Transcriptional changes during isoproterenol-induced cardiac fibrosis in mice

Figures

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  FIGURE 1. Induction of cardiac hypertrophy with a subcutaneous dose of isoproterenol. (A) Overview of the experimental setup where 12-week-old C57BL6 male mice were administered with 50 mg/kg/day isoproterenol for 4, 8, and 11 days, and the heart samples were collected for histology and RNA isolation. (B) Representative images of the hearts that were collected at different time points after isoproterenol treatment. (C) Graph showing heart weights normalized to body weight (BW) and tibia length (TL) at definitive timepoints. (D) Representative WGA staining and cardiomyocyte size analysis. (E) Picrosirius red staining indicative of ECM remodeling among the three groups and its quantification (n>=3) (marked in white arrow). (F) qRT PCR showing the levels of heart failure markers like ANP and β-MHC. * p value < 0.05, ** p value < 0.01, *** p value < 0.001, and **** p value < 0.0001. One way ANOVA followed by Dunnett’s test/Fishers test was performed to compute statistical differences between saline and isoproterenol groups.

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  FIGURE 2. Transcriptomic analysis of the isoproterenol-treated hearts. RNA sequencing shows differential expression of common genes (DEGs) across the three groups. (A) Heatmap showing the DEGs across the three timepoints compared to control (LFC = 0, p < 0.05). (B) Gene Ontology showing the common pathways affected during isoproterenol-induced cardiac remodeling.

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  FIGURE 3. Time-course analysis of DEGS (A) Time-course analysis shows significant changes in gene expression over time. The gene expression profile was grouped into four clusters with distinct temporal profiles. (B) Gene Ontology (GO) of the genes obtained from the aforementioned four clusters. (C) Heatmap depicting the gene expression patterns of few pathways from each clusters.

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  FIGURE 4. Expression profile of novel long non-coding RNA transcripts. (A) Schematic representation of the novel lncRNA prediction pipeline. (B) RNA sequencing shows differential expression of different novel lncRNAs across three groups. (C) Analysis of coding potential of Isolncs by CPAT compared to known coding and non-coding RNAs. (D) qRT PCR validation of Isolncs 2 and 4 in isoproterenol-treated animals. * p value < 0.05, ** p value < 0.01, and *** p value < 0.001. One-way ANOVA followed by Dunnett’s test was performed to compute statistical differences between saline and isoproterenol groups.

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  FIGURE 5. UCSC screenshots of ATAC-seq and qRT-PCR showing expression patterns of Isolncs 2 and 4 in different tissues and subcellular fractions. (A) UCSC Genome Browser (mouse mm10) screenshots using “open chromatin track” showing ATAC sequencing across the heart at E11 to E16 and P0 along with the liver and stomach. (B) Tissue profiling of Isolncs 2 and 4 in adult mice. (C) Expression of the novel Isolncs in a subcellular fraction of the heart (CM vs. CF).