BCKDK protects against obesity-induced cardiac remodelling and dysfunction by alleviating mitochondrial oxidative stress and ROS-driven MAPK signalling

Fig. 1. Cardiac BCAAs accumulate in HFD-fed mice. A. Schematic view of experimental design. Eight-week-old WT mice were fed with ND or HFD for 28 weeks with cardiac function and metabolic analyses conducted at the study endpoint. B. Body weight gain over 28-week feeding period (n = 6 per group). C. IPGTT in ND and HFD groups, with corresponding area under the curve (AUC) analysis (n = 6 per group). D. Detection of serum lipid levels (TG, TC, HDL, and LDL) (n = 5 per group). E-F. Representative echocardiographic images and quantitated data of left ventricular ejection fraction (EF) and mitral valve E/E′ ratio (n = 5-9 per group). G-H. Statistical analyses of HW and HW/TL ratio of the above mice (n = 5-9 per group). I. Representative immunoblot and quantification of heart p-BCKDHA (Ser 293) to BCKDHA ratio (n = 5-8 per group). J. Cardiac BCAA levels in ND and HFD groups at 28 weeks post diet intervention (n = 7-8 per group). Data are presented as mean ± SEM. Statistical analyses were performed by unpaired two-tailed Student's t-test (C, D, and F-J).

Fig. 2. Cardiomyocyte-specific BCKDK deletion enhances BCAA catabolism but fails to improve HFD-induced ventricular dysfunction and remodelling. A. Schematic view of experimental design for the knockout cohort. Six- to eight-week-old BCKDKflox/flox/Myh6-CreEsr1- and BCKDKflox/flox/Myh6-CreEsr1+ mice were intraperitoneally injected with tamoxifen at a dosage of 75 mg/kg body weight for five consecutive days. Two weeks after the final tamoxifen injection, mice were fed with ND or HFD for 28 weeks. Cardiac function and metabolic assessment were performed at the study endpoint. B. Body weight gain over 28-week feeding period (n = 6-7 per group). C. IPGTT with corresponding AUC analysis (n = 6 per group). D. Representative immunoblot and quantification of heart BCKDK levels, and p-BCKDHA (Ser 293) to BCKDHA ratio (n = 4 per group). E. Cardiac BCAA levels at 28 weeks post diet intervention (n = 6-7 per group). F-G. Representative echocardiographic images and quantitated data of left ventricular EF, left ventricular fractional shortening (FS), and mitral valve E/E′ ratio (n = 6 per group). H. Representative gross morphologies of the hearts. I. Statistical analyses of HW and HW/TL ratio of the…

Fig. 3. BCKDK is downregulated in cardiomyocytes under HFD. A. Schematic view of experimental design. Eight-week-old WT mice were fed ND or HFD and were sacrificed every four weeks starting from week 8 of dietary intervention for serial cardiac tissue collection. Cardiomyocytes were isolated at week 28 of the dietary intervention. B–C. Representative immunoblot and quantification of BCKDK protein levels in the hearts at indicated time points (n = 3 per group). D. Real-time quantitative PCR (qRT-PCR) analysis of the mRNA expression of BCKDK in the hearts at week 28 of the dietary intervention (n = 5-8 per group). E. Representative immunoblot and quantification of BCKDK protein levels in the isolated cardiomyocytes at week 28 of the dietary intervention (n = 4 per group). F. Representative immunoblot and quantification of BCKDK expression in primary cardiomyocytes after 40 h of 200 μM PA treatment (n = 3 per group). G. Representative immunoblot and quantification of BCKDK expression in H9c2 cells after 40 h of 200 μM PA treatment (n = 4 per group). H. Representative immunofluorescence staining showing reduced BCKDK (red) fluorescence in H9c2 cells following 200 μM PA treatment. Nuclei…

Fig. 4. Cardiomyocyte-specific BCKDK overexpression alleviates HFD-induced ventricular dysfunction and remodelling. A. Schematic view of experimental design for the overexpression cohort. Eight-week-old WT mice were injected via the tail vein with cardiomyocyte-specific AAV9 vectors (cTNT promoter): cTNT-BCKDK-3×Flag (AAV-BCKDK) or cTNT-CON-3×Flag (AAV-CON), at a dose of 5 × 1011 v.g. per mouse per injection. Injections were administered at an interval of 14 weeks. Immediately after the first viral injection, mice were fed with ND or HFD for 28 weeks. Cardiac function and metabolic assessment were performed at the study endpoint. B. Body weight gain over 28-week feeding period (n = 6 per group). C. IPGTT with corresponding AUC analysis (n = 6 per group). D. Representative immunoblot and quantification of heart BCKDK levels, and p-BCKDHA (Ser 293) to BCKDHA ratio (n = 4 per group). E. Cardiac BCAA levels at 28 weeks post diet intervention (n = 6-7 per group). F-G. Representative echocardiographic images and quantitated data of left ventricular EF, left ventricular FS, and mitral valve E/E′ ratio (n = 6 per group). H. Representative gross morphologies of the hearts. I. Statistical analyses…

Fig. 5. Cardiomyocyte-specific BCKDK overexpression reverses the HFD-induced transcriptional signature. A. Schematic view of experimental design for the overexpression cohort. Left ventricular tissues (three biological replicates per group) were used for Bulk-RNA sequencing at the study endpoint. B. Principal component analysis (PCA) of Bulk-RNA seq data. Ellipses represent the 95% dispersion of samples within each group. PERMANOVA confirmed significant group differences (R2 = 0.571, p = 0.004), while dispersion analysis indicated comparable within-group variability (p = 0.460). C. A majority of the differentially expressed genes (DEGs) altered by HFD responded to AAV-BCKDK intervention, exhibiting either full or partial reversal. Genes considered partially reversed were significantly altered in the opposite direction in HFD + AAV-BCKDK (versus HFD + AAV-CON), while remaining significantly different from ND + AAV-CON. Genes considered fully reversed were significantly altered in the opposite direction in HFD + AAV-BCKDK (versus HFD + AAV-CON), and without significantly difference from ND + AAV-CON. D-E. The Biological Process category of the Gene Ontology enrichment analysis was…

Fig. 6. HFD-induced mitochondrial damage and oxidative stress are exacerbated by cardiomyocyte-specific BCKDK knockout but attenuated by overexpression. A. Schematic view of experimental design for the knockout cohort. Transmission electron microscopy (TEM) analysis and reactive oxygen species (ROS) detection were performed at the study endpoint. B. Representative images of myocardial TEM in ND + BCKDKfl/fl, HFD + BCKDKfl/fl and HFD + BCKDKiKO mice (n = 3 per group, with quantification from at least five TEM sections per heart; ND + BCKDKfl/fl: total 656 mitochondria; HFD + BCKDKfl/fl: total 565 mitochondria; HFD + BCKDKiKO: total 628 mitochondria). C. Bar graph depicting the percentage of the individual score grades according to the degree of damage from 0 to 4 across the analysed mitochondrial population in ND + BCKDKfl/fl, HFD + BCKDKfl/fl and HFD + BCKDKiKO myocardia. Overall mitochondrial mean scores in each group. The 5-grade scoring criteria referred to SFig. 4A. Quantitative assessment of mitochondrial swelling in each group, based on the number of swollen mitochondria as a percentage of total mitochondria. D-E. Representative images and quantitated data of cytosolic ROS (cROS)…

Fig. 7. HFD-induced inflammatory response via JNK/p38 is exacerbated by cardiomyocyte-specific BCKDK knockout but attenuated by overexpression. A. Schematic view of experimental design for the knockout cohort. Left ventricular tissues were used to evaluate MAPK pathway activity by Western blotting and inflammatory factors by qRT-PCR at the study endpoint. B. Representative immunoblot and quantification of p-p38 to p38 ratio, p-JNK1/2 to JNK1/2 ratio in myocardial tissues from ND + BCKDKfl/fl, HFD + BCKDKfl/fl and HFD + BCKDKiKO groups (n = 5-6 per group). C. qRT-PCR analysis of the mRNA expression of Il-6, Il-1β, and Tnf-α in myocardial tissues from ND + BCKDKfl/fl, HFD + BCKDKfl/fl and HFD + BCKDKiKO groups (n = 5-6 per group). D. Schematic view of experimental design for the overexpression cohort. Left ventricular tissues were used to evaluate MAPK pathway activity by Western blotting and inflammatory factors by qRT-PCR at the study endpoint. E. Representative immunoblot and quantification of p-p38 to p38 ratio, p-JNK1/2 to JNK1/2 ratio in myocardial tissues from ND + AAV-CON, HFD + AAV-CON and HFD + AAV-BCKDK groups (n = 6-7 per group). F. qRT-PCR analysis of the mRNA expression of…

Fig. 8. MitoTEMPO restores mitochondrial function and suppresses JNK/p38 activation in BCKDK-deficient H9c2 cells. A. Workflow illustrating in vitro BCKDK knockdown and MitoTEMPO (MT) rescue in H9c2 cells treated with 200 μM PA for 40 h to assess ROS levels, MMP, ATP production, and MAPK pathway activity. B–C. Representative images and quantification of mROS (mtSOX staining) in each H9c2 cell groups (n = 15-16 fields per group from 3 independent batches of cells). D-E. Representative images and quantification of overall cROS (DHE staining) in each H9c2 cell groups (n = 16 fields per group from 3 independent batches of cells). F-G. Representative images and quantification of mitochondrial membrane potential (MMP) (MT-1 staining) in each H9c2 cell groups (n = 10-12 fields per group from 3 independent batches of cells). H. Quantification of ATP content in each H9c2 cell groups (n = 4 per group). I. Representative immunoblot and quantification of p-p38 to p38 ratio, p-JNK1/2 to JNK1/2 ratio in each H9c2 cell groups (n = 4 per group). Data are presented as mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Tukey's multiple comparisons (C, E, G, H, and I).

Fig. 9. Mitochondrial metabolic disruption under HFD is aggravated by cardiomyocyte-specific BCKDK knockout but mitigated by overexpression. A. Schematic view of experimental design for the knockout cohort. Left ventricular tissues (five hearts per group) were used for untargeted metabolomics at the study endpoint. B. PLS-DA score plot illustrating metabolic distinctions of ND + BCKDKfl/fl, HFD + BCKDKfl/fl, and HFD + BCKDKiKO groups. Ellipses indicate the 95% dispersion of samples within each group. Model performance parameters are shown as R2X and Q2, and model validity was evaluated by 500 permutation tests. C. Schematic model illustrating potential alterations in metabolic fate in HFD + BCKDKiKO hearts compared with HFD + BCKDKfl/fl. Color code: red, increased; blue, decreased; black, unchanged; grey, not detected. D. Differentially accumulated metabolites involved in “BCAA oxidation” in the knockout cohort were presented. E. Differentially accumulated metabolites involved in “Glycolysis” in the knockout cohort were presented. F. Differentially accumulated metabolites involved in “Citric Acid Cycle” and “Mitochondrial Electron Transport Chain” in the knockout cohort were presented.…

Fig. 10. BCKDK expression is decreased in cardiac specimens from obese patients. A-E. BMI, left ventricular EF, IVS, LVPW, and mitral valve E/E′ ratio are compared between non-obese individuals (n = 23) and obese patients (n = 14). F. Representative immunoblot and quantification of indicated protein expression in human atrial tissue from obese patients and normal weight controls. G. Representative immunoblot and quantification of p-BCKDHA (Ser 293) to BCKDHA ratio. H. Representative immunoblot and quantification of p-p38 to p38 ratio. Data are presented as mean ± SEM. Statistical analyses were performed by unpaired two-tailed Student's t-test (A-E).