Transcriptional analysis reveals that the intracellular lipid accumulation impairs gene expression profiles involved in insulin response-associated cardiac functionality

Figures

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  Figure 1. Bulk RNA-Seq was performed on HL-1 cardiomyocytes. The cells were either stimulated with aggLDL 100 µg/mL for 8 h or vehicle (Ctrl) or stimulated with insulin 100 nM for 2 h. (a) Principal Component Analysis (PCA), (b) Expression matrix-based Pearson’s correlation analysis, and (c) heatmap representation and unsupervised hierarchical clustering of the top 100 most expressed genes in the transcriptome profiles. Four replicates were performed per condition.

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  Figure 2. Differentially expressed genes (DEG) analysis between HL-1 treated with aggLDL or insulin compared to vehicle (control) conditions. Left panel corresponds to aggLDL compared to control and right panel correspond to insulin compared to control. (a) Volcano plot shows DEGs with a |logFC| ≥ 2; p-value < 0.01; and FDR < 0.05. The number of up and downregulated genes is shown in the graph. (b) Gene Ontology (GO) (Biological Process) enrichment analysis. Red nodes indicate upregulated processes and blue ones down-regulated. The size and intensity of the colours of the nodes are proportional to the number of genes enriching this biological process. The lines connecting the nodes represent the biological processes related to each other.

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  Figure 3. Network diagram of the main intracellular processes affected. Genes involved in different intracellular processes are shown as rectangles in red (upregulated) or blue (downregulated) according to their expression from RNA-Seq profiles under different conditions, insulin 100 nM for 2 h or aggregated LDL 100 µg/mL for 8 h. Conditions are displayed at the top of each rectangle as black (insulin) or white (aggLDL) squares. The network was manually adjusted to show the main genes obtained in the unsupervised GO enrichment and WikiPathways analysis.

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  Figure 4. Quantitative RT-qPCR assay to evaluate Insr, Ins1, Pik3ip1, Slc2a4, Cacna1s, Gjc2, Myh3 and Abca1 mRNA expression in HL-1 cardiomyocytes treated with 100 μg/mL aggLDL for 8 h or insulin 100 nM for 2 h. Bar graph showing the mean ± SEM of mRNA levels, and represented as fold change as compared to the mean of control condition. *p < 0.05 are considered significant. The RT-qPCR products of Gapdh transcripts were used as a housekeeping gene loading control.

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  Figure 5. Quantitative RT-qPCR assay to evaluate Insr, Ins1, Pik3ip1, Slc2a4, Cacna1s, Gjc2, Myh3 and Abca1 mRNA expression in the heart of ApoE-KO or wild-type (Wt) mice of 6 months of age. Bar graph showing the mean ± SEM of mRNA levels, and represented as fold change as compared to the mean of Wt group. *p < 0.05 are considered significant. The RT-qPCR products of Gapdh transcripts were used as a housekeeping gene.