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. 2024 Nov 7;14:27069. doi: 10.1038/s41598-024-76100-x

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

Asako Yanagisawa 1,#, Jun-Dal Kim 2,3,4,✉,#, Akira Naito 1,✉, Takayuki Kobayashi 1, Tomoko Misawa 1, Seiichiro Sakao 1,5, Takayuki Jujo-Sanada 1,6, Takeshi Kawasaki 1, Shin-ichi Muroi 2, So-Ichiro Sasaki 7, Takuji Suzuki 1, Yoshihiro Hayakawa 7, Yoshimi Nakagawa 2, Yoshitoshi Kasuya 1,8, Koichiro Tatsumi 1
PMCID: PMC11544210  PMID: 39511196

Abstract

Pulmonary hypertension (PH) is a devastating disease characterized by vascular remodeling, resulting in right ventricular failure and death. Dysregulation of energy metabolism is linked to PH pathogenesis. Trimetazidine (TMZ), a selective long-chain 3-ketoacyl coenzyme A thiolase inhibitor, is critical in maintaining energy metabolism. Despite the indicated TMZ’s inhibitory effect on pulmonary vascular remodeling in PH development, the integrated evaluation of the changes in biomolecules, such as metabolites and transcripts, that TMZ induces in the lung and heart tissues is largely unknown in vivo. For an improved understanding of the molecular mechanism involving the effects of TMZ on PH development, we performed a comprehensive analysis of the changes in cardiac metabolites and pulmonary transcripts of SU5416-Hypoxia (Su/Hx) rats treated with TMZ. Metabolomic analysis of the Su/Hx-induced PH hearts demonstrated that TMZ reduced the long-chain fatty acid concentration. Additionally, TMZ alleviated PH degree and excessive strain on the right heart functions in rats with Su/Hx-induced PH. We identified the candidate target genes for TMZ treatment during PH development. Interestingly, the mRNA levels of the fatty acid transporters were substantially downregulated by TMZ administration in the lungs with Su/Hx-induced PH. Notably, TMZ suppressed excessive proliferation of human pulmonary artery smooth muscle cells under hypoxic conditions. Our study suggests that TMZ ameliorates PH development by involving energy metabolism in the lungs and heart.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-76100-x.

Keywords: Pulmonary hypertension, Trimetazidine, Pulmonary transcriptome, Cardiac metabolome, SU5416-hypoxia

Subject terms: Chronic obstructive pulmonary disease, Transcriptomics, Metabolomics

Introduction

Pulmonary hypertension (PH) is a pathological state characterized by with elevated pulmonary artery pressure leading to right ventricular hypertrophy and failure1, which exhibits impaired vascular constituent cell regulation in the small-to-medium-sized distal pulmonary arteries, accompanied by inflammatory cell infiltration2. Thus, there is great interest to develop novel therapeutic strategy focused on right ventricular remodeling and pulmonary vascular.

On the other hand, metabolic abnormalities in the lungs and heart are involved in PH pathogenesis, although alterations in metabolism and bioenergetics vary by cell types (endothelial and smooth muscle cells) and organs (lungs and heart)3. Moreover, altered metabolic pathways, including fatty acid and glucose oxidation, redox reactions, the tricarboxylic acid cycle, and the electron transport chain, have been implicated in PAH pathogenesis4. It is also known that the involvement of metabolic remodeling in the mechanisms underlying pathological changes in PH vessels has been recently found5. Interestingly, a reciprocal relationship exists between fatty acid oxidation and glucose oxidation (Randle’s cycle) in human cells6. Additionally, carnitine, dicarboxylic acid, and long- and medium-chain free fatty acid products are increased in the lungs of patients with PAH7, and 13C-α-ketoglutarate from 13C-long-chain fatty acids and acetyl-CoA acetyltransferase 2 (ACAT2) are decreased in the cultured pulmonary artery endothelial cells8,9. Moreover, a metabolic shift from glucose oxidation to glycolysis occurs, suggesting that decreased energy efficiency is a mechanism underlying RV failure in RV cardiomyocyte10. Therefore, selective inhibitors of fatty acid oxidation may indirectly increase glucose oxidation and improve the RV function11.

Trimetazidine (TMZ), an inhibitor of long-chain 3-ketoacyl coenzyme A (CoA) thiolase, is involved in fatty acid beta-oxidation and indirectly activates glucose oxidation via the Randle’s cycle12,13. TMZ’s mechanism of action could be attributed to energy metabolism optimization, as TMZ decreases fatty acid oxidation and stimulates glucose oxidation14. TMZ improves energy efficiency in the setting of RV failure12, and attenuates PH in monocrotaline-induced PAH rats15, by restoring mitochondrial function. Additionally, non-metabolic effects of TMZ, such as anti-inflammatory16, apoptosis-promoting, and anti-cancer effects17, have been recently reported. Therefore, a decrease in long-chain fatty acid oxidation with TMZ leads to improved cardiac output in a rat model of RV hypertrophy as a therapeutic strategy to alter metabolic pathways12.

In the Su/Hx model, animals received an injection of SU5416 subcutaneously, an inhibitor of the vascular endothelial growth factor receptor, before being exposed to hypoxia. Su/Hx rats exhibit severe pulmonary artery remodeling, including plexiform lesions. Thus, Su/Hx models have been considered key preclinical models of PAH because of the formation of occlusive pulmonary vascular lesions in the lungs4,18. In the current study, we performed comprehensive cardiac metabolome and pulmonary transcriptome analyses in Su/Hx-induced PH rats to clarify TMZ’s inhibitory effects on PH development through the changes in biomolecules such as metabolites and transcripts. Furthermore, we confirmed the pathophysiological changes in this model with TMZ administration.

Results

TMZ inhibits cardiac dysfunction in the Su/Hx-induced rat PH model

We generated Su/Hx-induced rat PH models with and without TMZ administration (Fig. 1A). We measured the hemodynamic parameters using the Millar Mikro‑Tip catheter system to evaluate TMZ’s inhibitory effects in PH pathogenesis (Supplementary Table S1). The right ventricular systolic pressure (RVSP) in the Su/Hx rats was elevated compared to that in the control (CTRL) rats (Fig. 1B). Moreover, after TMZ administration, the end-systolic elastance (Ees)/EA ratio, a clinical index of ventricle-artery functional coupling, reduced in the Su/Hx group and recovered to normal levels in the CTRL group (Fig. 1C). Additionally, RV maximum (dP/dt max) and minimum (dP/dt min) pressure rates and RV systolic and diastolic performance indices were considerably increased in the Su/Hx group and were remarkably decreased via TMZ treatment (Fig. 1D and E). However, no substantial differences were observed in heart rate (HR), stroke volume, or cardiac index among the CTRL, Su/Hx, and Su/Hx + TMZ groups (Fig. 1F–H).

Fig. 1.

Fig. 1

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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.

TMZ treatment improves RV hypertrophy and remodeling in the Su/Hx-induced PH rat model

TMZ’s inhibitory effects on the Su/Hx-induced RV hypertrophy and remodeling were examined. The RV/ left ventricle and septum (LV + S) ratio was significantly lower in the Su/Hx + TMZ group than in the Su/Hx group (Fig. 2A). Subsequently, RV hypertrophy and fibrosis were evaluated using hematoxylin and eosin (H&E) and Masson’s trichrome staining, respectively (Fig. 2B). The RV cardiomyocyte and the fibrotic area sizes were increased in the Su/Hx group compared to those in the CTRL group and were remarkably inhibited after TMZ treatment (Fig. 2C and D).

Fig. 2.

Fig. 2

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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.

Metabolic changes in the right heart of the Su/Hx rats after TMZ treatment

Altered metabolic pathways are considered the key causes of PAH pathogenesis5. We performed a comprehensive mass spectrometry-based metabolic analysis to investigate TMZ’s effects on metabolic changes in the right heart of rats with Su/Hx-induced PH. Principal component 1 (PC1) and PC2 of the metabolites in the samples accounted for 45.1% and 22.5% of the total metabolites, respectively (Fig. 3A). The biological replicates were separated between the Su/Hx and TMZ groups as observed using hierarchical heatmap clustering analysis of the samples (Supplementary Fig. S1A). The partial least squares discriminant analysis (PLS-DA) model was used to compare the differences in metabolic profiles between the Su/Hx and Su/Hx + TMZ groups. The variable importance in projection (VIP) score of the PLS-DA analysis revealed that the levels of carnitine, inosinic acid, CoA, nicotinamide adenine dinucleotide phosphate, and glutamine were considerably higher in the right heart of TMZ-treated Su/Hx rats than in the Su/Hx rats (Fig. 3B). Contrastingly, glutathione, creatinine, urea, and amino acids, such as proline, serine, aspartic acid, tyrosine, and asparagine levels, were markedly decreased in the Su/Hx + TMZ group. Moreover, enrichment analysis revealed that beta-alanine metabolism, glycine and serine metabolism, pyruvaldehyde degradation, fatty acid biosynthesis, and tryptophan metabolism were lower in the SU/Hx with TMZ group than in the Su/Hx group (Fig. 3C). Additionally, pyruvic acid and the total adenylate concentration (the sum of AMP, ADP, and ATP) were higher in the Su/Hx + TMZ group than in the Su/Hx group, although the difference was statistically insignificant (Supplementary Fig. S1B–F). Contrastingly, the long-chain fatty acid (LCFA) levels such as palmitic acid and stearic acids were lower in the Su/Hx + TMZ group than in the Su/Hx group (Fig. 3D and E). These results suggest that TMZ reduces the cumulative energy storage and increases energy decomposition.

Fig. 3.

Fig. 3

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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.

Improvement of pulmonary vascular remodeling using TMZ in the Su/Hx-induced rat PH model

We further focused on pulmonary vascular remodeling, which was pathologically quantified using Elastica van Gieson (EVG) staining. Samples from the Su/Hx group demonstrated medial wall thickening and lesions with combined mesangial and intimal thickening, which are characteristic of pulmonary vascular remodeling. The medial wall thickening of the small pulmonary arteries was substantially attenuated in the Su/Hx + TMZ group (Fig. 4A). Percentage of grade 2 obstructive lesions was markedly lower in the Su/Hx + TMZ group than in the Su/Hx group (Fig. 4B), suggesting that TMZ alleviated pulmonary artery remodeling in the Su/Hx-induced rat PH model.

Fig. 4.

Fig. 4

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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 of variance followed by Tukey’s post-hoc test. (E, F) The proliferation of hypoxia-induced human pulmonary artery smooth muscle cells (hPASMCs). The relationship of O2 concentration to the promotion of hPASMC proliferation. Data are presented as mean ± standard errors of the mean for n = 6 per group. **** p < 0.0001 and ns (not significant) vs. normoxic condition (O2 = 21%) (E). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test used for multiple comparisons. (F) TMZ treatments attenuate hypoxia-induced hPASMC proliferation without cell toxicity, as assessed using two-way ANOVA followed by Tukey’s post-hoc test. (n = 6 per group).

Excessive pulmonary artery smooth muscle cell (PASMC) proliferation is a cellular process underlying pulmonary vascular occlusion in PAH. Additionally, hyper-proliferation and apoptosis resistance of PASMCs mirror a malignant phenotype, which are involved in pulmonary vascular remodeling in PH5. Interestingly, the results of Ki67, a prominent proliferation marker, staining showed that PASMC proliferation was significantly reduced by TMZ treatment in the Su/Hx-induced rat PH model (Fig. 4C and D). In order to determine the effect of TMZ on the apoptosis resistance of PASMC, we confirmed levels of PASMCs apoptosis by activated caspase-3, a maker for cells undergoing apoptosis, staining that there was no significant difference between the Su/Hx and Su/Hx + TMZ groups (Supplementary Fig. S2). Subsequently, cells were cultured under various O2 conditions to determine the level of hypoxia that induces cell proliferation in human (h)PASMCs. Hypoxia-induced death of hPASMCs occurred under extremely hypoxic conditions (1% O2). Meanwhile, hPASMCs exposed to 10% O2 substantially proliferated compared to those exposed to normoxia (21% O2) (Fig. 4E). Moreover, 100 µM of TMZ did not affect hPASMC proliferation and apoptosis under normoxic conditions (Supplementary Fig. S3). Cell proliferation assays were also performed to assess TMZ’s effects on PASMC proliferation under hypoxic conditions. TMZ (approximately 0–100 µM) inhibited hPASMC proliferation in a concentration-dependent manner under 10% O2 conditions without cytotoxicity, indicating that TMZ suppressed hypoxia-induced hPASMC proliferation (Fig. 4F). Taken together, these data suggest that TMZ suppresses PASMC proliferation in PH.

Transcriptomic changes in the lungs of Su/Hx rats with TMZ treatment

We conducted RNA sequencing of whole lung tissues from the CTRL, Su/Hx, and Su/Hx + TMZ groups to understand the molecular mechanisms underlying the effects of TMZ-sensitive metabolites and transcripts on PH pathobiology. Principal component analysis (PCA) was performed to assess the reproducibility of the biological replicates in the CTRL, Su/Hx, and Su/Hx + TMZ groups. Contrary to our expectations, the PCA score plot did not reveal any distinct clusters in the Su/Hx and Su/Hx + TMZ groups (Supplementary Fig. S4). However, the Su/Hx and Su/Hx + TMZ samples were considerably different from the CTRL samples (Fig. 5A and B). Subsequently, we calculated the difference in reads per kilobase per million reads (RPKM), and the differentially expressed genes (DEGs) were identified between the CTRL and Su/Hx groups and between CTRL and Su/Hx + TMZ groups using a false discovery rate (FDR) of < 0.05 with a fold change (FC) ≥ 2 or ≤ − 2. Hierarchical clustering analysis and volcano plots revealed that 357 (238 upregulated and 119 downregulated) and 435 (270 upregulated and 165 downregulated) unique genes were substantially altered in the Su/Hx and Su/Hx + TMZ groups, respectively, compared with the CTRL group (Fig. 5C–F and Supplementary Table S2).

Fig. 5.

Fig. 5

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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, log2 FC ≥ 2 or ≤ − 2; dotted horizontal line, the significance cut-off (false discovery rate: p = 0.05).

Identifying the candidate target genes of TMZ for PH improvement

We compared the DEGs between the Su/Hx and Su/Hx + TMZ groups to determine the candidate target genes of TMZ in pulmonary vascular remodeling. We identified 58 DEGs that displayed upregulated expression levels in the Su/Hx vs. CTRL group but not in the Su/Hx + TMZ group vs. CTRL group (Fig. 6A and Supplementary Table S3). These DEGs could represent a defensive mechanism affected by TMZ in PH development. We performed enrichment analysis of the gene set for gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to characterize the functional features of these 58 DEGs. Regarding the biological process, GO annotation of “Keratinocyte proliferation,” “Positive regulation of apoptotic cell clearance,” “Regulation of apoptotic cell clearance,” “Tetrahydrobiopterin metabolic process,” “Positive regulation of calcium-mediated signals,” “Pteridine-containing compound metabolic process,” “Macrophage chemotaxis,” and “Macrophage migration and nitric oxide (NO) biosynthetic process” were listed (Fig. 6B), suggesting that TMZ attenuates gene functions related to these GO terms in PH development. Additionally, KEGG pathway analysis revealed the GO terms involved in the inflammation stress, such as advanced “glycation end products (AGEs)/receptor for advanced glycation end products (RAGE) signaling pathway”, “sphingolipid signaling pathway” and “arginine biosynthesis.” Furthermore, cluster gram analysis suggested that in GO biological processes, C-C motif chemokine 2 (CCL2) (monocyte chemoattractant protein 1; MCP1), complement C4B (C4B), CKLF, CCL21, c-type lectin domain-containing 7 A (CLEC7A), CDH13, FERMT1, NO synthase 3 (NOS3, eNOS), and GTP cyclohydrolase 1 (GCH1) were suppressed by TMZ treatment (Supplementary Fig. S5A). TMZ treatment suppressed IL-6, CCL2, CCL21, PLCB2, NOS3, FCER1A, C4B, C1S, EGR2, CLEC7A, GTP cyclohydrolase (GCH), and aldo-keto reductase family 1 member B10 (AKR1B10) in the KEGG pathway analysis (Supplementary Fig. S5B).

Fig. 6.

Fig. 6

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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).

Among the transcripts, 119 DEGs in the Su/Hx group were downregulated relative to those in the CTRL group; of these, the expression levels of the 48 transcripts were not downregulated in the Su/Hx + TMZ vs. CTRL group (Fig. 6C). These DEGs could represent a part restored by TMZ during PH development. Regarding the biological process, terms of “Regulation of systemic arterial blood pressure by endothelin” and “Tonic smooth muscle contraction” were listed (Fig. 6D). Meanwhile, KEGG pathway analysis revealed terms such as “Insulin secretion,” “Vascular smooth muscle contraction,” and “Arginine and proline metabolism.”

TMZ’s effects on the energy metabolism-associated gene expression in the lungs of Su/Hx-induced PH rats

Finally, we performed quantitative reverse transcriptase polymerase chain reaction (RT-PCR) on the whole lungs obtained from each experimental group to explore TMZ’s effect on energy metabolism in the lungs. The mRNA levels of fatty acid transporters, including Cd36, Cpt1a, and Cpt2 were induced in the Su/Hx group compared with those in the CTRL group and were considerably reduced by TMZ treatment, indicating that TMZ downregulates Su/Hx-induced pulmonary fatty acid metabolism (Fig. 7A–C).

Fig. 7.

Fig. 7

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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.

Reprogramming energy metabolism with preferential aerobic glycolysis utilization plays a crucial role in PH development19. The mRNA levels of glycolytic enzymes, including Glut1, Glut4, Hk1, Hk2, Pfkm, and Pdk1, 2, 4 were considerably higher in the Su/Hx group than in the CTRL group (Fig. 7D–K). Interestingly, the expression of these eight genes was markedly lower in the Su/Hx + TMZ group than those in the Su/Hx group. Taken together, these results indicate that TMZ directly acts on the lungs to avoid energy metabolism reprogramming using aerobic glycolysis during PH development.

Discussion

Patients with PH exhibit marked impairments in pulmonary hemodynamics and vascular growth regulation20. Furthermore, metabolic abnormalities have been recognized in the hearts and lungs of patients, animal models of the disease, and in cells derived from the lungs of patients with PH3–5. Therefore, energy metabolic pathways are partly potential therapeutic targets for PH. TMZ is widely used as an anti-ischemic agent to clinically treat cardiovascular diseases, including angina pectoris, ischemia-reperfusion, diabetic cardiomyopathy, and heart failure20. By inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase, TMZ shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation12,13.

Herein, we investigated TMZ’s inhibitory effects on PH development in rats with Su/Hx-induced PH using a combination of pathological analysis and biomolecule profiling. Hemodynamic analysis revealed that TMZ administration decreased RVSP and recovered Ees/Ea, the ratio of ventricular to vascular elastance, suggesting that TMZ alleviated PH degree and excessive strain on the function of the right heart in the Su/Hx-induced PH model. Additionally, we confirmed that TMZ treatment reversed medial wall thickening, decreased the occlusive lesion ratio in the small pulmonary arteries, and reduced RV cardiomyocyte size and RV free-wall fibrosis degree.

Cardiac metabolome analysis using rats with Su/Hx-induced PH revealed that the total adenylate concentration tended to be higher in the TMZ-treated group (Supplementary Table S4). Moreover, TMZ administration alleviated right cardiac hypertrophy partly by releasing sustained pressure overload and improving energy efficiency. This was demonstrated by the increase in Ees/Ea in the Su/Hx rat model since Ees/Ea yields insight into the energy balance of the myocardium. Therefore, the improvement in cardiac function could be a secondary effect due to the decrease of pressure overload, although the protective effect of TMZ on the myocardium may partly contribute to energy optimization. Interestingly, it has been reported that RV-specific accumulation of LCFA can result in lipotoxicity21. The improved RV hypertrophy may also be secondary to pulmonary vascular remodeling, and there may be a metabolic modulatory effect of TMZ in the pulmonary vasculature. Notably, regarding mitochondrial and metabolic reprogramming in the right heart, the metabolomic analysis indicated that TMZ reduced LCFA concentration, especially palmitic and stearic acids. Therefore, our results indicate that the energy balance of the RV myocardium recovered in PH’s improved status after TMZ administration.

TMZ treatment attenuated pulmonary vascular remodeling in the Su/Hx-induced PH rats (Fig. 4). PASMC proliferation is a key pathophysiological component of vascular remodeling in PAH22. In addition to increased proliferation, pulmonary vascular cells in PH have resistance to apoptosis that is also considered as an important pathological component of pulmonary vascular remodeling23. Notably, TMZ treatment markedly inhibited PASMC proliferation in the Su/Hx-induced PH rats. By contrast, TMZ did not affect the apoptosis resistance of PASMC in the Su/Hx-induced rat PH model (Supplementary Fig. S2). It is known that the knockdown of the mammalian target of rapamycin (mTOR) using siRNA markedly inhibits the proliferation of hypoxia-induced PASMCs24. The mTOR pathway is activated in the lungs of rats with Su/Hx-induced PH25,26. As shown in terms of Biological Process in Fig. 6B, the upregulated 58 DEGs between the Su/Hx vs. CTRL group were enriched in “Positive regulation of AMPA receptor activity (GO:2000969)”. Additionally, TMZ is related to a decrease in the activation of the AMP-activated protein kinase (AMPK)/mTOR signaling pathway26, which supports our results.

The anti-inflammatory and anticancer effects of TMZ have been previously reported16,17. Analyses of human samples and PH rodent models have clearly demonstrated various immune cell infiltration, including monocytes, into the pulmonary perivascular regions and their involvement in PH development. Concurrently, the lung tissue displays increased chemokine transcript expression, including those responsible for monocyte recruitment, such as the CCL2. Our pulmonary transcriptome analysis revealed that Ccl2 (Mcp1) gene was one of the 58 DEGs in GO biological process enrichment and KEGG pathways, which were assumed to be suppressed by TMZ treatment during PH development (Supplementary Fig. S3A, B). CCL2, which is overexpressed in pulmonary endothelial cells, has been reported to function as a growth factor for PASMC in patients with PH27,28. Additionally, we confirmed that the levels of Ccl2 mRNA in the lungs of Su/Hx-induced PH rats were higher than those in the controls and tended to be lower in the TMZ-treated group (Supplementary Fig. S3C). This suggests that TMZ suppresses CCL2-mediated inflammatory processes via macrophage chemotaxis and migration during PH pathogenesis.

Contrastingly, nitric oxide synthase 3 (NOS3 and eNOS) is abundant in the vascular endothelium and produces nitric oxide (NO) from L-arginine and the coenzyme tetrahydrobiopterin, an important molecule involved in PAH pathogenesis29. Additionally, low NO production levels in patients with PAH and Su/Hx rats were due to NOS3-uncoupling with arginase 1 induction, leading to excessive ROS production30,31. TMZ has consistently been shown to have cardioprotective effects on coronary microcirculation through NO-dependent effects, in which eNOS mRNA and protein levels increase32,33. Notably, mRNA expression levels of Nos3 mRNA were elevated in the lung tissues of the Su/Hx group and were diminished by TMZ treatment (Supplementary Fig. S3D). Moreover, Nos3 gene was enriched in the biological process terms of the clustergram analysis like “Tetrahydrobiopterin metabolic process,” “Pteridine-compound metabolic process,” and “Nitric oxide biosynthetic process” (Supplementary Fig. S3A). Thus, Nos3 elevation in the Su/Hx group may compensate for poor NO production, which was restored in the Su/Hx + TMZ group. Contrastingly, a previous study suggested that NOS3 phosphorylation levels and NO production decreased in the lung cells of hypoxia-induced animals corresponding to a decrease in AMPK activity34,35. We identified 58 candidate target genes of TMZ in PH development that were enriched with respect to “positive regulation of AMPK receptor activity (GO:2000969)” (biological process terms of Fig. 6B). AMPK activation activates NOSs, which is widely accepted as one of the important regulatory mechanisms of NOS3 activity and subsequent vasodilation36, which supports our study.

One of the mechanisms of RV dysfunction in PAH is maladaptive fatty acid metabolism, stemming from an increase in fatty acid uptake by its transporter molecules like CD36, CPT1, and CPT2, and an imbalance between glucose and fatty acid oxidation in the mitochondria. These dysfunctions result in lipid accumulation in the form of triglycerides, diacylglycerol, and ceramides in the cytoplasm, which is characteristic of lipotoxicity37. In the present study, we demonstrated that the mRNA levels of fatty acid transporters, including Cd36, Cpt1a, and Cpt2 were downregulated by TMZ administration in the lungs of Su/Hx PH rats, suggesting that TMZ directly acts in the lungs to avoid reprogramming of energy metabolism using aerobic glycolysis during PH development. Intriguingly, the mRNA levels of glycolytic enzymes, including Glut1, Glut 4, Hk1, Hk2, Pfkm, and Pdk1, 2, 4, were increased in the lungs of Su/Hx PH rats; however, TMZ treatment decreased the activities of these glycolytic enzymes. These results suggest that TMZ decreases fatty acid oxidation and promotes glucose oxidation, correcting the metabolic imbalance in PH pathogenesis.

TMZ has been widely used to treat coronary artery disease and information regarding its tolerability and safety has been accumulated38–40. Its clinical application in PH is a potential treatment option, although further studies are required. Collectively, our profiles of altered biomolecules, including cardiac metabolites and pulmonary transcripts, with TMZ in the Su/Hx-induced PH rats with or without TMZ treatment may provide promising information to understand the therapeutic targets in PH.

Limitations

A limitation of our study was the lack of data on molecule expression at the protein level and the detailed signaling pathways involved in each biological process. Furthermore, in vitro experimental data obtained using hPASMCs reflect only limited aspects of PH pathobiology.

Materials and methods

Experimental design and animals

All animal procedures were approved by the Review Board for Animal Experiments of Chiba University and were performed in accordance with the guidelines of the Animal Research Committee of Laboratory Animal Center, Graduate School of Medicine, Chiba University. Five-week-old male Sprague–Dawley rats weighing 130–150 g were purchased from Japan SLC (Shizuoka, Japan). All the rats were housed in an animal experimental facility at Chiba University and had access to drinking water and food ad libitum. The rats were kept in cages at 24℃ under a 12 h light/dark cycle. All methods are reported in accordance with ARRIVE (Animal Research Reporting of In vivo Experiments) guidelines.

TMZ administration to the Su5416-hypoxia rat model

The Su/Hx rat model was established as previously described41,42. Briefly, SUGEN (20 mg/kg; SU5416, R&D Systems, Inc., MN, USA), a vascular endothelial growth factor receptor inhibitor, was dissolved in carboxymethyl cellulose, and the suspension was subcutaneously injected into rats. Subsequently, the rats were maintained under normobaric-hypoxic conditions (10% O2) for three weeks and then returned to normoxic conditions (21% O2) for five weeks.

TMZ was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). TMZ was administered for 3 and 2 weeks after the end of hypoxic exposure to investigate TMZ’s effect after PH development. The rats were divided into three groups according to the experimental design:1 vehicle administration control (CTRL)2, Su/Hx with vehicle administration (Su/Hx), and3 Su/Hx with TMZ administration (Su/Hx + TMZ).

Immunofluorescence staining

Immunofluorescence staining was performed to detect the expression of Ki67 and α-SMA in pulmonary arteries. Tissues were embedded in paraffin and sectioned into 6 μm slices. Then, the paraffin-embedded slices were incubated with primary antibodies against Ki67 (diluted 1:100; AF0198, Affinity Biosciences, Cincinnati, OH), the cleaved caspase-3 (Asp175) (diluted 1:100; #9661, Cell Signaling Technology, Danvers, MA) or α-SMA (diluted 1:100; AB7817, Abcam, Cambridge, UK) for 60 min at overnight at room temperature. Subsequently, the followed by staining with a secondary antibody, namely Donkey anti-mouse IgG Alexa Fluor™ Plus 594 (diluted 1:300; A32754, Invitrogen, Carlsbad, CA) or Donkey anti-rabbit IgG Alexa Fluor™ Plus 488 (diluted 1:800; A32766, Invitrogen, Carlsbad, CA) was added and incubated for 30 min at room temperature. Cell nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) (19178-91, Nacalai Tesque). Images were observed under an Axio Imager A2, (Zeiss, Oberkochen, DE).

RNA-sequencing

Total RNA (approximately 500 ng) from the heart and lungs was ribosomal RNA-depleted using the NEBNext rRNA Depletion Kit (New England Biolabs, MA, USA) and converted to an Illumina sequencing library using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). The libraries were validated using a Bioanalyzer (Agilent Technologies) and sequenced using NextSeq 500 (Illumina, CA, USA) with a paired-end 36-base read option. Reads were mapped to the Rattus norvegicus Rnor_6.0 (rn6) reference genome and quantified using the CLC Genomics Workbench (version 12.0, QIAGEN). The RNA-seq datasets were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo; accession number: GSE186996).

Identification of DEGs and functional enrichment analysis

The number of RPKM for each transcript in individual samples was calculated using CLC Genomics Workbench (version 11.0.1, QIAGEN) to normalize the read counts. DEGs were identified using FC − 2 to + 2 filtering analysis with an FDR of p < 0.05 between the two groups. Thereafter, the distinct gene expression was compared and visualized using the PCA plot and clustering heat map analyses. Volcano plots were used to visualize the significance of changes in gene expression between the − log10p-value and log2 FC.

K-means functional enrichment analysis of the DEGs was performed using the integrated differential expression and pathway analysis online tools (http://bioinformatics.sdstate.edu/idep/)43. The web-based Enrichr suite (http://amp.pharm.mssm.edu/Enrichr/)44 was used to assign the GO terms for biological processes and KEGG pathway45–47 enrichment of DEGs between sample groups.

Quantitative real-time PCR analysis of RNA from the lung

Total RNA was extracted from rat lungs using the RNeasy Fibrous Tissue Kit (QIAGEN, Hilden, Germany), and the primary hPASMCs were extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). The isolated RNA (0.2–0.5 µg) was reverse transcribed using an RT2 First Strand Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Quantitative analyses of the changes in expression levels were performed using SYBR Green ROX qPCR master mix (QIAGEN, Hilden, Germany) in an ABI 7300 system (Applied Biosystems, Life Technologies, USA). RT-PCR was performed using RT2 qPCR Primer Assays (QIAGEN, Hilden, Germany) for Cd36 (cat. no. PPR43233A), Cpt1a (cat. no. PPR45833A-200), Cpt 2 (cat.no PPR45410A-200), Glut1 (cat. no. PPR42999A-200), Glut4 (cat. no. PPR42463A-200), Hk1 (cat. no. PPR45173E-200), Hk2 (cat. no. PPR52902A-200), Pfkm (cat. no. PPR45165B-200), Pdk1 (cat. no. PPR45282F-200), Pdk2 (cat. no. PPR52731B-200), Pdk4 (cat. no. PPR48407B-200), Ccl2 (cat. no. PPR06714B-200), Nos3 (cat. no. PPR49724A-200), and β-actin (cat. no. PPR06570C). PCR was performed as follows: 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Data analyses were performed using the comparative Ct method and ABI (SDS version 1.4). β-actin was evaluated to normalize the mRNA expression levels.

hPASMC proliferation assay

The hPASMCs were purchased from Lonza (Cat. No. CC-2581; NJ, USA), and cultured in smooth muscle Growth Mediun-2 (SmGM-2) (Cat. No. CC-3182, Lonza) according to the provider’s instructions at 37 °C in 5% CO2 in a humidified air incubator. Experiments were performed using cells that had been passaged fewer than six times. The hPASMCs were plated in 96-well plates at a density of 1 × 104 cells/well after serum starvation for 12 h. PASMCs were cultured in SmGM for two days in 96-well plates and subsequently used for the experiments described below48.

The cells were cultured under various oxygen concentrations (1%, 3%, 5%, 10%, and normoxia) for 48 h to examine the effects of O2 concentration on hPASMCs proliferation. Furthermore, cells were cultured under various TMZ concentrations for 48 h in normoxia and 10% hypoxic condition, (TMZ was dissolved in medium and added at a 0–100 µM concentration) to investigate the effects of TMZ concentration on the hPASMC proliferation. Twenty microliters of 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and the cells were further incubated for 3 h. After removing the supernatants, the formazan crystals derived from MTT were dissolved in 100 µL dimethyl sulfoxide. Absorbance was measured at 570 nm using a microplate reader, and the results were expressed as the average absorbance.

Metabolomic analysis

The metabolites were extracted from the right heart of the Su/Hx and Su/Hx + TMZ groups, and metabolome analysis was conducted according to the manufacturer’s instructions. The details are described in a previous study4 and partly documented in Appendix S1. Processing and visualization of the metabolomic data, including PCA, PLS-DA, heatmap clustering, and enrichment analysis, were performed using the web-based software MetaboAnalyst (V6.0) (https://www.metaboanalyst.ca). Calibration and validation of the PLS-DA model were based on the leave-one-out cross-validation method via Q2 and R2 parameters, and the significance was demonstrated via a permutation test with 2000 iterations using separation distance and p-value < 0.05.

Invasive right heart catheterization

The details of invasive right heart catheterization have been previously described18. Briefly, pulmonary hemodynamics was measured in rats lightly anesthetized with isoflurane after measuring their blood pressure. The anesthetized rats underwent tracheostomy and were placed on a ventilator (Harvard Apparatus, MA, USA). A 2.0-F microtip pressure transducer (Millar Instruments, Houston, TX, USA) was inserted into the RV. The RVSP, stroke volume, heart rate, and dP/dt index were continuously monitored for over 10 min, and the data were analyzed using a Power Lab system (AD instruments, Denver CO, USA). At the end of the monitoring period, Ea and Ees were measured with vena cava compression. The rats were euthanized using pentobarbital sodium (150 mg/kg) after hemodynamic measurements. The hearts were removed, and the RV-free wall was dissected from the LV + S. The weight ratio of the RV to that of the LV + S was calculated as an RV hypertrophy index. Parts of the right heart and lung were stored in RNA protection tissue reagent for real-time PCR analysis and in formalin for pathological analysis, respectively.

Pathological analysis

Paraffin-embedded RV and lung specimens were sectioned (3 μm each). Lung specimens were stained with the EVG stain. The extent of pulmonary vessel luminal obstruction was evaluated as previously reported18,49: Grade 0, no evidence of neointimal formation; grade 1, partial (≤ 50%) luminal occlusion; and grade 2, severe (> 50%) luminal occlusion.

The myocyte cross-sectional area (CSA) was visualized using H&E) staining. Images of the H&E staining were digitally captured, and the CSAs were calculated using ImageJ (version 1.53k; National Institutes of Health, Bethesda, Maryland, USA). Five sections were randomly selected from every five animals in each group. At least 30 myocytes were counted in each group. The RV specimens were stained with Masson’s trichrome. Stained RV myocardial slides were randomly photographed at five locations at 400× magnification. The blue-stained fibrotic area was quantified using ImageJ. The details of this method have been previously described50.

Statistical analysis

The differences between groups were assessed using the unpaired two-tailed t-test and one-way ANOVA or two-way ANOVA considering the potential assumption violations (equal variance) using more standard tests. When significant differences were detected, the individual mean values were compared using post-hoc tests that allowed for multiple comparisons with adequate type I error control (Bonferroni or Dunnett’s test). P < 0.05 was considered statistically significant. Values are expressed as mean ± standard errors of the mean. Statistical analyses of RT-PCR, right heart catheterization, and pathology were performed using PRISM (version 9.1; GraphPad Software Inc.).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (19.3KB, xlsx)
Supplementary Material 2 (16.5KB, xlsx)
Supplementary Material 3 (17KB, xlsx)
Supplementary Material 4 (1.9MB, pdf)

Acknowledgements

We are grateful to Sumina Atarashi and Chieko Handa for their technical advice, assistance, and administrative support. We would like to thank Editage (https://www.editage.com/) for the English language editing. This work was supported by grants from AMED-CREST (JP21gm1410010 (to J.-D.K) and JP21gm1210003 (to T.S.)), AMED (JP223fa627003h0001(to T.S.)), JSPS KAKENHI (Grant Numbers 19H03664 (to S.S.), 19K17663 (to T.Ka.), 22K16163 (to T.Ka.), 22K16164 (to A.N.), 22H03076 (to T.S.), and 24K08827 (to J.-D.K.)) and a research grant from the Intractable Respiratory Diseases and Pulmonary Hypertension Research Group, the Ministry of Health, Labor and Welfare, Japan (Grant Numbers 20FC1027 (to K.T.) and 23FC1031 (to K.T.)).

Author contributions

A.Y., J.-D.K., and S.S. were involved in the study design and conceptualization. A.Y., J.-D.K., T.J.-S., T.M., S.M., So.S., T.Ko., and K.Y. were involved in the animal and laboratory experiments. A.Y., J.-D.K., and K.T. wrote the main manuscript, and A.N., T.Ka., S.M., Y.H., Y.N., Y.K. and T.S. critically revised the report and commented on the drafts of the manuscript. T.S. supervised this study. All authors have approved the final version of the manuscript.

Data availability

All data needed to evaluate the conclusions in the paper are present in this study or upon reasonable request from the correspondent authors. All raw data files for the RNA-seq analysis were deposited in the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo; accession number: GSE186996).

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Asako Yanagisawa and Jun-Dal Kim contributed equally to this work.

Contributor Information

Jun-Dal Kim, Email: jdkim@inm.u-toyama.ac.jp.

Akira Naito, Email: akira-n.390@chiba-u.jp.

References

  • 1.Voelkel, N. F. et al. Right ventricular function and failure: Report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure. Circulation114(17), 1883–1891. 10.1161/CIRCULATIONAHA.106.632208 (2006). [DOI] [PubMed] [Google Scholar]
  • 2.Funk-Hilsdorf, T. C., Behrens, F., Grune, J. & Simmons, S. Dysregulated immunity in Pulmonary Hypertension: From companion to composer. Front. Physiol.13, 819145. 10.3389/fphys.2022.819145 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Xu, W., Janocha, A. J. & Erzurum, S. C. Metabolism in pulmonary hypertension. Annu. Rev. Physiol.83, 551–576. 10.1146/annurev-physiol-031620-123956 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Sakao, S. et al. Metabolic remodeling in the right ventricle of rats with severe pulmonary arterial hypertension. Mol. Med. Rep.23(4), 227. 10.3892/mmr.2021.11866 (2021). [DOI] [PubMed] [Google Scholar]
  • 5.Sutendra, G. & Michelakis, E. D. The metabolic basis of pulmonary arterial hypertension. Cell. Metab.19(4), 558–573. 10.1016/j.cmet.2014.01.004 (2014). [DOI] [PubMed] [Google Scholar]
  • 6.Hue, L. & Taegtmeyer, H. The Randle cycle revisited: A new head for an old hat. Am. J. Physiol. Endocrinol. Metab.297(3), E578–E591. 10.1152/ajpendo.00093.2009 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Zhao, Y. et al. Metabolomic heterogeneity of pulmonary arterial hypertension. PLoS One9(2), e88727. 10.1371/journal.pone.0088727 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Xu, W. et al. Integrative proteomics and phosphoproteomics in pulmonary arterial hypertension. Sci. Rep.9(1), 18623. 10.1038/s41598-019-55053-6 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Hernandez-Saavedra, D. et al. Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment. Sci. Rep.10(1), 413. 10.1038/s41598-019-57200-5 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Agrawal, V., Lahm, T., Hansmann, G. & Hemnes, A. R. Molecular mechanisms of right ventricular dysfunction in pulmonary arterial hypertension: Focus on the coronary vasculature, sex hormones, and glucose/lipid metabolism. Cardiovasc. Diagn. Ther.10(5), 1522–1540. 10.21037/cdt-20-404 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Thenappan, T., Ormiston, M. L., Ryan, J. J. & Archer, S. L. Pulmonary arterial hypertension: Pathogenesis and clinical management. BMJ360, j5492. 10.1136/bmj.j5492 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Fang, Y. H. et al. Therapeutic inhibition of fatty acid oxidation in right ventricular hypertrophy: Exploiting Randle’s cycle. J. Mol. Med.90(1), 31–43. 10.1007/s00109-011-0804-9 (2012). Epub 2011 Aug 28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Randle, P. J., Garland, P. B., Hales, C. N. & Newsholme, E. A. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet1 (7285), 785–789. 10.1016/s0140-6736(63)91500-9 (1963). [DOI] [PubMed] [Google Scholar]
  • 14.Kantor, P. F., Lucien, A., Kozak, R. & Lopaschuk, G. D. The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme a thiolase. Circ. Res.86(5), 580–588. 10.1161/01.res.86.5.580 (2000). [DOI] [PubMed] [Google Scholar]
  • 15.Sutendra, G. et al. Fatty acid oxidation and malonyl-CoA decarboxylase in the vascular remodeling of pulmonary hypertension. Sci. Transl. Med.2(44), 44ra58. 10.1126/scitranslmed.3001327 (2010). [DOI] [PubMed] [Google Scholar]
  • 16.Tanoglu, A. et al. Appreciation of trimetazidine treatment in experimental sepsis rat model. Bratisl Lek Listy116(2), 124–127. 10.4149/bll_2015_024 (2015). [DOI] [PubMed] [Google Scholar]
  • 17.Atlı Şekeroğlu, Z., Şekeroğlu, V., Işık, S. & Aydın, B. Trimetazidine alone or in combination with gemcitabine and/or abraxane decreased cell viability, migration and ATP levels and induced apoptosis of human pancreatic cells. Clin. Res. Hepatol. Gastroenterol.45(6), 101632. 10.1016/j.clinre.2021.101632 (2021). [DOI] [PubMed] [Google Scholar]
  • 18.Kobayashi, T. et al. Multiomics analysis of right ventricles in rat models of pulmonary arterial hypertension: Consideration of mitochondrial biogenesis by chrysin. Int. J. Mol. Med.49(5), 69. 10.3892/ijmm.2022.5124 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Izquierdo-Garcia, J. L. et al. Metabolic reprogramming in the heart and lung in a murine model of pulmonary arterial hypertension. Front. Cardiovasc. Med.5, 110. 10.3389/fcvm.2018.00110 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Humbert, M. et al. Pathology and pathobiology of pulmonary hypertension: State of the art and research perspectives. Eur. Respir. J.53(1), 1801887. 10.1183/13993003.01887-2018 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Brittain, E. L. et al. Fatty acid metabolic defects and right ventricular lipotoxicity in human pulmonary arterial hypertension. Circulation133(20), 1936–1944. 10.1161/CIRCULATIONAHA.115.019351 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Liu, X. et al. Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector. Biomaterials35(14), 4401–4416. 10.1016/j.biomaterials.2014.02.009 (2014). [DOI] [PubMed] [Google Scholar]
  • 23.Tuder, R. M., Marecki, J. C., Richter, A., Fijalkowska, I. & Flores, S. Pathology of pulmonary hypertension. Clin Chest Med.28(1):23–42. 10.1016/j.ccm.2006.11.010 (2007). [DOI] [PMC free article] [PubMed]
  • 24.Wang, A. P. et al. A critical role of the mTOR/eIF2α pathway in Hypoxia-Induced Pulmonary Hypertension. PLoS One10(6), e0130806. 10.1371/journal.pone.0130806 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Pena, A. et al. Pharmacological inhibition of mTOR kinase reverses right ventricle remodeling and improves right ventricle structure and function in rats. Am. J. Respir Cell. Mol. Biol.57(5), 615–625. 10.1165/rcmb.2016-0364OC (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhang, W. B. et al. Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition. J. Biol. Chem.285(52), 40461–40471. 10.1074/jbc.M110.164046 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sanchez, O. et al. Role of endothelium-derived CC chemokine ligand 2 in idiopathic pulmonary arterial hypertension. Am. J. Respir Crit. Care Med.176(10), 1041–1047. 10.1164/rccm.200610-1559OC (2007). [DOI] [PubMed] [Google Scholar]
  • 28.Hosokawa, S. et al. Pathophysiological roles of nuclear factor kappaB (NF-kB) in pulmonary arterial hypertension: Effects of synthetic selective NF-kB inhibitor IMD-0354. Cardiovasc. Res.99(1), 35–43. 10.1093/cvr/cvt105 (2013). [DOI] [PubMed] [Google Scholar]
  • 29.Coggins, M. P. & Bloch, K. D. Nitric oxide in the pulmonary vasculature. Arterioscler. Thromb. Vasc Biol.27(9), 1877–1885. 10.1161/ATVBAHA.107.142943 (2007). [DOI] [PubMed] [Google Scholar]
  • 30.Christou, H. et al. Impaired pulmonary arterial vasoconstriction and nitric oxide-mediated relaxation underlie severe pulmonary hypertension in the Sugen-Hypoxia Rat Model. J. Pharmacol. Exp. Ther.364(2), 258–274. 10.1124/jpet.117.244798 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Yang, Z. & Ming, X. F. Arginase: The emerging therapeutic target for vascular oxidative stress and inflammation. Front. Immunol.4, 149. 10.3389/fimmu.2013.00149 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Di Napoli, P. et al. Trimetazidine improves post-ischemic recovery by preserving endothelial nitric oxide synthase expression in isolated working rat hearts. Nitric Oxide16(2), 228–236. 10.1016/j.niox.2006.09.001 (2007). [DOI] [PubMed] [Google Scholar]
  • 33.Wu, Q. et al. Mechanisms underlying protective effects of trimetazidine on endothelial progenitor cells biological functions against H2O2-induced injury: Involvement of antioxidation and Akt/eNOS signaling pathways. Eur. J. Pharmacol.707(1–3), 87–94. 10.1016/j.ejphar.2013.03.027 (2013). [DOI] [PubMed] [Google Scholar]
  • 34.Omura, J. et al. Protective roles of endothelial AMP-Activated protein kinase against Hypoxia-Induced Pulmonary Hypertension in mice. Circ. Res.119(2), 197–209. 10.1161/CIRCRESAHA.115.308178 (2016). [DOI] [PubMed] [Google Scholar]
  • 35.Abdelazeem, H. et al. AMPK activation by metformin protects against pulmonary hypertension in rats and relaxes isolated human pulmonary artery. Eur. J. Pharmacol.946, 175579. 10.1016/j.ejphar.2023.175579 (2023). [DOI] [PubMed] [Google Scholar]
  • 36.Oliveira-Paula, G. H., Lacchini, R. & Tanus-Santos, J. E. Endothelial nitric oxide synthase: From biochemistry and gene structure to clinical implications of NOS3 polymorphisms. Gene575(2 Pt 3), 584–599. 10.1016/j.gene.2015.09.061 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Talati, M. & Hemnes, A. Fatty acid metabolism in pulmonary arterial hypertension: Role in right ventricular dysfunction and hypertrophy. Pulm Circ.5(2), 269–278. 10.1086/681227 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kumthekar, P. et al. A phase II trial of arsenic trioxide and temozolomide in combination with radiation therapy for patients with malignant gliomas. J. Neurooncol133(3), 589–594. 10.1007/s11060-017-2469-x (2017). [DOI] [PubMed] [Google Scholar]
  • 39.Damato, A. R. et al. Temozolomide chronotherapy in patients with glioblastoma: A retrospective single-institute study. Neurooncol Adv.3(1), vdab041. 10.1093/noajnl/vdab041 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Verdejo, H. E. et al. Effects of Trimetazidine on right ventricular function and ventricular remodeling in patients with pulmonary artery hypertension: A Randomised Controlled Trial. J. Clin. Med.12(4), 1571. 10.3390/jcm12041571 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kato, F. et al. Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats. Am. J. Physiol. Lung Cell. Mol. Physiol.313(5), L899–L915. 10.1152/ajplung.00527.2016 (2017). [DOI] [PubMed] [Google Scholar]
  • 42.Takeuchi, T. et al. Pulmonary haemodynamics are correlated with intimal lesions in a rat model of severe PAH: Attenuation of pulmonary vascular remodelling with ambrisentan. Histol. Histopathol31(12), 1357–1365. 10.14670/HH-11-764 (2016). [DOI] [PubMed] [Google Scholar]
  • 43.Ge, S. X., Son, E. W. & Yao, R. iDEP: An integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinform.19(1): 534. 10.1186/s12859-018-2486-6 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kuleshov, M. V. et al. Enrichr: A comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res.44(W1), W90–W97. 10.1093/nar/gkw377 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Kanehisa, M. & Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res.28(1), 27–30. 10.1093/nar/28.1.27 (2000). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kanehisa, M. Toward understanding the origin and evolution of cellular organisms. Protein Sci.28(11), 1947–1951. 10.1002/pro.3715 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Kanehisa, M., Furumichi, M., Sato, Y., Kawashima, M. & Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res.51(D1), D587–D592. 10.1093/nar/gkac963 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Parra, V. et al. Inhibition of mitochondrial fission prevents hypoxia-induced metabolic shift and cellular proliferation of pulmonary arterial smooth muscle cells. Biochim. Biophys. Acta Mol. Basis Dis.1863(11), 2891–2903. 10.1016/j.bbadis.2017.07.018 (2017). [DOI] [PubMed] [Google Scholar]
  • 49.Shoji, H. et al. The isoquinoline-sulfonamide compound H-1337 attenuates SU5416/Hypoxia-Induced Pulmonary arterial hypertension in rats. Cells11(1), 66. 10.3390/cells11010066 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Deshmane, S. L., Kremlev, S., Amini, S. & Sawaya, B. E. Monocyte chemoattractant protein-1 (MCP-1): An overview. J. Interferon Cytokine Res.29(6), 313–326. 10.1089/jir.2008.0027 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (19.3KB, xlsx)
Supplementary Material 2 (16.5KB, xlsx)
Supplementary Material 3 (17KB, xlsx)
Supplementary Material 4 (1.9MB, pdf)

Data Availability Statement

All data needed to evaluate the conclusions in the paper are present in this study or upon reasonable request from the correspondent authors. All raw data files for the RNA-seq analysis were deposited in the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo; accession number: GSE186996).

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