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. 2019 Mar 15;20(1):219.
doi: 10.1186/s12864-019-5550-3.

Cardiomyogenic differentiation is fine-tuned by differential mRNA association with polysomes

Isabela Tiemy Pereira  1 , Lucia Spangenberg  2 , Anny Waloski Robert  1 , Rocío Amorín  2 , Marco Augusto Stimamiglio  1 , Hugo Naya  2 , Bruno Dallagiovanna  3
Affiliations

Cardiomyogenic differentiation is fine-tuned by differential mRNA association with polysomes

Isabela Tiemy Pereira et al. BMC Genomics. .

Abstract

Background: Cardiac cell fate specification occurs through progressive steps, and its gene expression regulation features are still being defined. There has been an increasing interest in understanding the coordination between transcription and post-transcriptional regulation during the differentiation processes. Here, we took advantage of the polysome profiling technique to isolate and high-throughput sequence ribosome-free and polysome-bound RNAs during cardiomyogenesis.

Results: We showed that polysome-bound RNAs exhibit the cardiomyogenic commitment gene expression and that mesoderm-to-cardiac progenitor stages are strongly regulated. Additionally, we compared ribosome-free and polysome-bound RNAs and found that the post-transcriptional regulation vastly contributes to cardiac phenotype determination, including RNA recruitment to and dissociation from ribosomes. Moreover, we found that protein synthesis is decreased in cardiomyocytes compared to human embryonic stem-cells (hESCs), possibly due to the down-regulation of translation-related genes.

Conclusions: Our data provided a powerful tool to investigate genes potentially controlled by post-transcriptional mechanisms during the cardiac differentiation of hESC. This work could prospect fundamental tools to develop new therapy and research approaches.

Keywords: Cardiomyogenesis; Polysome profiling; Post-transcriptional regulation; hESC-derived cardiomyocytes.

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Figures

Fig. 1
Fig. 1
Polysome profiling of hESC during cardiomyogenic differentiation. (a) Schematic representation of cardiomyogenic differentiation protocol indicating developmental stages and timing of sample collection. (b) Flow cytometry analysis of D4 (CD56), D9 (eGFP) or D15 (cTnT and eGFP) differentiating cells. Representative dot plots (n = 3). (c) Representative images of EBs during differentiation showing NKX2–5/eGFP expression on D15 (top panel) and immunostaining of cTNI on D20 (bottom panel). Cells were co-stained with DAPI to visualize the nucleus. (d) Representative polysome profile obtained by sucrose gradient of cells at distinct time-points of differentiation (n = 3). Ribosome-free (red) and polysome (blue) fractions are indicated. Correspondence analysis (COA) of (e) all sequenced samples (total 30 samples), (f) ribosome-free and (g) polysome-bound samples at days D0, D1, D4, D9 and D15 (n = 3). See also Additional file 2: Figure S1
Fig. 2
Fig. 2
Polysome-bound RNA-seq revealed massive regulation of developmental genes. (a) Expression of lineage marker genes quantitated by -log2 RPKM. Stage markers are color-coded. Blue: pluripotency. Green: mesoderm. Light red: cardiac progenitors. Dark red: cardiomyocytes. Uncolored: endoderm and ectoderm. (b) Numbers of differentially expressed genes at each differentiation time-point, compared to the preceding time-point (FDR < 0.05, − 2 > logFC> 2). Numbers of protein-coding and non-coding genes are also shown (bottom panel). (c) Gene Ontology analysis of EnrichR Biological Process (BP) enriched terms for up-regulated (FDR < 0.05, logFC> 2) genes during cardiomyocyte differentiation when compared to each preceding time-point. (d) Gene expression pattern clusters of pluripotency (top) and cardiac (bottom) related genes and their EnrichR BP enriched terms. Six terms with lower p-values are shown. See also Additional file 2: Figures S4 and S5
Fig. 3
Fig. 3
Mesoderm and cardiac progenitor commitment gene expression. (a) GO EnrichR BP enriched terms for D4 up-regulated (FDR < 0.05, logFC> 2) and D9 down-regulated genes (FDR < 0.05, logFC<− 2). Overlap is shown inside the bars for each term in each condition. (b) Venn diagram and GO EnrichR BP enriched terms of common D4 up-regulated (FDR < 0.05, logFC> 2) and D9 down-regulated genes (FDR < 0.05, logFC<− 2). Six terms with lower p-values are shown. (c) Gene expression pattern cluster of mesoderm-related genes and their GO EnrichR BP enriched terms. Six terms with lower p-values are shown
Fig. 4
Fig. 4
Post-transcriptionally regulated genes during cardiomyogenesis. (a) Gene categories based on the ribosome-free and polysome-bound comparison analysis. (b) Number of DEGs (FDR < 0.05, − 2 > logFC> 2, RPKM> 1) classified at the gene regulation categories. See also Additional file 2: Figure S6
Fig. 5
Fig. 5
Post-transcriptionally regulated genes during cardiomyogenic differentiation were involved in diverse biological processes. GO Reactome pathways enriched terms for up- (FDR < 0.05, logFC> 2, RPKM> 1) and down- (FDR < 0.05, logFC<− 2, RPKM> 1) buffered and loaded genes. Ten terms with lower p-values are shown
Fig. 6
Fig. 6
Non-DEGs showed differences on polysome recruitment and dissociation on D1 vs. D4 and D4 vs. D9. (a) Venn diagram and (b) GO EnrichR BP enriched terms of polysome recruitment (FDR < 0.05, logFC> 2) or dissociation (FDR < 0.05, logFC<− 2) for non-DEG based on polysome/ribosome-free ratio. (c) Polysome/ribosome-free ratio variation of the Notch and Wnt pathways genes during cardiomyocyte differentiation (FDR < 0.05, − 2 > logFC> 2). See also Additional file 2: Figure S8
Fig. 7
Fig. 7
Cardiomyocytes (D15) showed down-regulation of translation and RNA processing genes. (a) GO EnrichR BP RNA-related terms enriched for D15 down-regulated genes (FDR < 0.05, logFC<− 1) compared to hESC (D0). (b) Genes classified on RNA-related BP terms (a) according to the co-regulated, buffered and loaded classification as indicated. (c) KEGG pathway analysis of D15 down-regulated ribosomal proteins. Down-loaded proteins showed in solid color, down co-regulated proteins showed as a green outline. (d) Venn diagram and (e) GO EnrichR BP enriched terms of polysome recruitment (FDR < 0.05, logFC> 2) or dissociation (FDR < 0.05, logFC<− 2) for non-DEG based on polysome/ribosome-free ratio of D0 vs. D15. (f) Translation genes showed polysome/ribosome-free ratio decreasing on D15 (FDR < 0.05, logFC<− 2). (g) Representative images of D0 and D15 cells cultured with OPP to stain nascent proteins. (h) Quantification of Alexa488 fluorescence intensity (OPP incorporation) at the cytoplasm region around the nucleus. For each condition (D0 and D15), 1400 cells were randomly chosen for intensity analysis. Statistical analysis was performed using the Mann-Whitney test (nonparametric t test). ****p < 0.0001
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