Determination of the human cardiomyocyte mRNA and miRNA differentiation network by fine-scale profiling

Abstract

To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation. Nevertheless, analysis of cells continuously cultured for 120 days indicated that the cardiomyocytes continued to mature toward a more adult-like gene expression pattern. Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed an miRNA pattern indicative of stem cell to cardiomyocyte specification. A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a cardiomyocyte differentiation miRNA network and identified putative mRNAs targeted by multiple miRNAs. Together, these data reveal the miRNA network in human heart development and support the notion that overlapping miRNA networks re-enforce transcriptional control during developmental specification.

FIG. 1.

The differentiation time course from human-induced pluripotent stem cells (hiPSCs) to cardiomyocytes. Three independent differentiations were performed (Run 1, 2, and 3), and RNA was sampled at days 0, 3, 7, 10, 14, 20, 25, 35, 45, 60, 90, and 120 days. (A) Dendogram of all independent samples derived from unsupervised hierarchical clustering. Arrowhead shows location of important bifurcation. (B) Heat map of expression of the 298 differentially expressed genes.

FIG. 2.

Cardiomyocytes continue to mature in extended culture. (A) R-squared values were computed between each of the 5 human heart RNAs and the differentiation days for the 203 heart-specific genes. Note the continued increase in R-squared after day 28. (B) Direct comparison of the heart-specific genes between a single total heart sample (total 1) and day 28 and day 120 of differentiation. (C) Analysis of 4 genes that continued to increase toward the adult level of expression in extended culture: CASQ2, CKMT2, COX6A2, and CRYAB. Shown are the array signal intensities for each of the days of differentiation and the 2 total adult heart RNAs. Arrowheads highlight day 28 expression. (D) Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmation of the 4 genes. Expression is relative to PPIA. Each bar represents 16 wells (average±standard error of the mean).

FIG. 3.

miRNA expression profiles during cardiomyocyte differentiation. (A) Dendogram of all independent samples derived from unsupervised hierarchical clustering. Arrowhead shows location of important bifurcation. (B) The expression profile of the pluripotency-associated hsa-mir-302 cluster. (C) The expression profile of miRNAs involved in mammalian heart development (hsa-mir-1, hsa-mir-133a/b, hsa-mir-208a/b, hsa-mir-143, and hsa-mir-145).

FIG. 4.

The cardiomyocyte miRNA differentiation network. Differentially expressed miRNAs and mRNAs were identified, and the computationally predicted miRNA/mRNA targets were computed for anticorrelated sets. The shared target overlap between miRNAs was calculated. (A) Heat map of expression of the 119 miRNAs that were differentially expressed segregates into 2 groups: pluripotent and cardiomyocyte differentiated. (B) The network of miRNAs in cardiomyocyte differentiation. Two large, independent networks were assembled—1 pluripotent and 1 differentiated. Shown are the top 5% of overlaps, with the top 1% shaded red.