Shared splicing dysregulation in heart failure associated with dilated and ischemic cardiomyopathy and spatial specificity across cardiac regions

Figures

• 

  Figure 1. Study design. (A) Cardiac samples were collected from 54 end-stage heart failure patients with dilated cardiomyopathy (DCM) and 45 patients with ischaemic cardiomyopathy (ICM), along with 23 samples from non-failing control hearts. (B) RNA-seq data was generated from the left ventricle of 10 DCM, 11 ICM patients and 5 controls. The formula used to calculate the Percent Spliced In (PSI) value is indicated. (C) Selected splicing events in the indicated genes were quantified by qRT-PCR in all available samples. Primer pairs (arrows) were designed to amplify transcripts that include or skip the differentially spliced target exon. Expression levels were normalized to a constitutive exon present in all transcript isoforms of the gene. (D) Dysregulated splicing events detected in the left ventricle (LV) were analysed by qRT-PCR in matched samples from the right ventricle (RV) and interventricular septum (IVS).

• 

  Figure 2. RNA-seq analysis using rMATS identifies shared splicing changes in DCM and ICM hearts. (A) Principal component analysis (PCA) based on PSI values demonstrates clustering of DCM and ICM splicing profiles, distinct from controls (n = 10 DCM, n = 11 ICM and n = 5 controls). (B) Distribution and types of events identified as differentially spliced in pairwise comparisons between controls and diseased groups (DCM and ICM). Differential splicing events were defined using a false discovery rate (FDR) < 0.05 and an absolute change in Percent Spliced In (ΔPSI) > 0.1. (C) UpSet plot showing the percentage of differentially spliced events identified in pairwise comparisons: DCM vs. Control, ICM vs. Control, and DCM vs. ICM, as well as the subset of events shared between DCM and ICM when compared to Control. (D) Pearson's correlation analysis of ΔPSI values between DCM and ICM relative to controls, indicating a high degree of overlap in splicing changes. (E) Pathway enrichment analysis of genes containing differentially spliced events identified in DCM and ICM compared to controls, as well as in the subset of events shared between both cardiomyopathy subtypes.

• 

  Figure 3. Gene expression dysregulation in heart failure. (A) Principal Component Analysis (PCA) based on rlog-transformed count data. Analysis was performed using the 1000 genes with the highest variance across samples. (B) Number of differentially expressed genes identified for each pairwise comparison between controls, DCM, and ICM samples. (C) Venn diagram illustrating the overlap between differentially expressed genes and genes containing at least one significant splicing event, as detected by rMATS. (D) Normalized gene expression (log2) of a curated list of cardiac-associated splicing factors. (E) Log-transformed, normalized expression values of RBM20 calculated using DESeq2's median of ratios method and levels of RBM20 mRNA relative to U6 snRNA measured by qRT-PCR. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests, followed by Games-Howell's multiple comparisons test. Differences were not statistically significant, ns (P > 0.05). (F,G) RNA-binding protein enrichment analysis using rMAPS2 for differentially spliced events in DCM (F) and ICM (G) for QKI. (H) Western blot analysis of QKI protein levels in human heart tissue. Representative Western blot…

• 

  Figure 4. Validation of selected dysregulated splicing events. For each mRNA, the dysregulated alternative exon is highlighted. In ESRRG, an arrow denotes the transcription start site (AFE, alternative first exon). Left panels: Sashimi plots. Middle panels: PSI values from RNA-seq data. Right panels: qRT-PCR quantification of target exon inclusion normalized to the level of a constitutive exon from the same gene (Figure 1C) across the entire cohort (n = 54 DCM, n = 45 ICM and n = 23 controls), with statistical significance indicated as follows: ns (P > 0.05), *P < 0.05, **P < 0.01 and ****P < 0.0001. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests to account for unequal variances, followed by Games-Howell's multiple comparisons test.

• 

  Figure 5. Dysregulated splicing across cardiac regions. For each mRNA, the dysregulated alternative exon is highlighted. Results of qRT-PCR analysis from the indicated regions of the heart (21 controls, 25 DCM, and 15 ICM patients). Statistical significance is indicated as follows: ns (P > 0.05), *P < 0.05, **P < 0.01, and ****P < 0.0001. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA tests to account for unequal variances, followed by Games-Howell's multiple comparisons test.