Methylmalonate accumulation contributes to myocardial vulnerability post-reperfusion: a novel therapeutic target and prognostic biomarker

Figures

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  Fig. 1. MMA predicts myocardial injury and incident heart failure compared with its coenzyme vitamin B12 and the isomer succinate. A Flow of the study (cohort 1), including 11,410 general adults aged ≥ 20 years after excluding pregnant women or those with a history of CVD. B The weighted prevalence and 95% CI of elevated hs-cTnT across serum vitamin B12 levels. C The weighted prevalence and 95% CI of elevated hs-cTnT (Roche assay) and hs-cTnI (3 independent Abbott, Siemens, and Ortho assays) across serum MMA levels. D Flow of the study (cohort 2), including 49 pairs of patients with AMI and incident heart failure within a 1-year follow-up and those without HF events to conduct widely targeted metabolomics in baseline plasma after pPCI. E Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). F Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially abundant metabolites in both groups. G Molecular structure and schematic overview of MMA- and succinate (SA)-related metabolic processes. H Relative levels of MMA/SA-related metabolites. I Flow of the study (cohort 3), including 300 age- and sex-matched patients with or…

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  Fig. 2. Prolonged elevation of cardiac MMA in mice post-ischemia/reperfusion. A The levels of MMA and SA in the heart tissues of mice 24 h post-I/R (n = 6). B The levels of MMA and SA in serum of mice 24 h post-I/R (n = 6). C MMA and SA levels in the heart tissues of mice subjected to I/R surgery at serial time points (n = 6). D MMA and SA levels in serum of mice after reperfusion at serial time points (n = 6). E Western blots of the MMA metabolism-related enzyme MMUT in the heart tissues of mice after reperfusion for 24 h (n = 6). F The levels of MMA and SA in other organs 24 h after myocardial reperfusion (n = 6). G Cellular-specific expression of the MMUT protein in the heart tissues of mice 24 h after I/R injury, determined by immunofluorescence staining (top panel bar = 50 μm, bottom panel bar = 100 μm). H Methylmalonyl-coenzyme A mutase (Mmut) gene expression across multiple organs, tissues, and cells. The data were acquired from http://biogps.org/#goto=genereport&id=17850. Statistical significance was determined using Student’s t-test (A, B, E, F) and 1-way ANOVA with Tukey’s post hoc test (C, D); data are presented as the means ± SDs (*P < 0.05; **P < 0.01; ***P < 0.001; ****P <…

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  Fig. 3. Myocardial-specific Mmut knockout-induced endogenous MMA accumulation increases myocardial vulnerability during I/R. A Construction of myocardial-specific Mmut knockout mice. B MMUT protein expression in the heart tissues of Mmutfl/fl and Mmutcko mice (n = 6). C MMA and SA contents in the hearts of Mmutfl/fl and Mmutcko mice (n = 6). D Serum levels of MMA and SA in Mmutfl/fl and Mmutcko mice (n = 6). E Evans blue and triphenyltetrazolium chloride (TTC) staining of heart sections from Mmutfl/fl and Mmutcko mice after reperfusion for 24 h (bar = 2 mm). F Quantification of the infarct area (IF), at-risk area (AAR), and left ventricle (LV) (n = 6). G Representative images of left ventricular echocardiography. H The left ventricular ejection fraction (EF) and fractional shortening (FS) of Mmutfl/fl and Mmutcko mice 24 h after I/R surgery (n = 6). I, J Representative TUNEL staining of heart sections and quantification of the apoptosis index (bar = 20 μm) (n = 6). K Serum levels of the myocardial injury biomarkers CKMB and LDH in Mmutfl/fl and Mmutcko mice 24 h after I/R surgery (n = 6). L, M Masson trichrome staining of heart sections from Mmutfl/fl and Mmutcko mice 7 days after I/R…

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  Fig. 4. MMA promotes mitochondrial damage and oxidative stress in myocardial I/R injury. A PTMs of total proteins in AC16 cells treated with methylmalonic acid (MMA) for 4 h. B The top 16 enriched KEGG pathways associated with the differentially acetylated proteins. C, D Gene Ontology analysis and heatmap of mitochondria-related differentially expressed proteins from acetylproteomics. E Representative electron microscopy images (15,000 ×) of heart sections. Representative impaired mitochondria are indicated by red arrows (bar = 500 nm). F Quantification of mitochondria with disorganized cristae (n = 6). G ATP contents in heart tissues 24 h after I/R (n = 6 per group). H Relative cardiac activities of mitochondrial respiratory chain complexes I, II, III, IV, and V in Mmutfl/fl and Mmutcko mice 24 h after I/R (n = 6 per group). I, J The mitochondrial oxygen consumption rate (OCR) in AC16 cells treated with low (50 μM/24 h) or high (100 μM/24 h) DiMMA concentrations was measured via a Seahorse trace (n = 4). K Representative images of JC-1 staining for the detection of the mitochondrial membrane potential in AC16 cells (n = 6). L Oxylipin profiles in the heart tissues of Mmutfl/fl and…

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  Fig. 5. MMA accumulation aggravates the disruption of mitochondrial quality control in myocardial I/R injury. A, B Representative electron microscopy images of heart sections 24 h after I/R surgery (top panel bar = 500 nm, bottom panel bar = 250 nm), and quantification of the average number of mitochondria and the number of mitochondria with disorganized cristae from A (n = 6). C, D Mitochondrial mass assessed by the mtDNA/nDNA ratio and MitoTracker Green staining in AC16 cells after treatment with each metabolite for 24 h (n = 3). E mRNA expression of nuclear-encoded and mitochondrial-encoded electron transport chain genes in heart tissues of Mmutfl/fl and Mmutcko mice 24 h after I/R surgery (n = 4). F Quantitative polymerase chain reaction analysis of the mRNA levels of the mitochondrial biogenesis-related genes PGC-1α, TFAM, NRF1, ND6 and ATP6 in AC16 cells (n = 4–6). G, H Western blot analysis and quantification of the mitochondrial autophagy/mitophagy markers LC3 and p62 in isolated heart mitochondria from Mmutfl/fl and Mmutcko mice 24 h after I/R surgery (n = 6). I Immunofluorescence staining of TOM20/LC3 in the heart tissues of mice 24 h after myocardial reperfusion injury (bar =…

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  Fig. 6. BNIP3 overexpression ameliorates impaired mitochondrial quality in Mmut-deficient hearts after I/R injury. A Western blot analysis and quantification of classical and alternative mitophagy pathway-related proteins (BNIP3, PARKIN, FUNDC1, NIX, and RAB9) in Mmutfl/fl and Mmutcko mice after I/R surgery (n = 6). B BNIP3 mRNA expression in AC16 cells with MMUT inhibition and hypoxia reoxygenation treatment (n = 6). C Experimental design of this study. Eight-week-old Mmutfl/fl and Mmutcko littermates were injected via the caudal vein with AAV9-BNIP3 or AAV9–empty vector. After 4 weeks, I/R injury was induced for further experiments. D Western blots showing BNIP3 expression in the heart tissues of wild-type mice subjected to I/R surgery after reperfusion for 24 h. E Representative M-mode and B-mode echocardiographic images of the left ventricle from Mmutfl/fl and Mmutcko mice that received AAV9-BNIP3 or AAV9–empty vector 24 h after I/R surgery. F Quantification of left ventricle ejection fraction (EF) and fractional shortening (FS) (n = 8). G Serum levels of the myocardial injury biomarkers lactate dehydrogenase (LDH) and CKMB in Mmutfl/fl and Mmutcko mice with BNIP3 overexpression 24 h…

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  Fig. 7. MMA inhibits BNIP3-mediated mitochondrial quality control by promoting CREBK309 hyperacetylation to reduce its transcriptional activity. A Dual luciferase activity of BNIP3 in AC16 cells treated with or without dimethyl methylmalonate (DiMMA) and subjected to H/R injury. B The top 10 of upregulated acetylated proteins. C Chromatin immunoprecipitation-sequencing data showing the binding of CREB and H3K27ac on the BNIP3 promoter according to silica analysis (accession codes #GSE99895 for CREB and GSE73769 for H3K27Ac). D Western blot analysis of BNIP3 in AC16 cells after treatment with CREB siRNA or a plasmid containing the MMUT gene for 48 h and then treatment with 50 μM DiMMA for another 24 h (n = 6). E The acetylation levels of CREB were measured in lysates of heart tissue from Mmutfl/fl and Mmutcko mice 24 h after I/R (n = 6). F The mass spectrum of the acetylized peptide from CREB identified different residues, K309 and K325, in DiMMA-treated AC16 cells. G Sequence alignment analysis of K309 and K325 of CREB across species. H Luciferase activity of the BNIP3 promoter in HEK 293 cells with different types of CREB mutants (n = 6). Both K residues (K309 and/or K325) in CREB were…

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  Fig. 8. MMUT delivery via adeno-associated virus serotype 9 significantly improves methylmalate metabolism and cardiac phenotypes in pigs subjected to I/R injury. A Schematic study design. Adeno-associated virus serotype 9 (AAV9), encoding the human MMUT gene, or an empty vector was locally injected into the hearts of male Bama miniature pigs (25 to 30 kg, 4 months of age) to induce cardiac overexpression. Four weeks after transfection, a porcine model of I/R injury was established. B Western blot analysis and quantification of MMUT protein expression (n = 5). C Serum MMA levels at baseline and at different time points after I/R injury (n = 5). D, E Representative cardiac magnetic resonance imaging (CMR) images of edema and the infarct size (day 3). Quantification of the infarct mass and infarct size (n = 5). F Quantification of the ejection fraction based via CMR (n = 5). G, H Serum levels of the myocardial injury biomarkers CKMB and hs-cTnI in pigs at baseline and 72 h after I/R surgery (n = 5). I Gross pathological images of heart sections (bar = 1 cm) and quantification of the scar area (n = 5). J Representative electron microscopy images (20,000 ×) of heart sections from pigs 4…