Cytochrome P450 2E1 aggravates DXR-induced myocardial injury through imbalanced mitochondrial OPA1

Figures

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  Fig. 1. CYP2E1 Expression in myocardial mitochondria under various heart diseases. (A) CYP2E1 expression in left ventricular myocardial tissue from clinical dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) samples. Data were analyzed using GSE116250 dataset from GEO and presented as RPKM (Reads Per Kilobase per Million mapped reads); n = 14 for non-heart failure donor group, n = 37 for DCM group, and n = 13 for ICM group. (B) Schematic representation of animal model establishment and experimental strategy for analyzing expression characteristics. Abbreviations: DXR, doxorubicin; AngII, angiotensin II; ISO, isoprenaline. (C) CYP2E1 protein levels in total protein and mitochondrial protein extracts from cardiac tissues in the animal models, determined via immunoblotting. (D-E) Quantification of CYP2E1 expression in total and mitochondrial protein extracts, normalized to GAPDH or VDAC1. Data shown are mean ± SEM; n = 4 per group. (F) CYP2E1 distribution in myocardial tissue from DXR-induced myocardial injury rat model and ISO-induced myocardial hypertrophy rat model via immunofluorescence. Colocalization of CYP2E1 (red) and ATP5A (mitochondrial marker, green) is indicated by…

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  Fig. 2. Establishment of myocardium-specific Cyp2e1 overexpression and knockout rats. (A) Experimental strategy for generating myocardium-specific CYP2E1 tool rats. (B-C) generating of conditional Cyp2e1 knockout (Cyp2e1 cKO) rats using the Cre-loxP recombination system. Crossbreeding with α-MHC-Cre rats produced myocardium-specific CYP2E1 knockout rats (Cyp2e1-KO; Cyp2e1flox/flox/α-MHC-Cre). LA: left arm; RA: right arm. (D-E) Immunoblotting of CYP2E1 in cardiac tissues from Cyp2e1-KO rats to assess knockout efficiency. Quantitative analysis normalized to GAPDH. (F) Strategy for constructing myocardium-specific CYP2E1 overexpression rats (Cyp2e1-OV using a-MHC (a-myosin heavy chain)-driven transgene expression. (G) Generation of Cyp2e1-OV rats through crossing with SD wild-type rats. (H-J) Validation of CYP2E1 overexpression efficiency in total and mitochondrial protein extracts from Cyp2e1-OV cardiac tissues via immunoblotting. Quantitative analysis was normalized to GAPDH or VDAC1. Data shown are mean ± SEM; n = 9 per group. Two-group comparisons were executed using unpaired two-tailed Student’s t-tests. **P < 0.01, ***P < 0.001

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  Fig. 3. CYP2E1 enhances susceptibility to pathological myocardial injury induced by DXR treatment. (A) Schematic diagram of pathological myocardial injury stimulation and experimental strategy in myocardium-specific Cyp2e1-KO and Cyp2e1-OV rats. (B) Transthoracic echocardiographic images (M-mode) of left ventricle (LV) morphology in six groups: control-saline (CON-saline), Cyp2e1-OV-saline (OV-saline), Cyp2e1-KO-saline (KO-saline), control-DXR (CON-DXR), Cyp2e1-OV-DXR (OV-DXR), and Cyp2e1-KO-DXR (KO-DXR). (C-J) Echocardiographic parameters, including LVPWs, LVPWd, LVAWs, LVAWd, LVIDs, LVIDd, ejection fraction, and fractional shortening, were analyzed across the six groups. Data shown are mean ± SEM; n = 8–12 rats per group. (K-L) Representative images of whole-heart transverse sections and magnified view of the left ventricle with H&E staining. Scale bars: blue = 5 mm; black = 40 μm. (M) Degree of fibrosis in myocardial tissue across the six groups, assessed by Masson trichrome staining. Scale bars: black = 40 μm. (N) Quantification of collagen area fraction in cardiomyocytes. Data shown are mean ± SEM; n = 3 rats and 18 fields per group. Multiple comparisons between groups were…

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  Fig. 4. CYP2E1 enhances susceptibility to pathological cardiac remodeling induced by ISO treatment. (A) Schematic representation of pathological cardiac hypertrophy stimulation and the experimental strategy in myocardium-specific Cyp2e1-KO and Cyp2e1-OV rats. (B) Transthoracic echocardiographic images (M-mode) illustrating left ventricle (LV) morphology in six groups: control-saline, Cyp2e1-OV-saline, Cyp2e1-KO-saline, control-ISO, Cyp2e1-OV-ISO (OV-ISO), and Cyp2e1-KO-ISO (KO-ISO). (C-J) Echocardiographic parameters, including LVPWs, LVPWd, LVAWs, LVAWd, LVIDs, LVIDd, ejection fraction, and fractional shortening, were analyzed across the six groups. Data are presented as mean ± SEM; n = 6–13 rats per group. (K-L) Representative images of whole-heart transverse sections and magnified view of the left ventricle with H&E staining. scale bars: blue = 5 mm; black = 40 μm. (M) Myocardial fiber morphology in myocardial tissue was assessed using immunofluorescence staining with wheat germ agglutinin (green). Paraffin sections were imaged using a confocal microscope. DAPI (blue) indicates nuclei. Scale bars: white = 40 μm. (N) Quantification of cross-sectional cardiomyocyte area across the six…

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  Fig. 5. CYP2E1 expression affects myocardial injury, mitochondrial metabolism, and myocardial apoptosis under DXR stimulation. (A) Experimental strategy for transcriptomic analysis and protein interaction mass spectrometry using myocardium-specific CYP2E1 animal models and stable H9c2 cell lines with high expression of Flag-CYP2E1. (B) Transcriptomic analysis of myocardium from Cyp2e1-KO or Cyp2e1-OV rats. Volcano plot shows up-regulated (pink) and down-regulated (green) genes. The x-axis represents log2fold-change, indicating the direction of gene expression changes (negative for downregulation, and positive for upregulation). The y-axis illustrates the negative log of the FDR-adjusted p-value, indicating the significance of changes. (C) Heatmap of differentially expressed genes involved in cardiomyopathy, mitochondrial metabolism, and apoptosis. (D) Enrichment analysis of RNA sequencing data, highlighting enriched pathways. Bubble color indicates significance, while bubble size corresponds to the number of DEGs in each pathway. (E) Enrichment analysis of CYP2E1-bound proteins identified by mass spectrometry following CYP2E1 immunoprecipitation. Key gene ontology terms and KEGG pathways…

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  Fig. 6. CYP2E1 expression affects the morphology and function of mitochondria under DXR stimulation. (A) Schematic diagram illustrating the experimental strategy to analyze the role of CYP2E1 in mitochondrial morphology and OPA1 processing in myocardium-specific Cyp2e1-KO and Cyp2e1-OV rats under pathological myocardial injury. (B) TEM images of myocardium from control-DXR (CON-DXR), Cyp2e1-OV-DXR (OV-DXR), and Cyp2e1-KO-DXR (KO-DXR) groups. Blue and purple arrowheads indicate smaller and elongated mitochondria, respectively. (C-D) Histogram and violin plot presenting the frequency distribution and median surface area of mitochondria across the three groups (49–67 mitochondria were analyzed per group). CSA, cell cross-sectional area. (E-G) Levels of CYP2E1 proteins, phosphorylated Drp1 (phosphor-Drp1) at Ser616 and Ser637), and Drp1 translocation (mitochondria and cytoplasm) were quantified via immunoblotting and standardized to GAPDH or VDAC1. Data shown are mean ± SEM; n = 4–6 per groups. (H-I) Activity of mitochondrial Complex I and mitochondrial Complex II. Data shown are mean ± SEM; n = 4–7 per groups. (J-K) Levels of NDUFS1, SDHA, SDHB and CYTB were quantified via immunoblotting,…

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  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…

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  Fig. 8. Unbalanced mitochondrial dynamics mediated by dysregulated CYP2E1 contributes to myocardial injury and heart failure. Dysregulated CYP2E1 expression in cardiomyocytes, induced by multiple factors, acts as a sensor of various pathophysiological conditions in the myocardium. Under pathological stimulation by DXR, CYP2E1 promotes mitochondrial fragmentation and the formation of smaller mitochondria, as demonstrated by TEM observations and analysis of Drp1 translocation and phosphorylation. CYP2E1 colocalizes with OPA1 in the inner mitochondrial membrane, where their interaction alters long and short OPA1 balance. This imbalance, mediated by CYP2E1, triggers mitochondrial fragmentation and mitochondria-dependent apoptosis, ultimately causing cardiomyocyte apoptosis and contributing to myocardial injury and heart failure