, we performed integrative genomic analysis, using whole exome sequencing (WES) and RNA-seq in a female infant with confirmed pathological diagnosis of pEFE. Within regions of homozygosity in the proband genome, WES analysis revealed novel parent-transmitted homozygous mutations affecting three genes with known roles in cilia assembly or function. Among them, a novel homozygous variant [c.1943delA] of uncertain significance in ALMS1 was prioritized for functional genomic and mechanistic analysis. Loss of function mutations of ALMS1 have been implicated in Alstrom syndrome (AS) [OMIM 203800], a rare recessive ciliopathy that has been associated with cardiomyopathy. The variant of interest results in a frameshift introducing a premature stop codon. RNA-seq of the proband's dermal fibroblasts confirmed the impact of the novel ALMS1 variant on RNA-seq reads and revealed dysregulated cellular signaling and function, including the induction of epithelial mesenchymal transition (EMT) and activation of TGFß signaling. ALMS1 loss enhanced cellular migration in patient fibroblasts as well as neonatal cardiac fibroblasts, while ALMS1-depleted cardiomyocytes exhibited enhanced proliferation activity. Herein, we present the unique pathological features of pEFE compared to DCM and utilize integrated genomic analysis to elucidate the molecular impact of a novel mutation in ALMS1 gene in an AS case. Our report provides insights into pEFE etiology and suggests, for the first time to our knowledge, ciliopathy as a potential underlying mechanism for this poorly understood and incurable form of neonatal cardiomyopathy. KEY MESSAGE: Primary endocardial fibroelastosis (pEFE) is a rare form of neonatal cardiomyopathy that occurs in 1/5000 live births with significant consequences but unknown etiology. Integrated genomics analysis (whole exome sequencing and RNA sequencing) elucidates novel genetic contribution to pEFE etiology. In this case, the cardiac manifestation in Alstrom syndrome is pEFE. To our knowledge, this report provides the first evidence linking ciliopathy to pEFE etiology. Infants with pEFE should be examined for syndromic features of Alstrom syndrome. Our findings lead to a better understanding of the molecular mechanisms of pEFE, paving the way to potential diagnostic and therapeutic applications.

A rare cause of sudden unexpected death syndrome (SUDS) in the first year of life: endomyocardial fibroelastosis (EFE) due to two compound heterozygous MYBPC3 mutations.

BACKGROUND: Autopsies regularly aim to clarify the cause of death; however, relatives may directly benefit from autopsy results in the setting of heritable traits ("mortui vivos docent"). CASE PRESENTATION: A case of a sudden unexpected cardiac death of a 5.5-months-old child is presented. Autopsy and thorough postmortem cardiac examinations revealed a massively enlarged heart with endomyocardial fibroelastosis. Postmortem molecular testing (molecular autopsy) revealed an unusual combination of two biparental MYBPC3 gene mutations likely to underlie the cardiac abnormalities. Thus, the molecular autoptic findings also had consequences for the relatives of the deceased child and impact on further family planning. CONCLUSIONS: The presented case highlights the need for clinical autopsies including cardiac examinations and postmortem molecular testing; it also paves the way for further cascade screening of family members for cardiac disease, if a distinct genetic disorder is suspected.

[HYPOPLASTIC LEFT HEART SYNDROME: MORPHOGENESIS OF PATOMORPHOLOGICAL TYPES OF THE LEFT VENTRICLE].

The purpose of the study was to investigate the morphogenesis of the left ventricle in the hypoplastic left heart syndrome (HLHS). There are five types of hypoplastic left ventricles were identified: with a slit-like shape and hypoplasia of LV wall, with a slit-like cavity shape and wall hypertrophy and types with endocardial fibroelastosis (with a cylindrical cavity shape, with lacunar cavities and lacunar-cylindrical cavity of the left ventricle), as a result of differences in the wall structure, cavity shape, presence or absence of endocardial fibroelastosis. The analysis of morphometric data of pathomorphological types of the left ventricle in the HLHS revealed the possible ways of their morphogenesis. Left displacement of interventricular septum in embryogenesis at 4-5 weeks of intrauterine development is associated with the occurrence of atresia of the left atrioventricular orifice and aortic valve and the appearance of a slit-like shape and hypoplasia of LV wall in the HLHS. The displacement of only the conotruncus septum leads to the appearance of a slit-like shape of cavity and hypertrophy of LV wall in the HLHS. The pathomorphological types with endocardial fibroelastosis in the HLHS depends on the stage of embryogenesis of myocardium at which fibroelastosis appears: before the myocardial compaction (up to 4th week of gestation) - the lacunar shape of LV cavity with thin compact layer of myocardium; during the compaction of myocardium (5-6th week of gestation) - the lacunar-cylindrical shape of LV cavity and after compaction (after 7-8th week of fetal development) - a cylindrical shape of LV cavity.

Flow disturbances and the development of endocardial fibroelastosis.

OBJECTIVES: Endothelial-to-mesenchymal transition (EndMT) has been identified as the underlying mechanism of endocardial fibroelastosis (EFE) formation. The purpose of this study was to determine whether hemodynamic alterations due to valvar defects promote EndMT and whether age-specific structural changes affect ventricular diastolic compliance despite extensive surgical resection of EFE tissue. MATERIAL AND METHODS: We analyzed EFE tissue from 24 patients with hypoplastic left heart syndrome (HLHS) who underwent left ventricular (LV) rehabilitation surgery at Boston Children's Hospital between December 2011 and March 2018. Six patients with flow disturbances across the aortic valve and/or mitral valve but no HLHS diagnosis and macroscopic appearance of "EFE-like tissue" in the LV were included for comparison. All samples were examined for amount of collagen/elastin production and degradation, and presence of active EndMT by histologic analysis. RESULTS: EFE tissue from patients with and without HLHS consisted predominantly of elastin and collagen fibers. There was no alteration in degradation activity for collagen or elastin as shown by in situ zymography. Active EndMT was found in all patients with and without HLHS with flow disturbances ("EFE-like"). In patients with HLHS, EFE infiltrated into the underlying myocardium with increasing age. CONCLUSIONS: Patients with and without HLHS with flow disturbances due to stenotic or incompetent valves develop EndMT-derived fibrotic tissue covering the LV. When EFE recurs, it is directly associated with flow disturbances and switches to an infiltrative growth pattern with increasing age, leading to increased diastolic stiffness of the LV.

Flow disturbances and progression of endocardial fibroelastosis - a case report.

Endocardial fibroelastosis (EFE) is described as thickening of the endocardium and is associated with hypoplastic left heart syndrome (HLHS). The stimulus for EFE and the mechanism for recurrence and/or progression need to be investigated. In this report, we describe the case of a 4-year-old HLHS patient who underwent several surgeries with EFE resections due to recurrence of EFE. EFE recurrence was associated with flow disturbances due to valvar defects. At her latest follow-up 7 months after the last surgery, competent valves and no EFE were identified on all imaging study.

Endocardial fibroelastosis in two related tiger cubs (Panthera tigris).

Two tiger cubs (Panthera tigris) from the same litter were evaluated for suspected cardiac disease. Two cubs with a dilated cardiomyopathy phenotype were diagnosed with endocardial fibroelastosis based on necropsy and histopathologic examinations. Echocardiography revealed salient anatomic and functional aspects of this cardiac disorder. This is the first report of endocardial fibroelastosis in this species.

Fibroblasts in an endocardial fibroelastosis disease model mainly originate from mesenchymal derivatives of epicardium.

Endocardial fibroelastosis (EFE) refers to the thickening of the ventricular endocardium as a result of de novo deposition of subendocardial fibrous tissue layers during neonatal heart development. The origin of EFE fibroblasts is proposed to be postnatal endocardial cells that undergo an aberrant endothelial-to-mesenchymal transition (EndMT). Genetic lineage tracing of endocardial cells with the inducible endocardial Cre line Npr3-CreER and the endothelial cell tracing line Cdh5-CreER on an EFE-like model did not reveal any contribution of neonatal endocardial cells to fibroblasts in the EFE-like tissues. Instead, lineage tracing of embryonic epicardium by Wt1-CreER suggested that epicardium-derived mesenchymal cells (MCs) served as the major source of EFE fibroblasts. By labeling MCs using Sox9-CreER, we confirmed that MCs of the embryonic heart expand and contribute to the majority of neonatal EFE fibroblasts. During this pathological process, TGFß signaling, the key mediator of fibroblasts activation, was highly upregulated in the EFE-like tissues. Targeting TGFß signaling by administration of its antagonist bone morphogenetic protein 7 effectively reduced fibroblast accumulation and tissue fibrosis in the EFE-like model. Our study provides genetic evidence that excessive fibroblasts in the EFE-like tissues mainly originate from the epicardium-derived MCs through epicardial to mesenchymal transition (EpiMT). These EpiMT-derived fibroblasts within the EFE-like tissues could serve as a potential therapeutic target.

Re-evaluation of hypoplastic left heart syndrome from a developmental and morphological perspective.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) covers a spectrum of rare congenital anomalies characterised by a non-apex forming left ventricle and stenosis/atresia of the mitral and aortic valves. Despite many studies, the causes of HLHS remain unclear and there are conflicting views regarding the role of flow, valvar or myocardial abnormalities in its pathogenesis, all of which were proposed prior to the description of the second heart field. Our aim was to re-evaluate the patterns of malformation in HLHS in relation to recognised cardiac progenitor populations, with a view to providing aetiologically useful sub-groupings for genomic studies. RESULTS: We examined 78 hearts previously classified as HLHS, with subtypes based on valve patency, and re-categorised them based on their objective ventricular phenotype. Three distinct subgroups could be identified: slit-like left ventricle (24%); miniaturised left ventricle (6%); and thickened left ventricle with endocardial fibroelastosis (EFE; 70%). Slit-like ventricles were always found in combination with aortic atresia and mitral atresia. Miniaturised left ventricles all had normally formed, though smaller aortic and mitral valves. The remaining group were found to have a range of aortic valve malformations associated with thickened left ventricular walls despite being described as either atresia or stenosis. The degree of myocardial thickening was not correlated to the degree of valvar stenosis. Lineage tracing in mice to investigate the progenitor populations that form the parts of the heart disrupted by HLHS showed that whereas Nkx2-5-Cre labelled myocardial and endothelial cells within the left and right ventricles, Mef2c-AHF-Cre, which labels second heart field-derived cells only, was largely restricted to the endocardium and myocardium of the right ventricle. However, like Nkx2-5-Cre, Mef2c-AHF-Cre lineage cells made a significant contribution to the aortic and mitral valves. In contrast, Wnt1-Cre made a major contribution only to the aortic valve. This suggests that discrete cardiac progenitors might be responsible for the patterns of defects observed in the distinct ventricular sub-groups. CONCLUSIONS: Only the slit-like ventricle grouping was found to map to the current nomenclature: the combination of mitral atresia with aortic atresia. It appears that slit-like and miniature ventricles also form discrete sub-groups. Thus, reclassification of HLHS into subgroups based on ventricular phenotype, might be useful in genetic and developmental studies in investigating the aetiology of this severe malformation syndrome.

A case of cardioembolic stroke due to intracardiac papillary fibroelastoma evaluated by using transesophageal echocardiography.

A 62-year-old woman had a prior ischemic stroke in the right temporal lobe with dysarthria and dysesthesia of the left hand. Embolic stroke of undetermined source (ESUS) was diagnosed and warfarin was administered. However, transient ischemic attack recurred upon admission to our hospital. Paroxysmal atrial fibrillation and cerebral arterial stenotic lesions were absent. Transesophageal echocardiography revealed a mobile hyperechoic structure on the aortic valve indicating papillary fibroelastoma. She was diagnosed with a brain embolism due to the intracardiac tumor which was surgically excised and pathologically confirmed as papillary fibroelastoma. This type of tumor is relatively rare but it is important as an embolic source especially in ESUS. Transesophageal echocardiography was indispensable for detecting the embolic source in this patient with ESUS.

Successful Biopsy and Removal of a Tricuspid Valve Papillary Fibroelastoma in Cardiac Catheterization Laboratory: A Case Report.

Papillary fibroelastomas are rare benign cardiac tumours with a predilection for cardiac valves. Because of the rarity of these tumours, management is individualized, but some recommend surgical removal of all papillary fibroelastomas due to the increased risk of embolization. We report a case of a 71-year-old man who presented with a sessile mass on the tricuspid valve. The mass, a papillary fibroelastoma, was successfully biopsied and removed in the cardiac catheterization laboratory. This report demonstrates a unique minimally invasive way of approaching a cardiac tumour wherein a major surgery was avoided.

Knock-out of nexilin in mice leads to dilated cardiomyopathy and endomyocardial fibroelastosis.

Cardiomyopathy is one of the most common causes of chronic heart failure worldwide. Mutations in the gene encoding nexilin (NEXN) occur in patients with both hypertrophic and dilated cardiomyopathy (DCM); however, little is known about the pathophysiological mechanisms and relevance of NEXN to these disorders. Here, we evaluated the functional role of NEXN using a constitutive Nexn knock-out (KO) mouse model. Heterozygous (Het) mice were inter-crossed to produce wild-type (WT), Het, and homozygous KO mice. At birth, 32, 46, and 22 % of the mice were WT, Het, and KO, respectively, which is close to the expected Mendelian ratio. After postnatal day 6, the survival of the Nexn KO mice decreased dramatically and all of the animals died by day 8. Phenotypic characterizations of the WT and KO mice were performed at postnatal days 1, 2, 4, and 6. At birth, the relative heart weights of the WT and KO mice were similar; however, at day 4, the relative heart weight of the KO group was 2.3-fold higher than of the WT group. In addition, the KO mice developed rapidly progressive cardiomyopathy with left ventricular dilation and wall thinning and decreased cardiac function. At day 6, the KO mice developed a fulminant DCM phenotype characterized by dilated ventricular chambers and systolic dysfunction. At this stage, collagen deposits and some elastin deposits were observed within the left ventricle cavity, which resembles the features of endomyocardial fibroelastosis (EFE). Overall, these results further emphasize the role of NEXN in DCM and suggest a novel role in EFE.

A mouse model of endocardial fibroelastosis.

BACKGROUND: Endocardial fibroelastosis (EFE) is a pathologic condition of abnormal deposition of collagen and elastin within the endocardium of the heart. It is seen in conjunction with a variety of diseases including hypoplastic left heart syndrome and viral endocarditis. While an experimental model using heterotopic heart transplant in rats has been described, we sought to fully describe a mouse model that can be used to further elucidate the potential mechanisms of and treatments for EFE. MATERIALS AND METHODS: The hearts of 2-day-old C57BL/6 mice were transplanted into the abdomen of 7-week-old C57BL/6 mice. At 2 weeks, the hearts were harvested and histologic analysis was performed using hematoxylin and eosin, Masson's trichrome, Russell-Movat's pentachrome, Picrosirius red, Hart's, Verhoeff-Van Gieson, and Weigert's Resorcin-Fuchsin stains. Additionally, one heart was analyzed using transmission electron microscopy (TEM). RESULTS: Specimens demonstrated abnormal accumulation of both collagen and elastin within the endocardium with occasional expansion into the myocardium. Heterogeneity in extracellular matrix deposition was noted in the histologic specimens. In addition, TEM demonstrated the presence of excess collagen within the endocardium. CONCLUSIONS: The heterotopic transplantation of an immature heart into a mouse results in changes consistent with EFE. This model is appropriate to investigate the etiology and treatment of EFE.

Endocardial fibroelastosis is caused by aberrant endothelial to mesenchymal transition.

RATIONALE: Endocardial fibroelastosis (EFE) is a unique form of fibrosis, which forms a de novo subendocardial tissue layer encapsulating the myocardium and stunting its growth, and which is typically associated with congenital heart diseases of heterogeneous origin, such as hypoplastic left heart syndrome. Relevance of EFE was only recently highlighted through the establishment of staged biventricular repair surgery in infant patients with hypoplastic left heart syndrome, where surgical removal of EFE tissue has resulted in improvement in the restrictive physiology leading to the growth of the left ventricle in parallel with somatic growth. However, pathomechanisms underlying EFE formation are still scarce, and specific therapeutic targets are not yet known. OBJECTIVE: Here, we aimed to investigate the cellular origins of EFE tissue and to gain insights into the underlying molecular mechanisms to ultimately develop novel therapeutic strategies. METHODS AND RESULTS: By utilizing a novel EFE model of heterotopic transplantation of hearts from newborn reporter mice and by analyzing human EFE tissue, we demonstrate for the first time that fibrogenic cells within EFE tissue originate from endocardial endothelial cells via aberrant endothelial to mesenchymal transition. We further demonstrate that such aberrant endothelial to mesenchymal transition involving endocardial endothelial cells is caused by dysregulated transforming growth factor beta/bone morphogenetic proteins signaling and that this imbalance is at least in part caused by aberrant promoter methylation and subsequent transcriptional suppression of bone morphogenetic proteins 5 and 7. Finally, we provide evidence that supplementation of exogenous recombinant bone morphogenetic proteins 7 effectively ameliorates endothelial to mesenchymal transition and experimental EFE in rats. CONCLUSIONS: In summary, our data point to aberrant endothelial to mesenchymal transition as a common denominator of infant EFE development in heterogeneous, congenital heart diseases, and to bone morphogenetic proteins 7 as an effective treatment for EFE and its restriction of heart growth.

Primary endocardial fibroelastosis and nonimmune hydrops fetalis: case report with autopsy.

Endocardial fibroelastosis is an important cause of congestive heart failure and death in infancy and early childhood. When present, it is commonly associated with non immune hydrops fetalis. The aim of this study is to draw attention for possible cardiac abnormalities in cases of fetal hydrops, and report a case of premature death by primary endocardial fibroelastosis with autopsy.

[Congenital atresia of left main coronary artery in 4 children: case report and literature review].

OBJECTIVE: To investigate the clinical manifestations and treatment of congenital atresia of the left main coronary artery (CLMCA-A). METHOD: Four patients were diagnosed to have CLMCA-A from June 2010 to June 2012 in Beijing Anzhen Hospital. Clinical manifestations, ultrasound, ECG and angiographic characteristics were analyzed and summarized. RESULT: Of the 4 cases, age of onset was 3 months to 2 yrs. Three cases were diagnosed by angiography, and 1 case by CTA . All 4 cases had chronic heart failure symptoms and signs, such as sweating, shortness of breath, easily choked by milk, predispose to pneumonia, activity intolerance. ECG showed abnormal Q wave and other ischemic signs such as ST-T segment depression. Ultrasonography showed left ventricular enlargement, left ventricular systolic function was normal or slightly reduced, and there was moderate to large amount of mitral valve regurgitation. Left ventricular trabeculations increased. Coronary collateral circulation increased. Left coronary artery appeared to be slender and disconnected with left coronary artery sinus. Aortic root angiography was the golden diagnostic standard. Angiography was performed in 3 patients and showed that left main coronary artery had a blind end, diameter 1.1-2.0 mm. The right coronary artery was found rising from the right coronary sinus and visible on coronary collateral circulation. Contrast agent developing sequence: right coronary artery-collateral vessels-left coronary artery distal branches-left main coronary artery. CTA exam was performed in 2 cases and in 1 case the diagnoses was confirmed. All the 4 patients are currently in the close follow-up, digoxin and diuretics were taken everyday and clinical symptoms were improved. CONCLUSION: CLMCA-A is not rare, its clinical manifestations should be differentiated from those of cardiomyopathy, endocardial fibroelastosis, congenital valvular disease and abnormal left coronary artery originating from pulmonary artery etc. For pediatric patients with cardiac enlargement, abnormal heart function, mitral valve regurgitation etc, attention must be paid to consider the developmental abnormality of coronary artery, particularly the CLMCA-A diagnosis.