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

Background: 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. Methods: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed: i) whole-exome sequencing of 87 HLHS parent-offspring trios, ii) nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, iii) single cell RNA sequencing and iv) 3D modeling in iPSCs from 3 patients with HLHS and 3 controls. Results: Gene set enrichment and protein network analyses of damaging de-novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell-cycle and cardiomyocyte maturation. Single-cell and 3D modeling with iPSCs demonstrated intrinsic defects in the cell-cycle/UPR/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte-subtype differentiation/maturation in HLHS. Additionally, premature cell-cycle exit of ventricular cardiomyocytes from HLHS patients prevented normal tissue responses to developmental signals for growth leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. Conclusions: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

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

Circulation ◽

10.1161/circulationaha.121.056198 ◽

2021 ◽

Vol 144 (17) ◽

pp. 1409-1428

Author(s):

Markus Krane ◽

Martina Dreßen ◽

Gianluca Santamaria ◽

Ilaria My ◽

Christine M. Schneider ◽

...

Keyword(s):

Cell Cycle ◽

3D Modeling ◽

Pluripotent Stem Cells ◽

Heart Development ◽

Left Ventricular ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Cellular Processes ◽

Induced Pluripotent ◽

Left Heart Syndrome

Background: 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. Methods: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent–offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. Results: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. Conclusions: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

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Abstract 238: Single-Cell Transcriptomic Analysis and Patient-Specific IPSCs Reveal Dysregulated Cell Cycle in Coronary Endothelial Cell in Hypoplastic Left Heart Syndrome

Circulation Research ◽

10.1161/res.127.suppl_1.238 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Mingxia Gu ◽

Yifei Miao ◽

Xin Zhou ◽

Lei Tian ◽

Marcy Martin ◽

...

Keyword(s):

Cell Cycle ◽

Endothelial Cell ◽

Left Ventricle ◽

Single Cell ◽

Hypoplastic Left Heart Syndrome ◽

Human Heart ◽

G1 Phase ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is a single ventricle congenital heart disease that results in severe underdevelopment of the left ventricle, mitral valve, aortic valve, and ascending aorta. Early serial postmortem examinations also revealed a high rate of coronary anomalies in HLHS, which included multiple ventriculo-coronary arterial connections as well as thick-walled and kinked coronary arteries. A previous study showed that fetal hypoplastic left hearts had a reduced endothelial cell (EC) population and lower capillary density compared with normal hearts. However, the mechanism underlying coronary abnormalities associated with HLHS remains unknown. Thus, we generated induced pluripotent stem cells derived ECs (iPSC-ECs) from three HLHS patients and three age-matched controls. Single Cell RNA-Seq (scRNA-seq) profiling identified both endocardial (NPR3 + /CDH5 + ) and coronary endothelial populations (APLN + /CDH5 + ) from the heterogeneous iPSC-ECs. Intriguingly, a subcluster of the coronary endothelial cells (CECs) with cell cycle arrest was specifically enriched in HLHS patients. Further cell cycle analysis showed that 30.6% of the HLHS cells were trapped in the G1 phase, while the majority of the control CECs entered cell cycle normally. Additionally, the cell cycle differences between control and HLHS was only seen in CECs, not in the endocardial population. To verify our transcriptomic analysis, we applied negative cell sorting (NPR3 - /CDH5 + ) on iPSC-ECs to purify CECs (iCECs) and confirmed that HLHS iCECs showed profound reduction of cell cycle/proliferative genes ( KI67, PCNA, CCNA2, CCNB1 ) and abnormal induction of CCND2 , which is the hallmark of G1 phase. BrdU assays also indicated suppressed proliferation in HLHS iCECs. Furthermore, we profiled the transcriptome from a human heart with an underdevelopment left ventricle (ULV) at single cell resolution. When compared to the normal human heart, pathway enrichment analysis of differentially expressed genes in ULV hearts demonstrated reduced cell proliferation in the CEC subpopulation. Here, we identified that CECs from HLHS patients exerted proliferative defects that can potentially impede the development of vascular/capillary structure and cause related functional deficiencies. Reformation of the coronary defect provides a promising therapeutic strategy to prevent HLHS deterioration.

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Abstract 12937: Single-cell Transcriptomic Analysis Reveals Developmentally Impaired Endocardial Population in Hypoplastic Left Heart Syndrome

Circulation ◽

10.1161/circ.142.suppl_3.12937 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Yifei Miao ◽

Lei Tian ◽

Marcy Martin ◽

Sharon Paige ◽

Francisco X Galdos ◽

...

Keyword(s):

Single Cell ◽

Hypoplastic Left Heart Syndrome ◽

Heart Development ◽

Heart Diseases ◽

Critical Role ◽

Abnormal Development ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Mesenchymal Transition ◽

Left Heart Syndrome

Hypoplastic left heart syndrome (HLHS) is one of the most challenging forms of congenital heart diseases. Previous studies were mainly focused on intrinsic defects in myocardium. However, this does not sufficiently explain the abnormal development of the cardiac valve, septum, and vasculature, known to originate from the endocardium. Here, using single-cell transcriptomic profiling, induced pluripotent stem cells (iPSC) derived endocardial cells (iEECs), human fetal heart tissue with underdeveloped left ventricle, as well as a Xenopus model, we identified a developmentally impaired endocardial population in HLHS. The intrinsic endocardial deficits contributed to abnormal endothelial to mesenchymal transition, NOTCH signaling, and extracellular matrix organization, all of which are key factors in valve formation. Consequently, in an endocardium-myocardium co-culture system, we found that endocardial abnormalities conferred reduced proliferation and maturation of iPSC derived cardiomyocyte (iPSC-CMs) judged by Ki67 staining, contractility, sarcomere organization, and related gene expressions through a disrupted fibronectin (FN1)-integrin interaction. Several recently described HLHS de novo mutations such as ETS1 and CHD7 showed reduced binding to FN1 promoter and enhancer in HLHS vs. control iEECs based on ChIP-qPCR analysis. Additionally, we found that suppression of the ETS1 in Xenopus caused reduced endocardial FN1 expression and impaired heart development. Supplementation of FN1 or ETS1 over-expression in HLHS iEECs could rescue dysfunctions in both endocardium and myocardium in HLHS. Our studies reveal a critical role of endocardial abnormality in causing HLHS, and provide a rationale for improving endocardial function in future regenerative strategies. Schematic illustration of the endocardial and myocardial defects in HLHS.

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The genetics of hypoplastic left heart syndrome

Cardiology in the Young ◽

10.1017/s1047951100005722 ◽

1999 ◽

Vol 9 (6) ◽

pp. 627-632 ◽

Cited By ~ 31

Author(s):

Paul D. Grossfeld

Keyword(s):

Hypoplastic Left Heart Syndrome ◽

Heart Defects ◽

Left Ventricular ◽

Left Ventricular Cavity ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Term Survival ◽

Surgical Options ◽

Systemic Ventricle ◽

Left Heart Syndrome

Hypoplastic left heart syndrome is one of the most therapeutically challenging congenital cardiac defects. It accounts for as many as 1.5% of all congenital heart defects, but is responsible for up to one quarter of deaths in neonates with heart disease.1The management of hypoplastic left heart syndrome is controversial. Two surgical options exist:2,3the Norwood procedure, is a three stage repair in which the morphologically right ventricle is converted to function as the systemic ventricle. Alternatively, orthotopic transplantation can be performed. Although both surgical options have had improved outcomes, the prognosis for long-term survival is guarded, with a five year survival for either approach reported to be in the region of 50–60%. In this review, I explore the evidence for a genetic etiology for the “classic” hypoplastic left heart syndrome, defined as mitral and/or aortic atresia with hypoplasia of the left ventricular cavity and the other left-sided structures.

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1212 LEFT VENTRICULAR AND CORONARY ARTERY MORPHOLOGY IN THE HYPOPLASTIC LEFT HEART SYNDROME

Pediatric Research ◽

10.1203/00006450-198104001-01238 ◽

1981 ◽

Vol 15 ◽

pp. 644-644

Author(s):

Willian O'Connor ◽

James Cash ◽

Carol Cottrill ◽

Gregory Johnson ◽

Jacqueline A Noonan

Keyword(s):

Coronary Artery ◽

Hypoplastic Left Heart Syndrome ◽

Left Ventricular ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Left Heart Syndrome

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Epidemiology of noncomplex left ventricular outflow tract obstruction malformations (aortic valve stenosis, coarctation of the aorta, hypoplastic left heart syndrome) in Texas, 1999-2001

Birth Defects Research Part A Clinical and Molecular Teratology ◽

10.1002/bdra.20169 ◽

2005 ◽

Vol 73 (8) ◽

pp. 555-561 ◽

Cited By ~ 52

Author(s):

Kim L. McBride ◽

Lisa Marengo ◽

Mark Canfield ◽

Peter Langlois ◽

David Fixler ◽

...

Keyword(s):

Aortic Valve ◽

Hypoplastic Left Heart Syndrome ◽

Left Ventricular Outflow Tract ◽

Coarctation Of The Aorta ◽

Ventricular Outflow Tract ◽

Left Ventricular ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Left Ventricular Outflow ◽

Left Heart Syndrome

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Hypoplastic left heart syndrome: Progression of left ventricular dilation and dysfunction to left ventricular hypoplasia in utero

American Heart Journal ◽

10.1016/0002-8703(92)90834-i ◽

1992 ◽

Vol 123 (6) ◽

pp. 1712-1713 ◽

Cited By ~ 55

Author(s):

David A. Danford ◽

Patrick Cronican

Keyword(s):

Hypoplastic Left Heart Syndrome ◽

In Utero ◽

Left Ventricular ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Ventricular Dilation ◽

Left Ventricular Dilation ◽

Left Heart Syndrome

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Assessment of Structural and Functional Abnormalities of the Myocardium and the Ascending Aorta in Fetus with Hypoplastic Left Heart Syndrome

BioMed Research International ◽

10.1155/2016/2616729 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9

Author(s):

Yan Jiang ◽

Yali Xu ◽

Jinliang Tang ◽

Hongmei Xia

Keyword(s):

Hypoplastic Left Heart Syndrome ◽

Left Ventricular ◽

Ascending Aorta ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Histological Changes ◽

Left Heart Syndrome ◽

Rv Function ◽

Normal Controls ◽

Pulmonary Artery Diameter

Aims. To detect anatomical and intrinsic histopathological features of the ascending aorta and left ventricular (LV) myocardium and evaluate right ventricular (RV) function in fetuses with hypoplastic left heart syndrome (HLHS).Methods. Twenty-five fetuses diagnosed with HLHS were followed up in the antenatal and postpartum periods. 12 necropsy heart specimens were analyzed for morphological and histological changes.Results. Prenatal echocardiography and pathologic anatomy displayed the typical characteristics of HLHS as a severe underdevelopment of the LV in the form of mitral stenosis or atresia or as aortic atresia or stenosis, with a decreased ratio of aortic diameter to pulmonary artery diameter (median of 0.49 with a range of 0.24 to 0.69,p≤0.001) and a higher ratio of RV diameter to LV diameter (median of 2.44 with a range of 1.33 to 6.25,p≤0.001). The RV volume, stroke volume, and cardiac output in HLHS fetuses were increased compared with the gestational age-matched normal controls (p<0.01). Histological changes in the 12 HLHS specimens included LV myocardial fibrosis, aortic elastic fragmentation, and fibrosis.Conclusions. In addition to severe anatomical deformity, distinct histological abnormalities in the LV myocardium and aortic wall were identified in the fetuses with HLHS. RV function damage may be potentially exists.

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Improvement in right ventricular function by mitral valve closure in hypoplastic left heart syndrome

Interactive Cardiovascular and Thoracic Surgery ◽

10.1093/icvts/ivab037 ◽

2021 ◽

Author(s):

Dai Asada ◽

Yoko Kawai ◽

Yoshinobu Maeda ◽

Masaaki Yamagishi

Keyword(s):

Mitral Valve ◽

Right Ventricular ◽

Ventricular Function ◽

Hypoplastic Left Heart Syndrome ◽

Right Ventricular Function ◽

Left Ventricular ◽

Valve Closure ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Left Heart Syndrome

Abstract A male neonate presented with the aortic/mitral stenotic variant of hypoplastic left heart syndrome, wherein the suprasystemic left ventricular pressure and relatively large left ventricle had shifted the intraventricular septum. Despite bilateral pulmonary artery banding, the stroke volume was difficult to maintain owing to the compressed right ventricle, causing heart failure symptoms. Percutaneous balloon aortic valvuloplasty decreased the left ventricular pressure, restoring the right ventricular function. Norwood procedure with mitral valve closure after catheter intervention reduced the left ventricular size and improved the right ventricular function. This paper refers to the potential of mitral valve closure for hypoplastic left heart syndrome.

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