High-fat stimulation induces atrial structural remodeling via the TPM1/P53/SHISA5 Axis

Fig. 1. Identification of TPM1 as a putative target protein. Masson staining to assess the degree of tissue fibrosis after high-fat diet (A). Analysis of differentially expressed proteins by LC-MS/MS (B). Volcano plot showing up- and down-regulated proteins (C). WB detection of TPM1 protein expression changes after high-fat diet (D). WB detection of TPM1 protein expression abundance differences between AC16 cells and HCF cells (E). Changes in TPM1 protein expression in AC16 cells after stimulation with different concentrations of PA (F)

Fig. 2. PA stimulation increases the secretion of EVs-TPM1 from AC16 cells. Verification of TPM1 changes in AC16 cells after PA stimulation using confocal microscopy (40X), Scale bar = 10 μm (A). WB verification of EVs marker proteins (B). Representative morphological analysis by TEM (C). Particle size distribution detected by NTA (D). Immunofluorescence tracing of exosomes (40X), Scale bar = 10 μm (E)

Fig. 3. TPM1 stimulation induces proliferation and fibrosis in HCF cells. WB detection of changes in Fibronectin, Collagen I, MMP2, TGF-β expression in HCF cells after rTPM1 stimulation (A). EdU assay of HCF cell proliferation levels after rTPM1 stimulation (20X), Scale bar = 100 μm (B). Flow cytometry verification of HCF cell proliferation levels after rTPM1 stimulation (C)

Fig. 4. RNA-seq analysis on HCF cells stimulated with rTPM1. Analysis of differentially expressed genes by Transcriptome sequencing (A,B). qPCR validation of transcriptomics results (C). Gene Ontology (GO) enrichment analysis of differentially expressed proteins (DEPs) in NC and rTPM1-stimulated groups. The Y-axis represents the number of DEP genes (D)

Fig. 5. TPM1 induces ER stress and autophagy in HCF cells. WB detection of P62, P53, SHISA5, LC3-II, p-IRE1α, p-JNK protein expression after rTPM1 and BafA1 stimulation (A). TEM observation of autophagosomes and endoplasmic reticulum structures in HCF cells with or without rTPM1 stimulation (B). Immunofluorescence co-localization analysis of autophagy levels in HCF cells after rTPM1 and BafA1 stimulation, Scale Bar = 10 μm (C). Immunofluorescence observation of P53 and SHISA5 protein expression in HCF cells after rTPM1 stimulation, Scale Bar = 10 μm (D). WB detection of Collagen I, p-IRE1α, GRP78, MMP2, P62, p-JNK, LC3-II proteins after 4-PBA inhibition of ERS (E)

Fig. 6. The TPM1/P53/SHISA5 axis mediates ER stress and autophagy. WB detection of cytoplasmic and nuclear P53 protein expression in HCF cells after rTPM1 stimulation (A). WB detection of P53, SHISA5, Collagen I, MMP2, p-IRE1α, GRP78, p-JNK, P62, LC3-II protein expression in HCF cells after activation of P53 by C16-ceramide stimulation (B). WB detection of Collagen I, p-IRE1α, GRP78, MMP2, P62, p-JNK, SHISA5, LC3-II protein expression after ADV-SHISA5 transfection in HCF cells (C)

Fig. 7. In vivo animal experiment validation. Optical microscopy (20X) observation of rat atrial myocardial tissue, HE staining to observe tissue morphology, Masson staining to observe the degree of tissue fibrosis, IHC to observe the expression proportion of GRP78 and p-IRE1 proteins in the tissue, fluorescence microscopy (20x) observation of AAV virus transfection efficiency of SHISA5 in rat atrial myocardial tissue (A). WB detection of Collagen I, p-IRE1α, GRP78, MMP2, P62, p-JNK, P53, SHISA5, LC3-II expression in atrial tissue (B). Immunofluorescence observation of P53 protein (Red) expression in PDGFRα-marked cardiac fibroblasts (Green) (Yellow) (C)

Fig. 8. Proposed mechanism of the TPM1/P53/SHISA5 axis. Under high-fat stimulation, AC16 cells secrete EVs-TPM1, which act on HCF cells. Within HCF, TPM1 inhibits P53 activation, leading to downregulation of SHISA5 expression in the ER. Reduced SHISA5 triggers ERS and subsequent dysregulation of autophagy, ultimately promoting myocardial fibrosis (By Figdraw)