Palmitoylated SARM1 targeting P4HA1 promotes collagen deposition and myocardial fibrosis: A new target for anti-myocardial fibrosis

Figures

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  Figure 1. SARM1 upregulated expression in cardiac fibrosis, and knockdown of SARM1 attenuates myocardial fibrosis. (A) GEO datasets analyzed the differential expression of SARM1. (B) Western blot was used to detect the protein expression of SARM1 in the heart tissue of MI mice. n = 4. ∗P < 0.05 vs. Sham. (C) Immunofluorescence was used to determine the expression of SARM1 in the heart tissue of MI mice. n = 3. ∗P < 0.05 vs. Sham. Scale bar, 200 μm. (D) Western blot is used to detect the expression of SARM1 in neonatal mouse cardiac fibroblasts (NMCFs) after TGF-β1 (10 ng/mL) treatment. n = 3. ∗P < 0.05 vs. CTL. (E) Immunofluorescent staining to detect the expression and localization of SARM1 in NMCFs after TGF-β1 treatment. n = 3. Scale bar, 400 μm. (F) Western blot was used to detect the protein level of SARM1 after tail vein injection of SARM1 knockdown virus. n = 4. ∗∗∗P < 0.001 vs. Sham, #P < 0.05 vs. +VP-shNC. (G) Echocardiographic detection of cardiac function changes. n = 15–21. ∗∗∗P < 0.001 vs. Sham, ###P < 0.001 vs. +VP-shNC. (H) The effects of SARM1 knockdown on the heart weight/body weight ratio of mice. n = 10–16. ∗∗P < 0.01 vs. Sham, ##P < 0.01 vs. +VP-shNC. (I) Masson…

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  Figure 2. Knockdown of SARM1attenuates TGF-β1 induced fibrosis in NMCFs. (A) Experimental flow chart. (B) Western blot was used to detect the efficiency of SARM1 knockout plasmid (Sh-Sarm1). n = 3, ∗P < 0.05 vs. Vector. (C) CCK8 to detect the effect of cell viability. n = 7–9. ∗∗P < 0.01 vs. CTL, ###P < 0.001 vs. +Vector. (D, E) Wound healing assay to detect the effect of cell migration ability. n = 3. ∗∗P < 0.01 vs. CTL, ##P < 0.01 vs. +Vector. Scale bar, 100 μm. (F, G) EdU staining was used to detect the effect of cell proliferation. n = 3. ∗∗P < 0.01 vs. CTL, #P < 0.05 vs. +Vector. Scale bar, 200 μm. (H, I) Application of immunofluorescence in the determination of fibrosis-related protein expression of α-SMA. n = 3, ∗∗P < 0.01 vs. CTL, ##P < 0.01 vs. +Vector. (J, K) Western blot was used to detect the protein expression of Collagen I (n = 4) and Collagen III (n = 3) in fibroblasts. ∗P < 0.05, ∗∗P < 0.01 vs. CTL, #P < 0.05 vs. +Vector. (L) The hydroxyproline assay kit is used to detect the secretion of collagen. n = 5. ∗∗∗P < 0.001 vs. CTL, ###P < 0.001 vs. +Vector. Data are presented as mean ± SEM.

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  Figure 3. SARM1 overexpression aggravates cardiac fibrosis. (A) A schematic diagram of the generation of fibroblasts-specific Sarm1 knock-in mice. (B) Reproduction diagram of transgenesis mice. (C) Immunofluorescent staining to detect the protein expression of SARM1 in myocardial fibroblasts. n = 3. ∗P < 0.05, ∗∗P < 0.01 vs. Sarm1fl/fl. Scale bar, 200 μm. (D) Echocardiographic detection of cardiac function changes. n = 7. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Sarm1fl/fl. (E) Immunofluorescent staining to detect the protein expression of Collagen I. n = 4. ∗P < 0.05 vs. Sarm1fl/fl. Scale bar, 200 μm. (F) Experimental flow chart. (G) Western blot was used to detect the efficiency of Sarm1 overexpression plasmid (Sarm1). n = 3. ∗P < 0.05 vs. Vector. (H) CCK8 to detect the effect of cell viability. n = 6–10. ∗∗∗P < 0.001 vs. Vector. (I, J) Wound healing assay was used to detect the influence of cell migration ability. n = 3. ∗∗P < 0.01 vs. Vector. Scale bar, 100 μm. (K, L) EdU staining was used to detect the effect of cell proliferation. n = 3. ∗∗∗P < 0.001 vs. Vector. Scale bar, 200 μm. (M) The hydroxyproline assay kit is used to detect the secretion of collagen. n = 5. ∗∗P < 0.01 vs. Vector. (N–P)…

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  Figure 4. SARM1 targeting P4HA1 affects collagen synthesis in fibroblasts. (A) Co-IP/MS sequencing to screen the downstream effector proteins of SARM1. (B) Western blot was used to detect the expression of P4HA1 in neonatal mouse cardiac myocytes (NMCMs) and neonatal mouse cardiac fibroblasts (NMCFs). n = 3, ∗P < 0.05 vs. NMCMs. (C) qRT-PCR is used to detect the knockdown efficiency of P4ha1. n = 4. ∗∗P < 0.01 vs. siNC. (D) CCK8 to detect the effect of cell viability. n = 12. ∗∗∗P < 0.001 vs. CTL, ###P < 0.001 vs. +siNC. (E) Wound healing assay was used to detect the influence of cell migration ability. n = 4. ∗P < 0.05 vs. CTL, ##P < 0.01 vs. +siNC. Scale bar, 100 μm. (F) The hydroxyproline assay kit is used to detect the secretion of collagen. n = 4. ∗∗P < 0.01 vs. CTL, ###P < 0.001 vs. +siNC. (G, H) Western blot was used to detect the protein expression of Collagen I and Collagen III in fibroblasts after P4HA1 knockout. n = 4. ∗∗P < 0.01, ∗∗∗P < 0.001 vs. CTL, #P < 0.05 vs. +siNC. (I) Western blot was used to detect the protein expression of P4HA1 in fibroblasts after overexpression of SARM1. n = 3. ∗P < 0.05 vs. Vector. (J) Immunofluorescent staining to detect the protein…

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  Figure 5. Palmitoylation of SARM1effects fibroblast activation. (A) Predicting the palmitoylation sites of SARM1 protein. (B) Experimental flow chart. (C) Click-iT reaction was used to detect the palmitoylation level of SARM1. n = 3. (D) Western blot was used to detect the protein expression of SARM1 in fibroblasts after administration of palmitoyl inhibitor (2-BP). n = 4. ∗∗P < 0.01 vs. CTL, ##P < 0.01 vs. TGF-β1. (E) CCK8 to detect the effect of cell viability. n = 6–9. ∗∗∗P < 0.001 vs. CTL, ###P < 0.001 vs. TGF-β1. (F) The hydroxyproline assay kit is used to detect the secretion of collagen. n = 4. ∗P < 0.05 vs. CTL, ###P < 0.001 vs. TGF-β1. (G, H) Western blot was used to detect the protein expression of Collagen I and Collagen III in fibroblasts after 2-BP administration. n = 4. ∗∗P < 0.01 vs. CTL, #P < 0.05 vs. TGF-β1. (I) Application of immunofluorescence staining to detect the expression of α-SMA. n = 3. ∗∗∗P < 0.001 vs. Vector, ###P < 0.001 vs. Sarm1. Scale bar, 200 μm. (J) Western blot was used to detect the protein expression of P4HA1 in fibroblasts after 2-BP administration. n = 3. ∗P < 0.05 vs. CTL, #P < 0.05 vs. TGF-β1. Data are presented as mean ± SEM.

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  Figure 6. ZDHHC17 mediates palmitoylation of SARM1 in myocardial fibroblasts after MI. (A, B) Venn diagram was used to screen the palmitoyl acylase acting on SARM1 (https://www.proteinatlas.org/). (C) qRT-PCR is used to detect the knockdown efficiency of Zdhhc3, Zdhhc9, Zdhhc16, Zdhhc17, and Zdhhc21. n = 4–6. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. siNC. (D) Western blot was used to detect the protein expression of SARM1 after the deletion of candidate palmitoylase. n = 3. ∗∗P < 0.01 vs. CTL, ##P < 0.01 vs. TGF-β1. (E) Click-iT reaction was used to detect the palmitoylation level of SARM1. n = 3. (F) qRT-PCR is used to detect the expression of Zdhhc17 in fibroblasts after TGF-β1 treatment. n = 5. ∗P < 0.05 vs. CTL. (G) CCK8 to detect the effect of cell viability. n = 12. ∗∗∗P < 0.001 vs. CTL, ###P < 0.001 vs. +siNC. (H) The hydroxyproline assay kit is used to detect the secretion of collagen. n = 4. ∗∗∗P < 0.001 vs. CTL, ###P < 0.001 vs. +siNC. (I) Multiplex immunohistochemistry is used to detect coexpression between ZDHHC17 and SARM1 in myocardial fibroblasts in the infarct margin area of MI tissue. n = 3. Scale bar, 200 μm. Data are presented as mean ± SEM.

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  Figure 7. The mutation of SARM1 in the palmitoylation site weakens its role in promoting cardiac fibrosis. (A) The amino acid sequence of C13S/C350S after the SARM1 palmitoylation site (CYS13, CYS350) mutation. (B) CCK8 to detect the effect of cell viability. n = 12. ∗∗∗P < 0.001 vs. Vector, ##P < 0.01 vs. Sarm1. (C) The hydroxyproline assay kit is used to detect collagen secretion. n = 3. ∗∗∗P < 0.001 vs. Vector, ###P < 0.001 vs. Sarm1. (D, E) Wound healing assay to examine the effect of cell migration ability. n = 4. ∗∗∗P < 0.001 vs. Vector, ###P < 0.001 vs. Sarm1. Scale bar, 100 μm. (F) Amino acid sequences of C13S and C350S after SARM1 palmitoylation site mutation of Cys13 and Cys350, respectively. (G) CCK8 to detect the effect of cell viability. n = 10. ∗∗P < 0.01 vs. Vector, #P < 0.05 vs. Sarm1. (H)The hydroxyproline assay kit is used to detect collagen secretion. n = 6. ∗∗∗P < 0.001 vs. Vector, ###P < 0.001 vs. Sarm1. (I, J) Wound healing assay to examine the effect of cell migration ability. n = 3. ∗∗P < 0.01 vs. Vector, #P < 0.05 vs. Sarm1. Scale bar, 100 μm. Data are presented as mean ± SEM.

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  Figure 8. Screening of anti-cardiac fibrosis drugs targeting SARM1. (A) CCK8 was used to detect the effects of disulfiram, BBC, DSRM-3716, and ZnCl2 on cell viability. n = 10. ∗∗∗P < 0.001 vs. Vector, ##P < 0.01, ###P < 0.001 vs. Sarm1. (B) The hydroxyproline assay kit was used to detect the secretion of fibroblast collagen by BBC. n = 4. ∗∗∗P < 0.001 vs. Vector, ###P < 0.001 vs. Sarm1. (C, D) Immunofluorescence in the determination of fibroblast expression of SARM1. n = 3. Scale bar, 400 μm. (E) CCK8 to detect the effect of cell viability. n = 10. ∗∗∗P < 0.001 vs. Vector, ##P < 0.01 vs. Sarm1. (F) The hydroxyproline assay kit is used to detect collagen secretion. n = 4. ∗∗P < 0.01 vs. Vector, ###P < 0.001 vs. Sarm1. (G) Echocardiographic detection of cardiac function changes. n = 3–4. ∗P < 0.05 vs. Sham, #P < 0.05 vs. MI. (H) The hydroxyproline assay kit is used to detect the secretion of collagen in mouse heart tissue. n = 3. ∗∗∗P < 0.001 vs. Sham, ##P < 0.01 vs. MI. (I) The flowchart of the bioinformatics analysis. (J)Volcano plot of GSE115031. (K–N) TCGA database analyzed the expression of SARM1 in different cancers. ∗P < 0.05, ∗∗∗P < 0.001 vs. Normal. Clear cell renal cell carcinomas…