Abstract
Primitive mammalian heart transforms from a single tube to a four-chambered muscular organ during a short developmental window. We found that knocking out global microRNA by deleting Dgcr8 microprocessor in Mesp1 cardiovascular progenitor cells lead to the formation of extremely dilated and enlarged heart due to defective cardiomyocyte (CM) differentiation. Transcriptome analysis revealed unusual upregulation of vascular gene expression in Dgcr8 cKO hearts. Single cell RNA sequencing study further confirmed the increase of angiogenesis genes in single Dgcr8 cKO CM. We also performed global microRNA profiling of E9.5 heart for the first time, and identified that miR-541 was transiently highly expressed in E9.5 hearts. Interestingly, introducing miR-541 back into microRNA-free CMs partially rescued their defects, downregulated angiogenesis genes and significantly upregulated cardiac genes. Moreover, miR-541 can target Ctgf and inhibit endothelial function. Our results suggest that microRNAs are required to suppress abnormal angiogenesis gene program to maintain CM differentiation.
Highlights
The heart is one of the first functional organs to form during mammalian development and is composed of multiple cell types, including cardiomyocytes (CMs), endothelial cells (ECs), smooth muscle cells (SMCs) and mesenchymal cells (MCs) such as fibroblasts (Buckingham et al, 2005; Xin et al, 2013)
Student’s t test was used to determine statistical significance: **P < 0.01, ***P < 0.0001. (E) Principle component analysis (PCA) of genes expressed in E8.5 and E9.5 control and Dgcr8 conditional knock-out (cKO) hearts. (F) Venn diagram showing 527 genes were downregulated from E8.5 to E9.5 in the heart, but became upregulated in E9.5 Dgcr8 cKO hearts compared with control hearts. (G) Gene Ontology analysis of 527 overlapped genes in (F). (H) Expression heatmap of angiogenesis genes that downregulated from E8.5 to E9.5 in the control hearts, but upregulated in E9.5 Dgcr8 cKO hearts compared with E9.5 control
We established an in vitro culture system of E9.5 heart cells from Dgcr8 cKO embryos and used it to dissect the function of individual miRNA
Summary
The heart is one of the first functional organs to form during mammalian development and is composed of multiple cell types, including cardiomyocytes (CMs), endothelial cells (ECs), smooth muscle cells (SMCs) and mesenchymal cells (MCs) such as fibroblasts (Buckingham et al, 2005; Xin et al, 2013). Many miRNAs, such as miR-1 and miR-133, have been shown to control CM maturation and function (Heidersbach et al, 2013; Ivey et al, 2008; Liu and Olson, 2010). Despite their interesting functions, knocking-out individual miRNA in mice rarely caused lethality (Liu and Olson, 2010), and very few showed severe phenotype at early embryonic stages possibly due to that miRNAs often function redundantly and exist at saturating levels (Wang et al, 2008b)
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