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.
Highlights
Single ventricle (SV) physiology including hypoplastic left heart syndrome (HLHS) is characterized by obstruction of the left ventricle (LV) in association with absence or underdevelopment of the left ventricular chamber morphogenesis that is not capable of supporting systemic cardiac output
A number of studies have uncovered that heterozygous mutations in cardiac regulatory genes caused congenital heart defects in humans, the identified genetic variants may not be directly correlated with biological insights that potentially contribute to disease development [20]
The key regulatory mechanisms involved in early heart morphogenesis have been investigated extensively, but our understanding of the causal genes responsible for the development of such complex disease is still limited in humans [16]
Summary
Single ventricle (SV) physiology including hypoplastic left heart syndrome (HLHS) is characterized by obstruction of the left ventricle (LV) in association with absence or underdevelopment of the left ventricular chamber morphogenesis that is not capable of supporting systemic cardiac output. Infants with SV physiology theoretically undergo two major medical treatments: cardiac transplantation and three-stage palliative reconstruction [1,2]. Their long-term survival has been shown to be remarkably lower than that of patients with all other congenital heart diseases [3]. HLHS has been associated with chromosome anomalies, but no single genetic basis has been found to be linked to this syndrome [4]. The majority of cases are sporadic, rare patients who inherited multiple gene variants suggest that HLHS may be pathophysiologically heterogeneous and caused by cumulative effects that are poorly understood [5]
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