Mitochondrial Transplantation Rejuvenates Aging Heart by Restoring Mitophagy Flux via the HIF ‐3α‐ BNIP3 Axis

Figures

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  FIGURE 1. Establishment and characterization of the murine model of aging heart. (A) Schematic showing the study design of DOX‐induced cardiac aging in animals. (B) Representative echocardiographic images of cardiac structure and function. (C) Calculations and quantifications of FS, EF, LVESV, and LVESD in each group (n = 8 per group). (D) KEGG analysis for the DEGs. X‐axis indicated the enrichment score (−log10 [p value]) of DEGs. Y‐axis showed the names of enriched pathways. Node size represented the number of enriched differential genes. The p‐value was represented by a color scale. (E) SA‐β‐gal staining was performed on frozen sections of cardiac tissues from young and aged groups, and the corresponding quantitative analysis is shown on the right. Scale bars, 50 μm. (F) Expression and quantification of senescence‐associated proteins (APOE and p16) in cardiac tissue by Western blot analysis. (G) SA‐β‐Gal staining of HL‐1 cells, with corresponding quantitative analysis shown on the right (n = 3 per group). Scale bars, 100 μm. (H) Expression and quantification of senescence‐associated proteins (APOE and p16) in HL‐1 cells by Western blot analysis. Bars represent mean ± SD. *p < 0.05, **p…

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  FIGURE 2. Mitophagy flux congestion and mitochondrial dysfunction in the aging heart. (A) Representative TEM images of myocardial tissue. Left panels: Scale bar = 1 μm (× 25,000); right panels: Scale bar = 500 nm (× 50,000). Red arrows indicate mitophagosomes. (B) Quantification of Mitophagosome numbers per area and the ratio of intact mitochondria (n = 3 per group). (C) Expression and quantification of LC3B in cardiac tissues by Western blot analysis (n = 3 per group). (D) Expression and quantification of p62 in cardiac tissues by Western blot analysis (n = 3 per group). (E) Representative fluorescence images of mitochondria (red) and LC3B (green) in HL‐1 cells. Zoomed panels (right) correspond to the boxed areas in the merged images. Scale bars, 10 μm (main and zoomed panels). (F) Quantification analysis of the colocalization percentage between mitochondria and LC3B (n = 5 per group). (G) Representative live‐cell SIM images showing mitochondria (PK Mito, red) and lysosomes (Lyso Tracker, green) in young and aged groups. Zoomed panels (right) correspond to the boxed areas in the merged images. Scale bars: 10 μm (main); 2 μm (zoomed). (H) Quantification of mitochondrial parameters (branch…

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  FIGURE 3. Mitochondrial transplantation rejuvenates aging heart in mice. (A) Schematic showing the study design for BMSC‐derived mitochondrial transplantation therapy in aging heart. (B) Representative echocardiographic images of cardiac structure and function. (C) Calculations and quantifications of FS, EF, LVESV and LVESD in each group. (D) Representative results of WGA and SA‐β‐gal staining on frozen sections of cardiac tissues are presented. Scale bars, 10 μm (WGA staining) and 50 μm (SA‐β‐gal staining). (E) Statistical analysis of cardiomyocyte cross‐sectional area per μm2 and the percentage of SA‐β‐gal‐positive area in frozen heart sections from aged and aged+mito groups (n = 3 per group). (F) Representative TEM images of cardiac tissue. Left panels: Scale bar = 1 μm (× 25,000); right panels: Scale bar = 500 nm (× 50,000). Red arrows indicate mitophagosome. (G) Quantification of Mitophagosomes number per area and the ratio of intact mitochondria. (n = 3 per group). (H) Flow cytometry analysis of MitoSOX levels in the Aged and aged+mito HL‐1 cells. Statistical analysis of these data is provided on the right. (n = 3 per group). (I, J) Expression and quantification of…

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  FIGURE 4. Multi‐omics screening identifies senescence associated mitophagy genes. (A) Analysis of mitochondrial‐related DEmRNAs in young and aged groups. The expression values were represented by a color scale. A1, A2, A3: Aged tissue, C1, C2, C3: Young tissue. (B) Analysis of mRNA profiling for differentially expressed genes in young versus aged groups (n = 3 per group). (C, D) The correlation analysis between LC3B (Map1lc3b) and Bnip3/Pink1. (E, F) Expression and quantification of BNIP3 protein in doxorubicin‐induced senescent myocardium by Western blot (n = 3 per group). (G) GSEA on the “TABULA_MURIS_SENIS_HEART_AND_AORTA_CARDIOMYOCYTE_AGEING” gene set, with genes ranked by Pearson correlation to Bnip3. (H) Statistical analysis of BNIP3 mRNA expression in the cardiac tissues of naturally aged mice (n = 3 per group). (I, J) Expression and quantification of BNIP3 protein in the cardiac tissues of naturally aged mice by Western blot (n = 3 per group). (K) Cell type annotation of single‐cell transcriptomes from human heart tissues was performed via UMAP dimensionality reduction and clustering. Datasets were from the GSE156703 and Heart_Cell_Atlas datasets…

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  FIGURE 5. Mitochondrial transplantation downregulates BNIP3 and restores mitophagy flux in aging heart. (A) Volcano plot of proteomic sequencing for the young and old groups of HL‐1 cells. (B) Expression of BNIP3 protein in HL‐1 cell by Western blot analysis, with corresponding quantitative analysis shown on the right (n = 3 per group). (C) Representative images of SA‐β‐Gal staining in HL‐1 cells, Scale bars, 100 μm. (D) Quantitative analysis of SA‐β‐Gal staining in HL‐1 cells (n = 3 per group). (E) Representative fluorescence images of mitochondria and LC3B in HL‐1 cells. Zoomed panels (right) correspond to the boxed areas in the merged images. Scale bars, 10 μm (main and zoomed panels). (F) Representative live‐cell SIM images showing mitochondria (PK Mito, red) and lysosomes (Lyso Tracker, green) in young and aged groups. Zoomed panels (right) correspond to the boxed areas in the merged images. Scale bars: 10 μm (main); 2 μm (zoomed). (G) Quantitative analysis of co‐localization of LC3B and mitochondria in HL‐1 cells. (n = 3 per group) (H) Quantification of mitochondrial parameters (branch end points and number per cell) (n = 8 per group). (I, J) Expression and quantification of…

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  FIGURE 6. Abnormally upregulated HIF‐3α drives the high expression of BNIP3. (A) Interaction analysis of DEmRNAs and transcriptional factor (TF) subsets. The expression values were represented by a color scale. A1, A2, A3: Aged tissues, B1, B2, B3: Aged+Mito tissues, C1, C2, C3: Young tissues. (B) Statistical analysis of Hif3α mRNA expression in cardiac tissue and HL‐1 cells (n = 3 per group). (C) Expression and quantification of HIF‐3α protein in cardiac tissue and HL‐1 cells, with corresponding quantitative analysis shown on the right (n = 3 per group). (D) JASPAR prediction result shows HIF‐3α shares a specific motif (CGCGTG) with Bnip3. (E) Chip‐qPCR results indicated the binding of HIF‐3α on the site 2 of BNIP3 promoter. (F) Bnip3 and Hif3α mRNA expression in HL‐1 cells with HIF‐3α overexpression or knockdown. (n = 3 per group). (G) Expression and quantification of BNIP3 and HIF‐3α in Hif3α overexpressing HL‐1 cells by Western blot analysis (n = 3 per group). (H) Expression and quantification of HIF‐3α and BNIP3 in Hif3α knockdown HL‐1 cells by Western blot analysis (n = 3 per group). (I) Expression and quantification of p62 and LC3B‐II/I in Hif3α knockdown and Bnip3 overexpression…