Figure 1.
Summary of differentially expressed genes (DEGs) in pathological hypertrophy (PAH) and physiological hypertrophy (PHH).
(A) Venn diagrams to show the number of expressed genes detected at least once for each biological replicate of sham, TAC, sedentary and exercise-training mouse models. (B) Comparisons of DEGs between the different mouse models. Genes having at least 2 RPKM value and differentially expressed at (p<0.05, |log1.5 fold change (FC)|≥1) were considered for comparisons.
Figure 2.
Network of the genes turned on in PAH.
(A) Top-scoring network derived from the 417 genes dramatically increased in PAH, despite their extremely low expression (<2 RPKM) in either normal adult heart or exercise-trained heart. The red-colored nodes represent up-regulated genes. (B) The histograms for the reads that were mapped by the UCSC isoforms of Plk1, Foxm1 and E2f1 in the different animal models. The blue lines underneath represent UCSC gene structures and the boxes show the exons of the genes. Expression of the 3 genes is only dominant in TAC. (C) The predicted motifs enriched in 1,000 bp upstream of 417 genes. The motifs shown on the bottom were predicted by MEME and the motifs shown above are the consensus motifs for the TFs (FOXM1 and PU.1). Y-axis indicates the amount of information at each position in the motif. The heatmaps (unit: RPKM) show the expression levels of the predicted TFs in sham (Sh), TAC (T), Se (Sedentary) and E (exercise). (D) Degree of expression (unit: RPKM) of the known targets of FOXM1 in the animal models are shown along with the previous evidence (PMID).
Table 1.
List of genes and isoforms derived from different alternative splicing variants in PAH and PHH.
Figure 3.
Experimentally verified isoforms alternatively spliced in PAH and PHH.
(A) The histograms of mapped reads for 8 genes. The red asterisks shown at UCSC isoforms (blue lines underneath) indicate the exons (shown as blue boxes) alternatively spliced in hypertrophic signal-specific manners. The degree of expression is shown as vertical length. (B) Distribution of UCSC isoforms for the matching 8 genes characterized in different hypertrophy models. Different distribution of isoforms in either PAH or PHH was analyzed using NEUMA. The average RPKM values are shown with standard errors (N = 3). (C) Experimental confirmation of exon variants for the matching 8 genes using RT-PCR. For each RT-PCR results, the amplified regions with the specific primers are described. The detailed information for the primers used for the detection of the exons is available in Table S2. Fhl1, Rcan1, Ndrg2, Synpo and Ttll1 were experimentally confirmed in PAH and the splicing pattern of Cxxc5 exon variants was confirmed in PHH. For Egfl7 and Tmpo, the opposite splicing patterns were confirmed for both PAH and PHH. The isoform distributions of Ttll1 and Cxxc5 were not analyzed, since the UCSC isoforms for Ttll1 is not reported and there were no mappable reads for Cxxc5-2. The exon numbers amplified are indicated on the right. For instance, E5∼6 means exon 5 and 6 were amplified with specific primers shown in Table S2. For Ndrg2, two isoforms (E2∼5, +E3 and E2∼5, −E3) were shown in the same gel.
Figure 4.
Critical pathways associated with DEGs and exon variants changed in PAH or PHH.
Significantly enriched pathways (p<0.05) with at least 5 DEGs or 5 exon variants for PAH and PHH are shown. Color intensity represents degree of enrichment (−log10[p-value]). The significant pathways were categorized. (Group I) pathways for muscle contraction and metabolism. (Group II) pathways mainly for immune and cell cycle. (Group III) pathways for autoimmunity. (Group IV) pathways for cell signaling. (Group V) pathways mainly for cardiac diseases.