Identification of Differentially Regulated Pathways in Cardiac Development and Cardiac Gene Expression during In Vitro Cardiac Differentiation of HLHS-derived Human Induced Pluripotent Stem Cells using Transcriptome Analysis

Objectives: Little is known about molecular processes leading to the development of Hypoplastic Left Heart Syndrome (HLHS). To understand gene regulatory networks affected during early embryogenesis, we conducted a comparative transcriptome analysis between human induced pluripotent stem cell (hiPS) lines derived from HLHS patients and healthy controls.

Methods: HLHS (n = 3) and control (n = 3) iPS lines were differentiated using a chemically defined cardiac differentiation protocol. RNA samples were harvested at D0, D6, D8, D10 and D14. Barcoded RNA-Seq libraries were prepared by depleting ribosomal RNA, reverse transcription and adapter ligation. Paired-end sequencing of 2x 100 bp was performed using an Illumina HiScanSQ sequencer. Pre-processed reads were mapped to GRCh38 and counted by RefSeq annotation. Normalization of counts and calculation of differential expression used DESeq2. Pathway analyses (Ingenuity Pathway Analysis, QIAGEN Bioinformatics) considered differentially expressed genes with an absolute log2 fold-change ≥ 1.

Results: Principal components analysis (PCA) of HLHS and control samples revealed clustering at D0 with no difference in the two groups. PCA of HLHS and control samples during cardiac differentiation (D6 to D14) visualized parallel shifting over time. Pathway analysis showed down-regulation of canonical pathways starting at D8: LXR/RXR-Activation (Activation z-score: D8 = −2.0, D10 = −1.3, D14 = −2.2), Actin Cytoskeleton Signaling (z-score: D8 = −0.82, D10 = 0.38, D14 = −1.90) and Wnt/b-catenin Signaling (z-score: D8 = −1.89, D10 = −1.16). In contrast Protein Kinase A Signaling was up-regulated upon differentiation (z-score: D8 = 0.45, D10 = 0.73, D14 = 1.51). Several genes essential for cardiac development and function were dysregulated. Thus, down-regulation of disease related pathways as function of muscles (p = 6.25⁻⁴, D14), formation of muscle cells (p = 1.18⁻³, D14) and contractility of cardiac muscle (p = 1.04⁻², D14) was identified. Furthermore, pathways related to morphology of heart cells (p = 7.14⁻⁵, D14) and morphology of cardiomyocytes (p = 1.76⁻⁴, D14) were shown to be influenced during HLHS cardiac differentiation.

Conclusion: RNA-seq identified differentially regulated pathways essential for cardiac development, therefore possibly influencing HLHS development. In addition, genes regulating cardiogenesis and cardiac function showed altered expression in HLHS differentiation.