Morphometric, Hemodynamic, and Multi-Omics Analyses in Heart Failure Rats with Preserved Ejection Fraction

Figure 1. Experimental protocol and evaluation of heart function of Dahl salt-sensitive (DSS) rats. (A) A schematic representative of experimental protocol, where 7-week-old Dahl salt-sensitive (DSS) rats were fed a high-salt (n = 17) or low-salt diet for 7 weeks (n = 10). (B) Ejection fraction (EF (%) mean ± SE) in the two groups at the age of 7 and 14 weeks. (C) A schematic representative of Doppler-mode tracings in rat hearts, and (D) E/A and (E) E/E’ in the two groups at the ages of 7 and 14 weeks. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 2. Severely impaired systolic contractile function in free wall (FW) and interventricular septum (IVS) of high-salt (HS)-fed DSS rat hearts. (A–D) Representative images of radial strain curves in FW and IVS in low-salt (LS)-fed and HS-fed rat hearts at the age of 14 weeks. The color lines represent the radial strain curves corresponding to the six myocardial segments, and the black line refers to the averaged strain curve over free wall or interventricular septum, where the arrow points to the peak value. (E and F) Peak radial strains in FW and IVS in LS-fed and HS-fed rat hearts. (G–J) Peak longitudinal strains on the endocardium and epicardium of FW and IVS in LS-fed and HS-fed rat hearts (n = 10 in LS group and n = 15 in HS group). * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 3. Electrocardiogram analysis showed prolonged myocyte repolarization in HS-fed DSS rat hearts. (A–D) QRS width, PR interval, QT interval, and QTc interval in the two groups at the ages of 7 and 14 weeks (n = 10 in LS group and n = 12 in HS group). ** p < 0.01, **** p < 0.0001.

Figure 4. Histological evaluation showed increase in size and apoptosis of myocytes of HS-fed DSS rats. (A,B) Representative immunofluorescence images of myocytes stained by WGA and TUNEL (arrow refers to apoptotic cells) and (C,D) cross-sectional area of a myocyte and apoptosis rate (TUNEL-positive nuclei as percentage of total nuclei) in the two groups at the age of 14 weeks (n = 5 for each group). ** p < 0.01, *** p < 0.001.

Figure 5. Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) on dysregulated proteins identified by proteomics. (A) Heatmap of the proteome, where hierarchical clustering of samples (columns) and proteins (rows) is based on Pearson’s correlation coefficient to measure the distance and the mean to cluster the samples. (B) Gene Ontology (GO) analysis of 21 significantly downregulated and 59 significantly upregulated proteins. (C) Gene Set Enrichment Analysis (GSEA) of all detected proteins, where nodes represent significantly enriched (p < 0.01, q < 0.05) gene sets of canonical pathways from the molecular signature database (MSigDB). The colors of node borders refer to upregulated (red) and downregulated (blue) in HS group, while the node color represents the FDR q-values

Figure 6. GO analyses and validation of dysregulated proteins identified by proteomics. (A,B) GO analysis of a gene network constructed from 80 differential expression proteins (21 significantly downregulated and 59 significantly upregulated proteins). The color gradient of the cluster distribution network shows the p-value of each cluster with darker (orange) color for a lower p-value. (A) Molecular functions and (B) cellular components. (C) Western blot results showing expression levels of Itga9, Mff, and Map1lc3a in the two groups, where all data were normalized to Gapdh expression. (D) Statistical analysis of the intensity of the bands in Figure 6C using ImageJ software (n = 3 for each group). * p < 0.05.

Figure 7. GO, GSEA, and real-time PCR analyses on the dysregulated genes identified by transcriptome. (A) Heatmap of the transcriptome, where hierarchical clustering of samples (columns) and genes (rows) is based on Pearson’s correlation coefficient to measure the distance and the mean to cluster the samples. (B) GO Analysis of 465 significant downregulated and 142 upregulated genes. (C) Real-time PCR results showing expression levels of Btk, Tlr7, Plcb2, and Diaph3 in the two groups, where all data were normalized to Gapdh expression. Fold change for expression level comparison was calculated with 2^(-ddCT). Primers are listed in Supplementary Table S1. * p < 0.05. (D) GSEA of all detected genes, where nodes represent significantly enriched (p < 0.01, q < 0.05) gene sets of canonical pathways from the MSigDB. The colors of node borders refer to upregulated (red) and downregulated (blue) in HS group, while the node color represents the FDR q-values.