Directed Differentiation of Patient-Specific Induced Pluripotent Stem Cells Identifies the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 in Hypoplastic Left Heart Syndrome

Abstract

The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.

Compound heterozygous NOTCH1 mutations underlie impaired cardiogenesis in a patient with hypoplastic left heart syndrome

“…While these data did not achieve statistical significance when comparing the mother and father, it is plausible that a single heterozygous mutation in NOTCH1 confers subtle changes in its expression and activity that require more sensitive assays and/or additional clones. Our findings are consistent with two previous studies that have reported reduced cardiomyocyte yields and a higher prevalence of myofibril disorganization in iPSCs derived from patients with HLHS compared to unrelated controls (Jiang et al 2014;Kobayashi et al 2014). Uniquely, our study is the first to model genetically defined HLHS with iPSC-derived cardiomyocytes using parental cells as controls, providing further evidence for impaired myogenic potential as a mechanism for HLHS.…”

Epigenetic modification in congenital heart diseases by using stem cell technologies

“…Some studies have proved the significant differences in differentiation potential among designed cells to differentiate into other types of cell [66][67][68] . In our experiments, we generated iPS cells from CPCs and differentiated them into cardiomyocytes [44] . This strategy might be reasonable, especially for research using patient-specific iPS cells, which are often resistant to differentiation.…”

“…Investigations of epigenetic regulation in patient-specific iPS cells during cardiac development might enable us to dissect the pathogenesis of congenital heart diseases. By using patient-specific iPS cells, we attempted to determine the epigenetic regulation during the cardiogenesis of HLHS, which is one of the severest congenital heart diseases with single ventricular circulation [44] . Some cases of HLHS are related to the mutation of cardiac-specific genes or associated chromosomal abnormalities; however, most of the cases are sporadic and the genetic basis has remained unknown.…”

Morphogenetic clarity in considerations of hypoplastic left heart syndrome

“…Supporting the concept of a broader developmental disorder that also affects the cardiomyocyte is work demonstrating fundamental differences in gene expression4 and function5 in cardiomyocytes generated from induced pluripotent stem cells derived from patients with HLHS. It will be some time before the extent of such functional deficits is fully characterised.…”