Application of compound for regulating and controlling BAF170 protein in preparation of medicine for preventing, relieving and / or treating cardiovascular diseases

Abstract

The invention relates to application of a compound for regulating and controlling BAF170 protein in preparation of a medicine for preventing, relieving and / or treating cardiovascular diseases. According to the invention, WWP2 is promoted to be ubiquitinated into BAF170 at the K874 site, and degradation of BAF170 is promoted, so that myocardial cells are protected from oxidative stress damage. The applicant also finds that the small molecule compound BFH772 can enhance the combination of WWP2 and BAF170 and promote the ubiquitination and degradation of BAF170, thereby alleviating myocardial remodeling after myocardial infarction.

Description

Technical Field

[0001] The present invention relates to use of a compound for regulating BAF170 protein in preparing a medicament for preventing, alleviating and / or treating cardiovascular diseases. Background Art

[0002] Cardiovascular disease, particularly coronary artery disease (CAD), remains a leading global health challenge. Coronary heart disease (CHD) is a clinical syndrome associated with the formation of plaque in the arterial lining, which gradually leads to vascular narrowing and, ultimately, vascular occlusion. Long-term coronary artery obstruction can cause irreversible ischemic damage to the heart, leading to myocardial infarction.

[0003] According to the Chest Pain Center Guidelines, the reference standard for door-to-wire (D-to-W) time is 90 minutes. Failure to provide timely and effective drug intervention during this period can lead to continued cardiomyocyte death. In-depth research into the mechanisms of cardiomyocyte apoptosis is crucial to delay its progression and develop effective targeted therapeutics.

[0004] Myocardial infarction (MI) initially leads to the accumulation of oxidative stress (ROS), which further triggers cardiomyocyte death. Both apoptosis and necrosis contribute to myocardial injury caused by coronary artery occlusion, but apoptosis is the primary mechanism. Although the pathogenesis of cardiomyocyte apoptosis is well understood, further research into how to mitigate cardiomyocyte apoptosis is crucial for developing targeted therapeutic interventions to prevent cardiac remodeling after MI and ultimately improve patient prognosis and survival.

[0005] At the molecular level, BAF170 is a key player in cardiac development. Predominantly expressed in cardiomyocytes, it is crucial for cardiac differentiation and stage-specific gene expression. BAF170 deficiency impedes cardiomyocyte differentiation and delays the first heartbeat. Despite the crucial role of BAF170 in cardiomyocyte differentiation and cardiac development, its pathophysiological significance in ischemic heart disease remains unclear. Summary of the Invention

[0006] Through in-depth research, the inventors of this application found that BAF170 is closely related to cardiomyocyte death and may play an important role in cardiovascular diseases, such as myocardial infarction. WWP2 is the ubiquitination E3 ligase of BAF170. By ubiquitinating BAF170 at the K874 site, it promotes its degradation, thereby protecting cardiomyocytes from oxidative stress damage. During myocardial infarction, BAF170 expression increases, but WWP2 binding to it decreases. The absence of WWP2 aggravates cardiomyocyte damage, while overexpression reduces the damage. In addition, the applicant also found that the small molecule compound BFH772 can enhance the binding of WWP2 to BAF170, promote the ubiquitination and degradation of BAF170, and thus reduce myocardial remodeling after myocardial infarction.

[0007] The present invention is achieved through the following technical solutions.

[0008] The present invention provides use of a compound that regulates BAF170 protein in the preparation of a medicament for preventing, alleviating and / or treating cardiovascular diseases.

[0009] Preferably, the compound that regulates BAF170 protein is a compound that degrades BAF170 protein.

[0010] Preferably, the compound that degrades BAF170 protein is a compound that promotes WWP2 to ubiquitinate BAF170 at the K874 site.

[0011] Preferably, the compound that degrades BAF170 protein is a WWP2 protein agonist.

[0012] Preferably, the compound that degrades BAF170 protein is BFH772, and its structural formula is as follows:

[0013]

[0014] Preferably, the cardiovascular disease is selected from coronary artery disease, coronary heart disease, and myocardial infarction.

[0015] In a specific embodiment, the present invention provides use of BFH772 in the preparation of a medicament for preventing, alleviating and / or treating cardiovascular diseases, such as myocardial infarction.

[0016] Through a myocardial infarction model and high-depth proteomic analysis, the present invention found that Smarcc2 (BAF170) in the SWI / SNF superfamily complex may play an important role in myocardial infarction, with the lowest p-value. Hypoxia and serum deprivation experiments showed that as the treatment time increased, cell apoptosis increased and BAF170 expression increased. Further experiments confirmed that WWP2 is the ubiquitination E3 ligase of BAF170, and in myocardial infarction, the binding of the two is reduced. WWP2 regulates its stability by ubiquitinating the K874 site of BAF170, affecting the transcription of key genes, thereby protecting myocardial cells from oxidative stress damage and preventing cardiac remodeling. The study revealed a new role of the WWP2-BAF170 ubiquitination axis in myocardial infarction protection. By screening small molecule compounds, it was found that BFH772 can promote BAF170 ubiquitination and degradation, alleviating myocardial infarction. This discovery provides new opportunities for the development of targeted drugs for myocardial infarction.

[0017] This application confirms for the first time that BFH772 can promote the ubiquitination of BAF170-K874 site by WWP2 and promote the degradation of BAF170 protein, thereby alleviating myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 showed that the expression of BAF170 was significantly increased during MI.

[0019] A, Volcano plot comparing changes in protein expression in control and MI-conditioned mouse hearts, highlighting significant fold changes and P values.

[0020] B, Gene ontology enrichment analysis was performed to compare differentially expressed proteins between the control group and the MI group. Statistical significance was determined by Fisher's exact test (-log10 P value).

[0021] C, Heat map visualization comparing the expression of the SWI / SNF chromatin remodeling complex in heart tissues of control and MI mice.

[0022] D, Western blot analysis to evaluate the expression of BAF170, Cleaved-PARP1, and Cleaved-Caspase3 proteins in H9C2 cells after hypoxia / serum deprivation (H / SD) treatment at different time points (0-24 h) (n = 3 independent experiments).

[0023] E, The number of apoptotic cells was detected by Hoechst 33342 staining (blue) at 0, 2, 4, and 6 h after H / SD treatment (n = 3 independent experiments).

[0024] F, Western blotting analysis to evaluate the expression levels of WWP2 in H9C2 cell lines at different time points (0-24 h) and H / SD (n = 3 independent experiments).

[0025] G, Cell lysates were immunoprecipitated with anti-BAF170 antibody, followed by Western blot analysis using anti-WWP2 antibody.

[0026] H, Cell lysates were immunoprecipitated with anti-WWP2 antibody, followed by Western blot analysis using anti-BAF170 antibody.

[0027] I, Direct interaction between exogenous WWP2 and endogenous BAF170 in cell lysates was detected by overexpression of HA-tagged WWP2 and immunoprecipitation.

[0028] J, BAF170 contains four functional domains, including the N-terminal, C-terminal, SWIRM and SANT domains.

[0029] K, HEK293T cells were transfected with full-length or truncated BAF170 plasmids carrying a Flag tag. Cell lysates were then immunoprecipitated with an anti-Flag antibody and analyzed by Western blotting using an anti-WWP2 antibody.

[0030] D, E, and F, three independent experiments were performed. Data were assumed to be normally distributed according to the central limit theorem. Relative protein levels were calculated as the fold change compared with the first group. Quantitative data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparison test (D, F, P values ​​adjusted for 15 comparisons; E, P values ​​adjusted for 6 comparisons). H / SD denotes hypoxia and serum deprivation; IB, immunoblot.

[0031] Figure 2 WWP2 regulates BAF170 polyubiquitination at the K874 site and promotes its degradation.

[0032] A, Analysis of BAF170 and WWP2 expression levels in H9C2 cells under four conditions: normal control and treatment with WWP2 short hairpin RNA variants (shWWP274451, 74452, and 74453) (n=3 independent experiments).

[0033] B, Western blot analysis to evaluate BAF170 expression in HEK293T and H9C2 cells following dose-dependent overexpression of HA-tagged WWP2 (n = 3 independent experiments).

[0034] C and G, Western blot analysis to assess BAF170 protein levels in control and shWWP2 cells at different time points after cycloheximide treatment (n = 3 independent experiments).

[0035] D and H, Western blot analysis to assess BAF170 protein expression levels in control cells and cells overexpressing HA-tagged WWP2 at different time points after cycloheximide treatment (n = 3 independent experiments).

[0036] E and I, Western blot analysis to evaluate the expression level of BAF170 in normal control cells and shWWP2 cells treated with MG132 at different time points (n = 3 independent experiments).

[0037] F and J, Western blot analysis to assess the expression level of BAF170 in normal control cells and cells expressing HA-tagged WWP2 treated with MG132 at different time points (n = 3 independent experiments).

[0038] K, BAF170 ubiquitination levels were analyzed by immunoprecipitation using anti-HA antibody after co-transfection of HA-tagged WWP2 or HA-tagged vector control with HA ubiquitin (HA-Ub) in the presence or absence of the proteasome inhibitor MG132.

[0039] L, BAF170 ubiquitination was analyzed by coimmunoprecipitation with anti-HA antibody after overexpression of HA-Ub in control and shWWP2 cells, with or without MG132 treatment.

[0040] M, Diagram illustrating the workflow for proteomic and ubiquitination analysis in cardiac tissues of WWP2 cKO, WWP2-TG, and WT mice with MI (n=7 per group).

[0041] N, Nine-quadrant graphs of proteomics and ubiquitination histology in heart tissues of WWP2 knockout (WWP2 cKO) and transgenic (WWP2-TG) mice and wild-type (WWP2-WT) control mice.

[0042] O, Proteomic Muffz analysis of WWP2 cKO, WWP2-WT, and WWP2-TG.

[0043] P, Ubiquitinomics Muffz analysis of WWP2 cKO, WWP2-WT, and WWP2-TG.

[0044] Q, Biological function enrichment analysis of upregulated ubiquitination sites and proteins in WWP2 transgenic and wild-type tissue samples.

[0045] R, Biological function enrichment analysis of downregulated ubiquitination sites and proteins in WWP2 gene knockout and wild-type tissue samples.

[0046] S, BAF170 ubiquitination site sequence.

[0047] Mass spectrometry identification of the ubiquitination site of T and BAF 170-K874

[0048] H9C2 cells were transfected with Flag-BAF170 variants (WT, K874R, K694R, or K704R) and HA-Ub or HA-WWP2. Immunoprecipitation was performed with anti-Flag antibody, followed by Western blotting with anti-HA antibody.

[0049] A, B, GJ, three independent experiments were performed. Data were assumed to be normally distributed according to the central limit theorem. Relative protein levels were calculated as the fold change compared with the first group. Quantified data are mean ± SD. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparison test (A and B, P values ​​adjusted for 6 comparisons); unpaired t-test (2-tailed Student's t-test; GJ). Ub indicates ubiquitin antibody; CHX indicates cycloheximide; IB, immunoblot. WWP2 cKO: Myh6 Cre+; Wwp2f / f; WWP2-WT: Wwp2f / f; WWP2-TG: R26-LSL-WWP2+ / +; Myh6CreER.

[0050] Figure 3 Cardiac-specific WWP2 knockout mice showed increased BAF170 expression and significantly aggravated MI-induced cardiomyocyte injury.

[0051] A, shows the construction method of myocardium-specific WWP2 knockout (Myh6-Cre+; Wwp2f / f) mice.

[0052] B, Schematic diagram of the MI mouse model construction. Proteins extracted from control (Wwp2f / f) and cardiac-specific knockout (Myh6-Cre+; Wwp2f / f) hearts at 28 days post-MI.

[0053] C, Total lysates of heart tissue were immunoprecipitated (IP) with anti-BAF170 antibody and Western blotted with anti-WWP2 antibody.

[0054] D, Total lysates of heart tissue were subjected to IP with anti-BAF170 antibody and Western blotting with anti-ubiquitination (Ub) antibody in Myh6-Cre+;Wwp2f / f mice.

[0055] EG, EF% and FS% of Wwp2f / f and Myh6-Cre+; Wwp2f / f mice.

[0056] HI, HW / BW and HW / TL of Wwp2f / f and Myh6-Cre+; Wwp2f / f mice.

[0057] JK, Western blot analysis of WWP2 expression levels.

[0058] L, Heart tissue was stained using a DHE (dihydroethidium) detection kit (red). Scale bar = 20 μm.

[0059] MN, Western blot analysis to evaluate the expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1), and SOD2 (superoxide dismutase 2).

[0060] OP, Western blot analysis of BCL2 expression levels.

[0061] Q. The Oroboros O2K system is used to assess the activity of mitochondrial oxidative phosphorylation components.

[0062] R, Representative images of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

[0063] ST and Western blot analysis were used to evaluate the expression levels of apoptosis markers Cleaved-PARP1 and Cleaved-Aspase3.

[0064] UW, hematoxylin and eosin (H&E), wheat germ agglutinin (WGA), and Masson staining were performed to assess the extent of myocardial hypertrophy and fibrosis. (Above, scale bar = 800 μm; below, scale bar = 20 μm).

[0065] FN, PW, For data with normality and equal variance, two-way ANOVA with Bonferroni test was used to compare the groups. If normality or homogeneity of variance was not met, treatment differences were assessed by Kruskal-Wallis test and Dunn's multiple comparison test (n = 7 mice per group). Relative protein levels were calculated as fold change compared with the first group. Quantitative data are mean ± SD. Statistical significance was assessed by two-way ANOVA with Bonferroni's multiple comparison test (HI, K, L, N, P, Q, T, V, W values ​​were adjusted for 6 comparisons); Kruskal-Wallis and Dunn's multiple comparison test.

[0066] Figure 4 WWP2 overexpression leads to decreased BAF170 expression and reduced myocardial infarction-induced cardiomyocyte injury.

[0067] A, shows the construction method of Rosa26-WwP2-Flag (WWP2-TG, R26-LSL-Wwp2+ / +; Myh6-CreER) mice.

[0068] B, Schematic diagram of the construction of the MI mouse model. Protein samples were extracted from the hearts of wild-type (WWP2-WT, R26-LSL-WWP2+ / +; Myh6-CreER-) and WWP2-TG mice 28 days after infarction.

[0069] C, Total lysates of heart tissues from WWP2-TG and WWP2-WT mice were immunoprecipitated (IP) with anti-BAF170 antibody and Western blotted with anti-WWP2 antibody.

[0070] D, Total lysates of heart tissues from WWP2-TG and WWP2-WT mice were IP-treated with anti-BAF170 antibody and Western blotted with anti-ubiquitinated (Ub) antibody.

[0071] EF% and FS% of EG, WWP2-TG mice, and WWP2-WT mice.

[0072] HW / BW and HW / TL of HI, WWP2-TG, and WWP2-WT mice.

[0073] JK, Western blot analysis of WWP2 expression levels.

[0074] L, Heart tissue was stained using a DHE (dihydroethidium) detection kit (red). Scale bar = 20 μm.

[0075] MN, Western blot analysis to evaluate the expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1), and SOD2 (superoxide dismutase 2).

[0076] OP, Western blot analysis of BCL2 expression levels.

[0077] Q. The Oroboros O2K system is used to assess the activity of mitochondrial oxidative phosphorylation components.

[0078] R, Representative images of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

[0079] ST and Western blot analysis were used to evaluate the expression levels of Cleaved-PARP1 and Cleaved-Aspase3.

[0080] UW, hematoxylin and eosin (H&E), wheat germ agglutinin (WGA), and Masson staining were performed to assess the extent of myocardial hypertrophy and fibrosis. (Above, scale bar = 800 μm; below, scale bar = 20 μm).

[0081] FN, PW, For data with normality and equal variance, two-way ANOVA with Bonferroni test was used to compare the groups. If normality or homogeneity of variance was not met, treatment differences were assessed by Kruskal-Wallis test and Dunn's multiple comparison test (n = 7 mice per group). Relative protein levels were calculated as fold change compared with the first group. Quantitative data are mean ± SD. Statistical significance was assessed by two-way ANOVA with Bonferroni's multiple comparison test (HI, K, L, N, P, Q, T, V, W, values ​​adjusted for 6 comparisons); Kruskal-Wallis and Dunn's multiple comparison test.

[0082] Figure 5 BAF170-K874R disrupts the ubiquitination of BAF170 and significantly aggravates MI-induced cardiomyocyte injury.

[0083] A, Schematic diagram of the construction method of K874R heterozygous mutant mice. Protein samples were extracted from the hearts of wild-type (WT) and K874R heterozygous mutant mice 28 days after infarction.

[0084] B, Diagram showing the K874R mutation site and the N-terminal, SWIRM, SANT, and C-terminal functional domains.

[0085] C, Heart tissue lysates were immunoprecipitated with anti-BAF170 antibody in BAF170-K874R mice and analyzed by western blot using anti-ubiquitin (Ub) antibody.

[0086] EF% and FS% of DF, WT, and BAF170-K874R mice.

[0087] HW / BW and HW / TL of GH, WT, and BAF170-K874R mice.

[0088] IJ, Western blot analysis of BAF170 expression levels

[0089] K, Heart tissue was stained using a DHE (dihydroethidium) detection kit (red). Scale bar = 20 μm.

[0090] LM, Western blot analysis to evaluate the expression levels of 33-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1), and SOD2 (superoxide dismutase 2).

[0091] Western blot analysis of NO, BCL2 expression levels.

[0092] P, The activity of mitochondrial oxidative phosphorylation components was assessed using the Oroboros O2K system.

[0093] Q, Representative images of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

[0094] RS and Western blot analysis were used to evaluate the expression levels of Cleaved-PARP1 and Cleaved-Aspase3.

[0095] TV, hematoxylin and eosin (H&E), wheat germ agglutinin (WGA), and Masson staining were performed to assess the extent of myocardial hypertrophy and fibrosis. (Above, scale bar = 800 μm; below, scale bar = 20 μm).

[0096] EF, OV, For normally distributed and equal variance data, two-way ANOVA with Bonferroni test was used to compare the groups. If normality or homogeneity of variance was not met, treatment differences were assessed by Kruskal-Wallis test and Dunn's multiple comparison test (n = 7 mice per group). Relative protein levels were calculated as fold change compared with the first group. Quantitative data are mean ± SD. Statistical significance was assessed by two-way ANOVA with Bonferroni's multiple comparison test (GH, J, K, M, O, P, S, U, V, values ​​adjusted for 6 comparisons); Kruskal-Wallis and Dunn's multiple comparison test.

[0097] Figure 6 .BFH772 significantly alleviated myocardial cell damage induced by myocardial infarction.

[0098] A, Graph shows wild-type mice treated with BFH772 at concentrations of 0, 20, 30, and 40 mg / kg. Total protein extracted from heart tissue was analyzed 28 days after infarction.

[0099] BD, EF% and FS% of mice treated with different concentrations of BFH772 (including 0 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg).

[0100] E, HW / BW and HW / TL of mice treated with different concentrations of BFH772 (including 0 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg).

[0101] FG, Western blot analysis to evaluate the expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1), and SOD2 (superoxide dismutase 2).

[0102] HI, Western blot analysis of BCL2 expression levels.

[0103] JK, Oroboros O2K system for assessing the activity of mitochondrial oxidative phosphorylation components.

[0104] L, Representative images of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

[0105] MN and Western blot analysis were performed to evaluate the expression levels of Cleaved-PARP1 and Cleaved-Aspase3.

[0106] OQ, hematoxylin and eosin (H&E), wheat germ agglutinin (WGA), and Masson staining were used to detect myocardial hypertrophy and fibrosis. Above, scale bar = 800 μm; below, scale bar = 20 μm.

[0107] CE, G, IK, N, PQ. For data with normality and equal variance, one-way ANOVA with Tukey's multiple comparison test was used to compare groups (N = 7 mice per group). Quantitative data are mean ± SD. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test (CE, JK, PQ, adjusted values ​​for 6 comparisons; G, I, N, adjusted values ​​for 15 comparisons).

[0108] Figure 7 .Quality control results of WWP2 ubiquitin proteomic analysis.

[0109] A, Principal component analysis (PCA) showing the first two principal components of protein intensities, with samples connected by centroids, according to sample type.

[0110] B. Pearson's correlation analysis; each value represents the correlation coefficient between two samples.

[0111] C. Tolerance distribution of peptide mass

[0112] D. Box plot based on relative standard deviation (RSD), with each point representing an RSD value.

[0113] E, Histogram of mass spectrometry results.

[0114] F, Protein molecular weight statistics; the height of each bar represents the amount of protein.

[0115] G. Density distribution map.

[0116] H, Bar graph of intensity value distribution.

[0117] I, Box plot based on intensity values ​​(WWP2-cKO: Myh6-Cre+; Wwp2f / f; WWP2-WT: Wwp2f / f; WWP2-TG: R26-LSL-Wwp2+ / +; Myh6-CreER).

[0118] Figure 8 .WWP2 alleviates ischemia- and hypoxia-induced cardiomyocyte damage.

[0119] A, Lysates from H9C2-shWWP2 cell lines treated with or without H / SD for 12 h were subjected to IP with anti-BAF170 antibody and Western blotting with anti-WWP2 antibody.

[0120] B, Lysates from H9C2-shWWP2 cell lines treated with or without H / SD for 12 h were subjected to IP with anti-BAF170 antibody and Western blotting with anti-ubiquitination (Ub) antibody.

[0121] C, Lysates from cells overexpressing HA-tagged WWP2 treated with or without H / SD for 12 h were subjected to IP with anti-BAF170 antibody and Western blotting with anti-WWP2 antibody.

[0122] D, Lysates from cells overexpressing HA-tagged WWP2 treated with or without H / SD for 12 h were subjected to IP with anti-BAF170 antibody and Western blotting with anti-Ub antibody.

[0123] E and F, Western blot analysis of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1), and SOD2 (superoxide dismutase 2) expression levels after HA-WWP2 NTm re-expression in the H9C2-shWWP2 cell line with or without H / SD treatment for 12 h (n = 3 independent experiments).

[0124] GK, Mitochondrial ROS levels were assessed using MitoSOX Red and CellROX Green staining in H9C2-shWWP2 cells expressing HA-WWP2-NTm after treatment with or without H / SD for 12 hours, and apoptotic cells were quantified using Hoechst 33342 (blue). (n = 3 independent experiments).

[0125] L and M, Immunoblot analysis of BCL2 protein expression in H9C2-shWWP2 cells transfected with HA-WWP2 NTm, with or without 12-h hypoxia / serum deprivation (H / SD) treatment (n = 3 independent experiments).

[0126] N and O, JC-1 staining to evaluate changes in mitochondrial membrane potential in H9C2-shWWP2 cells expressing HA-WWP2-NTm with and without H / SD treatment for 12 h (n = 3 independent experiments).

[0127] P and Q, Western blot analysis of cleaved-PARP1 and cleaved-Aspase3 expression in H9C2-shWWP2 cells after re-expression of HA-WWP2 NTm with and without H / SD treatment for 12 h (n = 3 independent experiments).

[0128] F, IK, M, O, Q: Three independent experiments were performed. Data were assumed to be normally distributed according to the central limit theorem. Relative protein levels were calculated as the fold change compared with the first group. Quantitative data are presented as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparison test (F, O, Q; P values ​​adjusted for 3 comparisons) and two-way ANOVA with Bonferroni's multiple comparison test (M, IK; P values ​​adjusted for 15 comparisons). NC, normal control group, no treatment; H / SD, oxygen / glucose deprivation; IB, immunoblotting.

[0129] Figure 9 .BAF170-K874R inhibits BCL2 transcription and promotes Casp3 transcription by binding to the enhancer.

[0130] A. BAF170 binding density was analyzed using the Deep tool to generate a heatmap showing the distribution of CUT and Tag binding peaks. Samples from WT and K874R heterozygous mutant mice were compared in microglia and total heart tissue, and peaks were arranged by signal intensity.

[0131] B. Statistical analysis of differentially expressed genes.

[0132] C. Statistical analysis of differential gene distribution in KEGG pathways

[0133] D and E, Visualization of CUT and Tag signal tracks at BCL2 and Casp3 genomic loci, showing enrichment patterns.

[0134] Figure 10 BAF170-K905R aggravates myocardial cell injury induced by ischemia and hypoxia

[0135] A, Alignment of the sequences surrounding K874 in BAF170 homologs from different species. The ubiquitin lysine residue at BAF170-K874 is highlighted (bold and red).

[0136] B and C, Western blot analysis was used to evaluate BAF170 expression levels in HEK293T cells after treatment with three different short hairpin RNA constructs (shBAF170 121254, 121255, and 121256) compared with normal controls (n = 3 independent experiments).

[0137] D and E, Western blot analysis to evaluate the expression of oxidative stress markers (3-Nitrotyrosine, 8-oxo-dG) and antioxidant enzymes (SOD1, SOD2) in H9C2-shBAF170 cells expressing WT-BAF170 NTm or K905R-BAF170 NTm with or without 12 h of H / SD treatment (n = 3 independent experiments).

[0138] FJ, Mitochondrial ROS levels were assessed using MitoSOX Red and CellROX Green staining, and apoptotic cells were quantified using Hoechst 33342 (blue) after re-expression of WT-BAF170 NTm or K905R-BAF170 NT in H9C2-shBAF170 cells, with or without H / SD treatment for 12 h (n = 3 independent experiments).

[0139] K and L, Western blot analysis to evaluate BCL2 expression in H9C2-shBAF170 cells after reintroduction of WT-BAF170 NTm or K905R-BAF170 NTm with and without 12-h H / SD treatment (n = 3 independent experiments).

[0140] M and N, JC-1 staining to evaluate changes in mitochondrial membrane potential in H9C2-shBAF170 cells expressing WT-BAF170 NTm or K905R-BAF170 NTm treated with hypoxia / serum deprivation (H / SD) or without hypoxia / serum deprivation (H / SD, 12 h) (n = 3 independent experiments).

[0141] O and P, Western blot analysis of cleaved-PARP1 and cleaved-Aspase3 expression in H9C2-shBAF170 cells after recovery of WT-BAF170 NTm or K905R-BAF170 NTm with and without 12-h H / SD treatment (n = 3 independent experiments).

[0142] Three independent experiments were performed in C, E, HJ, L, N, and P. Data were assumed to be normally distributed according to the central limit theorem. Relative protein levels were calculated as the fold change compared with the first group. Quantitative data are presented as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparison test (C, P values ​​adjusted for 6 comparisons; E, N, P values ​​adjusted for 3 comparisons) and two-way ANOVA with Bonferroni's multiple comparison test (H, J, L, P values ​​adjusted for 15 comparisons). H / SD denotes hypoxia-ischemia; IB, immunoblot; NS, not statistically significant.

[0143] Figure 11 Purification of WWP2 protein and truncated BAF170 peptide fragments

[0144] A, Sensorgram showing the concentration-dependent binding kinetics of BAF170 truncation mutant (1-647) to immobilized WWP2 protein in the range of 7.8-125 nM.

[0145] B, Sensorgram showing the binding kinetics between BAF170 truncation (1-595) and immobilized WWP2, measured in the range of 15.63-250 nM.

[0146] CF, Sensorgrams of BAF170 peptide fragments (1-423, 424-1214, 569-1214, and 648-1214) binding to WWP2 protein. Peptide concentrations ranging from 0.06 to 1 μM were used to assess the binding interaction by microarray.

[0147] Figure 12 .Involves virtual screening of the small molecule compound BFH772.

[0148] A, Surface plasmon resonance analysis (SPR) to determine the binding affinity between ten candidate small molecules and BAF170-WWP2 immobilized on the chip surface.

[0149] B. WWP2 was immobilized on the chip surface. SPR was used to evaluate the binding interaction between BAF170 and WWP2 in the presence of 10 candidate small molecule compounds or controls.

[0150] C, SPR analysis to evaluate the interaction between immobilized WWP2 and BAF170 in the presence or absence of the small molecule inhibitor BFH772. The molecular structure of BFH772 is shown.

[0151] D. Three-dimensional structure of the small molecule BFH772.

[0152] E. Binding of the BFH772 small molecule to the BAF170 protein is visualized in a surface representation. Key interactions are highlighted in red, showing that residue K874 (yellow) interacts with BFH772 (cyan).

[0153] F, The binding status of BFH772 and BAF170 is shown in the figure.

[0154] G, Three-dimensional interaction diagram of BFH772 and BAF170.

[0155] H, Two-dimensional interaction diagram of BFH772 and BAF170.

[0156] Figure 13 .The small molecule compound BFH772 alleviates ischemia- and hypoxia-induced cardiomyocyte damage.

[0157] AB, Western blot analysis to assess protein expression levels of BAF170, WWP2, and apoptosis markers (Cleaved-PARP1 and Cleaved-Aspase3) after 48 h of exposure to BFH772 (0-100 μM). (n=3 independent experiments).

[0158] C, HA-tagged WWP2 was overexpressed in the presence or absence of 1 μM BFH772 after 48 h of induction. Lysates were immunoprecipitated with anti-HA antibody and analyzed by Western blotting using anti-BAF170.

[0159] D, Cells were co-transfected with HA-tagged WWP2 or vector control and HA-Ub, and then induced with or without 1 μM BFH772 for 48 h. BAF170 ubiquitination was assessed by anti-HA immunoprecipitation.

[0160] EF and H9C2 cells were pretreated with BFH772 (36 hours) and then co-treated with H / SD (12 hours). Western blot analysis was performed to assess the expression of 3-Nitrotyrosine, 8-oxo-dG, and SOD1. (n = 3 independent experiments).

[0161] GH and H9C2 cardiomyocytes were treated with BFH772 for 36 hours and then with H / SD for 12 hours. Western blot analysis was performed to assess the protein levels of BAF170, WWP2, BCL2, and apoptosis markers (Cleaved-PARP1, Cleaved-Aspase3) (n=3 independent experiments).

[0162] B, F, and H, three independent experiments were performed. Data were assumed to be normally distributed according to the central limit theorem. Relative protein levels were calculated as the fold change compared with the first group. Quantified data are presented as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparison test (B, P values ​​adjusted for 15 comparisons) and two-way ANOVA with Bonferroni test (F, H, P values ​​adjusted for 6 comparisons). Ub denotes ubiquitin antibody; IB, immunoblot. DETAILED DESCRIPTION

[0163] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0164] The following non-standard abbreviations and acronyms are used in this disclosure:

[0165] 8-oxo-dG 8-oxo-dG

[0166] SOD1 superoxide dismutase 1

[0167] SOD2 Superoxide dismutase 2

[0168] H&E Hematoxylin and Eosin

[0169] WGA wheat germ agglutinin

[0170] CM cardiomyocytes

[0171] CHD congenital heart disease

[0172] CHX cycloheximide

[0173] H / SD hypoxia and serum deprivation

[0174] ROS reactive oxygen species

[0175] EF% ejection fraction

[0176] FS% Fractional Shortening

[0177] HW / BW Heart Weight / Body Weight

[0178] HW / TL Heart weight / Tibia length

[0179] TEM transmission electron microscope

[0180] PARP1 poly ADP-ribose polymerase family member 1

[0181] Example

[0182] 1. Raw materials

[0183] The antibodies and reagents used in the following methods are listed in Table 1:

[0184] Table 1 Antibodies and reagents

[0185]

[0186]

[0187] 2. Experimental methods

[0188] Proteomics and Ubiquitination Proteomics

[0189] Protein extraction

[0190] The sample was ground into a cell powder using liquid nitrogen and then transferred to a 5 mL centrifuge tube. Next, four volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail) were added to the cell powder, followed by three sonications on ice using a high-intensity ultrasonic processor (Scientz). The remaining fragments were centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was collected and protein concentration was determined using a BCA assay kit according to the manufacturer's instructions.

[0191] Trypsin digestion

[0192] For digestion, the protein solution was reduced with 5 mM dithiothreitol at 56°C for 30 minutes and then alkylated with 11 mM iodoacetamide for 15 minutes at room temperature in the dark. The protein sample was then diluted with 100 mM TEAB to reduce the urea concentration to below 2 M. Finally, trypsin was added at a trypsin-to-protein ratio of 1:50 for the first overnight digestion and 1:100 for the second 4-hour digestion.

[0193] Enrichment of post-translationally modified peptides

[0194] Peptides were dissolved in immunoprecipitation (IP) buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and mixed with pre-washed anti-lysine ubiquitination (PTM-1104, Jingjie Biolabs (Hangzhou) Co., Ltd.) residual antibody resin. The mixture was incubated overnight at 4°C with gentle shaking. The antibody resin was washed with IP buffer and deionized water. Finally, the enriched peptides were eluted three times with 0.1% trifluoroacetic acid and purified using C18 ZipTips.

[0195] LC-MS / MS (liquid chromatography-tandem mass spectrometry) analysis

[0196] Tryptic peptides were dissolved in liquid chromatography mobile phase A and separated on a NanoElute ultra-high performance liquid chromatography system. Mobile phases A and B were 0.1% formic acid in water and 2% acetonitrile, respectively. The peptide elution gradient was set at a constant flow rate of 450 nL / min: 7% to 24% B (0–72 min); 24% to 32% B (72–84 min); 32% to 80% B (84–87 min); and 80% B (87–90 min). After separation on a capillary column (internal diameter, particle size), the peptides were injected into the capillary ion source for ionization and analyzed on a TIMS-TOF Pro mass spectrometer (ion source voltage, 1.6 kV; scan range, 100–1700 Da). Data were acquired in parallel accumulation serial fragmentation (PASEF) mode. Precursor ions with charge states 0 to 5 were fragmented, and 10 PASEF MS / MS scans were acquired per cycle. A dynamic exclusion time of 30 seconds was used for the MS / MS scans to avoid multiple scans of the same precursor ion.

[0197] Database search

[0198] The raw mass spectrometry data were searched against the SwissProt protein sequence database (Mus_musculus_10090_SP_20210721.fasta) using MaxQuant (v1.6.15.0), including reverse bait entries and common contaminating proteins. Trypsin / P digestion allowed up to two missed cleavage sites, and each peptide required at least 7 amino acids. The mass error tolerance for precursor ions was 10 ppm, and the mass error tolerance for product ions was 20 ppm. Cysteine ​​alkylation (carbamidomethyl [C]) was set as a fixed modification. Variable modifications included methionine oxidation and N-terminal ubiquitination. Lysine ubiquitination and diglycine on lysine were also set as variable modifications for corresponding modification enrichment analysis. The FDR for protein and PSM identification was 1%.

[0199] CUT&Tag experimental method

[0200] CUT&Tag assays were performed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903-01) according to the manufacturer's instructions. The steps were summarized as follows: cells were harvested and bound to concanavalin A-coated magnetic beads. After permeabilization with digitonin, the cells were incubated with a BAF170 antibody (ab243634, Abcam, USA). pA-Tn5 transposase was then added and incubated with the sample. After DNA extraction, amplification, and purification, libraries were created by transposon activation and tagmentation, and analyzed on the Illumina NovaSeq 150PE platform.

[0201] Cell culture and hypoxia and serum deprivation (H / SD) treatment

[0202] H9C2 cells were purchased from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, UT, USA). HEK293T cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in high-glucose Dulbecco's Modified Eagle Medium. Cells were cultured at 37°C in a humidified atmosphere with 5% CO₂ in a medium containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA), penicillin (100 U), and streptomycin (100 μg / ml). Under hypoxic and serum-deprived conditions, cells were cultured in an atmosphere of 1% O₂, 5% CO₂, and 94% N₂.

[0203] Plasmid construction and transfection

[0204] Table 2 below lists various plasmids and small hairpin RNAs (shRNAs). Full-length human BAF170 carrying the K874R / K704R / K694R mutations was cloned into the 3XFlag GV712 vector, and six truncated BAF170 plasmids containing different domains were constructed: Flag-tagged BAF170 N-terminus with SWIRM and SANT domains; Flag-tagged BAF170 N-terminus with SWIRM domains; Flag-tagged BAF170 N-terminus with SWIRM domains; Flag-tagged BAF170 C-terminus with SANT domains; and Flag-tagged BAF170 C-terminus with SANT and SWIRM domains. HA-WWP2 and HA-Ub were purchased from Sangon Biotech (Shanghai) Co., Ltd. Plasmid transfection was performed using Lipofectamine 3000 according to the manufacturer's instructions, and cells were harvested 48 hours after transfection.

[0205] Table 2 Plasmids and small hairpin RNA (shRNA) used in the present invention

[0206]

[0207]

[0208] Lentivirus production

[0209] BAF170 and WWP2 shRNA lentivirus was purchased from GeneCare Gene. Lentivirus was harvested from HEK293T cells according to the manufacturer's instructions. Lentivirus particles were mixed with 5× PEG-it™ solution. Cells in 6-well culture plates were infected with lentivirus. Stable cell lines were selected with puromycin (10 μg / ml) for 7 days. Finally, the infection efficiency of target cells was determined by Western blotting.

[0210] Western blotting and immunoprecipitation

[0211] Incubate cell lysates with anti-Flag magnetic beads overnight at 4°C or with appropriate antibodies for 3 hours at 4°C, followed by incubation with protein A / G immunoprecipitation magnetic beads for 12 hours at 4°C. Wash the protein-antibody complexes three times with ice-cold lysis buffer at 4°C and elute by boiling with SDS loading buffer for 10 minutes.

[0212] BAF170 ubiquitination analysis

[0213] Mouse myocardial tissue samples and cells transfected for 48 hours were lysed in 200 μL of 1% SDS buffer (Tris pH 7.5, containing 0.5 mM EDTA and 1 mM DTT), boiled for 10 minutes, and then diluted with 800 μL of Tris-HCl (pH 8.0). Endogenous proteins were immunoprecipitated using anti-BAF170 antibody (1 μg / mg cell lysate) for 2–3 hours at 4°C, followed by incubation with protein A / G immunoprecipitation beads for 12 hours at 4°C, or by incubation of lysates with anti-Flag (B26302; Biotool) immunoprecipitation beads for 12 hours at 4°C. Ubiquitinated BAF170 was detected using anti-HA antibody.

[0214] JC-1

[0215] The mitochondrial membrane potential (ΔΨm) of H9C2 cells was detected using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-iodinated amidine carbocyanine (JC-1) fluorescent probe. According to the instructions, 1×105 H9C2 cells were cultured per well and placed in a 24-well microplate overnight. After approximately 6 hours of treatment, each well was washed twice with PBS. 2 μL of JC-1 fluorescent probe was added to each well and incubated at 37°C in the dark for 20 minutes. After rinsing three times with PBS, DMEM was added and fluorescence was observed using a fluorescence microscope. When ΔΨm changes, JC-1 aggregates (red) will transform into monomers (green), indicating that the mitochondrial membrane of the H9C2 cells is damaged.

[0216] Hoechst 33342

[0217] H9C2 cells were mounted on slides and incubated with Hoechst 33342 dye solution at 37°C for 15 minutes. After washing three times with PBS, the cells were incubated with PI dye solution on ice for 15 minutes in the dark. The slides were then washed three times with PBS and mounted.

[0218] Cell oxidative stress staining

[0219] To detect intracellular ROS levels, cells were washed three times with PBS and incubated with 5 μM CellROX Green or 5 μM MitoSOX Red at 37°C for 30 minutes in the dark. After thorough washing, the fluorescence intensity of the cells was observed using a fluorescence microscope.

[0220] Wwp2 knockout, transgenic and BAF170-K874R mutant mouse models

[0221] Myh6 Cre+Wwp2f / f mice, WWP2-TG mice, BAF170-K874R mutant mice, and corresponding control mice were all established by Shanghai Model Organisms Science Co., Ltd. BAF170-K874R mutant mice were derived from the fourth generation of mice, in which lysine 874 (AAG) is mutated to arginine (CGC). All animals were maintained under pathogen-free conditions. All experiments were performed using mice aged 8-10 weeks. At appropriate ages, a permanent myocardial infarction model was established in these mice by ligating the left anterior descending coronary artery, and the modeling effect was stable. During the modeling process, cardiac ultrasound examinations were performed on days 0, 7, 14, and 28 in each group to verify the success of the modeling. All animal procedures complied with the Animal Welfare Regulations of China Medical University, and the animal research protocol was approved by the Animal Experimentation Ethics Committee of China Medical University (CMU20241520, CMU20241518, CMU20241909, and CMU20251150).

[0222] BFH772 pretreatment

[0223] BFH772 (6-((6-(hydroxymethyl)pyrimidin-4-yl)oxy)-N-(3-(trifluoromethyl)phenyl)-1-naphthamide, APE, China) has an IC50 value of 3 nM (PMID: 26629594). In in vivo experiments, BFH772 powder was diluted with dimethyl sulfoxide and corn oil, and 80 μL was administered to each mouse at a dimethyl sulfoxide:corn oil ratio of 1:9. Mice were dosed at concentrations of 0, 20, 30, and 40 mg / kg. Dosing began 48 hours after myocardial infarction and continued once daily for 28 days. In in vitro experiments, BFH772 powder was diluted with DMSO to final concentrations of 0.01, 0.1, 1, 10, and 100 μM, and the drug exposure lasted for 48 hours.

[0224] Myocardial infarction model

[0225] Target gene expression was induced by intraperitoneal injection of tamoxifen (75 mg / kg body weight, once every other day, for a total of 5 times). Myocardial infarction modeling was performed 30 days after injection. A permanent myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD) of C57BL / 6J mice. Mice were anesthetized by inhaling 1.5-2% isoflurane using an isoflurane delivery system. The left ventricle was exposed after the left chest cavity was incised between the third and fourth intercostal spaces. The left ventricular aorta was found, sutured and ligated approximately 3 mm from its origin. When the color of the anterior wall of the left ventricle became lighter, it was confirmed that myocardial ischemia had been successfully induced. After ligation, the heart was immediately returned to the chest cavity.

[0226] Echocardiography and left ventricular function assessment

[0227] Echocardiography was performed on mice at baseline (day 0), 7 days, 14 days, and 28 days. Cardiac function was assessed in each group using a VisualSonics Vevo 2100 real-time, high-resolution intravital microscopy system (Visualsonic, Canada; VINNO6 Lab, China). Mice were anesthetized with 1.5% isoflurane, and cardiac function analysis was performed using a 40 MHz transducer under continuous oxygen supply. Left ventricular function was assessed using two-dimensional M-mode recordings. Cardiac function was measured based on interventricular septum thickness (IVSd), left ventricular posterior wall thickness (PWTd), systolic left ventricular internal diameter (LVDs), diastolic left ventricular internal diameter (LVDd), and left ventricular mass. In addition, left ventricular ejection fraction (EF%) and fractional shortening (FS%) were determined. After 28 days, mice were euthanized, and their body weight (BW) and tibial length (TL) were measured. Tibial length was measured from the mid-proximal articular surface to the most distal projection of the medial malleolus [29915560]. The heart was removed, washed with PBS and the heart weight (HW) was measured.

[0228] Histopathological evaluation

[0229] Myocardial tissue samples were fixed in 4% fixative for 48 hours, embedded in paraffin, and cut into 4 μm sections. After dewaxing in xylene and rehydration with graded ethanol, sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome stain (G1340; Solarbio, China). Myocardial cell cross-sectional area was assessed by staining images with 5 μM wheat germ agglutinin (WGA) (Genetex, USA).

[0230] electron microscope

[0231] Transmission electron microscopy (TEM) was used to observe the morphology of cardiac mitochondria. Heart tissue was pre-fixed with EM-grade 2.5% glutaraldehyde in 0.1 mol / l sodium dimethylarsenate buffer. The fixed tissue was incubated with 1% osmium tetroxide in 0.1 mol / l sodium dimethylarsenate buffer for 2 hours. The fixed tissue was then dehydrated and embedded stepwise. Heart tissue sections were stained with uranyl acetate. A Hitachi H-7650 transmission electron microscope was used to observe and photograph the changes in mitochondria in myocardial tissue, with 15 to 24 images randomly taken for each group of mice.

[0232] Oxidative stress staining of animal myocardial tissue

[0233] In vitro, ROS production was detected using dihydroethidium (DHE) staining. Paraffin-embedded heart sections were mixed with dihydroethidium (5 μmol / L) and incubated at 37°C in the dark for 1 hour. Images were captured blindly using a fluorescence microscope, and fluorescence intensity was calculated using ImageJ software. Results are expressed as the fold change relative to the corresponding control group.

[0234] Mitochondrial extraction for high-resolution respirometry O2k

[0235] Mouse hearts were extracted and rapidly minced into small pieces within 4 minutes. The tissue was incubated for 2 minutes at 4°C in mitochondrial isolation buffer (Sigma Aldrich, P8038) containing 0.1 mg / ml protease. The mitochondrial isolation buffer consisted of 50 mM Tris × HCl (pH 7.4), 100 mM KCl, 100 mM sucrose, 1 mM KH2PO4, 0.1 mM EGTA, and 0.2% bovine serum albumin. The tissue was gently homogenized six times using a glass homogenizer. After multiple centrifugations at different speeds, the resulting pellet was resuspended in 10 mM buffer consisting of 10 mM Tris × HCl (pH 7.4), 225 mM mannitol, 75 mM sucrose, and 0.1 mM EDTA. All experimental procedures were performed at 4°C.

[0236] Mitochondrial respiratory activity measurement

[0237] Mitochondrial respiration was assessed at 37°C using a high-resolution respirometry system, O2k (Oroboros Instruments, Innsbruck, Austria), with a 2 mL reaction chamber volume. Oxygen was calibrated prior to the experiment using respiration medium MIR05 (110 mM sucrose, 60 mM potassium lactobionate, 0.5 mM EGTA, 1 g / L fatty acid-free BSA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, and 20 mM HEPES; pH 7.1, 37°C). The medium was equilibrated with air in the oxygen meter chamber and stirred at 750 rpm for 20 minutes until the signal stabilized. 70 μg of mitochondrial extract was added to each chamber, and substrate-uncoupler-inhibitor titration (SUIT) was performed in the following order: pyruvate / malate / glutamate (PMG) → MgCl2 / ADP (D) → succinate (S) → oligomycin (O) → carbonyl cyanide m-chlorophenylhydrazone (CCCP) → rotenone (R) → antimycin A (AmA) → N,N,N',N'-tetramethyl-p-phenylenediamine / ascorbic acid (TMPD / Asc). Oxygen consumption rate (OCR) was calculated as the negative time derivative of oxygen concentration. Data acquisition and analysis were performed using Software version 7.4.0.4 (Oroboros Instruments) was used.

[0238] Reagents

[0239] Proteasome inhibitors MG132 (A2585) (50 μmol / L) and cycloheximide (CHX, A8244) (50 μmol / L) were purchased from Apexbio (USA) and dissolved in dimethyl sulfoxide.

[0240] Protein purification-WWP2

[0241] The recombinant plasmid containing the WWP2 gene was transformed into competent Escherichia coli BL21(DE3) cells, cultured, and induced to produce high-quality protein. The cells were harvested by centrifugation. For affinity purification, the cells were lysed with a buffer (50mM Tris, 300mM NaCl, 0.1% TritonX-100, 0.2mM PMSF, pH 8.0), sonicated, and the supernatant collected by centrifugation to obtain the crude protein. A 5ml Ni-NTA column was then equilibrated with five volumes of binding buffer (PBS-NaCl, pH 7.4). The crude protein was incubated with the equilibrated column packing for 1 hour, and the flow-through was collected. The column was then washed with binding buffer and then with wash buffer (PBS-NaCl, 20mM imidazole, pH 7.4), and the flow-through was collected. The protein was eluted with elution buffer (PBS-NaCl, 500mM imidazole, pH 7.4), and the eluate was collected. The eluted sample was further purified by ion exchange chromatography (Q column) and analyzed by SDS-PAGE. The purified eluted sample was subjected to gel filtration chromatography (Superdex200) using a buffer solution (PBS, 0.12% SKL, pH 7.4). The purified fraction was dialyzed into protein storage buffer (PBS, 0.12% SKL, pH 7.4), concentrated, filtered, and sterilized. The purified protein was aliquoted and stored at -80°C.

[0242] Protein purification-BAF170

[0243] A series of recombinant expression vectors encoding different SMARCC2 domains were constructed through molecular cloning: SMARCC2 (1-647, 1-423, 1-595, 648-1214, 424-1214, and 596-1214). Recombinant plasmids were verified for correct construction by restriction enzyme analysis and then recombined and amplified. The plasmids were transfected into 293 cells, and the protein was purified. First, cells were lysed with buffer C, sonicated, and centrifuged to collect the crude protein supernatant. A 5 ml Ni-NTA column was equilibrated with five times the column bed volume of binding buffer. The crude protein was incubated with the equilibrated column packing for 1 hour, and the flow-through was collected. The equilibrated column was washed with binding buffer and then with wash buffer (PBS-NaCl, 20 mM imidazole, pH 7.4). The protein was eluted with elution buffer (PBS-NaCl, 500 mM imidazole, pH 7.4). The eluate sample was purified by ion exchange chromatography (Q column) and analyzed by SDS-PAGE. The high-purity eluate sample was purified by gel filtration chromatography (Superdex 200) (PBS, 0.12% SKL, pH 7.4). The purified fraction was dialyzed into protein storage buffer (PBS, 0.12% SKL, pH 7.4), concentrated, filter-sterilized, and stored in aliquots at -80°C. For SDS-PAGE analysis, protein samples were processed and prepared using 12% separating gel and 5% stacking gel, and molecular weight was determined.

[0244] Compound library preparation

[0245] The SMILES format of the selected compounds (15,000 molecules) was obtained from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) and these compounds were normalized and converted using RDKit (2023.09.1) to generate a compound library. All selected compounds were converted to the desired structure (SDF format).

[0246] Virtual screening

[0247] The SMARCC2 protein structure model was predicted by AlphaFold to confirm the binding site of k874. The central coordinates were fixed to The grid box size is fixed to Flexible ligand and rigid receptor docking was performed using AutoDockTools 1.5.6 and Vina 1.1.2, with an energy range of 4 kcal / mol and an exhaustiveness of 12. Genetic algorithms (GA) and particle swarm optimization (PSO) were used to identify the optimal binding mode, and the affinity of each binding mode was calculated using a scoring function based on an empirical formula. The binding modes of each compound with the target protein were ranked according to affinity, and the results were visualized using PyMOL 2.2.0.

[0248] Surface plasmon resonance (SPR)

[0249] Binding analysis of BAF170 truncated with different domains and WWP2

[0250] SPR experiments were performed on a Biacore T200 system (Cytiva) for BAF170(1-426), BAF170(1-595), BAF170(1-647), BAF170(424-1214), BAF170(569-1214), and BAF170(648-1214) at 25°C and a flow rate of 30 μl / min. Purified wild-type WWP2 protein was immobilized on an S CM5 series sensor chip (Cytiva) using amine coupling chemistry. BAF170 proteins with different domain truncations were serially diluted in running buffer to various concentrations and passed over the chip at a constant flow rate from low to high concentration for 4-6 minutes. Data were analyzed using Biacore T200 Evaluation Software 3.0, and affinity constants were calculated.

[0251] SPR analysis of the effect of BFH772 on the binding of BAF170 and WWP2

[0252] SPR experiments were performed on a Biacore T200 system (Cytiva) with conivaptan hydrochloride, NVP-BHG712, U-74389G, 1-{[4-({4-[(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)methyl]phenyl}methyl)phenyl]methyl}-2,3-dihydro-1H-indole-2,3-dione, a citrulline-specific probe, BFH772, BMS195614, LIMKi 3, N-methylprotoporphyrin IX, and L-689,560. The information for these compounds is shown in Table 3. The temperature was 25°C and the flow rate was 30 μl / min. Purified wild-type WWP2 protein was immobilized on an S CM5 series sensor chip (Cytiva) using amine coupling chemistry. 50 μM of various small molecule complexes were flowed through the chip at a constant flow rate as binding substrates, with the equilibrium state serving as the signal baseline before injection into BAF170. Using the ternary interaction system, the binding curves and signal changes between WWP2 and BAF170 before and after the addition of the small molecule compound were visually compared to determine its specific effects.

[0253] Table 3 Information of small molecule compounds

[0254]

[0255] Relative value

[0256] Immunoblot images were analyzed using ImageJ software. The intensity of the internal reference band was used as a reference for the loaded protein. First, in an independent experiment, the grayscale value of each band was divided by the grayscale value of the internal reference to calculate the relative expression of the target protein. Then, the relative expression of each band was divided by its average value to obtain a ratio. This calculation process was repeated in other independent experiments. Next, the ratios of the first group (control group) were averaged, and each ratio in the first group was divided by the average value to obtain a relative value. Finally, the ratios of the other groups were divided by this average value to obtain the final value for statistical analysis. The fluorescence intensity of DHE (oxidative stress marker), CellROX-Green, and MitoSOX-Red represents the level of oxidative stress, and the relative fluorescence units were calculated using ImageJ software. First, the average value of the fluorescence unit values ​​of the first group (control group) was calculated, and each value in the first group was divided by this average value to obtain the relative fluorescence intensity. Finally, the values ​​of the other groups were divided by this average value to obtain the fluorescence intensity value for statistical analysis.

[0257] Statistical analysis

[0258] Data are presented as mean ± standard deviation (SD). For in vitro experiments, if the cell number was sufficiently large (hundreds of thousands of cells), the ratio (assay mean index) was calculated as the protein expression level divided by the internal control. According to the central limit theorem, the assay mean index approximately followed a normal distribution, and the mean index of three independent replicates followed a normal distribution. For in vivo experiments (sample size ≥ 7), normality was tested using the Shapiro-Wilk test and the F test (for comparisons between two groups) or the Bartlett test (for comparisons between more than two groups) for homogeneity of variance, with a threshold P value of 0.05. For data with normal distribution and homogeneity of variance, unpaired t-tests (two-tailed Student's t-tests) were used for two-group comparisons, and one-way analysis of variance with Tukey's multiple comparison test was used for multiple group comparisons. When two conditions were considered between two groups, two-way analysis of variance with Bonferroni's multiple comparison test was used. For data that did not pass normality or homogeneity of variance tests, the Mann-Whitney test was used to compare two groups, and the Kruskal-Wallis test with Dunn's multiple comparison test was used for multiple group comparisons. Multiple comparisons were adjusted for type I error inflation using the Bonferroni correction, and P values ​​were adjusted when applicable. Each figure legend contains detailed significance assessment methods and the number of biological replicates for each set of experiments. The exact P value for significant changes is indicated in the figure legend. Statistical analysis was performed using SPSS 22.0 software (SPSS, USA) and GraphPad Prism 10.0 software (GraphPad, Bethesda, MD, USA), and P < 0.05 indicated statistical significance. Representative images reflecting the average results of each experiment were selected in the figure. "N" or "n" in the figure legend indicates biological replicates.

[0259] 3. Results

[0260] The experimental results are as follows Figure 1-13 And shown in Table 4.

[0261] BAF170 protein expression increases during MI

[0262] To investigate which proteins and functions in the heart change during myocardial infarction (MI), we created an MI mouse model by ligating the left anterior descending coronary artery. We then performed a high-depth proteomic analysis of the heart tissue and compared it with the heart tissue of control mice. The results showed that among the differentially expressed proteins, 1079 proteins were upregulated (FC>1.5, P value <0.05) and 225 proteins were downregulated (FC<0.667, P value <0.05) ( Figure 1 A). GO functional enrichment analysis showed that among cellular components, the SWI / SNF superfamily complex had the highest score, except for cellular structural proteins ( Figure 1 B). Among the SWI / SNF chromatin remodeling complex family members identified among the differentially expressed proteins, SMARCC2 (also known as BAF170) had the lowest p-value ( Figure 1 C). This suggests that BAF170 may play an important role in the heart during myocardial infarction.

[0263] Hypoxia and serum deprivation (H / SD) is a widely accepted in vitro model used to simulate ischemic hypoxic conditions. The H / SD model has been widely used to study the response of cardiomyocytes to ischemic hypoxia. We established an H / SD model in H9C2 cells to explore the role of BAF170 under these conditions and found a direct correlation between H / SD exposure time and cell apoptosis rate and BAF170 expression level ( Figure 1 Using Hoechst 33342, a commonly used nuclear stain, we observed that the number of apoptotic cells gradually increased with the extension of H / SD treatment time, as evidenced by enhanced blue fluorescence ( Figure 1 E).

[0264] To explore the mechanism of increased BAF170 expression, we performed immunoprecipitation (IP) using a BAF170 antibody to enrich interacting proteins from cardiac tissues of control and myocardial infarction (MI) mice. By mass spectrometry analysis, we detected changes in downstream protein interactions. The results showed that the binding of BAF170 to WWP2 was significantly reduced in the MI model (Table 4). In addition, in the H / SD model, the expression of WWP2 gradually decreased with the extension of induction time ( Figure 1 F).

[0265] After myocardial infarction, the interaction between BAF170 and WWP2 is weakened, indicating that there is a regulatory relationship between the two. Endogenous immunoprecipitation experiments showed that there is a significant interaction between WWP2 and BAF170 ( Figure 1 G, 1H), this finding was further confirmed by exogenous immunoprecipitation studies ( Figure 1 I). To identify the specific regions of interaction, the researchers performed binding experiments using endogenous WWP2 and various Flag-tagged BAF170 constructs, including full-length and truncated versions. The results showed that WWP2 specifically interacts with three regions of BAF170: the SANT domain, the SWIRM domain, and the N-terminal region ( Figure 1 J, 1K).

[0266] WWP2 regulates BAF170 degradation through K874 ubiquitination

[0267] To explore the potential role of WWP2 in regulating BAF170 levels, we investigated whether reducing WWP2 would affect BAF170 expression levels. To this end, we generated a stable WWP2 knockdown (shWWP2) H9C2 cell line using three different shRNA fragments ( Figure 2 A). Of the three fragments, fragment 74453 exhibited the highest knockdown efficiency and was therefore selected for subsequent experiments. Consistent with a role for WWP2 in regulating BAF170 levels, silencing WWP2 resulted in an increase in BAF170 levels.

[0268] To further verify this relationship, we performed a gradient overexpression experiment of WWP2 in HEK293T and H9C2 cells. The results showed that as the expression of WWP2 increased, the level of BAF170 protein decreased in a dose-dependent manner ( Figure 2 B) These findings strongly suggest that WWP2 acts as a negative regulator of BAF170 protein levels.

[0269] WWP2 may reduce the level of BAF170 by promoting its degradation ( Figure 2 C-2H). To test this hypothesis, we used cycloheximide (CHX) as a translation inhibitor for time-gradient treatment. This inhibitor can prevent the synthesis of new proteins, allowing us to monitor the steady-state levels of proteins at different time points. The experiment was performed in normal control and shWWP2 cells. After the addition of cycloheximide, the BAF170 level in the control group decreased faster, while the shWWP2 group maintained a high level ( Figure 2C, 2G). This suggests that the reduction in WWP2 levels is associated with an increase in BAF170 stability. Similarly, when comparing cells expressing HA-Vector or HA-WWP2, we found that gradient overexpression of HA-WWP2 resulted in an accelerated decrease in BAF170 levels upon cycloheximide treatment ( Figure 2 D, 2H).

[0270] Next, we tested whether BAF170 is recruited to the ubiquitin-proteasome-mediated degradation pathway. To test this, we performed a time course experiment using the proteasome inhibitor MG132 ( Figure 2 E-2J). Compared with the control group, the BAF170 protein level in cells transfected with shWWP2 was significantly increased ( Figure 2 E, 2I). In addition, after MG132 treatment, the accumulation of BAF170 in cells expressing HA-WWP2 increased significantly, while this phenomenon was not seen in the control group expressing HA-Vector ( Figure 2 F, 2J). These results support the conclusion that WWP2 regulates BAF170 protein levels through the proteasome pathway. Similarly, WWP2 overexpression significantly increased the ubiquitination level of BAF170 ( Figure 2 K), while WWP2 knockdown significantly reduced the ubiquitination level of BAF170 ( Figure 2 L). Taken together, these findings indicate that WWP2 promotes BAF170 degradation through polyubiquitination followed by proteasome-dependent degradation.

[0271] To identify the BAF170 ubiquitination sites targeted by WWP2, we conducted a comprehensive analysis using multiple mouse models and advanced proteomics technologies. Two key experimental groups were established: conditional myocardium-specific WWP2 knockout mice (WWP2-cKO: Myh6-Cre+; Wwp2 f / f ) and its corresponding WWP2-WT (Wwp2 f / f ) control group, as well as transgenic Rosa26-WwP2-Flag mice (WWP2-TG) and their corresponding control groups. Through complex quantitative proteomics and ubiquitination modification genomics analysis, the specific signaling pathways regulated by WWP2 in myocardial infarction response were explored ( Figure 2 M). Four-dimensional label-free high-depth proteomics identified 7,020 ubiquitination sites and 2,202 ubiquitinated proteins, of which 4,091 sites and 1,082 proteins showed quantitative differences.

[0272] We then integrated the differentially expressed and ubiquitinated proteins using a nine-quadrant plot, focusing on two key groups: proteins that were downregulated in ubiquitination and highly expressed in WWP2 knockout compared with wild-type controls; and proteins that were upregulated in ubiquitination and low expressed in WWP2 transgenic compared with wild-type controls ( Figure 2 N). Through expression pattern cluster analysis, we found that these two proteins ( Figure 2 O) and ubiquitination sites ( Figure 2 P) showed significant trends. Of particular note, as WWP2 levels increased, the expression of some proteins decreased (Cluster 3), while the modification of some ubiquitination sites increased (Cluster 6). Systematic analysis revealed a significant negative correlation between the expression levels of BAF170 protein and WWP2, accompanied by evidence of ubiquitination modification.

[0273] The role of WWP2 in myocardial infarction (MI) was investigated by biological function enrichment analysis. In the WWP2 transgenic / wild-type model, upregulated ubiquitinated proteins were analyzed and found to be involved in cardiac contractility regulation, myofilament sliding, and myocardial function ( Figure 2 Q). In the WWP2 conditional knockout / wild-type model, analysis of downregulated ubiquitinated proteins revealed that these proteins were involved in nicotinamide nucleotide metabolism, NADP metabolism, and ADP metabolism ( Figure 2 R).

[0274] We paid special attention to the role of WWP2 in regulating BAF170 ubiquitination. Through mass spectrometry data analysis, we found six ubiquitination sites on BAF170: K694, K704, K872, K874, K897, and K902 ( Figure 2 Among these sites, only K694, K704, and K874 are directly correlated with the expression level of WWP2, that is, when WWP2 expression increases, the ubiquitination degree of these sites also increases accordingly; and when WWP2 expression decreases, the ubiquitination degree of these sites decreases ( Figure 2 These findings indicate that K694, K704, and K874 are specific sites for WWP2-mediated BAF170 ubiquitination.

[0275] To further explore these potential ubiquitination sites, we analyzed the K694, K704, and K874 sites of BAF170 by creating lysine to arginine (KR) mutations. We observed changes in BAF170 ubiquitination levels after exogenous overexpression of HA-tagged vectors or HA-tagged WWP2 variants, and when co-transfected with HA-Ub in the presence of MG132. Wild-type BAF170 and its mutants (K694R, K704R, or BAF170-K874R) were overexpressed separately. Co-immunoprecipitation experiments showed that only the overexpression of BAF170-K874R significantly reduced the ubiquitination level of BAF170 compared to the wild type ( Figure 2 These findings suggest that WWP2 specifically mediates polyubiquitination of BAF170 through the K874 site, leading to BAF170 degradation via the proteasome.

[0276] Myocardial-specific WWP2 deletion leads to increased BAF170 expression and aggravates cardiomyocyte injury

[0277] The specific role of WWP2 in regulating BAF170 during myocardial infarction (MI) remains unclear. To further investigate this relationship, we used Wwp2 f / f and Myh6-Cre+;Wwp2 f / f The left anterior descending coronary artery was ligated for 28 days to establish a mouse model of MI. Western blot analysis confirmed that Myh6-Cre+;Wwp2 f / f In mice, exon 3 of WWP2 has been deleted ( Figure 3 AB). and Wwp2 f / f Compared with the control group, Myh6-Cre+; Wwp2 f / f WWP2 expression was significantly reduced in mice, and this reduction was more pronounced under MI conditions ( Figure 3 J-3K).

[0278] We confirmed the regulatory role of WWP2 on BAF170 by analyzing the interaction in cardiac tissue in a myocardial infarction (MI) model. f / f Compared with the control group, Myh6-Cre+; Wwp2 f / f In mouse heart tissue, the interaction between BAF170 and WWP2 was significantly reduced after MI ( Figure 3 C). In addition, under MI conditions, Myh6-Cre+; Wwp2 f / f The ubiquitination level of BAF170 in mice was significantly decreased ( Figure 3D) IP analysis in shWWP2 cell lines showed that WWP2 bound to BAF170 similarly regardless of H / SD treatment. H / SD treatment attenuated the interaction between WWP2 and BAF170 in H9C2-shWWP2 cells ( Figure 8 A), and reduced WWP2-mediated BAF170 ubiquitination ( Figure 8 B).

[0279] Mitochondrial dysfunction in cardiomyocytes is considered an important pathogenic factor in myocardial infarction (MI). Myocardial infarction triggers an acute burst of reactive oxygen species (ROS), leading to mitochondrial damage and respiratory dysfunction, which in turn triggers cardiomyocyte apoptosis, cardiac fibrosis and cardiac dysfunction, ultimately leading to worsening of heart failure. f / f Compared with mice, Myh6-Cre+; Wwp2 f / f The cardiac dysfunction of mice after myocardial infarction was significantly aggravated, as shown by the decrease of ejection fraction (EF%) and fractional shortening (FS%) ( Figure 3 E-3G). Assessment of cardiac hypertrophy markers showed that Myh6-Cre+; Wwp2 f / f The heart weight to body weight ratio (HW / BW) and heart weight to tibia length ratio (HW / TL) of mice were higher than those in the control group ( Figure 3 H-3I). Simultaneous analysis of oxidative stress markers in cardiac tissue revealed that Myh6-Cre+; Wwp2 f / f Endogenous ROS levels in mice increased ( Figure 3 L), increased levels of 3-Nitrotyrosine and 8-oxo-dG, decreased expression of SOD1 and SOD2, and Wwp2 f / f Compared with mice ( Figure 3 M-3N).

[0280] Next, we investigated mitochondrial respiratory function, structural abnormalities, oxidative stress damage, and fibrosis in an established MI mouse model. First, we examined BCL2, a protein associated with mitochondrial apoptosis, because its downregulation is directly associated with mitochondrial damage and dysfunction. By CUT&Tag and chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis, we found that BAF170 binding to the BCL2 enhancer was reduced in MI tissues compared with controls ( Figure 9 D). It is noteworthy that Myh6-Cre+; Wwp2 f / f In MI mice, BCL2 expression was significantly lower than Wwp2 f / f control group, which indicated enhanced mitochondrial apoptosis ( Figure 3 O-3P).

[0281] To assess mitochondrial respiratory function, we isolated cardiac mitochondrial tissue and analyzed oxygen consumption of different mitochondrial complexes using the Oroboros O2K system. f / f In Myh6-Cre+;Wwp2f / f mice, after myocardial infarction, the oxidative phosphorylation capacity of complex I, the oxidative phosphorylation capacity of complex I and II, and ATP production were all reduced, while the oxidative phosphorylation capacity of complex II remained unchanged. f / f Compared with the Myh6-Cre+; Wwp2 f / f The three parameters of the group were significantly reduced ( Figure 3 Q).

[0282] Transmission electron microscopy (TEM) is widely considered the gold standard for assessing mitochondrial content and is capable of measuring mitochondrial volume density. f / f In mice, mitochondria exhibit dense, tightly packed, and well-organized cristae. However, mice in the MI-treated group showed signs of mitochondrial damage, as evidenced by loose, swollen, damaged, and vacuolated cristae. This damage was not observed in Myh6-Cre+;Wwp2 f / f It is particularly severe in mice ( Figure 3 R).

[0283] To investigate whether cardiomyocyte apoptosis is associated with changes in the expression of apoptosis-related proteins, we f / f mice and Myh6-Cre+;Wwp2 f / f The results showed that in Myh6-Cre+;Wwp2 f / f In mice, the levels of cleaved-PARP1 and cleaved-Caspase3 were significantly increased ( Figure 3 In addition, histological analysis by hematoxylin and eosin (H&E), wheat germ agglutinin (WGA), and Masson staining showed that Myh6-Cre+; Wwp2 f / f Cardiac hypertrophy and fibrosis in mice were significantly aggravated ( Figure 3 U-3W).

[0284] Next, we investigated the levels of oxidative stress markers and endogenous ROS in the H9C2-shWWP2 cell line with and without H / SD treatment. First, we analyzed several proteins associated with oxidative stress, including 3-Nitrotyrosine, 8-oxo-dG, SOD1, and SOD2. The results showed that the levels of oxidative stress-related proteins in the H9C2-shWWP2 cell line, whether treated with H / SD or not, were higher than those in the control group. Notably, re-expression of HA-WWP2 NTm was able to effectively reduce these elevated levels ( Figure 8 E-8F). Subsequently, we detected ROS levels using a fluorescence method. The CellRox-Green fluorescent probe detects ROS production in intact tissues by staining the cell nucleus, producing green fluorescence, while the Mitosox-Red fluorescent probe is used to measure ROS in mitochondria, showing red fluorescence. Quantitative analysis of the staining intensity of the CellRox-Green and Mitosox-Red probes showed that the ROS production in the H9C2-shWWP2 cell line was significantly higher than that in the control group, regardless of whether it was treated with H / SD ( Figure 8 G-8J).

[0285] To investigate the effect of WWP2 on mitochondrial function in the H / SD model, we transfected HA-WWP2 NTm into the H9C2-shWWP2 cell line. The results showed that in shWWP2 cells, BCL2 levels were significantly decreased in both the control and H / SD models, indicating increased mitochondrial apoptosis. In contrast, when HA-WWP2 NTm was re-expressed in shWWP2 cells, BCL2 levels were significantly increased, indicating decreased mitochondrial apoptosis ( Figure 8 L-8M). Changes in mitochondrial membrane potential were detected by JC-1 staining. Regardless of whether H / SD treatment was performed, the ratio of green JC-1 monomers to red JC-1 aggregates increased in the H9C2-shWWP2 cell line, indicating a decrease in mitochondrial membrane potential. Importantly, re-expression of HA-WWP2 NTm alleviated this change ( Figure 8 N-8O).

[0286] Finally, we investigated the relationship between apoptosis and changes in the expression of apoptosis-related proteins in H9C2 cells. Comparison of the H / SD group and the control group revealed that the levels of cleaved-PARP1 and cleaved-Caspase3 were significantly increased in the H9C2-shWWP2 cell line, regardless of whether or not H / SD treatment was performed. Notably, re-expression of HA-WWP2 NTm was able to mitigate this increase ( Figure 8P-8Q). In addition, Hoechst33342 staining showed that the number of apoptotic cells in the H9C2-shWWP2 cell line increased significantly under H / SD and control conditions. This effect was also attenuated by re-expression of HA-WWP2 NTm ( Figure 8 G-8H,8K).

[0287] WWP2 overexpression downregulates BAF170 and alleviates cardiomyocyte injury after myocardial infarction

[0288] Given the negative effects of reduced WWP2 expression, we tested whether overexpression of WWP2 could alleviate cardiomyocyte injury. We generated Rosa26-WwP2-Flag transgenic mice (WWP2-TG, R26-LSL-Wwp2+ / +; Myh6-CreER) by crossing F0 mice with Myh6-CreER mice ( Figure 4 A). Subsequently, myocardial infarction was induced in wild-type (WWP2-WT, R26-LSL-Wwp2+ / +; Myh6-CreER-) and WWP2-TG mice by ligating the left anterior descending coronary artery and monitored for 28 days ( Figure 4 B). The analysis results confirmed the successful generation of WWP2-TG mice, which showed that WWP2 expression levels were significantly lower than those in WWP2-WT mice and decreased significantly after myocardial infarction ( Figure 4 J-4K).

[0289] To investigate the regulatory role of WWP2 on BAF170 in this model, we analyzed protein interactions in cardiac tissue. Under myocardial infarction (MI) conditions, enhanced interactions between BAF170 and WWP2 were observed in cardiac tissue of WWP2 transgenic (WWP2-TG) mice compared to wild-type (WWP2-WT) mice. Figure 4 C). In addition, we found that the ubiquitination level of BAF170 was increased in WWP2 transgenic mice after myocardial infarction ( Figure 4 D). Similarly, in the H / SD model, after transfection with HA-WWP2, the interaction between WWP2 and BAF170 was enhanced compared with the HA vector control group, and the ubiquitination level of BAF170 by WWP2 was also increased ( Figure 8 C-8D).

[0290] Cardiac function of WWP2 transgenic mice was significantly improved after myocardial infarction (MI), as confirmed by measurements of ejection fraction (EF%) and fractional shortening (FS%) ( Figure 4Furthermore, WWP2 transgenic mice exhibited reduced cardiac hypertrophy markers during myocardial infarction, as demonstrated by decreased heart weight to body weight ratio (HW / BW) and heart weight to tibia length ratio (HW / TL) compared to WWP2 wild-type mice ( Figure 4 H-4I). In addition, WWP2 transgenic mice also showed a decrease in markers of oxidative stress, including a decrease in endogenous reactive oxygen species ( Figure 4 L), and the levels of 3-Nitrotyrosine and 8-oxo-dG decreased, while the expression of antioxidant enzymes SOD1 and SOD2 remained high ( Figure 4 M-4N), which was higher than that of the WWP2 wild-type control group.

[0291] We then investigated the effects of mitochondrial respiratory dysfunction, structural abnormalities, oxidative stress damage, and fibrosis observed in an established MI mouse model. The results showed that WWP2 transgenic mice had significantly increased BCL2 expression after MI compared with wild-type WWP2 mice, indicating reduced mitochondrial apoptosis ( Figure 4 Using the Oroboros O2K system, we found that after MI, both wild-type WWP2 mice and WWP2 transgenic mice showed decreased complex I oxidative phosphorylation capacity, complex I and II oxidative phosphorylation capacity, and ATP production compared to the sham-operated group. Notably, complex II oxidative phosphorylation capacity was unaffected. WWP2 transgenic mice performed better than wild-type WWP2 mice in all three parameters ( Figure 4 Q).

[0292] Electron microscopy showed that mitochondrial cristae in the sham-operated group of WWP2-WT mice were dense, compact, and well-organized. In contrast, mitochondrial cristae in MI-treated mice appeared loose, swollen, disorganized, and vacuolated. However, this structural damage was significantly reduced in WWP2-TG mice after MI treatment ( Figure 4 R). Compared with WWP2-WT mice, the levels of cleaved-PARP1 and cleaved-Caspase3 were significantly decreased in WWP2-TG mice ( Figure 4 In addition, histological analysis by H&E staining, WGA staining, and Masson staining showed that cardiac hypertrophy and fibrosis were significantly improved in WWP2-TG mice ( Figure 4 U-4W).

[0293] To determine the specific effects of WWP2 on BAF170 ubiquitination in myocardial infarction (MI), we used three different short hairpin RNA (shRNA) fragments to establish stable BAF170 knockdown cell lines. The 121254 fragment showed the best knockdown efficiency and was therefore used in subsequent experiments (Figures S4B-S4C). Subsequently, we expressed wild-type BAF170 or its mutant forms in these knockdown cell lines. Sequence analysis showed that K874 is an evolutionarily conserved site from rats to mammals and may be a BAF170 ubiquitination site regulated by WWP2. This site corresponds to K874 in mice and K905 in rats ( Figure 10 A). Therefore, we focused specifically on the K874 / K905 residues to explore the importance of BAF170 ubiquitination sites in the oxidative stress response. We analyzed multiple oxidative stress indicators, including 3-Nitrotyrosine, 8-oxo-dG, SOD1, and SOD2. Under hypoxia / serum deprivation (H / SD) conditions, cells expressing K905R-BAF170 NTm showed higher levels of 3-Nitrotyrosine and 8-oxo-dG than H9C2-shBAF170 cells expressing WT-BAF170 NTm. In contrast, the levels of SOD1 and SOD2 were lower in cells expressing K905R-BAF170 NTm ( Figure 10 D-10E).

[0294] We used the fluorescent probes MitoSOX-Red and CellRox-Green to assess mitochondrial reactive oxygen species (ROS) levels in the experiments with and without H / SD treatment. The results showed that mitochondrial ROS levels in cells expressing K905R-BAF170 NTm were consistently higher than those in cells expressing WT-BAF170 NTm, regardless of whether H / SD treatment was performed ( Figure 10 F-10I). These results strongly suggest that K905R-BAF170 can aggravate cellular damage caused by oxidative stress.

[0295] Next, we investigated the effect of BAF170 on mitochondrial respiratory function. By analyzing the H9C2-shBAF170 cell line after re-expression of WT-BAF170 NTm or K905R-BAF170 NTm, whether or not it was treated with H / SD, we found that re-expression of K905R-BAF170 NTm significantly reduced BCL2 levels compared with re-expression of WT-BAF170 NTm, regardless of whether it was treated with H / SD, indicating enhanced mitochondrial apoptosis ( Figure 10JC-1 staining results showed that under H / SD conditions, the ratio of green JC-1 monomers to red JC-1 aggregates increased significantly after K905R-BAF170 NTm was re-expressed ( Figure 10 These results indicate that the K905R-BAF170 mutation exacerbates mitochondrial dysfunction and impairs mitochondrial function in cardiomyocytes under ischemic hypoxia stimulation.

[0296] Consistent with the increased mitochondrial apoptosis, the apoptosis markers Cleaved-PARP1 and Cleaved-Caspase3 showed significantly higher expression after K905R-BAF170NTm re-expression compared with WT-BAF170NTm re-expression in H9C2-shWWP2 cells ( Figure 10 Hoechst33342 assay confirmed that apoptotic cells were significantly increased in the H9C2-shBAF170 cell line under normal and H / SD conditions compared with the control group, and re-expression of K905R-BAF170 NTm further amplified this effect ( Figure 10 F-10G, 10J).

[0297] In summary, our results indicate that mutation of the lysine (K) residue at position 874 in BAF170 to arginine (R) prevents its WWP2-mediated proteasomal degradation, leading to increased mitochondrial dysfunction, elevated oxidative stress, and enhanced cardiomyocyte apoptosis.

[0298] BAF170-K874R disrupts BAF170 ubiquitination and aggravates cardiomyocyte injury

[0299] To directly demonstrate the role of BAF170-K874 in myocardial infarction, we constructed a heterozygous K874R mutant within the SWIRM domain of mouse Smarcc2 exon 28. This mutation involves a codon change from AAG (lysine) to CGC (arginine), which occurs in a functionally critical region ( Figure 5 A-5B). Compared with the wild-type BAF170 control group, the BAF170 expression level in BAF170-K874R mice was significantly increased. In addition, the expression of BAF170 was also significantly increased after myocardial infarction ( Figure 5 I-5J).

[0300] We used an established MI point mutation mouse model to investigate the role of WWP2 in BAF170 regulation. The results showed that compared with wild-type BAF170 mice, BAF170-K874R mice had significantly reduced BAF170 ubiquitination levels in cardiac tissue after MI ( Figure 5C). Importantly, BAF170-K874R mice exhibited significantly worse cardiac dysfunction following MI compared to WT-BAF170 mice, as measured by lower ejection fraction (EF%) and fractional shortening (FS%) ( Figure 5 Assessment of cardiac hypertrophy markers, including heart weight / body weight (HW / BW) and heart weight / tibia length (HW / TL) ratios, showed elevated values ​​in BAF170-K874R mice with MI compared with WT-BAF170 controls ( Figure 5 G-5H).

[0301] Endogenous ROS levels were measured simultaneously in heart tissues of WT-BAF170 and BAF170-K874R mice, along with Western blot analysis of oxidative stress markers. The markers tested included 3-Nitrotyrosine, 8-oxo-dG, SOD1, and SOD2. The results showed that endogenous ROS levels were significantly elevated in BAF170-K874R mice ( Figure 5 K), and 3-Nitrotyrosine and 8-oxo-dG levels were also increased. In contrast, the levels of SOD1 and SOD2 in these mice were lower than those in WT-BAF170 mice ( Figure 5 L-5M). Mitochondrial apoptosis was monitored by detecting the expression level of BCL2 protein. Compared with wild-type BAF170 mice, BAF170-K874R mice showed a significant decrease in BCL2 levels after myocardial infarction, indicating that their mitochondrial apoptosis was enhanced ( Figure 5 N-5O).

[0302] Analysis using the Oroboros O2K system revealed significant changes in mitochondrial function between experimental groups. Compared with the sham group, the WT-BAF170 and BAF170-K874R groups showed a decrease in complex I oxidative phosphorylation capacity, complex I and II oxidative phosphorylation capacity, and ATP production after myocardial infarction. Notably, complex II oxidative phosphorylation capacity was not affected. Compared with the WT-BAF170 group, the BAF170-K874R group had significantly lower values ​​for these three parameters ( Figure 5 P).

[0303] Electron microscopy revealed significant morphological differences between the groups. In sham-operated WT-BAF170 mice, mitochondria were neatly arranged, with dense and compact cristae, exhibiting an ordered structure. In contrast, mice in the MI group showed signs of mitochondrial dysfunction, characterized by loose, swollen, and damaged cristae, accompanied by vacuolation. These structural abnormalities were particularly pronounced in BAF170-K874R mice after MI ( Figure 5 Q).

[0304] To investigate whether cardiomyocyte apoptosis is associated with changes in the expression of apoptosis-related proteins, we compared wild-type BAF170 mice and BAF170-K874R mutant mice. The study found that the levels of cleaved-PARP1 and cleaved-Caspase3 were significantly increased in BAF170-K874R mutant mice ( Figure 5 R-5S). In addition, these mice showed more severe cardiac hypertrophy and fibrosis, as confirmed by H&E, WGA, and Masson staining tests ( Figure 5 These findings indicate that when lysine (K) at position 874 of the BAF170 protein is replaced by arginine (R), WWP2 can no longer promote BAF170 degradation through proteasomal ubiquitination. This mutation leads to exacerbated mitochondrial dysfunction, increased oxidative stress, and aggravated cardiomyocyte apoptosis.

[0305] BFH772 can significantly reduce myocardial cell damage caused by myocardial infarction

[0306] Our results suggest that the BAF170-WWP2 axis is crucial for protecting the heart from myocardial infarction (MI). To identify small molecule compounds that could enhance the binding of WWP2 to BAF170 and thus potentially mitigate the effects of MI, we screened the APE database of bioactive compounds (L-CO-020). From a pool of 30,000 small molecule compounds, we identified 10 candidate compounds with significant binding affinity to BAF170-K874 (Table 3, Figure 12 B).

[0307] These candidates underwent rigorous in vitro validation to test their effects on the interaction between WWP2 and SMARCC2 domains. We first expressed the full-length WWP2 protein in E. coli BL21 and purified it ( Figure 11 A). Subsequently, we generated and purified different domains of SMARCC2 protein (including 1-647, 1-423, 1-595, 648-1214, 424-1214, and 596-1214) by transfection in HEK293T cells ( Figure 11 B). We performed surface plasmon resonance (SPR) analysis on WWP2 immobilized on a surface plasmon resonance (SPR) chip. The results showed that the binding of BAF170 truncated peptides 1-647 and 1-595 to WWP2 was concentration-dependent, with affinity constants of 176 nM and 58.7 nM, respectively ( Figure 11C-11D). Notably, the affinity of the BAF170 truncated peptide 1-595 for WWP2 was significantly reduced compared to the 1-647 peptide, which is consistent with our binding assay results ( Figure 1 K). Other BAF170 truncated peptides showed no detectable binding to WWP2 ( Figure 11 SPR analysis consistently confirmed that WWP2 interacts with the SANT, SWIRM, and N-terminal domains of BAF170.

[0308] We performed surface plasmon resonance (SPR) experiments to evaluate 10 pre-screened small molecule compounds, aiming to find compounds that could enhance the binding of WWP2 to the BAF170 truncated peptide (1-647). Using a ternary interaction system, we immobilized WWP2 on the chip, then mixed the candidate compounds (and a blank control) with BAF170 and incubated the mixture with the chip ( Figure 12 A). Compounds 5, 6, and 7 showed enhanced signal transduction, indicating improved WWP2-BAF170 binding ( Figure 12 B). Notably, compound 6 exhibited the most robust and consistent signal enhancement, making it a prime candidate for further investigation ( Figure 12 C). This compound, named BFH772, has shown significant efficacy in inhibiting melanoma growth. BH772 is currently undergoing clinical trials for the treatment of rosacea, demonstrating promising anti-inflammatory effects. However, the potential therapeutic application of BH772 in cardiovascular disease, particularly myocardial infarction (MI), has not been fully explored.

[0309] The three-dimensional structure of the small molecule BFH772 ( Figure 12 D) A virtual screening analysis was performed that simulated the binding of BFH772 to BAF170 protein, using surface maps ( Figure 12 E) and cartoon images ( Figure 12 The interaction between BFH772 and BAF170 was visualized in 3D and 2D ( Figure 12 G-12H) revealed multiple binding points: hydrophobic interactions with residues A870, V871, K874, and A877; a hydrogen bond with K874; a π-π electronic interaction with the amide group of A873; and halogen bonds with residues A877 and E881.

[0310] To further validate our findings, we investigated the effects of BFH772 on H9C2 cells in vitro. We treated cells with different concentrations of BFH772 (0, 0.01, 0.1, 1, 10, and 100 μM) for 48 hours. The results showed that the expression of apoptosis markers Cleaved-PARP1 and Cleaved-Caspase3 gradually decreased with increasing doses. The expression of WWP2 peaked at 1 μM BFH772, while the expression of BAF170 decreased significantly at this concentration and remained stable at higher concentrations. Based on these results, we determined 1 μM as the optimal BFH772 concentration for subsequent experiments ( Figure 13 A-13B).

[0311] Subsequently, we used H9C2 cells transfected with HA-WWP2 (with or without 1 μM BFH772 treatment) to study the interaction between WWP2 and BAF170. After 48 hours, BFH772 treatment significantly enhanced the interaction between WWP2 and BAF170 and promoted the ubiquitination of BAF170 ( Figure 13 C-13D). To investigate the effects of BFH772, H9C2 cells were pretreated with the compound for 36 hours and then co-treated with H / SD for 12 hours. The results showed that compared with the control group, oxidative stress markers (3-Nitrotyrosine and 8-oxo-dG) were significantly reduced in BFH772-treated cells, while the levels of antioxidant enzymes (SOD1 and SOD2) were significantly increased ( Figure 13 E-13F). In addition, BFH772 treatment resulted in decreased expression of apoptotic markers (BAF170, Cleaved-PARP1, and Cleaved-Caspase3) and increased expression of protective proteins (WWP2 and BCL2) ( Figure 13 G-13H). These findings suggest that BFH772 exerts its cardioprotective effects through multiple mechanisms: it enhances WWP2-mediated BAF170 ubiquitination and subsequent degradation, reduces oxidative stress, and inhibits mitochondrial apoptosis. Taken together, these actions result in significant protection against cardiomyocyte apoptosis, even in vitro.

[0312] To further verify the effect of BFH772 in myocardial infarction (MI) in vivo, we established an MI mouse model by ligating the left anterior descending coronary artery for 28 days. 48 hours after surgery, different concentrations of BFH772 (20, 30, or 40 mg / kg) were administered by intraperitoneal injection ( Figure 6A). MI-induced cardiac dysfunction was ameliorated in mice treated with 30 and 40 mg / kg BFH772 compared to the DMSO-treated control group. This improvement was demonstrated by increases in ejection fraction (EF%) and fractional shortening (FS%) on days 14 and 28 ( Figure 6 B-6D). However, the 20 mg / kg dose did not show a significant therapeutic effect. In addition, only mice treated with 30 mg / kg and 40 mg / kg BFH772 showed a significant decrease in the HW / BW and HW / TL ratios ( Figure 6 E).

[0313] Analysis of oxidative stress-related proteins showed that mice treated with 30 mg / kg and 40 mg / kg BFH772 exhibited lower levels of 3-Nitrotyrosine and 8-oxo-dG, and higher levels of SOD1 and SOD2 compared with DMSO-treated mice ( Figure 6 F-6G). In addition, the expression of the mitochondrial apoptosis-related protein BCL2 was significantly increased in mice receiving 30 and 40 mg / kg BFH772, indicating improved mitochondrial function ( Figure 6 H-6I).

[0314] Using the Oroboros O2K system, we observed that mice treated with 30mg / kg and 40mg / kg BFH772 had significantly higher complex I oxidative phosphorylation capacity, complex I and complex II oxidative phosphorylation capacity, and ATP production than the DMSO-treated control group and the 20mg / kg group. Complex II oxidative phosphorylation capacity remained unchanged ( Figure 6 J). It is noteworthy that these parameters did not show statistically significant changes in the sham group at different BFH772 concentrations ( Figure 6 K).

[0315] Electron microscopy showed that the mitochondrial cristae in the sham group were dense, tight, and orderly arranged at all BFH772 doses. In contrast, the mitochondrial damage in the surgical group varied, manifested as loosening, swelling, destruction, and vacuolization of the cristae. Notably, the 30 mg / kg and 40 mg / kg treatment groups showed significant improvement compared to the DMSO group ( Figure 6 L).

[0316] The therapeutic effect of BFH772 was further confirmed by the decreased levels of apoptosis-related proteins (Cleaved-PARP1 and Cleaved-Caspase3) in mice treated at 30 mg / kg and 40 mg / kg doses. Figure 6M-6N). Histological analysis, including H&E, WGA, and Masson staining, showed that myocardial hypertrophy and fibrosis were significantly improved in these high-dose groups compared with the DMSO-treated control group and the 20 mg / kg group ( Figure 6 O-6Q).

[0317] Our results suggest that BFH772 enhances the binding of WWP2 to BAF170, promotes BAF170 ubiquitination, and inhibits oxidative stress and mitochondrial apoptosis. These mechanisms contribute to the alleviation of myocardial remodeling after myocardial infarction, thereby effectively alleviating myocardial infarction symptoms.

[0318] BAF170-K874R inhibits BCL2 transcription and promotes Caspase3 transcription by binding to enhancers

[0319] Total CUT&Tag analysis using BAF170 antibodies showed significant peak enrichment in hearts from both BAF170-K874R and WT-BAF170 mice ( Figure 9 A). Differential analysis showed that compared with the wild-type control group, 1,281 gene fragments were up-regulated and 820 gene fragments were down-regulated in the K874R mutant group ( Figure 9 B). To elucidate the role of BAF170 ubiquitination, we performed KEGG pathway analysis on the differentially expressed genes. Notably, several key apoptosis regulators appeared in multiple signaling pathways—Caspase3 appeared in the MAPK signaling pathway and the apoptosis pathway, while BCL2 appeared in the PI3K-Akt pathway ( Figure 9 C). These findings suggest that BAF170 ubiquitination influences the apoptotic program by regulating the transcription of target genes. Indeed, ChIP-seq peak analysis revealed that BAF170 has direct binding sites at the regulatory regions of BCL2 and Caspase3. Specifically, the K874R mutation enhances BAF170 binding at the Caspase3 enhancer while reducing binding at the BCL2 enhancer, leading to increased and decreased transcription of these genes, respectively ( Figure 9 D-9E).

[0320] Table 4. Mass spectrometry results of proteins with reduced binding to BAF170 in heart tissues of mice with heart failure after myocardial infarction

[0321]

[0322] 4. Conclusion

[0323] Using high-resolution proteomics, we discovered that the SWI / SNF chromatin remodeling complex family, specifically the BAF170 subunit, plays a key role in myocardial infarction. We identified WWP2 as the physiological E3 ubiquitin ligase responsible for regulating BAF170 through polyubiquitination at K874, triggering BAF170 degradation. Notably, inhibition of this polyubiquitination process, either through point mutation (BAF170-K874R) or WWP2 knockout, significantly increased BAF170 expression and exacerbated cardiomyocyte injury after myocardial infarction. Using CUT&Tag experiments, we identified BCL2 and Cleaved-Caspase3 as novel downstream apoptotic proteins regulated by BAF170. Our studies reveal a novel model in which ubiquitination of BAF170, a key component of the SWI / SNF chromatin remodeling complex, plays a key role in coordinating mitochondrial function, oxidative stress responses, and apoptotic signaling pathways. Furthermore, these findings shed light on how chromatin remodeling mechanisms influence the development of cardiovascular health and disease.

[0324] This study provides novel mechanistic insights by linking the regulation of BAF170, a component of the SWI / SNF chromatin remodeling complex, to sustained cardiomyocyte apoptosis induced by myocardial infarction (MI). By demonstrating a role for WWP2-mediated polyubiquitination in BAF170 degradation, we introduce a new axis by which oxidative stress and mitochondrial dysfunction cooperate to sustain cardiomyocyte apoptosis. Our findings not only reveal the molecular pathways involved but also suggest potential therapeutic targets to mitigate sustained cardiomyocyte apoptosis, providing a promising strategy to combat the progression of heart failure.

[0325] Our study found that BAF170 expression is significantly increased during cardiac remodeling after myocardial infarction. This increase is associated with three negative effects: increased mitochondrial dysfunction, elevated oxidative stress, and enhanced cardiomyocyte apoptosis. These findings represent a significant advance in our understanding of the role of BAF170 in cardiovascular disease.

[0326] This study further confirmed that WWP2, as a physiological E3 ubiquitin ligase, is responsible for the polyubiquitination and subsequent degradation of BAF170-K874. The study found that during cardiac remodeling after myocardial infarction, WWP2-mediated BAF170-K874 polyubiquitination and degradation is impaired, leading to BAF170 accumulation. This accumulation triggers a series of adverse consequences, including increased mitochondrial dysfunction, oxidative stress, and increased cardiomyocyte apoptosis. These findings reveal the mechanistic role of ubiquitination in chromatin remodeling and the progression of cardiovascular disease.

[0327] By elucidating the relationship between chromatin dynamics and apoptosis, our findings open new avenues for studying the potential role of chromatin remodeling in other cell death pathways, particularly in terminally differentiated cells. This insight significantly advances our understanding of the mechanisms regulating cell death and provides guidance for future research in this area.

[0328] Encouragingly, through extensive screening of small molecule compounds, we discovered that BFH772, known for its remarkable efficacy in cancer treatment, significantly mitigates cardiomyocyte damage caused by heart failure after myocardial infarction. This finding effectively improved mitochondrial dysfunction, reduced oxidative stress, alleviated cardiomyocyte apoptosis, and ultimately reduced fibrosis in myocardial infarction and heart failure. Our study highlights the importance of the WWP2-BAF170 axis in protecting cardiomyocytes from damage after myocardial infarction and heart failure.

[0329] In summary, we have discovered a novel ubiquitination-dependent mechanism that regulates chromatin remodeling after myocardial infarction. Our studies suggest that targeting BAF170-K874 ubiquitination could be a promising strategy for treating cardiac remodeling after myocardial infarction. Furthermore, we identified a promising small molecule, BFH772, which provides a solid foundation for the development of targeted therapies for cardiac remodeling after myocardial infarction. This discovery opens new avenues for therapeutic intervention and warrants further investigation in preclinical and clinical settings.

Claims

1. Use of a compound that regulates BAF170 protein in the preparation of a medicament for preventing, alleviating and / or treating cardiovascular diseases.

2. The use according to claim 1, wherein The compound that regulates BAF170 protein is a compound that degrades BAF170 protein.

3. The use according to claim 1, wherein The compound that degrades BAF170 protein is a compound that promotes WWP2 to ubiquitinate BAF170 at the K874 site.

4. The use according to claim 1, wherein The compound that degrades BAF170 protein is a WWP2 protein agonist.

5. The use according to claim 1, wherein The compound that degrades BAF170 protein is BFH772, and its structural formula is as follows:

6. The use according to claim 1, wherein The cardiovascular disease is selected from the group consisting of coronary artery disease, coronary heart disease, and myocardial infarction.

7. Use of BFH772 in the preparation of a medicament for preventing, alleviating and / or treating cardiovascular diseases.

8. The use according to claim 7, wherein The cardiovascular disease is selected from the group consisting of coronary artery disease, coronary heart disease, and myocardial infarction.

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