Deciphering the inhibitory effects of trimetazidine on pulmonary hypertension development via decreasing fatty acid oxidation and promoting glucose oxidation

Figures

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  Fig. 1. Trimetazidine’s (TMZ) inhibitory effects on the cardiac dysfunction in the Su/Hx-induced rat pulmonary hypertension (PH) model. (A) Schematic representation of the experimental design and the timeline of the experimental procedures. (B–H) TMZ’s hemodynamic effects for PH in the SU5416-Hypoxia with vehicle administration (Su/Hx) rats. Right ventricular systolic pressure (RVSP) (B), Ees/EA ratio (C), RV maximum pressure (dP/dt max) (D), RV minimum pressure (dP/dt min) (E), Heart Rate (HR) (F), Stroke volume (G), and Cardiac index (H). Data are presented as mean ± standard errors of the mean for n = 3–8 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s post-hoc test.

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  Fig. 2. Improvement of right ventricular (RV) hypertrophy and remodeling using trimetazidine (TMZ) treatment in the Su/Hx-induced rat pulmonary hypertension (PH) model. (A) RV weight-to-left ventricular (LV) plus septum (S) weight ratio (RV/(LV + S)). (B) Histological analysis of the right heart. Representative photographs of hematoxylin and eosin (H&E) (upper panel) and Masson’s trichrome (lower panel) stains in the RV free wall of the control (CTRL), SU5416-Hypoxia with vehicle administration (Su/Hx), and Su/Hx with TMZ administration (Su/Hx + TMZ) groups (scale bars, 50 μm). (C) Quantified cardiomyocyte RV cross-sectional area in the H&E-stained sections. (D) Quantified fibrosis of the RV free wall (blue-stained areas expressed as the percentage of total RV surface area) in the Masson’s trichrome-stained sections. Data are presented as mean ± standard errors of the mean for n = 3–6 per group. * p < 0.05, ** p < 0.01, **** p < 0.0001, and ns (not significant). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s post-hoc test.

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  Fig. 3. Metabolic changes in the right heart of the Su/Hx rats after trimetazidine (TMZ) treatment. (A) The principal component analysis (PCA) score plot of the metabolic profiles of SU5416-Hypoxia with vehicle administration (Su/Hx) (red) and Su/Hx with TMZ administration (Su/Hx + TMZ) (green) samples. (B) The variable influence on projection (VIP) score plot of each distinctive metabolite (VIP > 1 and Q-value < 0.05) between the two groups. (C) Enrichment analysis based on the human metabolic pathways in the Small Molecule Pathway Database (SMPDB) that displayed substantial variation in the Su/Hx and Su/Hx + TMZ groups. (D, E) Concentration of long chain fatty acids (LCFA), palmitic acid (D), and stearic acid (E). Data are presented as mean ± standard errors of the mean for n = 4–5 per group. * p < 0.05. Statistical analysis was performed using an unpaired two-tailed t-test.

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  Fig. 4. Attenuation of the pulmonary vascular remodeling with trimetazidine (TMZ) administration. (A) Representative Elastica van Gieson (EVG)-stained images of the pulmonary arteries of control (CTRL), SU5416-Hypoxia with vehicle administration (Su/Hx), and Su/Hx with TMZ administration (Su/Hx + TMZ) groups (scale bars, 50 μm). (B) Quantified data from pulmonary vascular remodeling. Data are presented as mean ± standard errors of the mean for the control (CTRL) (n = 5), Su/Hx (n = 8), and Su/Hx + TMZ (n = 6) groups. * p < 0.05, ** p < 0.01, and ns = not significant vs. Su/Hx group. Statistical analysis was performed using an unpaired two-tailed t-test. (C) Representative immunofluorescence images of lung sections stained with anti-Ki67 antibodies (red) and anti-α-SMA antibodies (green). DAPI was used to stain cell nuclei (blue). Arrows indicate the colocalization of α-SMA and Ki67 in pulmonary arteries (scale bars, 20 μm). (D) The percentage of Ki67 positive cells with stained α-SMA in pulmonary arteries was quantified. Data are presented as mean ± standard errors of the mean for n = 6 per group. ** p < 0.01 and **** p < 0.0001. Statistical analysis was performed using one-way analysis…

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  Fig. 5. Comprehensive analysis of the pulmonary transcriptome among the control (CTRL), Su/Hx, and Su/Hx + TMZ groups. (A, B) Principal component analysis (PCA) of the RNA-seq data. principal component 1 (PC1) (x-axis) and PC2 (y-axis) represent 24.3% and 16.3% of the variation, respectively, in comparison between the CTRL and SU5416-Hypoxia with vehicle administration (Su/Hx) (A) groups. PC1 (x-axis) and PC2 (y-axis) represent 29.2% and 16.3% of the variation in comparison between CTRL and Su/Hx with TMZ administration (Su/Hx + TMZ) groups (B). Each dot denotes a single biological replicate, and the dashed circles represent three replicates for each sample. Black dots, control (CTRL) group; blue dots; Su/HX group, and red dots; Su/Hx + TMZ group. (C, D) Hierarchical clustering of the expression profiles between the CTRL and Su/Hx (C), and CTRL and Su/Hx + TMZ groups (D). Individual samples are provided in columns and genes in rows. Heatmaps represent the relative expression (red; high, white; intermediate, blue; low expression). (E, F) Volcano plots represent the differentially expressed genes (DEGs) between CTRL and Su/HX (E), and CTRL and Su/Hx + TMZ groups (F). Dotted vertical lines,…

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  Fig. 6. Identifying the candidate target genes of trimetazidine (TMZ). (A) Venn diagram identifying the differences in the upregulated differentially expressed genes (DEGs) (SU5416-Hypoxia with vehicle administration (Su/Hx) vs. CTRL and Su/Hx with TMZ administration (Su/Hx + TMZ) vs. CTRL group) between the groups. (B) Functional enrichment analysis of 58 upregulated DEGs only in the Su/Hx vs. CTRL group, the negative log10 of the p-value. The top 10 enriched gene ontology (GO) terms associated with biological process (red) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (https://www.kegg.jp/kegg/kegg1.html) analysis (orange). (C) Venn diagram identifying the differences in the downregulated DEGs between groups (Su/Hx vs. CTRL and Su/Hx + TMZ vs. CTRL group). (D) Functional enrichment analysis of the 48 downregulated DEGs only in the Su/Hx vs. CTRL group, the negative log10 of the p-value. The top 10 enriched GO terms associated with biological process (dark blue) and KEGG pathway analysis (light blue).

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  Fig. 7. Effects of trimetazidine (TMZ) on fatty acid- and glucose metabolisms-related gene expression. (A) Relative expression of fatty acid metabolism-related genes, Cd36 (A), Cpt1a (B), and Cpt2 (C) normalized to b-actin in the lungs of the control (CTRL), SU5416-Hypoxia with vehicle administration (Su/Hx), and Su/Hx with TMZ administration (Su/Hx + TMZ) groups analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Relative expression of the glucose metabolism-related genes, Glut1 (D), Glut (E), Hk1 (F), Hk2 (G), Pfkm (H), Pdk1 (I), Pdk2 (J), and Pdk4 (K) normalized to b-actin in the lungs of the control (CTRL), Su/Hx and Su/Hx + TMZ groups analyzed using qRT-PCR. Data are presented as mean ± standard errors of the mean for n = 5 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant). Statistical analysis was performed using one-way analysis of variance followed by Tukey’s post-hoc test.