Revealing new mouse epicardial cell markers through transcriptomics

Abstract

Background: The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.

Methodology: Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.

Principal findings: Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.

Conclusion: This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.

Figure 1. Unsupervised analysis of epicardial gene expression.

A self organizing map was used as an unsupervised analysis tool to simplify complex expression information. B) x-axis are relative normalized expression of epicardium samples, y-axis are relative normalized expression of muscle samples. The four resulting clusters represent generally different expression patters rather than their absolute expression values. Cluster 1 contains genes that were higher in the epicardial samples than in the muscle controls. Clusters 2 and 3 contains genes that were similar in expression in epicardium and muscle. Cluster 4 contains genes that were higher in the muscle controls than in the epicardial samples. B) Starting from Cluster 1, we applied conditions as described in Materials and Methods to select 197 epicardial signature genes shown as a heat map. The top four rows are epicardial samples, and the lower four rows are muscle control samples, where green colour represents upregulation and red colour represents downregulation of a given gene.

Figure 2. Confirmation of epicardial gene expression.

A) qRT-PCR was used to confirm gene array data, normalized against 18 S expression. Statistical significance was tested using the student t-test. Expression levels for uroplakin 3b, basonuclin 1, proline rich 15, dermokine, Efemp1, and sulfatase were significantly upregulated in the epicardial samples. GAPDH, RMPS2, and CD5L serve as controls; GAPDH was expressed higher in muscle tissue while RMPS2 and CD5L showed no statistically relevant expression in the different samples as expected. B,C) Using immunohistochemical localization of glycoprotein M6A (B), dermokine (C), and basonuclin-1 (D) proteins in the epicardial or subepicardial layer. (* denotes statistical significance with p<0.05, **: p<0.005, ***: p<0.0005; Error bars are S.E.M., scale bars are 100 microns).

Figure 3. Unsupervised analysis of epicardial gene expression post infarction.

A) Self- organizing maps were used to derive six clusters two of which, (3 and 4) contain genes that are strongly regulated. Cluster 3, includes epicardial signature genes whose expression decreases sharply in the epicardium but remains low in muscle post-infarction. Cluster 4 contains epicardial signature genes that are largely unchanged in the epicardium but are increased in muscle post-infarction. B) Heat map of data in A).

Figure 4. Unsupervised analysis of epicardial gene expression changes post infarction in IGF-1 animals.

Self- organizing maps were used to derive six clusters of epicardial genes for IGF-1 overexpressing mice. Unlike for the wildtype animals, no cluster contained extensive expression changes post infarction.