Fig 2: Metformin prevents CHMP2B accumulation in H/R-treated cardiomyocytes in AMPK dependent manner. H9c2 cells were treated with metformin (2.5 mmol/L) or vehicle for 12 h and subjected to hypoxia for 15 h followed by reoxygenation for 1 h. (A) Representative immunoblotting and quantification data of p-AMPKα, AMPKα, atrogin-1, and CHMP2B for each group. (B) H9c2 cells were treated with metformin (2.5 mmol/L) or compound C (20 μmol/L) for 12 h. Representative immunoblotting and quantification data of p-AMPKα, AMPKα, atrogin-1, and CHMP2B for each group. (C) Relative mRNA expression levels of atrogin-1 as determined by qRT-PCR. (D) H9c2 cells were treated with metformin (2.5 mmol/L) or vehicle and simultaneously subjected to hypoxia for 15 h followed by reoxygenation for 1 h. Representative photomicrographs for CHMP2B (green) and atrogin-1 (red) co-localization staining by immunofluorescence. Scale bar = 50 μM. Values are means ± SEM, n = 6 per group. Except as otherwise noted, *p ≤ 0.05 vs. control group, #p ≤ 0.05 vs. H/R + vehicle or compound C group.

Fig 3: Overexpressed CHMP2B impairs autophagic flux leading to myocardial susceptibility to ischemia. H9c2 cells were infected with control adenovirus (Ad-Con) or CHMP2B-transfected adenovirus (Ad-CHMP2B) for 48 h. (A) Representative immunoblotting and quantification data of CHMP2B, LC3 II/I, LAMP1, and p62 were compared with normal control, Ad-Control, or Ad-CHMP2B treatment. (B) Ad-Con or Ad-CHMP2B cardiomyocytes were subjected to bafilomycin A1 (10 nM) or DMSO vehicle treatment. Representative immunoblotting and quantification data of CHMP2B, LC3 II/I, and p62. (C) Cell death rate was determined by annexin V-FITC/PI staining assay. B2 + B4 means apoptotic cells. Representative images obtained by flow cytometric analysis are shown on left side. Statistical analysis of percentage of apoptotic cells in different groups is shown on the right. Values are the means ± SEM, n = 6 per group. Except as otherwise noted, *p ≤ 0.05 vs. control or Ad-Con group, #p ≤ 0.05 vs. control + HR or Ad-Con + HR group. BAf-A1, Bafilomycin A1; DMSO, dimethylsulfoxide.

Fig 4: Impaired autophagic flux and expression levels of ESCRT-III components in cortical neurons after CA. a Representative Western blot showing Beclin-1, SQSTM1/p62 and LC3 in the cortical tissue lysates of sham and CA-ROSC rats at the indicated timepoints. b Quantification of Beclin-1, SQSTM1/p62 and LC3 with respect to the loading control β-actin. c Representative Western blot showing CHMP2A, CHMP2B, CHMP3, CEP55 and CHMP7 in the cortical tissue lysates of sham and CA-ROSC rats at the indicated timepoints. d Quantification of ESCRT-III components with respect to the loading control β-actin. e, f Representative immunofluorescence images of SQSTM1/p62 and LC3 double-staining and the quantification of LC3-positive cells (yellow bars) and LC3 and SQSTM1/p62 double-positive cells (red bars) in the cortex of sham-operated and injured rats. g, h Representative immunofluorescence images and the quantification of CHMP2A and NeuN double-staining. Scale bar, 50 μm. i, j Representative immunofluorescence images and quantification of LC3 and NeuN double staining. k, l Representative immunofluorescence images and quantification of SQSTM1/p62 and NeuN double staining. Scale bar, 50 μm. The data are expressed as means ± SEM (n = 4–6), one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. sham

Fig 5: CHMP2B accumulated in hypoxia/reoxygenation and in patients with coronary heart disease. (A) Representative immunoblotting and quantification data of CHMP2B levels in control cells and those subjected to H/R with or without metformin treatment. (B) Total RNA samples from three CAD patients and three healthy people of the Chinese Han population were extracted from peripheral blood and hybridized with Affymetrix microarrays. Volcano plot shows that mRNA expression levels of CHMP2B were upregulated in CAD patients, ~1.9-fold. (C) Fold change of CHMP2B mRNA from three CAD patients and three healthy people. We reanalyzed data from GEO database (GSE71226) for two microarray sites. Values are the means ± SEM, n = 6 per group, *p ≤ 0.05 vs. control group, #p ≤ 0.05 vs. H/R + vehicle group.