Fig. 7

CYP2E1 participates in myocardial injury through imbalanced mitochondrial OPA1 processing. (A) Interaction between CYP2E1 and OPA1 in myocardium from the OV-DXR group was confirmed using reciprocal Co-IP assays. (B) Immunoelectron microscopy of rat myocardium from the control-DXR (CON-DXR) group. Yellow and blue arrowheads indicate CYP2E1 and OPA1 localization, respectively. Scale bars: 200 nm. (C-D) Protein levels in total, cytoplasmic, and mitochondrial extracts from the three groups were analyzed via immunoblotting. Assessed proteins included mitochondrial OPA1, cytoplasmic cytochrome c, procaspase-3, and activated caspase-3. GAPDH, β-actin, or VDAC1 was employed for normalization. Data shown are mean ± SEM (n = 4–6 per group). (E-F) Fluorescence microscopic images of DHE (red) using frozen sections of cardiac tissues from above 3 groups. DAPI (blue) was used to stain the cell nucleus. White bar = 50 μm. Data shown are mean ± SEM (n = 9 per group). (G) ROS levels in rat left ventricular myocardial tissue were measured by ELISA. Data shown are mean ± SEM (n = 8 per group). (H-I) Fluorescence microscopic images of TUNEL assay on paraffin sections of cardiac tissues from the three groups. Confocal imaging shows colocalization of TUNEL-positive nuclei (green) with DAPI (red), indicated by white arrows. Scale bars: 10 μm. Quantitative analysis of TUNEL-positive nuclei was conducted (n = 3 rats per group, n = 6 fields per rat). Multiple comparisons between groups were analyzed using ANOVA with Tukey correction. *P < 0.05, **P < 0.01, ***P < 0.001