Cardiac Outflow Research Articles

The myoarchitecture of the vertebrate cardiac ventricles: evolution and classification.

The ventricle of the vertebrate heart is the main segment of the cardiac outflow region. Compared with other cardiac components, it shows remarkable histomorphological variation among different animal groups. This variation is especially apparent in the myocardium, which is generally classified into three main types: trabeculated, compact and mixed. The trabeculated or 'spongy' myocardium is characterized by the existence of trabeculae and deep recesses or intertrabecular spaces, lined by the endocardium. The compact type is composed of condensed myocardial fibers, with almost no trabeculated layer. The mixed type consists of an outer compact layer and an inner trabeculated layer. Among vertebrates, fishes show a great diversity of myocardial types. On this basis, the ventricular myoarchitecture has been categorized into four groups of varying complexity. This classification is made according to (i) the proportion of the two types of myocardium, trabeculated versus compact, and (ii) the vascularization of the heart wall. Here, we review the morphogenetic mechanisms that give rise to the different ventricular myoarchitecture in gnathostomes (i.e. jawed vertebrates) with special emphasis on the diversity of the ventricular myocardium throughout the phylogeny of ancient actinopterygians and teleosts. Finally, we propose that the classification of the ventricular myoarchitecture should be reconsidered, given that the degrees of myocardial compactness on which the current classification system is based do not constitute discrete states, but an anatomical continuum.

Mesodermal fibronectin controls cell shape, polarity, and mechanotransduction in the second heart field during cardiac outflow tract development.

Failure in the elongation of the cardiac outflow tract (OFT) results in congenital heart disease due to the misalignment of the great arteries with the left and right ventricles. The OFT lengthens via the accretion of progenitors from the second heart field (SHF). SHF cells are exquisitely regionalized and organized into an epithelial-like layer, forming the dorsal pericardial wall (DPW). Tissue tension, cell polarity, and proliferation within the DPW are important for the addition of SHF-derived cells to the heart and OFT elongation. However, the genes controlling these processes are not completely characterized. Using conditional mutagenesis in the mouse, we show that fibronectin (FN1) synthesized by the mesoderm coordinates multiple cellular behaviors in the anterior DPW. FN1 is enriched in the anterior DPW and plays a role in OFT elongation by maintaining a balance between pro- and anti-adhesive cell-extracellular matrix (ECM) interactions and controlling DPW cell shape, polarity, cohesion, proliferation, and mechanotransduction.

Study on ultrasound diagnosis and pathological anatomy of fetal complex congenital heart disease in the first trimester.

To assess the feasibility of using the stereo-microscope to identify the pathological anatomy of the congenital heart diseases in the first trimester. Fifteen fetuses of 8-12 weeks aborted due to prevent miscarriage failure and 42 fetuses of 11-14 weeks with congenital heart diseases were included in the study, we dissected their hearts through a stereo-microscope, then compared with the prenatal ultrasonographic diagnosis. Using stereomicroscopy, the positive view of the heart and the great arteries, the long axis view of the aortic arch, the inflow tract view of the bottom heart, the semilunar valve view of the bottom heart, and the transverse section of the ventricle were showed contented and obtained satisfactory images, but the structure of atrioventricular valve and venous system had a lower rate of display. The characteristic pathological changes of cardiac inflow and outflow tract can be obtained by dissecting the heart sequential under the stereo-microscopy. However it is often difficult to obtain satisfactory pathological sections for pulmonary venous abnormalities and Ebstein anomaly.

Deficient GATA6-CXCR7 signaling leads to bicuspid aortic valve.

The cardiac outflow tract (OFT) transiently links the ventricles to the aortic sac and forms the arterial valves. Abnormalities in these valves, such as bicuspid aortic valve (BAV), are common congenital anomalies. GATA6-inactivating variants cause cardiac OFT defects and BAV, but their mechanisms are unclear. We generated Gata6STOP/+ mice using CRISPR-Cas9, which show highly penetrant BAV (70%) and membranous ventricular septal defects (43%). These mice exhibited decreased proliferation and increased ISL1-positive progenitor cells in the OFT, indicating abnormal cardiovascular differentiation. Gata6 deletion with the Mef2cCre driver line recapitulated Gata6STOP/+ phenotypes, indicating a cell-autonomous role for Gata6 in the second heart field. Gata6STOP/+ mice showed reduced OFT length and caliber, associated with deficient cardiac neural crest cell contribution, which may cause valvulo-septal defects. RNA-sequencing analysis showed depletion in pathways related to cell proliferation and migration, highlighting Cxcr7 (also known as Ackr3) as a candidate gene. Reduced mesenchymal cell migration and invasion were observed in Gata6STOP/+ OFT tissue. CXCR7 agonists reduced mesenchymal cell migration and increased invasion in wild-type but not in Gata6STOP/+ explants, indicating the GATA6-dependent role of CXCR7 in OFT development and its potential link to BAV.

Abstract We088: Spatiotemporal analysis of cardiac progenitor cells reveals migratory characteristics during epithelial-mesenchymal transition

Introduction: Congenital heart defects (CHD) are one of the most common types of birth defects, with a rising prevalence in mild types. The most frequent CHDs include anomalies in the cardiac outflow tract (OFT), which mainly derives from multipotent cardiac progenitor cells termed second heart field (SHF). Hence, dysregulation of highly synchronized, chronological sequence of proliferation and differentiation of SHF cells could lead to severe CHDs. As the SHF contributes entirely to the OFT, mutations in the SHF-related genes lead to major arterial pole defects which account for one third of CHDs in newborn children. Investigating how these genes regulate cell-cell behavior and communication is of vital importance. Hypothesis and Methods: In this study, we employed a multidimensional approach to assess the hypothesis that the dynamic behavior of SHFs is regulated by key genes implicated in CHD pathogenesis. Leveraging single-cell RNA sequencing (RNA-seq) data obtained from experimentally dissected mouse OFT, we delineated distinct epithelial-to-mesenchymal transition states within SHF cell populations. Additionally, we developed a quantitative metric to assess SHF cell’s migratory level and integrated it with spatial transcriptomic analyses to map the developmental trajectory of SHFs. Results and Conclusion: Through spatiotemporal analysis, we identified a panel of key genes associated with SHF migration, shedding light on the molecular mechanisms underlying OFT development during early embryonic stages. Furthermore, we leveraged the data from The Pediatric Cardiac Genomics Consortium (PCGC) study (phs001194.v3.p2) which comprises a large cohort of CHD patients (13716 participants) and a proportion of 21.68% identified with OFT obstruction in its sub-study. In conclusion, using these datasets and quantitative metric being developed, we validated the SHF-derived genes in the PCGC participants and revealed specific expression patterns of these genes associated with OFT-specific CHDs. These results would benefit the understanding of the genetic regulation of SHF proliferation and differentiation, which is crucial for early detection and precise surgical intervention in CHD patients.

The chromatin regulator Ankrd11 controls cardiac neural crest cell-mediated outflow tract remodeling and heart function

ANKRD11 (Ankyrin Repeat Domain 11) is a chromatin regulator and a causative gene for KBG syndrome, a rare developmental disorder characterized by multiple organ abnormalities, including cardiac defects. However, the role of ANKRD11 in heart development is unknown. The neural crest plays a leading role in embryonic heart development, and its dysfunction is implicated in congenital heart defects. We demonstrate that conditional knockout of Ankrd11 in the murine embryonic neural crest results in persistent truncus arteriosus, ventricular dilation, and impaired ventricular contractility. We further show these defects occur due to aberrant cardiac neural crest cell organization leading to outflow tract septation failure. Lastly, knockout of Ankrd11 in the neural crest leads to impaired expression of various transcription factors, chromatin remodelers and signaling pathways, including mTOR, BMP and TGF-β in the cardiac neural crest cells. In this work, we identify Ankrd11 as a regulator of neural crest-mediated heart development and function.

Significant improvement of cardiac outflow tract septation defects in a DiGeorge syndrome model after minoxidil treatment

The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation.We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1−/− mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant.We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

Epigenetics of conotruncal congenital heart disease: Protocol for a systematic review and meta-analysis.

Conotruncal congenital heart defects (CTD) are a subset of congenital heart diseases (CHD) that involve structural anomalies of the right, left, or both cardiac outflow tracts. CHD is caused by multifactorial inheritance and changes in the genes or chromosomes. Recently, CHD was found to be due to epigenetic alterations, which are a combination of genetic and other environmental factors. Epigenetics is the study of how a gene's function changes as a result of environmental and behavioral influences. These causative factors can indirectly cause CHD by altering the DNA through epigenetic modifications. This is a protocol for a systematic review and meta-analysis that aims to explore whether the strength of association between various epigenetic changes and CTD types varies by race. Furthermore, to determine and compare the changes in gene expression of each mutation. Our protocol follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocol (PRISMA-P) guidelines. A comprehensive pre-search has been developed in PubMed and PubMed's Medical Subject Headings (MeSH). The final search will be performed in June 2023 in PubMed, Embase, Scopus, Web of Science, Cochrane Library, CIANHL, and PsycInfo, without restrictions on publication years. The Covidence systematic review software will be used for blinded screening and selection. Conflicts will be resolved by a third, independent reviewer. The risk of bias in selected studies will be assessed using the National Heart, Lung, and Blood Institute (NHLBI) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. The data to be extracted will cover basic information on the included studies, study sample size, number of patients with various types of epigenetic changes, number of patients with various CTD types, measures of association and their 95% confidence interval between each epigenetic change and each CTD. The protocol has been registered with the International Prospero Register of Systematic Review (PROSPERO) [CRD42023377597]. To the best of our knowledge, this protocol outlines the first systematic review and meta-analysis of the epigenetics of CTD. There is a growing body of evidence on epigenetics and its indirect involvement in disease by altering the DNA through epigenetic modifications in the genes associated with the causative factors for CHD. We will conduct a comprehensive and systematic search for literature in the above-mentioned seven core biomedical databases. It is very important to identify population-specific risk factors for CHD, which will have significant creative, custom-made, and effective prevention programs for the future generation.

Variants in FOXC1 and FOXC2 identified in patients with conotruncal heart defects

Conotruncal heart defects (CTD), subtypes of congenital heart disease, result from abnormal cardiac outflow tract development (OFT). FOXC1 and FOXC2 are closely related members of the forkhead transcription factor family and play essential roles in the development of OFT. We confirmed their expression pattern in mouse and human embryos, identifying four variants in FOXC1 and three in FOXC2 by screening these two genes in 605 patients with sporadic CTD. Western blot demonstrated expression levels, while Dual-luciferase reporter assay revealed affected transcriptional abilities for TBX1 enhancer in two FOXC1 variants and three FOXC2 variants. This might result from the altered DNA-binding abilities of mutant proteins. These results indicate that functionally impaired FOXC1 and FOXC2 variants may contribute to the occurrence of CTD.

Differential associations between abnormal cardiac left ventricular geometry types and cerebral white matter disease

ObjectivesReduced cardiac outflow due to left ventricular hypertrophy has been suggested as a potential risk factor for development of cerebral white matter disease. Our study aimed to examine the correlation between left ventricular geometry and white matter disease volume to establish a clearer understanding of their relationship, as it is currently not well-established. MethodsConsecutive patients from 2016 to 2021 who were ≥18 years and underwent echocardiography, cardiac MRI, and brain MRI within one year were included. Four categories of left ventricular geometry were defined based on left ventricular mass index and relative wall thickness on echocardiography. White matter disease volume was quantified using an automated algorithm applied to axial T2 FLAIR images and compared across left ventricular geometry categories. ResultsWe identified 112 patients of which 34.8 % had normal left ventricular geometry, 20.5 % had eccentric hypertrophy, 21.4 % had concentric remodeling, and 23.2 % had concentric hypertrophy. White matter disease volume was highest in patients with concentric hypertrophy and concentric remodeling, compared to eccentric hypertrophy and normal morphology with a trend-P value of 0.028. Patients with higher relative wall thickness had higher white matter disease volume (10.73 ± 10.29 cc vs 5.89 ± 6.46 cc, P = 0.003), compared to those with normal relative wall thickness. ConclusionOur results showed that abnormal left ventricular geometry is associated with higher white matter disease burden, particularly among those with abnormal relative wall thickness. Future studies are needed to explore causative relationships and potential therapeutic options that may mediate the adverse left ventricular remodeling and its effect in slowing white matter disease progression.

The c.1617del variant of TMEM260 is identified as the most frequent single gene determinant for Japanese patients with a specific type of congenital heart disease

Although the molecular mechanisms underlying congenital heart disease (CHD) remain poorly understood, recent advances in genetic analysis have facilitated the exploration of causative genes for CHD. We reported that the pathogenic variant c.1617del of TMEM260, which encodes a transmembrane protein, is highly associated with CHD, specifically persistent truncus arteriosus (PTA), the most severe cardiac outflow tract (OFT) defect. Using whole-exome sequencing, the c.1617del variant was identified in two siblings with PTA in a Japanese family and in three of the 26 DNAs obtained from Japanese individuals with PTA. The c.1617del of TMEM260 has been found only in East Asians, especially Japanese and Korean populations, and the frequency of this variant in PTA is estimated to be next to that of the 22q11.2 deletion, the most well-known genetic cause of PTA. Phenotype of patients with c.1617del appears to be predominantly in the heart, although TMEM260 is responsible for structural heart defects and renal anomalies syndrome (SHDRA). The mouse TMEM260 variant (p.W535Cfs*56), synonymous with the human variant (p.W539Cfs*9), exhibited truncation and downregulation by western blotting, and aggregation by immunocytochemistry. In situ hybridization demonstrated that Tmem260 is expressed ubiquitously during embryogenesis, including in the development of cardiac OFT implicated in PTA. This expression may be regulated by a ~ 0.8 kb genomic region in intron 3 of Tmem260 that includes multiple highly conserved binding sites for essential cardiac transcription factors, thus revealing that the c.1617del variant of TMEM260 is the major single-gene variant responsible for PTA in the Japanese population.

Dynamic load modulation predicts right heart tolerance of left ventricular cardiovascular assist in a porcine model of cardiogenic shock.

Ventricular assist devices (VADs) offer mechanical support for patients with cardiogenic shock by unloading the impaired ventricle and increasing cardiac outflow and subsequent tissue perfusion. Their ability to adjust ventricular assistance allows for rapid and safe dynamic changes in cardiac load, which can be used with direct measures of chamber pressures to quantify cardiac pathophysiologic state, predict response to interventions, and unmask vulnerabilities such as limitations of left-sided support efficacy due to intolerance of the right heart. We defined hemodynamic metrics in five pigs with dynamic peripheral transvalvular VAD (pVAD) support to the left ventricle. Metrics were obtained across a spectrum of disease states, including left ventricular ischemia induced by titrated microembolization of a coronary artery and right ventricular strain induced by titrated microembolization of the pulmonary arteries. A sweep of different pVAD speeds confirmed mechanisms of right heart decompensation after left-sided support and revealed intolerance. In contrast to the systemic circulation, pulmonary vascular compliance dominated in the right heart and defined the ability of the right heart to adapt to left-sided pVAD unloading. We developed a clinically accessible metric to measure pulmonary vascular compliance at different pVAD speeds that could predict right heart efficiency and tolerance to left-sided pVAD support. Findings in swine were validated with retrospective hemodynamic data from eight patients on pVAD support. This methodology and metric could be used to track right heart tolerance, predict decompensation before right heart failure, and guide titration of device speed and the need for biventricular support.

Exploring the origins of neurodevelopmental proteasomopathies associated with cardiac malformations: are neural crest cells central to certain pathological mechanisms?

Neurodevelopmental proteasomopathies constitute a recently defined class of rare Mendelian disorders, arising from genomic alterations in proteasome-related genes. These alterations result in the dysfunction of proteasomes, which are multi-subunit protein complexes essential for maintaining cellular protein homeostasis. The clinical phenotype of these diseases manifests as a syndromic association involving impaired neural development and multisystem abnormalities, notably craniofacial anomalies and malformations of the cardiac outflow tract (OFT). These observations suggest that proteasome loss-of-function variants primarily affect specific embryonic cell types which serve as origins for both craniofacial structures and the conotruncal portion of the heart. In this hypothesis article, we propose that neural crest cells (NCCs), a highly multipotent cell population, which generates craniofacial skeleton, mesenchyme as well as the OFT of the heart, in addition to many other derivatives, would exhibit a distinctive vulnerability to protein homeostasis perturbations. Herein, we introduce the diverse cellular compensatory pathways activated in response to protein homeostasis disruption and explore their potential implications for NCC physiology. Altogether, the paper advocates for investigating proteasome biology within NCCs and their early cranial and cardiac derivatives, offering a rationale for future exploration and laying the initial groundwork for therapeutic considerations.

KCTD1/KCTD15 complexes control ectodermal and neural crest cell functions, and their impairment causes aplasia cutis.

Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a long-standing enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we found that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss and that disease mutations are dominant negative, resulting in lack of KCTD1/KCTD15 function. We demonstrated that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes resulted in abnormal skin appendages but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells resulted in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells, giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 complexes as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.

Dgcr8 functions in the secondary heart field for outflow tract and right ventricle development in mammals

The DGCR8 gene, encoding a critical miRNA processing protein, maps within the hemizygous region in patients with 22q11.2 deletion syndrome. Most patients have malformations of the cardiac outflow tract that is derived in part from the anterior second heart field (aSHF) mesoderm. To understand the function of Dgcr8 in the aSHF, we inactivated it in mice using Mef2c-AHF-Cre. Inactivation resulted in a fully penetrant persistent truncus arteriosus and a hypoplastic right ventricle leading to lethality by E14.5. To understand the molecular mechanism for this phenotype, we performed gene expression profiling of the aSHF and the cardiac outflow tract with right ventricle in conditional null versus normal mouse littermates at stage E9.5 prior to morphology changes. We identified dysregulation of mRNA gene expression, of which some are relevant to cardiogenesis. Many pri-miRNA genes were strongly increased in expression in mutant embryos along with reduced expression of mature miRNA genes. We further examined the individual, mature miRNAs that were decreased in expression along with pri-miRNAs that were accumulated that could be direct effects due to loss of Dgcr8. Among these genes, were miR-1a, miR-133a, miR-134, miR143 and miR145a, which have known functions in heart development. These early mRNA and miRNA changes may in part, explain the first steps that lead to the resulting phenotype in Dgcr8 aSHF conditional mutant embryos.

Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Abstract 14239: Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve

Background: Extracellular matrix (ECM) is involved in aortic valve development and aortic valve anomaly. ADAMTS gene mediated ECM dysfunction was reported to be related to aortic valve diseases. Hypothesis: The ADAMTS gene family members can contribute to aortic valve diseases. Methods: Morpholino based adamts gene-targeted phenotype-screening of outflow tract anomaly in zebrafish and the 19 ADAMTS family gene-targeted exon sequencing on a cohort BAV patients (304 BAV and 301 normal controls) were performed. Accordingly, both specific genetic tracing and different gene modified mouse models were generated to evaluate the role of specific genes in the aortic valve formation process. Movat’s pentachrome staining was used to describe the altered ECM maturation process. Bulk RNA-seq was performed on both mouse heart tissue and human iPSC-induced ECs to explore the potential molecular pathways, which was further confirmed in ex vivo embryonic outflow tract tissue culture studies. Results: We identified some novel adamts genes that resulted in cardiac outflow valve defects in zebrafish, while exon sequencing identified ADAMTS16 p.H357Q variant in an inherited BAV family. The generated Ad amts16-Cre; Rosa26-tdTomato mice showed Adamts16 -expressing cells were primarily restricted to valvular endothelial cells (VECs) at E12.5 and then expressed in valvular mesenchymal cells (VICs) at E13.5. Both Adamts16 +/- and Adamts16 +/H355Q mice exhibited an R-NC-type BAV phenotype, with progressive aortic valve thickening. Conditional Adamts16 mutant mouse models demonstrated that endothelial lineage cell deficiency in Adamts16 recapitulated the BAV phenotype, which was associated with worse ECM deposition without ECM trilaminar structure. Bulk RNA sequencing unveiled enhanced FAK signaling, which was accompanied by elevated fibronectin levels. Both in vitro iPSC-ECs culture and ex vivo embryonic tissue explant studies validated the altered FAK signaling resulted in exaggerated cell proliferation. Conclusions: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

The Role of Endoplasmic Reticulum Stress in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD), which is involved in osteogenic reprogramming of valvular interstitial cells, is the most common form of valve disease. It still lacks effective pharmacologic intervention, as its cellular biological mechanisms remain unclear. Congenital abnormality (bicuspid valve) and older age are considered to be the most powerful risk factors for CAVD. Aortic valve sclerosis (AVS) and calcific aortic stenosis (CAS), 2 subclinical forms of CAVD, represent 2 distinct stages of aortic valve calcification. During the AVS stage, the disease is characterised by endothelial activation/damage, inflammatory response, and lipid infiltration accompanied by microcalcification. The CAS stage is dominated by calcification, resulting in valvular dysfunction and severe obstruction to cardiac outflow, which is life threatening if surgery is not performed in time. Endoplasmic reticulum (ER) stress, a state in which conditions disrupting ER homeostasis cause an accumulation of unfolded and misfolded proteins in the ER lumen, has been shown to promote osteogenic differentiation and aortic valve calcification. Therefore, identifying targets or drugs for suppressing ER stress may be a novel approach for CAVD treatment.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Cardiac sarcoma presenting as paraneoplastic arthritis and clubbing: a case report and literature review

BackgroundCardiac tumours are rare, and clinical manifestations depend on the anatomical location. Symptoms can be the result of cardiac outflow anomalies, constitutional features such as fever, loss of weight, and/or paraneoplastic manifestations such as arthritis. To date, there has only been one other case report in the literature of cardiac sarcoma presenting as paraneoplastic arthropathy.Case presentationA 52-year-old woman presented with acute onset corticosteroid-resistant inflammatory polyarthralgia, clubbing and a systolic murmur. Transthoracic echocardiogram revealed a dilated left atrium with an echogenic mass and brain magnetic resonance imaging revealed multiple embolic infarcts. Histopathology following emergency resection showed a Grade 3 left atrial intimal sarcoma. The polyarthralgia and clubbing resolved soon after tumour removal. The patient went on to receive chemotherapy and remains in remission.ConclusionsThis case highlights the rare paraneoplastic association of cardiac sarcoma and arthropathy.