RNA expression profiling of human iPSC-derived cardiomyocytes in a cardiac hypertrophy model

Abstract

Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.

Figure 1. Dose vs. response plot for hypertrophy markers.

RT-qPCR was used to measure the expression levels of (a) NPPB and (b) ACTA1 and NPPA as different ET-1 dose.

Figure 2. Cardiac hypertrophy marker expression.

Bar plot showing the expression levels for some of the canonical hypertrophy markers in ET1-CM when compared with control-CM. The y-axis represents the mean + SD log2 fold change values taken from triplicate control-CM and ET1-CM experiments.

Figure 3. PCA plot comparing in vitro hiPSC model with in vivo human myocardial biopsies.

Plot of first and second principal components (PC1 and PC2) for microarray expression data comparing ET-1 treated hiPSC-CMs with human myocardial biopsy with and without LVH data.

Figure 4. Differentially expressed known human miRNAs.

MA-plot showing the differentially expressed known human mature miRNAs between control-CMs and ET1-CMs. The red solid dots represent the significant differentially expressed (FDR< = 0.1 and 1.5 fold change) known human mature miRNAs. See also Table S4.

Figure 5. Known mature miRNA validation with RT-qPCR.

Comparison of RT-qpCR and miRNA-Seq derived log₂ fold change for a subset of known human mature miRNAs between control-CMs and ET1-CMs. The mean values taken from triplicate experiments are plotted with standard deviation error bars.