The solute carrier transporter SLCO5A1 as a novel therapeutic target for the treatment of myocardial infarction-induced heart failure

Figures

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  SLCO5A1 serves as a novel therapeutic target for heart failure. Enhancing SLCO5A1 expression protects against MI-induced HF by inhibiting TGF-β1/Smad3-mediated cardiomyocyte–fibroblast crosstalk.

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  Fig. 1. Screening of candidate genes based on DEG analysis and WGCNA. a PCA of GSE55296 and GSE57345 before batch correction. b PCA after batch correction. c Volcano map of DEGs (HF vs. control). Upregulated genes are shown in red, and downregulated genes are shown in green. d Module‒trait correlation heatmap from WGCNA. e Scatter plot showing the relationship between genetic significance for HF and module membership in the blue module. f Venn diagram showing overlap between DEGs and blue-module genes. g KEGG enrichment analysis of overlapping genes. h GO enrichment analysis of overlapping genes. DEG, differentially expressed gene; GO, Gene Ontology; HF, heart failure; KEGG, Kyoto Encyclopedia of Genes and Genomes; PCA, principal component analysis; WGCNA, weighted gene coexpression network analysis.

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  Fig. 2. SLCO5A1 is downregulated in the failing myocardium and cardiomyocytes and exerts a protective effect on HF. a Bidirectional two-sample MR results for candidate genes and HF risk (forward MR: SLCO5A1 toward HF; reverse MR: HF toward SLCO5A1). b, c Representative immunoblotting and quantification of SLCO5A1 protein levels in mouse hearts 4 weeks after MI or sham surgery (n = 6). d, e Representative immunoblotting and quantification of SLCO5A1 protein expression in NMCMs treated with ISO-PE or vehicle (n = 6). f, g Immunoblotting and quantification of SLCO5A1 protein expression in CFs, CMs and cMECs (n = 6). h, i Representative immunofluorescence images and quantification of SLCO5A1 expression in hearts after MI or sham surgery (scale bar = 50 μm). **P < 0.01, ***P < 0.001 vs. the indicated control. CF, cardiac fibroblast; CM, cardiomyocyte; cMEC, cardiac microvascular endothelial cell; ISO, isoprenaline; MI, myocardial infarction; NMCM, neonatal mouse cardiomyocyte; PE, phenylephrine.

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  Fig. 3. Cardiomyocyte-targeted SLCO5A1 overexpression attenuates post-MI cardiac dysfunction and remodeling. a Representative echocardiographic images from sham or HF mice injected with AAV9-cTnT-vector (Vec) or AAV9-cTnT-SLCO5A1 (OE) via tail-vein injection. b Quantitative analysis of EF and FS (n = 6). c Representative images of Masson’s trichrome staining of LV sections (scale bar = 1 mm). d Representative PSR staining images (scale bar = 1 mm). e, f Quantitative analysis of fibrosis and collagen areas from (c, d) (n = 6). g, h Western blotting and quantitative analysis of cardiac fibronectin, collagen I, and α-SMA expression. i, j Western blotting and quantitative analysis of cardiac β-MHC, ANP, and BNP expression. k, l WGA staining and quantitative analysis. **P < 0.01, ***P < 0.001, ##P < 0.01, and ### P < 0.001. Fibronectin, FN; Collagen I, Col.

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  Fig. 4. T1AM directly interacts with SLCO5A1 and increases its protein expression and thermal stability. a Chemical structure of T1AM. b Representative Western blotting images and quantitative analysis of SLCO5A1 expression in ISO-PE-induced NMCMs treated with T1AM at the indicated concentrations (n = 6). d Representative Western blotting images and quantitative analysis of SLCO5A1 expression in the hearts of mice with HF treated with vehicle or T1AM at the indicated doses (n = 6). c, e Quantitative analysis of the Western blotting results (b, d). f Representative molecular docking pose between SLCO5A1 and T1AM. g MM-GBSA analysis of energy catabolism showing the top 10 amino acids that contribute to the binding of T1AM and SLCO5A1. h RMSD of the SLCO5A1–T1AM complex during MD simulation. i RMSF of residues during MD simulation. j Number of hydrogen bonds between T1AM and SLCO5A1 during MD simulation. k Free energy landscape of the SLCO5A1–T1AM complex. l–m CETSA immunoblot showing SLCO5A1 levels after heating in vehicle-treated vs. T1AM-treated samples. n Quantitative analysis of the CETSA results (l, m). o SPR sensorgram showing the binding of T1AM to immobilize SLCO5A1 and model…

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  Fig. 5. T1AM increases survival and improves cardiac function and cardiac remodeling after MI. a Twenty-eight-day Kaplan‒Meier survival curves for sham or HF mice treated with NS or T1AM at the indicated doses (n = 30 mice per group). b Representative echocardiographic images at 28 days post-MI (n = 6). c Quantitative analysis of EF and FS. d Representative images of H&E-stained LV sections (scale bar = 50 μm). e Representative images following Masson’s trichrome staining (scale bar = 1 mm). f Representative images following PSR staining (scale bar = 1 mm). g Representative images following WGA staining of cross-sectional cardiac tissues (scale bar = 50 μm). h Representative images following immunofluorescence staining of α-SMA in cardiac sections (scale bar = 100 μm). i, j Quantitative analysis of fibrosis and collagen areas from (e, f) (n = 6). k Quantitative analysis of the cardiomyocyte cross-sectional area from (g) (n = 6). l Quantitative analysis of ɑ-SMA immunofluorescence staining data (n = 6). m Representative Western blotting images showing cardiac Collagen I, Fibronectin, and α-SMA expression in mice 28 days post-MI. n Representative Western blotting images showing β-MHC, ANP,…

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  Fig. 6. SLCO5A1 upregulation suppresses hypertrophic signaling in vitro. a Experimental T1AM treatment regimen applied to ISO-PE-treated NMCMs. b, c Representative Western blotting images and quantitative analysis of SLCO5A1, β-MHC, ANP, and BNP expression in ISO-PE-induced NMCMs treated with vehicle or T1AM under the indicated conditions (n = 6). d Experimental scheme for SLCO5A1 overexpression in ISO-PE-treated NMCMs. e, f Representative Western blot images and quantitative analysis of SLCO5A1, β-MHC, ANP, and BNP expression under the indicated conditions (n = 6). **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001.

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  Fig. 7. SLCO5A1 knockdown weakens the protective effects of T1AM on HF. a Representative echocardiographic images of HF mice treated with NS (HF-NS), T1AM (HF-T1AM), T1AM with Neg (HF-T1AM+Neg), or SLCO5A1 knockdown shRNA (HF-T1AM + sh). b Quantitative analysis of EF and FS (n = 6). c Representative images following Masson’s trichrome staining (scale bar = 1 mm). d Representative images following PSR staining (scale bar = 1 mm). e, f Quantitative analysis of the results in (c) and (d) (n = 6). g Representative Western blot images showing cardiac Fibronectin, Collagen I, and α-SMA expression. h Representative Western blot images showing cardiac β-MHC, ANP and BNP expression. i, j Quantitative analysis of the results in (g) and (h) (n = 6). k Experimental scheme used to combine T1AM treatment and SLCO5A1 knockdown in ISO-PE-treated NMCMs. l, m Representative images following immunofluorescence staining of α-actinin and quantitative analysis of cardiomyocyte size (n = 6). Scale bar = 25 μm. n, o Representative Western blotting images and quantitative analysis of SLCO5A1, β-MHC, ANP, and BNP expression in NMCMs under the indicated conditions (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, #P <…

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  Fig. 8. SLCO5A1 upregulation reduces TGF-β1 secretion in cardiomyocytes under HF conditions to alleviate NMCF fibrosis through the TGF-β1/Smad3 pathway. a ELISA of TGF-β1 levels in mouse cardiac tissue after MI and treatment with vehicle or T1AM at the indicated doses (n = 6). b ELISA of TGF-β1 levels in the supernatant from ISO-PE-induced NMCMs with or without SLCO5A1 overexpression (n = 6). c ELISA of TGF-β1 levels in the supernatant from ISO-PE-induced NMCMs treated with vehicle or T1AM (n = 6). d ELISA of TGF-β1 levels in the supernatant from ISO-PE-induced NMCMs treated with T1AM plus Neg or SLCO5A1 shRNA (n = 6). e Schematic diagram of conditioned-medium transfer from NMCMs to NMCFs. f–k Representative Western blotting images and quantitative analysis of Collagen I, Fibronectin, and α-SMA expression in NMCFs treated with conditioned medium from NMCMs under the indicated treatment conditions (n = 6). l–p Representative Western blot images and quantification of p-Smad3 (Ser423/425) and total Smad3 protein expression in NMCFs treated with the indicated conditioned medium (n = 6). **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001.