Lactylation of Mitochondrial Adenosine Triphosphate Synthase Subunit Alpha Regulates Vascular Remodeling and Progression of Aortic Dissection

Figures

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  Fig. 1.. Aortic dissection is associated with lactylation. (A) The pan-lactylation immunoblots of clinically normal vascular (n = 3) and AD tissue (n = 3). CBB, Coomassie brilliant blue staining; WB, Western blot. (B) The immunohistochemistry of pan-lactylation in normal vascular and AD tissue in clinical (n = 3) and mouse samples (n = 3) (10× and 40× lens; zoom level, 2×). Scale bar, 50 μm. (C and E) The immunofluorescence costaining for α-SMA (red) with Kla (green) in normal vascular (n = 5) and AD tissue (n = 5) in clinical (C) and mouse (E) samples. Scale bars, 100 μm. (D and F) Quantification of immunofluorescence (C and E). Data are means ± SEM from 5 independent experiments and were analyzed by Student’s t test (D and F). **P < 0.01 and ****P < 0.0001.

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  Fig. 2.. Lactylation regulates VSMC proliferation, migration, and phenotypic switching. (A) CCK-8 was performed to measure cell proliferation of VSMC after treatment with DCA (20 mM), 2-DG (10 mM), and oxamate (20 mM). OD450, optical density at 450 nm. (B) CCK-8 was performed to measure cell proliferation of VSMC after treatment with rotenone (10 nM) and Nala (25 mM). (C) Cell migration of VSMC under hypoxic conditions following the use of rotenone (10 nM), oxamate (20 mM), DCA (20 mM), and 2-DG (10 mM) was detected using a Transwell assay at 24 h. Scale bar, 500 μm. (D to G) Western blotting analyses and corresponding quantification were conducted to detect the protein expression level of contractile markers (D) and MMP-2/9 (E) after the use of rotenone (10 nM), oxamate (20 mM), 2-DG (10 mM), DCA (10 mM), and Nala (25 mM) in VSMC, SM-MHC, smooth muscle myosin heavy chain. (H) Detection of apoptosis in VSMC after different drug treatments using TUNEL staining. Scale bar, 500 μm. Data are means ± SEM from 3 independent experiments and were analyzed by Kruskal–Wallis test with Dunn post hoc test (A to C, E, and G). ****P < 0.0001.

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  Fig. 3.. Effect of lactylation on aortic dissection in mice. (A) AD mice were constructed by injecting BAPN and AngII, and PBS, FX-11 (4 mg/kg/d), or Nala (1 g/kg/d) was injected intraperitoneally for 7 days (n = 11). (B) Typical images showed macroscopic features of isolated mice aorta from different groups (n = 3). Scale bar, 1 cm. (C) Survival rate curve of each group of mice. (D and E) Detection of LDHA expression levels in different groups. (F) Western blot was used to detect the lactylation level of the aorta of mice in different groups. (G to I) Representative images of H&E, EVG, and Masson staining in thoracic arteries obtained from mice in different groups (n = 5). Scale bar, 200 μm. (J) Immunohistochemical (IHC) staining of MMP-9 in different groups. (K and L) Western blot was used to detect the marker of contractile phenotype mice in different groups. Data are means ± SEM from 3 or 5 independent experiments and were analyzed by Kruskal–Wallis test with Dunn post hoc test (D, H, and K). ***P < 0.001 and ****P < 0.0001.

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  Fig. 4.. Lactylation analysis. (A) The heatmap drawn by calculating the Pearson’s correlation coefficient between all samples in pairs (n = 3). This coefficient is a value that measures the degree of linear correlation between 2 sets of data: the closer the Pearson coefficient is to −1, the stronger the negative correlation; the closer it is to 1, the stronger the positive correlation; and the closer it is to 0, the weaker the correlation. (B) Modification omics differential analysis heatmap. (C) Statistical map of differential proteins and sites. (D) Cluster analysis of differentially expressed proteins, dividing them into 4 parts based on their differential expression multiples, called Q1 to Q4. (E) On the basis of the P value of Fisher’s exact test obtained from enrichment analysis, the relevant functions in different groups were clustered together using hierarchical clustering method and plotted as a heatmap. The horizontal direction of the heatmap represents the enrichment test results of different groups, while the vertical direction represents the description of differentially expressed enrichment-related functions (protein domain). The color blocks corresponding to different…

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  Fig. 5.. K531 lactylation of ATP5F1A regulates VSMC proliferation, migration, and phenotypic switching. (A to C) ATP5F1A expression levels were detected by Western blots after hypoxia (48 h) or Nala (25 mM) treatment. Then cell proteins were enriched by ATP5F1A antibody and detected with anti-lactyllysine antibody (n = 3). (D) Mutations at the K531 site. Lys, lysine; Arg, arginine; Glu, glutamate. (E and F) K531R, K531E site mutations, or wild-type Flag-ATP5F1A-overexpressed HAVSMCs were constructed respectively through overexpression plasmid. Flag-ATP5F1A-overexpressed HAVSMCs proteins were enriched by Flag antibody and detected with anti-lactyllysine antibody (n = 3). (G) Cell migrations of HAVSMCs following the ATP5F1A K531 site mutation with Nala treatment were detected using crystal violet dye solution. Scale bar, 500 μm. (H) Detection of apoptosis in HAVSMCs after the ATP5F1A K531 site mutation with Nala treatment using TUNEL staining. Scale bar, 500 μm. (I to L) Western Blots was conducted to measure the protein expression level of phenotypic switch markers (I) and MMP-2/9 (K) after ATP5F1A K531 site mutation with Nala treatment (n = 3), OPN, osteopontin. Data are means ± SEM from…

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  Fig. 6.. Lactylation of ATP5F1A at K531 affects mitochondrial function. Wild-type, K531R, or K531E site mutation ATP5F1A-overexpressed HAVSMCs were constructed through the pcDNA3.1 vector. (A) Mitochondrial tracing using mitochondria-specific fluorescent probes. Scale bars, 10 μm. GFP, green fluorescent protein. (B) Mitochondrial structure observed by mitochondrial scanning electron microscopy. Scale bar, 250 nm. Black arrows indicate mitochondria, and red arrows indicate broken mitochondria. (C) Left: Mitochondrial fragmentation ratio (proportion of mitochondria with long axis < 2 μm). Right: Number of mitochondrial cristae per unit area. (D and E) Detection of cellular ROS production by dihydrofluorescein diacetate (DCFH-DA) fluorescent staining (n = 3). Scale bar, 200 μm. (F) ATP production assay (n = 5). (G and H) Western Blots was conducted to measure the protein expression level of mitochondrial division (n = 3) Fis1, mitochondrial fission 1 protein. Data are means ± SEM from 3 independent experiments and were analyzed by Kruskal–Wallis test with Dunn post hoc test (D, E, and G). ***P < 0.001 and ****P < 0.0001.

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  Fig. 7.. Sirt3 regulates the lactylation of ATP5F1A. (A) Flag-ATP5F1A transfection of 293T cells was performed to achieve ATP5F1A overexpression, followed by treatment with NAM (5 mM). Western blots were used to detect Flag expression level. (B) Predicted force directed top 12 proteins that bind to ATP5F1A. (C) Prediction of the binding of acetylated enzymes to ATP5F1A. (D) Predicted 3D structure of the Sirt3–ATP5F1A complex. (E) Co-IP shows that Sirt3 can specifically bind to ATP5F1A. (F) Co-IP shows that ATP5F1A can specifically bind to Sirt3. (G) Immunofluorescence colocalization shows overlapping distribution of Sirt3 and ATP5F1A on mitochondria. Scale bar, 50 μm. (H and I) Characterization of colocalization using Manders’ colocalization coefficient and Pearson’s correlation coefficient (n = 5). (J) 293T cells were cotransfected with si-Sirt3 and FLAG-tagged overexpression vector for ATP5F1A, and the level of ATP5F1A lactylation was assessed. Data are means ± SEM from 3 independent experiments and were analyzed by Student’s t test (I and J). **P < 0.01 and ***P < 0.001.

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  Fig. 8.. Effect of ATP5F1A K531 lactylation on aortic dissection in vivo. (A) AD mice were constructed by injecting BAPN and AngII. K531R peptides and K531E peptides were injected into the tail vein for 14 days (n = 11). (B) Typical images showed macroscopic features of isolated mice aorta from different groups (n = 3). (C) Survival rate curve of each group of mice. WT, wild-type. (D and E) Representative images of H&E, EVG, and Masson staining in thoracic arteries obtained from mice in different groups (n = 5). Scale bar, 800 μm. (F) Immunohistochemistry of MMP-9 in different groups. Detection of LDHA expression levels in different groups. (G) Western blot was used to detect the marker of contractile phenotype mice in different groups. (H) Co-IP was used to detect the ATP5F1A lactylation level of the aorta of mice in different groups. (I) Western blot was used to detect the marker of contractile phenotype and mitochondrial division. Data are means ± SEM from 3 or 5 independent experiments. ****P < 0.0001.

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  Fig. 9.. Sirt3 regulates the lactylation of ATP5F1A in mitochondria and affects the progression of aortic dissection. The increased expression of LDHA in aortic dissection leads to an increase in lactate content in circulation and tissues. Lactate enters mitochondria and nuclei in the form of lactoyl groups, causing protein to undergo lactylation modification. The lactylation modification of ATP5F1A regulated by Sirt3 in the mitochondria of HAVSMCs affects the function of ATPase, resulting in mitochondrial damage, rupture, functional abnormalities, reduced ATP production, increased ROS production, and, ultimately, an increase in the number of synthetic HAVSMCs and the secretion of MMPs, thereby aggravating aortic dissection. MCT, monocarboxylate transporter; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase.