Dataset Information

Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Mario Oroshi

LAB HEAD: Alessandra Moretti

PROVIDER: PXD014812 | Pride | 2022-02-15

REPOSITORIES: Pride

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			Action	DRS
	20180115_QX3_SoDo_SA_iPS_C1D14.raw	Raw
	20180115_QX3_SoDo_SA_iPS_C1D8.raw	Raw
	20180115_QX3_SoDo_SA_iPS_C2D14.raw	Raw
	20180115_QX3_SoDo_SA_iPS_C2D8.raw	Raw
	20180115_QX3_SoDo_SA_iPS_C3D14_180118213128.raw	Raw

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Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome.

Krane Markus M Dreßen Martina M Santamaria Gianluca G My Ilaria I Schneider Christine M CM Dorn Tatjana T Laue Svenja S Mastantuono Elisa E Berutti Riccardo R Rawat Hilansi H Gilsbach Ralf R Schneider Pedro P Lahm Harald H Schwarz Sascha S Doppler Stefanie A SA Paige Sharon S Puluca Nazan N Doll Sophia S Neb Irina I Brade Thomas T Zhang Zhong Z Abou-Ajram Claudia C Northoff Bernd B Holdt Lesca M LM Sudhop Stefanie S Sahara Makoto M Goedel Alexander A Dendorfer Andreas A Tjong Fleur V Y FVY Rijlaarsdam Maria E ME Cleuziou Julie J Lang Nora N Kupatt Christian C Bezzina Connie C Lange Rüdiger R Bowles Neil E NE Mann Matthias M Gelb Bruce D BD Crotti Lia L Hein Lutz L Meitinger Thomas T Wu Sean S Sinnecker Daniel D Gruber Peter J PJ Laugwitz Karl-Ludwig KL Moretti Alessandra A

Circulation 20211025 17

<h4>Background</h4>Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role.<h4>Methods</h4>To identify perturbations in gene programs controlling ventricular ...[more]

PMID: 34694888

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Project description:The heart is a complex organ composed of multiple cell and tissue types. During early developmental stages, heart cells from different regions of the heart exhibit different patterns of gene expression, which are thought to significantly contribute to heart development and morphogenesis. Until recently, the lack of adequate tools prevented our ability to systematically profile gene expression in a cell type and anatomically-specific manner during heart development. Single cell RNA sequencing has emerged as a powerful approach that allows genome-wide analysis of gene expression in single cells. Using microfluidic-chip and droplet-based single-cell RNA sequencing platforms, we analyze cardiac cells during the early stages of heart development. We found, by principle component analysis, that the expression of cell cycle genes represents a major determinant of transcriptional variation in all analyzed cardiac cell types. Interestingly, when single cardiomyocytes (CMs) from different heart chambers were analyzed, we found that atrial ventricular canal derived CMs have reduced cell cycle activity compare with CMs from the left and right ventricular chambers. In addition, CMs in the trabecular myocardium exhibit reduced cell cycle activity than CMs in the compact myocardium. When CMs within the same chamber were segregated by their cell cycle phase, we found that CMs in the G2/M phase downregulate their sarcomeric and cytoskeletal markers. Finally, we revealed the expression of a signaling ligand from the endocardium that may repress trabecular CM proliferation, and an epicardial-derived ligand that can promote compact CM proliferation. Together these data highlight the variations in cell cycle activity to selectively promote cardiac chamber growth during development and the profound transcriptional shift within the same cell at different phases of cell cycle. Identifying alterations in the expression of key signaling molecules that drive CM proliferation may lead to greater understanding of the onset or progression of congenital heart disease.

Project description:Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart and high concentrations of RA have been shown to steer differentiation towards atrial CMs, whereas lower concentrations of RA promote a more ventricular-like CM profile. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of retinoic acid (RA) during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. The maturation and growth of EHTs were induced in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. The function of CMs and EHTs was analyzed under different conditions. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPRC and immunofluorescence staining analysis. RNA sequencing was conducted on EHTs to examine how RA affects both the function and structure of EHT. Results: In the HRA group, hiPSC-CMs exhibited the first onset of beating and showed the highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are the marker genes for left ventricular CMs, was also the highest in the HRA group. The transcription factor MEF2C associated with RA and ventricular development genes, NPPA and MYH7, were highly expressed in the HRA group, while GATA4 was less expressed in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was more functionally similar to the left ventricle. The expression of TBX5 and NKX2.5 was found to be the highest expression in the HRA group of EHT, while expression of TBX20 and ISL1 was found to be the highest expression in control group. When the electrical stimulation frequency of EHT in the HRA group was raised, it correspondingly increased its contractile force. The heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paved the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine.