Fig 2: Enrichment and identification of heart lysine malonylome by Label-Free quantitation. (A) Cardiac EF of sepsis rats measured by echocardiography (n=6 per group). (B) Representative immunoblot of lysine malonylated (Kmal) proteins in control and sepsis groups. GAPDH was used as loading control (n=3 independent experiments). (C) Workflow of the strategy for the malonylome analysis. (D) Principal components analysis (PCA) score plot of control and sepsis groups. (E) Pie chart showing the distribution of the number of identified Kmal sites per protein. (F) Histogram showing the ratio distribution of quantifiable Kmal sites between control and sepsis groups. (G) Scatterplot showing the quantification of Kmal sites in relation to peptide intensities. (H) Pathways enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG). (I) MCC analysis of malonylated proteins. The malonylated protein was represented by a concentric circle, with the outer ring color indicating the protein undergoes malonylation up or down, red indicating upregulation, and green indicating downregulation. The redder the inner ring color, the higher the importance of the protein, and the larger the shape, the more interacting proteins there are. (J) MS spectrum of the malonylated site Lys46. (K) Crystal structure of VDAC2. (L) VDAC2 K46 is evolutionarily conserved. The sequences of VDAC2 in thirteen species were aligned. Lysine 46 of VDAC2 was highlighted in red. (M) Representative western blots of immunoprecipitated (IP) endogenous malonylated proteins or VDAC2 and immunoblotted for the reciprocal proteins in control and sepsis groups. GAPDH was used as loading control (n=3 independent experiments). The results were analyzed by independent sample t-test. a: p<0.05 as compared with the control group.

Fig 3: Protective effects of nano material TPP-AAV on SIMD. (A) The expression of VDAC2 (K46) malonylation in cardiac tissues of sepsis rats after treatment with AAV(K46) or TPP-AAV (n=3 independent experiments). The level of cardiac (B) MDA and (C) Fe2+ after injection of TPP-AAV in sepsis rats (n=6 each group). (D) Cardiac output (CO) (n=8 each group). (E) Representative echocardiograms images and (F) quantitative results of cardiac EF of sepsis rats after being treated with AAV (K46Q) and TPP-AAV (n=6 each group). (G) HE staining of heart tissues after injection of TPP-AAV (Bar=25µm) (n=6 each group). (H) Survival rate of sepsis rats after treatment with TPP-AAV (n=16 each group). (I-L) Laser speckle technique was used to monitor time-lapse liver and kidney blood flow of rats (n=6 each group). (M) Schematic diagram of ferroptosis of cardiomyocytes in sepsis caused by VDAC2 malonylation and targeted treatment by TPP-AAV. The results were analyzed by independent sample t-test. Rat survival was assessed by Kaplan-Meier analysis. a: p<0.05 as compared with the K46Q group.

Fig 4: Effect of malonyl-CoA on VDAC2 malonylation and ferroptosis in sepsis myocardial injury. The malonyl-CoA levels were measured in (A) H9C2 cells (n=3 independent experiments) and (B) heart tissues (n=8 each group) detected using ELISA. (C) Western blot analysis was performed to determine the expression of VDAC2 K46mal in H9C2 cells after transfection with Ad-Vector and Ad-shACC2 (n=3 independent experiments). (D) Representative western blots of immunoprecipitated (IP) endogenous malonylated proteins or VDAC2 and immunoblotted for the reciprocal proteins in sepsis and ND-630 groups (n=3 independent experiments). (E) Representative images of H9C2 cells after treatment with MitoBright-Deep Red and Mito-FerroGreen (Bar=5µm) (n=3 independent experiments). (F) Representative images of Mito-SOX in H9C2 cells (Bar=10µm) (n=3 independent experiments). (G) Representative TEM images of mitochondria in cardiomyocytes of rat heart tissue (Bar=0.5µm) (n=6 each group). (H) The level of MDA (n=6 independent experiments). (I) Echocardiograms images of rats (n=6 each group). (J) Summary of cardiac EF of rats measured by echocardiography. (K) Cardiac output (CO) of rats (n=8 each group). The results were analyzed by one-way ANOVA. a: p<0.05 as compared with the normal or control group, b: p<0.05 compared with sepsis or LPS group.

Fig 5: Characterization of nano material TPP-AAV. (A) Synthesis flow chat of TPP-AAV. (B) Representative TEM images of TPP-COOH and TPP-AAV (Bar=500µm). The (C) size and (D) zeta potential of TPP-COOH and TPP-AAV. (E) Representative confocal images of TPP-AAV at different times (Bar=50µm). (F) Relative cellular uptake percentage of TPP-AAV (n=3 independent experiments). (G) Effect of TPP-AAV on cell viability (n=3 independent experiments). (H) Representative image of TPP-AAV and positive control (VDAC2) mitochondrial colocalization (Bar=10µm) (n=3 independent experiments). (I) Mitochondrial respiration in H9C2 cells (n=3 independent experiments). The results were analyzed by independent sample t-test and one-way ANOVA.