Integrated Transcriptomic Analyses and Experimental Validation Identify TSPO as a Mitochondria‐Associated Therapeutic Target in Myocarditis

Figures

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  Figure 1. Screening and validation of mitochondrial genes contribute to myocarditis. (A) Relationships between modules and disease status, and the corresponding p values in GSE248521. Yellow module (MEYellow) displays the highest correlation with myocarditis (red). (B) Top 10 enriched GO BPs and KEGG pathways of hub module. (C) Overlapping 1189 hub module genes and 3052 DEGs obtained from GSE248521, together with 1140 MtGs (from MitoCarta3.0), identified 11 upregulated MitoMYCGs. (D) Identification of top 5 genes through random forest analysis. (E) SHAP feature importance presented based on the mean absolute SHAP value of each gene. (F) Bar plots of Tspo between myocarditis and control in the 10 dpi and 90 dpi CVB3 induced myocarditis‐GSE35182 and TMEV‐induced myocarditis‐GSE53607. ∗∗∗ p value < 0.001; ∗∗∗∗ p value < 0.0001; BPs, biological processes; DEGs, differentially expressed genes; MitoMYCGs, mitochondrial genes contribute to myocarditis; MtGs, mitochondrial metabolism‐related genes; ns, nonsignificant; SHAP, SHapley Additive exPlanations.

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  Figure 2. Comprehensive analysis of scRNA‐seq data. (A) Cell type annotation of clusters. 8 dominating cell types were distinguished according to canonical cell markers. (B) The proportion of 8 cell types in myocarditis and control, in which macrophages are the immune cells that have the highest variation. (C) Violin plots of Tspo in all clusters and macrophages between myocarditis and control. (D) H&E staining of heart tissues from mice with and without CVB3 infection (scale bar = 50 μm). (E) Relative mRNA expression of Tspo in heart tissues of mice with and without CVB3 infection (n = 3). (F) Representative immunofluorescence images depicting the colocalization between Tspo (green) and the classical marker of macrophage‐Cd68 (red; scale bar = 75 μm). White arrows indicate macrophage in the PBS group and the colocalization of Cd68 and Tspo in the CVB3 group. (G) Flow cytometry was used to detect Cd45 and F4/80 double‐positive cells in mouse cardiac tissue. The proportion of Cd45+F4/80+ cells was significantly increased in the CVB3 group compared with the control group. ∗ p value < 0.05; ∗∗∗ p value < 0.001; ∗∗∗∗ p value < 0.0001; H&E, hematoxylin and eosin; scRNA‐seq, single‐cell RNA…

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  Figure 3. Relationship between Tspo and inflammation. (A) RT‐qPCR of Tspo in RAW264.7 cells between the IFN‐γ+LPS and PBS groups. (B) Western blot was performed to examine Tspo protein levels in the PBS group and IFN‐γ+LPS group. Densitometric quantification was conducted after normalization to β‐tubulin. (C–E) Macrophages with Tspo knockdown displayed enhanced inflammatory responses upon stimulation relative to si‐NC control (n = 3). (F–H) Treatment with the Tspo agonist Ro5‐4864 alleviates inflammatory responses in stimulated RAW264.7 macrophages (n = 3). ∗ p value < 0.05, ∗∗ p value < 0.01; ∗∗∗ p value < 0.001; ∗∗∗∗ p value < 0.0001; DMSO + sti, treatment with DMSO and subsequently stimulated with IFN‐γ and LPS; ns, nonsignificant; Ro5‐4864+sti, treatment with Ro5‐4864 and subsequently stimulated with IFN‐γ and LPS; si‐NC, siRNA negative control; si‐NC+sti, transfected with siRNA negative control and subsequently stimulated with IFN‐γ and LPS; si‐Tspo, siRNA targeting Tspo; si‐Tspo+sti, transfected with siRNA‐Tspo and subsequently stimulated with IFN‐γ and LPS.

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  Figure 4. Relationship between Tspo and mitochondrial function and morphology. (A) RAW264.7 were transfected with si‐NC or si‐Tspo and treated with or without inflammatory stimulation. Flow cytometry was applied to detect DCFH‐DA fluorescence intensity, followed by quantification of MFI. (B) RAW264.7 were treated with DMSO or Ro5‐4864 and with or without inflammatory stimulation. Flow cytometry was applied to detect DCFH‐DA fluorescence intensity, followed by quantification of MFI. (C) Representative TEM images depicting the morphological alterations in RAW264.7 mitochondria following treatment with DMSO + sti and Ro5‐4864+sti. The lower sections of the images depict magnified regions corresponding to the upper images, which are enclosed within black boxes (upper section scale bar = 5 μm; lower section scale bar = 500 nm). (D–E) Detection of ΔΨm using JC‐1 probe. Red indicated JC‐1 aggregates, while green indicated JC‐1 monomer. The ΔΨm was determined by calculating the ration of red‐to‐green fluorescence intensity (scale bar = 10 μm). (F) GSEA analysis identifies enrichment scores for gene set of “Regulation of oxidative stress induced apoptotic signaling pathway”. (G) Heatmap displaying…

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  Figure 5. The administration of Ro5‐4864 in mice with CVB3‐induced myocarditis confers cardioprotection and attenuates the inflammatory response. (A) Schematic of the administration workflow of Ro5‐4864 in mice with CVB3‐induced myocarditis. (B) The daily weight changes of mice receiving different treatments were monitored for 8 days in three groups: PBS (n = 3), CVB3 (n = 3), and CVB3+Ro (n = 3). Intergroup difference was analyzed by two-way ANOVA. (C) The levels of cTnT in the serum of mice from three groups were quantified using ELISA (n = 3). (D) Representative short‐axis M‐mode echocardiography images of mice assigned to PBS, CVB3, and CVB3+Ro groups. The cardiac function was presented as %LVEF and %LVFS (n = 3). (E) Representative immunofluorescence images depicting that the administration of Ro5‐4864 could diminished the infiltration of macrophage (scale bar = 125 μm). (F) Representative images of heart tissues, including gross morphology, H&E‐stained sections, and IHC images. Positive staining area proportion (%Area) was calculated on IHC sections (scale bar = 50 μm; n = 3). (G) Relative mRNA expression of TNF‐α, IL‐1β and IL‐6 in heart tissues of mice from three groups (n = 3).…