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Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency
Shiqiao Ye1,2, Cankun Wang3, Zhaohui Xu4,5
1Center for Cardiovascular Research, The Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH (S.Y., H.L., Y.Y., S.A., M.A., J.B., V.G., M.-T.Z.).
Insights
NOTCH1 is crucial for human ventricular cardiomyocyte development and proliferation. Its deficiency impairs first heart field progenitor differentiation, contributing to hypoplastic left heart syndrome.
Area of Science:
- Cardiovascular Biology
- Stem Cell Biology
- Developmental Biology
Background:
- NOTCH1 pathogenic variants are linked to congenital heart defects like hypoplastic left heart syndrome.
- The precise role of NOTCH1 in human cardiac cell lineage and cardiomyocyte proliferation remains unclear.
- Mechanisms underlying NOTCH1's role in ventricular hypoplasia are not well understood.
Purpose of the Study:
- To investigate the function of NOTCH1 in human cardiac cell differentiation and proliferation.
- To elucidate how NOTCH1 deficiency impacts cardiac mesoderm lineage determination.
- To understand the contribution of NOTCH1 variants to hypoplastic left heart syndrome.
Main Methods:
- Utilized CRISPR/Cas9 genome editing to create NOTCH1 knockout human induced pluripotent stem cells.
- Performed cardiac differentiation via WNT signaling modulation.
- Employed single-cell RNA sequencing at multiple time points to analyze transcriptomic changes.
Main Results:
- NOTCH1 knockout cells generated functional cardiomyocytes and endothelial cells, indicating no essential role in early mesoderm differentiation.
- NOTCH1 disruption blocked ventricular-like cardiomyocyte differentiation but promoted atrial-like cardiomyocyte generation.
- NOTCH1 deficiency resulted in impaired cardiomyocyte proliferation and biased cardiac mesoderm lineage determination towards epicardial and second heart field progenitors.
Conclusions:
- NOTCH1 is vital for human ventricular-like cardiomyocyte differentiation and proliferation by regulating cardiac mesoderm fate and cell cycle.
- Pathogenic NOTCH1 variants likely cause biased first heart field progenitor differentiation, hindering ventricular development.
- These NOTCH1-related defects contribute to the left ventricular hypoplasia observed in hypoplastic left heart syndrome.
Background:
NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive.
Methods:
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq.
Results:
Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations.
Conclusions:
NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.
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