RNA Sequencing Analysis of Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Congenital Heart Disease Patients

Congenital heart disease (CHD) is the most common congenital defect in newborns and remains the major cause of long-term morbidity and mortality among these patients.¹ Here, we generated induced pluripotent stem cell (iPSC) lines from 5 patients with single ventricle disease (SVD), 5 patients with tetralogy of Fallot (TOF), and 5 healthy individuals (non-CHD). We analyzed the mutation profile of these patients using a custom AmpliSeq panel and found no significant pathogenic mutations associated with CHD. We then differentiated all iPSC lines into cardiomyocytes (iPSC-CMs) as previously described² and found no significant difference in differentiation efficiency among the three groups (Fig. 1A).

Figure 1: Transcriptomic analysis of iPSC-CMs from patients with congenital heart disease.

(A) Schematic showing generation of iPSC-CMs and the experimental workflow. HLHS: hypoplastic left heart syndrome; TA: tricuspid atresia; DORV: double-outlet right ventricle; DILV: double inlet left ventricle. (B) PCA of gene expression levels in 5 non-CHD-iPSC-CMs (black), 5 SVD-iPSC-CMs (orange), and 5 TOF-iPSC-CMs (green). (C) Heatmap plot of all DEGs across all samples. (D) GO biological process analysis of DEGs in SVD-iPSC-CMs and TOF-iPSC-CMs. (E) Regulatory gene network in SVD-iPSC-CMs showing the relationship between the enriched TF (circle in pink) and their target genes (small nodes in light blue). (F) Venn diagram depicting the number of up-regulated and down-regulated genes in TOF-iPSC-CMs and TOF-heart tissue.

After metabolic selection, we then used highly enriched iPSC-CMs for RNA sequencing (RNA-seq). Due to modest difference between TOF-iPSC-CMs and non-CHD-iPSC-CMs, we found no clear segregation between CHD-iPSC-CMs and non-CHD-iPSC-CMs. However, analysis of variance shows significantly larger transcriptomic difference between SVD-iPSC-CMs and non-CHD-iPSC-CMs (p < 2.2e-16) (Fig. 1B).

We further examined the gene expression patterns in SVD-iPSC-CMs and TOF-iPSC-CMs compared to non-CHD iPSC-CMs. Using the threshold of fold-change > 2.0 and a false discovery rate-adjusted p-value (FDR-adj. p value) < 0.05, we obtained 919 differentially expressed genes (DEGs) in SVD-iPSC-CMs and 250 DEGs in TOF-iPSC-CMs. Hierarchical clustering of all DEGs in the heatmap shows similar results with the PCA (Fig. 1C). Interestingly, the top DEG-enriched biological processes in SVD-iPSC-CMs were related to development. We obtained similar but modest enrichment in developmental processes in TOF-iPSC-CMs (Fig. 1D).

Finally, we performed transcription factor (TF) binding site enrichment analysis and identified several TF binding motifs, including interferon regulatory factor (IRF), TGFβ-induced factor (TGIF), and NF-κB, enriched in SVD-iPSC-CMs (Fig. 1E). However, we found a modest enrichment of TFs’ motifs in TOF-iPSC-CMs.

The major limitations of our approach include the lack of three-dimensional environmental cues and absence of non-myocyte cardiac cells to simulate heart development in vivo. Indeed, we found a limited overlap of DEGs between TOF-iPSC-CMs and TOF heart tissue obtained from GEO (n = 18, GSE36761) (Fig. 1F). In addition, we performed bulk RNA-seq on terminally differentiated cardiomyocytes; therefore, our data do not have enough resolution to identify transcriptome at the level of cellular subpopulations and do not address the question whether the identified DEGs play causal roles in the development of CHD. Nevertheless, our results suggest that the CHD patient-specific cardiomyocytes have abnormalities at least at the transcriptomic level regardless of their hemodynamic status.

In conclusion, we provide genome-wide transcriptomic profiles of iPSC-CMs generated from CHD patients. We found that both SVD-iPSC-CMs and TOF-iPSC-CMs express distinctive transcriptomes compared to non-CHD-iPSC-CMs, but the significance of these findings and their correlation with the disease pathogenesis remain to be investigated. Further efforts are needed to utilize patient-specific iPSC-CMs as an effective tool for the CHD research.