Impaired myocardial contractility underlies an Rbfox-mediated zebrafish model of hypoplastic left heart syndrome
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ABSTRACT: Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, which was previously linked to HLHS in newborns, display cardiovascular defects overlapping those in HLHS patients. In contrast to current models, we demonstrate that co-existing ventricular, valve, and aortic deficiencies in rbfox mutant zebrafish arise secondary to impaired myocardial contractility. On a molecular and cellular level, we find diminished expression and alternative splicing of sarcomere and mitochondrial components that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 rescues ventricular structure and function in rbfox mutant zebrafish, demonstrating conservation of molecular function between humans and zebrafish Rbfox proteins. However, injection of RBFOX2 variants previously identified in newborns with HLHS do not. Taken together, our data suggest that mutations in RBFOX2 are causal for HLHS and challenge the obligate multigenic etiology of the disease. Instead, we provide a complimentary paradigm where HLHS can arise from a single gene mutation where ventricular, valve, and aortic deficiencies manifest secondary to a primary myocardial defect.
Project description:Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of left sided structures including the ventricle, valves, and aorta1. Although the mechanisms of disease pathogenesis remain elusive due to a paucity of candidate genes and animal models, prevailing paradigm suggests that HLHS is a multigenic disease of co-occurring phenotypes2,3. Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, a gene previously linked to HLHS in humans4,5, display cardiovascular defects overlapping those in HLHS patients. In contrast to current models, we demonstrate that co-existing ventricular, valve, and aortic deficiencies in rbfox mutant zebrafish arise secondary to impaired myocardial function as all three phenotypes are rescued when Rbfox is expressed specifically in the myocardium. On a molecular and cellular level, we find diminished expression and alternative splicing of sarcomere and mitochondrial components in rbfox-deficient hearts that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 mRNA restores ventricular structure and function in rbfox mutant zebrafish, while HLHS-linked RBFOX2 variants fail to rescue. Taken together, our data suggest that mutations in RBFOX2 are causal for HLHS pathogenesis and provide a complimentary paradigm for HLHS emergence where co-existing ventricular, valve, and aortic deficiencies have a monogenic etiology caused by myocardial dysfunction.
Project description:Affymetrix GeneChip Exon-1.0ST was used to study the differential gene profiles in RV (right ventricle) samples from neonates with HLHS (hypoplastic left heart syndrome) versus RV and LV (left ventricle) samples obtained from age-matched controls. Although few significant changes were observed in the genetic profiles between control LV and control RV, many genes passed the false discovery rate in comparing HLHS-RV to RV and LV control groups, with greater differential profiles noted between HLHS-RV and control RV. Myocardial samples were isolated from the RV of 6 HLHS neonates, diagnosed based upon clinical features including hypoplasia/atresia of the ascending aorta, various degrees of underdevelopment of the aortic valve, mitral valve, and LV cavity, and retrograde flow in the aortic arch as determined by conventional 2-D echocardiography. The mean gestational age at birth of all subjects was 38 weeks (range 36-39) and the mean body weight at surgery of 2.7 kg (range 2.1-3.4 kg) (3 males, 3 females). All subjects were undergoing stage 1 Norwood reconstruction. Children with HLHS and other complex cardiac anomalies entailing non-HLHS single ventricle circulation were excluded from our study. For control samples, RV and LV myocardial tissue was obtained from 5 newborns aged between 1-28 days (mean 18 days; 3 males and 2 females) with normal cardiac anatomy and expired from non-cardiac diseases processes.
Project description:To determine how the higher order assembly of Rbfox proteins affect Rbfox-dependent splicing regulation, we expressed Rbfox wildtype and its mutant protein in Flp-In™ T-REx™ 293 Rbfox2-/- cells and extracted RNA from these cells to perform RASL-seq which profiles thousands of alternative splicing event.
Project description:Myocardial left ventricular biopsies from male patients (n=6) with isolated aortic stenosis and pronounced left ventricular hypertrophy undergoing aortic valve replacement were harvested either from hearts with normal ejection fraction (EF,>50%) or with low EF (<30%). Biopsies were further obtained from non-hypertrophied hearts with normal EF (>60%) from coronary artery disease patients undergoing coronary artery bypass graft surgery (n=3). Total RNA isolated from biopsies was analyzed using Affymetrix HG-U133A and U133B GeneChip sets.
Project description:Pathological variants in NOTCH1 have been implicated in multiple types of congenital heart defects including bicuspid aortic valve and hypoplastic left heart syndrome (HLHS). To probe how NOTCH1 deficiency affects cardiac development, we generated homozygous NOTCH1 knockout (N1KO) human induced pluripotent stem cells (iPSCs). We then ran single-cell RNA-seq to temporally profile transcriptomic changes during cardiac differentiation in wild type (WT) and N1KO iPSCs. We collected differentiating cells at multiple time points corresponding to different development stages, i.e., Day 0 (D0: pluripotent stem cell), D2 (mesoderm), D5 (cardiac mesoderm), D10 (cardiac progenitor), D14 (early cardiomyocyte), and D30 (fetal cardiomyocyte). Single-cell transcriptomics analysis reveals that NOTCH1 disruption impairs human ventricular cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm toward the first heart field, second heart field, and epicardial lineages.
Project description:Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most severe congenital heart defect encompassing a spectrum of left-ventricular hypoplasia occurring in association with outflow-tract obstruction. The current clinical paradigm assumes HLHS is largely of hemodynamic origin. Here, by combining whole-exome sequencing of 87 HLHS parent-offspring trios and transcriptome of cardiomycytes (CMs) from healthy and patient native ventricles at different stages of development we identified perturbations in coherent gene programs controlling ventricular muscle lineage development. Single-cell and 3D molecular/functional modeling with iPSCs demonstrated intrinsic defects in the cell-cycle/ciliogenesis/autophagy hub resulting in disrupted differentiation of early cardiac progenitor (CP) lineages and ultimate defective CM-subtype differentiation/maturation in HLHS. Moreover, premature cellcycle exit of ventricular CM prevents tissue response to cues of developmental growth leading to multinucleation/polyploidy, accumulation of DNA damage, exacerbated apoptosis, and eventually ventricle hypoplasia. Our results highlight how genetic heterogeneity in HLHS converges in perturbations of sequential cellular processes driving cardiogenesis and facilitate potential novel nodes for therapy beside surgical intervention.
Project description:Rbfox proteins regulate alternative splicing, mRNA stability and translation. These proteins are involved in neurogenesis and have been associated with various neurological conditions. We generated expression profile in adult and developing mouse retinas which lacks in Rbfox2 expression by RNA sequencing. The goals of this study is to identify the affected pathways in Rbfox2 KO mouse retina as well as potential splicing targets of Rbfox2.
Project description:Calcific Aortic Valve Disease (CAVD) is a common heart valve condition, often characterized by severe narrowing of the aortic valve. It lacks pharmaceutical treatments and typically requires aortic valve replacement surgery, imposing a significant burden on healthcare resources.This study reports the expression profile of circRNAs in the aortic valve tissues of CAVD patients and a normal control group (non-CAVD). We collected aortic valve tissue samples from three CAVD patients who underwent aortic valve replacement surgery due to severe aortic valve stenosis, as well as aortic valve samples from non-CAVD patients who either received heart transplant surgery (recipient heart) or had their aortic valve removed due to aortic dissection. Overall, our research reveals the significant role of circRNAs in the progression of CAVD. CircRNAs, a class of circular non-coding RNA molecules, are actively studied for their functions and regulatory mechanisms within cells. These findings contribute to a deeper understanding of the molecular mechanisms underlying CAVD, particularly the potential involvement of circRNAs in this disease.
Project description:Continuous-flow left ventricular assist devices commonly lead to aortic regurgitation, which results in decreased pump efficiency and worsening heart failure. We hypothesized that non-physiological wall shear stress and oscillatory shear index alter the abundance of structural proteins in aortic valves of left ventricular assist device (LVAD) patients. Doppler images of aortic valves of patients undergoing heart transplants were obtained. Eight patients had been supported with LVADs, whereas 10 were not. Aortic valve tissue was collected and protein levels were analyzed using mass spectrometry. Echocardiographic images were analyzed and wall shear stress and oscillatory shear index were calculated. The relationship between normalized levels of individual proteins and in vivo echocardiographic measurements was evaluated. Of the 57 proteins of interest, there was a strong negative correlation between levels of 15 proteins and the wall shear stress (R < -0.500, p ? 0.05), and a moderate negative correlation between 16 proteins and wall shear stress (R ?0.500 to ?0.300, p ? 0.05). Gene ontology analysis demonstrated clusters of proteins involved in cellular structure. Proteins negatively correlated with WSS included those with cytoskeletal, actin/myosin, cell-cell junction and extracellular functions. In aortic valve tissue, 31 proteins were identified involved in cellular structure and extracellular junctions with a negative correlation between their levels and wall shear stress. These findings suggest an association between the forces acting on the aortic valve (AV) and leaflet protein abundance, and may form a mechanical basis for the increased risk of aortic leaflet degeneration in LVAD patients.
Project description:Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of expression Sox9 has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism. 3 samples of aortic valve interstitial cells treated with DAPT were compared with 3 samples of aortic valve interstitial cells treated with DMSO