Callie Cunningham

 Background: Hypoplastic Left Heart Syndrome (HLHS) is the leading cause of death due to cardiac defects for infants in the first year of life¹. Currently, long term outcomes for HLHS are poor; despite a 3-staged palliation surgery protocol there is a 50% failure rate by adulthood.  HLHS is a multifactorial disease with various genetic changes, chromosomal abnormalities, environmental influences, and epigenetic factors proposed as potential causes. This is due to, at least in part, a lack of successful animal models for HLHS.¹ Much of the recent knowledge surrounding HLHS has developed through studies using induced pluripotent stem cells^1. It is known that the NOTCH signaling pathway plays a role in the development of hypoplastic left heart syndrome in some cases^2, however the exact mechanism of this impact is not agreed upon. Some studies have implicated NOTCH’s role in cardiac valve development², the epithelial to mesenchymal transition³, cardiomyocyte development⁴, and trabeculation⁵. A better understanding of the genetic causes of HLHS has the potential to improve long term outcomes through therapeutic strategies that may be implemented alongside surgical intervention⁶.

Objective: In this narrative review, the role of NOTCH signaling on HLHS was explored in the context of how this understanding may inform future intervention.

Methods: An online search in the PubMed database was conducted from 2017 to 2023 using the following keywords: “hypoplastic left heart syndrome”, ” NOTCH”, ” etiology”, “genetics”.

 Results: Studies confirm the role of NOTCH signaling in HLHS phenotypes. In a study using whole exome gene sequencing, the only identified variant with a MAF of <1% was a premature stop codon in the LNR region of NOTCH 1 which encodes a transmembrane receptor.^7. This gene mutation demonstrated variable penetrance as it was present in a non-impacted family member as well. NOTCH 1, 3, and 4 signaling genes and NOTCH 2 and 4 receptor genes were implicated in an additional study, indicating that impactful NOTCH mutation is not limited to the NOTCH 1 pathway.⁸

In a study using induced pluripotent stem cells (iPSCs), it was noted that lower transcript levels of NOTCH 1 as well as downstream targets HES1 and HEY1 were present in HLHS probands.⁴ Custom conditions with increased Activin A growth factor were required for these HLHS proband iPSCs to differentiate into cardiomyocytes. When differentiated, they demonstrated disorganization as compared to samples derived from parent iPSCs. The beating activity and sarcomeres could be rescued through nitric oxide supplementation with Spermine Nonoate or Sildenafil. Beating activity of these cardiomyocytes could also be improved by exposure to a 5% O₂ environment, additional study confirmed reduced cardiomyocyte yield from iPSCs derived tissue with impaired NOTCH 1 signaling⁸. Cardiomyocytes from these samples demonstrated a reduced beating rate. Additionally, the iPSCs produced myofibroblasts in place of cardiomyocytes, stiffened ventricular walls with reduced contractility and conductance.  In a study using zebrafish models, hemodynamic shear stress was shown to induce NOTCH signaling and resultant gene expression⁵. Upregulating NOTCH signaling through sheer stress resulted in improved trabecular network and a more pronounced contractile ventricle. A phase II clinical trial demonstrated the ability to inject patient’s own stem cells during the Glenn surgery with no adverse effects, delays, or complications.⁹

 Conclusions:

While the etiology of HLHS is varied and elusive, for a specific subset of patients, impaired NOTCH signaling is shown to play a role in several different areas of cardiogenesis. The function of cardiomyocytes impacted by a NOTCH1 mutation can be rescued by nitric oxide in vitro⁴. If this result holds in vivo, there is potential for patient specific intervention through stem cell therapy during staged palliation surgeries. Using a patient’s own stem cells could inform these decisions through iPSC intervention. A better understanding of the role of NOTCH mutations in valvular development is needed³. Additionally, increased understanding of the mechanism of O₂ deprivation on cardiomyocyte beating rate rescue⁴ is an interesting direction for future research.

Works Cited:

1. Bejjani, A. T., Wary, N., & Gu, M. (2021). Hypoplastic left heart syndrome (HLHS): molecular pathogenesis and emerging drug targets for cardiac repair and regeneration. Expert opinion on therapeutic targets, 25(8), 621–632. https://doi-org.srv-proxy1.library.tamu.edu/10.1080/14728222.2021.1978069
2. Hall, B., Alonzo, M., Texter, K., Garg, V., & Zhao, M. T. (2022). Probing single ventricle heart defects with patient-derived induced pluripotent stem cells and emerging technologies. Birth defects research, 114(16), 959–971. https://doi-org.srv-proxy1.library.tamu.edu/10.1002/bdr2.1989
3. Miao Y, Tian L, Martin M, et al. Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome. Cell Stem Cell. 2020;27(4):574-589.e8. doi:10.1016/j.stem.2020.07.015
4. Hrstka SC, Li X, Nelson TJ; Wanek Program Genetics Pipeline Group. NOTCH1-Dependent Nitric Oxide Signaling Deficiency in Hypoplastic Left Heart Syndrome Revealed Through Patient-Specific Phenotypes Detected in Bioengineered Cardiogenesis. Stem Cells. 2017;35(4):1106-1119. doi:10.1002/stem.2582
5. Messerschmidt V, Bailey Z, Baek KI, et al. Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart. J Vis Exp. 2018;(138):57763. Published 2018 Aug 10. doi:10.3791/5776334
6. Birla, A. K., Brimmer, S., Short, W. D., Olutoye, O. O., 2nd, Shar, J. A., Lalwani, S., Sucosky, P., Parthiban, A., Keswani, S. G., Caldarone, C. A., & Birla, R. K. (2022). Current state of the art in hypoplastic left heart syndrome. Frontiers in cardiovascular medicine, 9, 878266. https://doi-org.srv-proxy1.library.tamu.edu/10.3389/fcvm.2022.878266
7.  Durbin MD, Cadar AG, Williams CH, et al. Hypoplastic Left Heart Syndrome Sequencing Reveals a Novel NOTCH1 Mutation in a Family with Single Ventricle Defects. Pediatr Cardiol. 2017;38(6):1232-1240. doi:10.1007/s00246-017-1650-5
8. Yang C, Xu Y, Yu M, et al. Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis. Hum Mol Genet. 2017 Aug 15;26 (16):3031–3045.
9. Burkhart HM, Qureshi MY, Rossano JW, et al. Autologous stem cell therapy for hypoplastic left heart syndrome: Safety and feasibility of intraoperative intramyocardial injections. J Thorac Cardiovasc Surg. 2019;158(6):1614-1623. doi:10.1016/j.jtcvs.2019.06.001