Mitophagy mitigates mitochondrial fatty acid β-oxidation deficient cardiomyopathy

Figures

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  Fig. 1. Cardiac-specific CPT2 deletion disrupts mitochondrial FAO. a The role of CPT1 located on the OMM and CPT2 on the IMM in transporting LCFA in the form of Acyl-CoA into the mitochondrial matrix for FAO. b Western blot analysis and (c) quantification of CPT2 expression in heart tissue derived from wild-type and CPT2H-KO mice (n = 4). Data represent mean ± s.d. GAPDH is shown as a loading control. d–f Lipidomic profiles of wild-type and CPT2H-KO hearts (n = 5). d Heatmap illustrating hierarchical clustering of differential features detected from wild-type and CPT2H-KO cardiac samples run in duplicate by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomic analysis. Normalized peak intensities were clustered in two dimensions based on Euclidean distance (column, samples; row, metabolites). Colors indicate the metabolite abundances. Higher and lower values are colored red and blue, respectively. Total lipid metabolite profiling in mouse hearts derived from wild-type and CPT2H-KO was assessed by principle-component analysis (PCA) (e) and Mummichog pathway analysis (f). All the matched pathways are displayed as circles. The color and size of each circle are based on…

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  Fig. 2. Cardiac phenotypes of CPT2H-KO mice. a Survival (both male and female) of a cohort of wild-type (n = 18) and CPT2H-KO (n = 29) mice. b Quantification of heart weight to body weight ratio (HW/BW) for 4-week-old wild-type (n = 8) or CPT2H-KO mice (n = 6). Data represent mean ± s.d. c Representative hematoxylin and eosin (H&E) staining of transverse heart sections from 4-week-old wild-type or CPT2H-KO mice. Scale = 1 mm. d–f Echocardiographic quantification of percentage fractional shortening (FS) (d), ejection fraction (EF) (e), and left ventricular internal dimension-diastole (LVIDd) (f) for wild-type and CPT2H-KO mice at indicated age (n = 12). Data represent mean ± s.d. The P-values were calculated using an unpaired two-tailed Student’s t test (b, d–f). Source data are provided as a Source Data file.

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  Fig. 3. Transcriptomics and proteomics analysis of CPT2H-KO mice. a–c Gene set enrichment analysis (GSEA) plots showing downregulated signatures related to oxidative phosphorylation (OXPHOS) (a), respiratory electron transport (b), and genes related to the TCA cycle (c). Gene expression was determined by RNA-seq data from heart samples collected from 4-week-old wild-type and CPT2H-KO mice (n = 5). NES denotes the normalized enrichment score. FDR (false discovery rate) < 0.001. d Volcano plot to compare the mean log2 fold change (CPT2H-KO/wild-type) of normalized spectral counts and the log10 of the P values obtained in the two-sided t-test comparison (n = 4 per group). The horizontal line represents the threshold of P = 0.05. e Gene ontology analysis of the proteomics data reveals enriched cellular components that are upregulated in the CPT2H-KO mice. Significantly enriched GO terms associated with mitochondria are highlighted in red. f Representative western blot and (g) quantification of IMM (TIM23, TIM22) and OMM (TOM40) protein expression in heart tissue derived from wild-type and CPT2H-KO mice. Data represent mean ± s.d. (n = 3). The P values were calculated using a one-sided…

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  Fig. 4. Loss of cardiac FAO inhibits mitophagy. a Representative images of heart tissue and (b) quantification of cardiac mitophagy in wild-type or CPT2H-KO mice expressing mt-Keima. The emission signal obtained after excitation with the 458-nm laser is shown in green (neutral), and that obtained after excitation with the 561 nm laser is shown in red (acidic). The fold change of mitophagy was quantified by comparing the red/green signal. Values are normalized to wild-type levels of mitophagy (n = 4). Scale bars, 20 μm. c Representative western blot and (d) quantification of mitophagy receptors, OPTN and p62, in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). e Representative western blot and (f) quantification of PINK1 and PDKs (PDK1, PDK2, PDK3, PDK4) expression in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). g Representative western blot and (h) quantification of PARL expression in heart tissue derived from wild-type or CPT2H-KO mice (n = 3). PARL knockout HeLa cell lysates are employed for antibody validation. i Representative western blot and (j) quantification of Cleaved (CL) PGAM5 expression in heart tissue derived from wild-type or CPT2H-KO mice (n =…

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  Fig. 5. Deletion of USP30 promotes cardiac mitophagy and rescues CPT2-deficient mice. a Representative western blot and (b) quantification of USP30 in heart tissue derived from wild-type or USP30H-KO mice (n = 3). Data represent mean ± s.d. c Representative images of heart tissue and (d) quantification of cardiac mitophagy in wild-type, CPT2H-KO, or CPT2/USP30H-KO mice expressing mt-Keima. Mitophagy fold change was calculated based on the red-to-green fluorescence and normalized to wild-type levels of mitophagy (n = 5). Data represent mean ± s.d. Scale bars, 20 μm. e Representative Transmission Electron Microscopy (TEM) images showing cardiomyocyte mitochondria from wild-type, CPT2H-KO, or CPT2/USP30H-KO mice. Scale bars, 500 nm. f Quantification of mitochondrial swelling and (g) cristae volume density, calculated as the percentage of cristae surface area relative to the outer mitochondrial membrane surface area. Data represent mean±s.d. from analysis of 100–150 mitochondria (n = 3). Data represent mean ± s.d. h–k Echocardiographic quantification of percentage fractional shortening (FS) (h), ejection fraction (EF) (i), left ventricular internal dimension-diastole (LVIDd) (j) and left…

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  Fig. 6. Deletion of USP30 improves mitochondrial metabolism in FAO-deficient hearts. a Total metabolite profiling of mouse hearts derived from wild-type, CPT2H-KO, and CPT2/USP30H-KO mice at 5 weeks of age (n = 3 each) assessed by 3D PCA. b Metabolic pathway activity using the Mummichog algorithm in heart tissue derived from CPT2H-KO, and CPT2/USP30H-KO mice. The P-values (one-sided) were determined by the Mummichog algorithm. c GSEA plot showing altered signatures related to amino acid metabolism and deubiquitination. Gene expression was determined by RNA-seq data from heart samples collected from 5-week-old CPT2H-KO and CPT2/USP30H-KO mice (n = 3). NES denotes the normalized enrichment score. FDR (false discovery rate) < 0.001. d Heat map showing levels of amino acids or TCA cycle intermediates in wild-type, CPT2H-KO, and CPT2/USP30H-KO hearts (n = 3). Metabolite levels in each sample were converted to a fold-change relative to the average metabolite level of the wild-type. Yellow and blue indicate higher and lower levels of metabolites, respectively. e A schematic of amino acid metabolic pathways. The metabolites indicated by blue on the map for amino acid metabolism were…