Pyruvate metabolism enzyme Dlat induces mitochondria protein hyperacetylation to limit fatty acid oxidation in the HFpEF heart

Figures

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  Fig. 1. Mitochondrial FAO-related protein hyperacetylation and lipid metabolism disorder were observed in the HFpEF heart. A–C Early to late diastolic transmitral flow velocity ratio (E/A) and early transmitral flow velocity to mitral annular early diastolic velocity ratio (E/e’) values were measured using echocardiography to assess cardiac diastolic function (n = 8 per group). D Treadmill running distance was measured to assess exercise tolerance; E Hematoxylin-eosin (HE) staining (n = 8 per group). F Ratio of heart weight (HW) to tibia length (TL) (n = 8 per group). G Immunohistochemistry for wheat germ agglutinin (WGA) staining of the left ventricle, scale bar, 30 μm (n = 8 per group). H quantitative analysis of cardiomyocyte cross-sectional area (n = 8 per group). I assessment of interstitial fibrosis was performed using Masson trichrome staining and quantification of fibrosis area, scale bar, 30 μm (n = 8 per group). J immunoblot analysis and quantification of LysAc in cardiac tissues from the Control and HFpEF hearts (n = 6 per group). K Volcano plots showing the hyperacetylated peptides (FDR-adjusted p < 0.05, fold change >1.5) in HFpEF heart as compared to Control group (n = 3…

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  Fig. 2. Activation of mitochondrial protein deacetylation restored FAO and attenuated diastolic dysfunction in the HFpEF heart. A Immunoblot analysis of LysAc in isolated mitochondria followed by treatment with vehicle, 3-TYP and 3-TYP + NR. B Seahorse XF Palmitate Oxidation Stress Test were employed to determine FAO in primary cardiomyocytes. C–E Basal respiration, maximal respiration and FAs oxidation capacity were calculated (n = 4 biological replicates). F Lipid droplet (LD) was determined by Bodipy 493/503 staining in cardiomyocytes, scale bar, 10 μm (n = 6 per group). G the number of LDs per cell were quantified. H graphical illustration of the experimental timeline. I NAD+ content was measured using a NAD+ assay kit (n = 6 per group). J, K immunoblot analysis and quantification of LysAc in isolated mitochondrial of cardiac tissue (n = 6 per group). L representative immunoblots and quantitative analysis of acetylated CPT1b, HADHA, HADHB, CrAT and Acadl in the HFpEF hearts with or without NR supplementation analyzed by immunoprecipitation/Western blot (IP-WB) (n = 3 per group). M respirational flux of cardiac tissue was determined using an Oroboros clark-type electrode (n = 4…

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  Fig. 3. Identify Dlat as the key transacetylase for mitochondrial protein hyperacetylation in HFpEF heart. A isolated mitochondria were subjected to quantitative proteomics analysis and the clustered data are represented on the heatmap, the arrowhead points to Dlat. B, C Relative protein level of Dlat at different time points during HFpEF progression (n = 6 per group). D Dlat expression were determined by immunofluorescence staining in isolated adult cardiomyocytes (left) and quantitative analysis of Dlat fluorescence density, scale bar, 20 μm (n = 6 per group). E recombinant adenovirus expressing Dlat (Ad-Dlat) were transfected into NRVCMs and overexpression efficiency were determined by western-blotting analysis (n = 6 per group). F mitochondrial Dlat expression were determined by co-staining of Dlat and mitochondria in the NRVCMs, scale bar, 20 μm (n = 6 per group). G Immunoblot analysis and quantification of LysAc in isolated mitochondrial of NRVCMs (n = 6 per group). H Representative immunoblots and quantitative analysis of acetylated CPT1b, HADHA, HADHB, CrAT and Acadl in the NRVCMs transfected with Ad-NC or Ad-Dlat (n = 3 per group). I Mitochondrial respiration was examined using…

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  Fig. 4. Cardiac-specific Dlat overexpression mimics FAO inhibition and cardiac pathological features of HFpEF heart. A Graphical illustration of the experimental timeline. B the overexpression efficiency of Dlat in the cardiac tissue was verified by western blot (n = 6 per group). C Immunoblot analysis and quantification of LysAc in cardiac tissues from the Dlat-Tg and NTg hearts (n = 6 per group). D, E Immunoblot analysis and quantification of LysAc in cytoplasm and isolated mitochondria from the DlatTg and DlatNTg hearts (n = 6 per group). F Volcano plots showing the hyperacetylated peptides (FDR-adjusted p < 0.05, fold change >1.5) in DlatTg heart as compared to DlatNTg group (n = 3 mice per group). G Subcellular distribution of the hyperacetylated proteins in DlatTg hearts (n = 3 mice per group). H Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the hyperacetylated proteins in the DlatTg hearts; I Respirational flux of cardiac tissue was determined using an Oroboros clark-type electrode (n = 4 biological replicates). J Basic respiratory measured by oxygen consumption (n = 4 per group). K FAO measured by oxygen consumption (n = 4 per group). L Intra-myocardial…

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  Fig. 5. Dlat knockdown restored FAO and ameliorated cardiac dysfunction in HFpEF mice. A Graphical illustration of the experimental timeline. B The knockdown efficiency of intramyocardial AAV9-sh-Dlat injection was verified by western blot (n = 6 per group). C Immunoblot analysis and quantification of LysAc in cardiac tissues (n = 6 per group). D Representative immunoblots and quantitative analysis of acetylated CPT1b, HADHA, HADHB, CrAT and Acadl in the HFpEF hearts transfected with AAV9-NC or AAV9-sh-Dlat (n = 3 per group). E Respirational flux of cardiac tissue was determined using an Oroboros clark-type electrode (n = 4 biological replicates). F Basic respiratory measured by oxygen consumption (n = 4 per group). G FAO measured by oxygen consumption (n = 4 per group). H, I Intra-myocardial lipid droplet (LD) were determined by Bodipy 493/503 staining in isolated adult cardiomyocytes and the number of LDs per cell were quantified, scale bar, 20 μm (n = 6 biological replicates). J Treadmill running distance was measured to assess exercise tolerance (n = 8 per group). K–M Early to late diastolic transmitral flow velocity ratio (E/A) and early transmitral flow velocity to mitral annular…

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  Fig. 6. Dlat functions as an acetyltransferase for the mitochondrial β-oxidation enzyme HADHA. A Schematic representation of the experimental protocol. B Top 15 candidates for Dlat-interacting proteins by LC-MS/MS analysis. C A Venn analysis was used to examine the intersection among up-acetylated proteins in HFpEF hearts, up-acetylated proteins in DlatTg hearts and Dlat-interacting proteins. D, E IP assay using different antibody to determine the binding of Dlat and HADHA (n = 6 per group). F Representative pictures from proximity ligation assay (PLA) experiments in cardiomyocytes. PLA signals are visible as red dots, scale bar, 10 μm (n = 4 biological replicates). G Representative immunofluorescence images of NRVCMs showing the location of Dlat (green), HADHA (red), and DAPI (blue) (n = 4 biological replicates), scale bar, 20 μm. H IP assay was performed to determine the binding of Dlat and HADHA in control and HFpEF hearts (n = 6 per group). I Representative immunoblots acetylated HADHA in the DlatNTg or DlatTg hearts (n = 4 biological replicates). J Immunoblots of Ac-K and HADHA from an in vitro acetylation assay using purified recombinant His-Dlat (mHis-Dlat) and Myc-HADHA in the…

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  Fig. 7. Dlat-mediated acetylation of HADHA at K728 site to impede FAO. A Acetylation modification level of HADHA at different sites in control and HFpEF hearts was displayed by heatmap. B Acetylation modification level of HADHA at different sites in DlatNTg and DlatTg hearts was displayed by heatmap. C Western blotting images showing the expression of Flag in HEK293 cells transfected with flag-tagged wild type (WT) or mutations of HADHA (n = 4 per group). D Western blotting images showing the acetylation level of Flag-HADHA in HEK293 cells transfected with Flag-tagged wild type (WT) or mutations of HADHA, the triple-mutant refers to that K350, 383, and 406 site of HADHA were all mutant to R (n = 4 per group). E Immunoblots of Ac-K and HADHA from an in vitro acetylation assay using purified recombinant His-Dlat (mHis-Dlat) and Myc-HADHA or Myc-HADHA K728R mutant proteins in the presence of acetyl-CoA (n = 4 biological replicates). F Western blotting images showing the effects of Dlat overexpression on the acetylation levels of Flag-HADHA in HEK293 cells transfected with Flag-tagged wild type (WT) or mutations of HADHA (n = 4 per group). G Real-time relative enzyme activity of HADHA was…

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  Fig. 8. Spermidine-induced HADHA activation restored cardiac lipid metabolism and cardiac function in HFpEF heart. A Real-time relative enzyme activity of HADHA in heart lysates from control and HFpEF mice with or without SPD supplementation (n = 4 biological replicates). B Relative HADHA activity was calculated (n = 4 per group). C Respirational flux of cardiac tissue was determined using an Oroboros clark-type electrode (n = 4 biological replicates). D Basic respiratory measured by oxygen consumption (n = 4 per group). E FAO measured by oxygen consumption (n = 4 per group). F, G Intra-myocardial lipid droplet (LD) were determined by Bodipy 493/503 staining in isolated adult cardiomyocytes and the number of LDs per cell were quantified, scale bar, 20 μm (n = 10 biological replicates). H–K Early to late diastolic transmitral flow velocity ratio (E/A) and early transmitral flow velocity to mitral annular early diastolic velocity ratio (E/e′) values were measured using echocardiography to assess cardiac diastolic function (n = 8 per group). L Treadmill running distance was measured to assess exercise tolerance (n = 8 per group). M, N Representative images of HE staining and ratio of heart…

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  Fig. 9. A pictorial illustration of Dlat-induced mitochondrial protein hyperacetylation in the pathogenesis of HFpEF. In the HFpEF hearts, Dlat function as the key transacetylase responsible for mitochondrial protein hyperacetylation. Dlat directly binds to the alpha subunit of mitochondrial trifunctional protein (HADHA) and triggers its acetylation at the K728 site, thereby inactivating HADHA enzymatic activity. Reactivating HADHA, either by cardiac-specific HADHA overexpression or oral administration of a biogenic polyamine, restored cardiac FAO capacity and prevented the development of HFpEF. The red arrow indicates pathological processes, while the green arrow indicates therapeutic strategies.