Characterization of Cardiomyocyte Differentiation Involving HLHS hiPS Cells Harboring a Mutation in the Promoter Region of NOTCH1

Student: Eric Baughman
Mentor: Frank Secreto (Mayo Clinic)

Hypoplastic Left Heart Syndrome is a congenital heart defect in which the left side of the heart fails to develop and function properly. Specific and definitive causes are yet to be identified with the disease, but studies of certain families, such as the 2H and 14H families, can provide insights into these relations. In the 14H family, the mother has a mutation in the gene for NOTCH1, which is vital during heart development, and she passed this mutation on to her child. Through the use of patient derived stem cells, called human induced pluipontent stem cells, we differentiated the cells into beating heart muscle under different conditions to test the influence of this specific mutation, and thus the overall gene, to the development of HLHS.

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect in which associated genetic mutations have yet to be established. The 14H family is of particular interest due to the afflicted child (proband) having inherited a single base pair mutation in the promoter region of NOTCH1. Notch/Activin/NO signaling is critical for cardiac development, and studying the potential dysregulation may provide an underlying mechanism explaining the development of HLHS. Previous studies conducted in the Nelson lab on 2H family indicated that human induced pluripotent stem cells (hiPSCs) carrying a mutation in the ORF of NOTCH1 failed to differentiate into cardiomyocytes without NO supplementation.  Based off this study design, we differentiated 14H family hiPSCs using spermine NONOate, an NO donor, and sildenafil, a protector of Notch/Activin/NO signaling. Beating activity was unaffected by the drugs in any of the clones.  However, no beating activity was recorded in the absence of Wnt modulation. Expression of genetic marker expression by RT-qPCR supported these conclusions, specifically that modulation of Wnt signaling enhanced cardiomyocyte differentiation.  Immunoblotting assays targeting NOTCH1 and the downstream Notch/Activin/NO pathway components Akt and eNOS showed no proband-specific differences compared to hiPSCs and hiPSC derived cardiomyocytes.  Overall, this pilot study demonstrated that the mutation present was insufficient to affect NOTCH1 protein or RNA expression as well as beating activity.  However, before a conclusion can be made regarding this mutation’s potential effect on cardiomyocyte and HLHS development, a broader study needs to be undertaken.