CN120818506A - BAF170 mutants and pharmaceutical uses thereof - Google Patents

Abstract

The invention relates to BAF170 mutant and pharmaceutical application thereof. Compared with the wild BAF170, the BAF170 mutant is a K874 locus mutation, and the amino acid sequence of the BAF170 mutant is shown as SEQ ID NO. 1. The invention promotes degradation of WWP2 by promoting ubiquitination of BAF170 at K874 site, thereby protecting myocardial cells from oxidative stress injury.

Description

BAF170 mutant and pharmaceutical application thereof

Technical Field

The invention relates to BAF170 mutant and pharmaceutical application thereof.

Background

Cardiovascular disease, and especially Coronary Artery Disease (CAD), remains a major health challenge worldwide according to the global disease burden study in 2019. Coronary Heart Disease (CHD) is a clinical syndrome associated with the formation of arterial intimal plaque, which progressively leads to vascular stenosis and ultimately may lead to vascular occlusion. Death statistics in 2019 show about 910 ten thousand people worldwide are going to be present each year due to coronary heart disease. Clinically, coronary heart disease is mainly manifested in two forms, myocardial Infarction (MI) and ischemic cardiomyopathy. Long-term coronary occlusion can lead to irreversible ischemic damage to the heart, which in turn can lead to myocardial infarction. In 2021, myocardial infarction resulted in 300 thousands of deaths. In the border zone of the myocardial infarction area, apoptosis is significantly increased, which has become a key factor in the development of chronic ischemic heart disease into heart failure.

According to China 2021 edition, chest pain center guide, the reference standard for door-to-guide wire (D-to-W) time is 90 minutes. If no effective pharmacological intervention is performed in time during this period, this can lead to continued death of the cardiomyocytes. More complicated, many patients arrive at the hospital after a treatment window period (i.e., 12 hours of gold), requiring stent implantation one week later. During this time, cardiomyocytes continued to die. Although the pathogenesis of myocardial apoptosis is well understood, the specific mechanism of myocardial apoptosis is still unknown in chronic ischemic heart disease. Therefore, it is particularly important to deeply study the mechanism of myocardial apoptosis to delay its progression and develop effective targeted therapeutic drugs.

Early stages of Myocardial Infarction (MI) lead to the accumulation of oxidative stress (ROS) and further to the initiation of cardiomyocyte death. Both apoptosis and necrosis cause myocardial damage due to coronary artery occlusion, but apoptosis is the primary mechanism. Mitochondrial apoptosis pathways play a key role in the process of myocardial apoptosis. Mitochondria occupy approximately one third of the volume of cardiomyocytes, providing 95% of the energy required for heart activity by oxidative phosphorylation. However, excessive ROS bursts damage mitochondria, affect respiratory function, disrupt oxidative phosphorylation processes, exacerbate mitochondrial damage. As the outer mitochondrial membrane becomes permeable, pro-apoptotic proteins are released, directly damaging heart tissue, inducing apoptosis in cardiomyocytes. This ultimately leads to cardiac fibrosis, dysfunction, and ultimately heart failure. Although the pathogenesis of myocardial apoptosis is well understood, further studies on how to reduce myocardial apoptosis are critical to develop targeted therapeutic interventions, preventing cardiac remodeling following myocardial infarction, and ultimately improving patient prognosis and survival.

At the molecular level, BAF170 is a key factor in cardiac development. It is expressed mainly in cardiac muscle cells and is critical for differentiation of the heart and gene expression at a specific stage. BAF170 deficiency can impede cardiomyocyte differentiation and delay the first heartbeat. Although BAF170 plays an important role in cardiomyocyte differentiation and cardiac development, the pathophysiology of ischemic heart disease is still unclear.

Disclosure of Invention

The inventors of the present application have found through intensive studies that BAF170 is closely related to myocardial cell death and may play an important role in cardiovascular diseases such as myocardial infarction. WWP2 is a ubiquitinated E3 ligase of BAF170, which promotes its degradation by ubiquitinating BAF170 at the K874 site, thereby protecting cardiomyocytes from oxidative stress injury. BAF170 expression increased upon myocardial infarction, but WWP2 binding thereto decreased. Loss of WWP2 exacerbates cardiomyocyte injury, while overexpression reduces injury.

The invention is realized by the following technical scheme.

In one aspect, the invention provides a BAF170 mutant, compared with wild BAF170, the BAF170 mutant is K874 locus mutation, and the amino acid sequence of the BAF170 mutant is shown as SEQ ID NO. 1.

SEQ ID NO:1

MAVRKKDGGPNVKYYEAADTVTQFDNVRLWLGKNYKKYIQAEPPTNKSLSSLVVQLLQFQEEVFGKHVSNAPLTKLPIKCFLDFKAGGSLCHILAAAYKFKSDQGWRRYDFQNPSRMDRNVEMFMTIEKSLVQNNCLSRPNIFLCPEIEPKLLGKLKDIIKRHQGTVTEDKNNASHVVYPVPGNLEEEEWVRPVMKRDKQVLLHWGYYPDSYDTWIPASEIEASVEDAPTPEKPRKVHAKWILDTDTFNEWMNEEDYEVNDDKNPVSRRKKISAKTLTDEVNSPDSDRRDKKGGNYKKRKRSPSPSPTPEAKKKNAKKGPSTPYTKSKRGHREEEQEDLTKDMDEPSPVPNVEEVTLPKTVNTKKDSESAPVKGGTMTDLDEQEDESMETTGKDEDENSTGNKGEQTKNPDLHEDNVTEQTHHIIIPSYAAWFDYNSVHAIERRALPEFFNGKNKSKTPEIYLAYRNFMIDTYRLNPQEYLTSTACRRNLAGDVCAIMRVHAFLEQWGLINYQVDAESRPTPMGPPPTSHFHVLADTPSGLVPLQPKTPQQTSASQQMLNFPDKGKEKPTDMQNFGLRTDMYTKKNVPSKSKAAASATREWTEQETLLLLEALEMYKDDWNKVSEHVGSRTQDECILHFLRLPIEDPYLEDSEASLGPLAYQPIPFSQSGNPVMSTVAFLASVVDPRVASAAAKSALEEFSKMKEEVPTALVEAHVRKVEEAAKVTGKADPAFGLESSGIAGTTSDEPERIEESGNDEARVEGQATDEKKEPKEPREGGGAIEEEAKEKTSEAPKKDEEKGKEGDSEKESEKSDGDPIVDPEKEKEPKEGQEEVLKEVVESEGERKTKVERDIGEGNLSTAAAAALAAAAVKARHLAAVEERKIKSLVALLVETQMKKLEIKLRHFEELETIMDREREALEYQRQQLLADRQAFHMEQLKYAEMRARQQHFQQMHQQQQQPPPALPPGSQPIPPTGAAGPPAVHGLAVAPASVVPAPAGSGAPPGSLGPSEQIGQAGSTAGPQQQQPAGAPQPGAVPPGVPPPGPHGPSPFPNQQTPPSMMPGAVPGSGHPGVAGNAPLGLPFGMPPPPPPPAPSIIPFGSLADSISINLPAPPNLHGHHHHLPFAPGTLPPPNLPVSMANPLHPNLPATTTMPSSLPLGPGLGSAAAQSPAIVAAVQGNLLPSASPLPDPGTPLPPDPTAPSPGTVTPVPPPQ

In another aspect, the invention provides an isolated nucleic acid molecule encoding the BAF170 mutant.

In yet another aspect, the invention provides a vector comprising said isolated nucleic acid molecule.

In yet another aspect, the invention provides a host cell, wherein said host cell comprises said vector.

In yet another aspect, the invention provides a pharmaceutical composition comprising said BAF170 mutant.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable diluent, excipient and/or carrier.

In yet another aspect, the invention provides the use of a BAF170 mutant or said pharmaceutical composition in the manufacture of a medicament for the prevention, alleviation and/or treatment of a cardiovascular disease.

Preferably, the cardiovascular disease is selected from coronary artery disease, coronary heart disease, myocardial infarction.

Compared with the prior art, the application has at least the following beneficial technical effects:

The application demonstrates for the first time that BAF170 mutants can prevent, alleviate and/or treat myocardial infarction.

The application combines proteomics, ubiquitination modification histology with WWP2 myocardial specificity knockout, WWP2 myocardial specificity over-expression mice and BAF170-K874 locus mutation cell line combined with BAF170-K874 locus mutation cell line for the first time, and proves that WWP2 can promote the ubiquitination of BAF170-K874 locus and degrade BAF170 on the cellular, molecular and animal level, thereby relieving myocardial infarction.

Drawings

Fig. 1 shows that BAF170 expression increases significantly during MI.

A volcanic plot compares changes in protein expression in mouse hearts under control and MI conditions, highlighting significant fold changes and P-values.

And B, comparing the differentially expressed proteins between the control group and the MI group through gene ontology enrichment analysis. Statistical significance was determined by Fisher's exact test (-log 10P values).

C, heat map visualization compares SWI/SNF chromatin remodeling complex expression in heart tissue of control and MI mice.

D, westernblot analysis evaluates the expression of BAF170, cleaved-PARP1 and Cleaved-Caspase3 proteins in H9C2 cells after various time points (0-24H) of hypoxia/serum deprivation (H/SD) treatment. (n=3 independent experiments).

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

F, westernblotting analysis evaluates the expression levels of WWP2 and H/SD at different time points (0-24H) in H9C2 cell lines. (n=3 independent experiments).

G, immunoprecipitation of cell lysates with anti-BAF 170 antibody, followed by Westernblot analysis using anti-WWP 2 antibody.

H, immunoprecipitation of cell lysates with anti-WWP 2 antibody, followed by Westernblot analysis using anti-BAF 170 antibody.

I, direct interactions between exogenous WWP2 and endogenous BAF170 in cell lysates were detected by HA-tagged WWP2 overexpression and immunoprecipitation experiments.

J, BAF170 comprises four functional domains including the N-terminal, C-terminal, SWIRM and SANT domains.

K, full-length or truncated BAF170 plasmid with Flag tag was transfected into HEK293T cells. Subsequently, the cell lysates were immunoprecipitated using anti-Flag antibodies and Western blot analysis was performed by anti-WWP 2 antibodies.

D. e, F three independent experiments were performed. The data is considered normally distributed according to the central limit theorem. The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance was assessed by one-way anova using Tukey multiple comparison test (D, F, P values were adjusted by 15 comparisons; E, P values were adjusted by 6 comparisons). H/SD indicates hypoxia and serum deprivation, IB, immunoblotting.

Figure 2WWP2 regulates BAF170 ubiquitination at the K874 site and promotes its degradation.

A. BAF170 and WWP2 expression levels in H9C2 cells were analyzed under four conditions, normal control and treatment with WWP2 short hairpin RNA variants (shWWP 274451, 74452 and 74453). (independent experiments with n=3).

B. Westernblot analysis to assess BAF170 expression in HEK293T and H9C2 cells following dose-dependent overexpression of the HA tag WWP 2. (independent experiments with n=3).

C and G, western blot analysis to assess BAF170 protein levels in control cells and shWWP cells at different time points after cycloheximide treatment. (independent experiments with n=3).

D and H, western blot analysis to assess the expression level of BAF170 protein in control cells and cells overexpressing HA-tagged WWP2 at different time points after cycloheximide treatment. (independent experiments with n=3).

E and I, western blot analysis to assess the expression levels of BAF170 in normal control cells treated with MG132 and shWWP2 cells at different time points. (independent experiments with n=3).

F and J, western blot analysis to assess the expression levels of BAF170 in normal control cells and cells expressing the HA marker WWP2 treated with MG132 at different time points. (independent experiments with n=3).

K. BAF170 ubiquitination levels were analyzed by co-immunoprecipitation using anti-HA antibodies after co-transfection of HA-tagged WWP2 or HA-tagged vector controls with HA ubiquitin (HA-Ub) in the presence or absence of the proteasome inhibitor MG 132.

After overexpression of HA-Ub in control cells and shWWP cells, BAF170 ubiquitination was analyzed by co-immunoprecipitation with anti-HA antibodies, whether or not MG132 treatment was performed.

M, workflow diagram illustrating proteomics and ubiquitination analysis in WWP2 cKO, WWP2-TG and WT mouse MI (7 per group) heart tissue.

Nine quadrant plots of proteomics and ubiquitination modified histology in heart tissue of N, WWP knockout (WWP 2 cKO) and transgenic (WWP 2-TG) mice versus wild type (WWP 2-WT) control mice.

O, WWP2 proteomic Muffz analysis of 2 cKO, WWP2-WT, WWP 2-TG.

Ubiquitin omics Muffz analysis of P, WWP2 cKO, WWP2-WT and WWP 2-TG.

And Q, WWP biological function enrichment analysis of the transgene and the upregulated ubiquitination site and protein in the wild type tissue sample.

R, WWP2 gene knockout and downregulated ubiquitination sites in wild type tissue samples and analysis of the biofunctionality enrichment of proteins.

U-W, H9C2 cells were transfected with Flag-BAF170 variants (WT, K874R, K694R or K704R) and HA-Ub or HA-WWP 2. Immunoprecipitation was performed with anti-Flag antibody, followed by western blotting with anti-HA antibody.

A. B, G-J, three independent experiments were performed. The data is considered normally distributed according to the central limit theorem. The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance was assessed by one-way anova using Tukey multiple comparison test (A and B, P values were adjusted by 6 comparisons), unpaired t test (2-measured Student t test; G-J). Ub represents ubiquitin antibody, CHX represents cycloheximide, IB, immunoblotting. WWP2 cKO Myh6 Cre+; wwp f/f, WWP2-WT Wwp f/f, WWP2-TG R26-LSL-WWP2+/+; myh6 Creer.

FIG. 3 myocardial-specific WWP2 knockout mice show increased BAF170 expression and MI-induced myocardial cell damage is markedly exacerbated.

A. shows the construction method of the myocardial specific WWP2 knockout (Myh 6-cre+; wwp f/f) mouse.

B. MI mouse model construction schematic. Proteins extracted from control (Wwp f/f) and heart-specific knockdown (Myh 6-cre+; wwp f/f) 28 day MI hearts.

C. total lysates of heart tissue were Immunoprecipitated (IP) with anti-BAF 170 antibody and Western blotted with anti-WWP 2 antibody.

D. total lysates of heart tissue were IP-blotted with anti-BAF 170 antibodies and Western blotted with anti-ubiquitinated (Ub) antibodies in Myh6-cre+; wwp f/f mice.

E-G Wwp f/fand Myh6-cre+; EF% and FS% in Wwp f/f mice.

HW/BW and HW/TL of H-I, wwp2f/fandMyh6-cre+; wwp f/f mice.

Western blot analysis of J-K, WWP2 expression levels.

L, heart tissue was stained using DHE (ethidium dihydrogen) assay kit (red). Scale bar = 20 μm.

M-N, western blot analysis to assess the expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1) and SOD2 (superoxide dismutase 2).

Western blot analysis of O-P, BCL2 expression levels.

The Q, oroboros O K system was used to evaluate mitochondrial oxidative phosphorylation component activity.

R, representative image of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

S-T, westernblot was analyzed to assess the expression levels of the apoptosis markers Cleaved-PARP1 and Cleaved-Aspase 3.

U-W, hematoxylin, eosin (H & E), wheat Germ Agglutinin (WGA) and Masson staining assays to assess the extent of myocardial hypertrophy and fibrosis. Above, scale = 800 μm, below scale = 20 μm.

F-N, P-W, for data with normal and equal variance, two-way anova with Bonferroni test was used to compare groups. If the normal or homogeneity of variance is not met, the treatment differences are assessed by the Kruskal-Wallis test and Dunn multiple comparison test (n=7 mice per group). The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance was assessed by two-way anova using Bonferroni multiple comparison test (6 comparisons of H-I, K, L, N, P, Q, T, V, W values), kruskal-Wallis and Dunn multiple comparison test.

FIG. 4WWP2 overexpression resulted in decreased BAF170 expression and decreased myocardial cell damage induced by myocardial infarction.

A. A method for constructing a Rosa26-WwP2-Flag (WWP 2-TG, R26-LSL-Wwp 2++; myh 6-Creer) mouse is shown.

B. MI mouse model construction schematic. Protein samples were taken from wild-type (WWP 2-WT, R26-LSL-WWP2+/+; myh 6-Creer-) and WWP2-TG mouse hearts 28 days post-infarct.

C. Total lysates of cardiac tissue from WWP2-TG mice and WWP2-WT mice were Immunoprecipitated (IP) with anti-BAF 170 antibody and Western blotted with anti-WWP 2 antibody.

D. Total lysates of cardiac tissue from WWP2-TG mice and WWP2-WT mice were IP treated with anti-BAF 170 antibody and Western blots with anti-ubiquitinated (Ub) antibody.

EF% and FS% in E-G, WWP2-TG mice and WWP2-WT mice.

HW/BW and HW/TL of H-I, WWP2-TG mice and WWP2-WT mice.

Western blot analysis of J-K, WWP2 expression levels.

L, heart tissue was stained using DHE (ethidium dihydrogen) assay kit (red). Scale bar = 20 μm.

M-N, western blot analysis to assess the expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1) and SOD2 (superoxide dismutase 2).

Western blot analysis of O-P, BCL2 expression levels.

The Q, oroboros O K system was used to evaluate mitochondrial oxidative phosphorylation component activity.

R, representative image of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

S-T, westernblot was analyzed to assess the expression levels of Cleaved-PARP1 and Cleaved-Aspase.

U-W, hematoxylin, eosin (H & E), wheat Germ Agglutinin (WGA) and Masson staining assays to assess the extent of myocardial hypertrophy and fibrosis. Above, scale = 800 μm, below scale = 20 μm.

F-N, P-W, for data with normal and equal variance, two-way anova with Bonferroni test was used to compare groups. If the normal or homogeneity of variance is not met, the treatment differences are assessed by the Kruskal-Wallis test and Dunn multiple comparison test (n=7 mice per group). The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance (H-I, K, L, N, P, Q, T, V, W, values after 6 comparisons) was assessed using two-way anova with Bonferroni multiple comparison test, kruskal-Wallis with Dunn multiple comparison test.

FIG. 5BAF170-K874R disrupted ubiquitination of BAF170, significantly exacerbating MI-induced myocardial cell injury.

A. schematic of the construction method of K874R heterozygous mutant mice. Protein samples were extracted from wild-type (WT) and K874R heterozygous mutant mouse hearts 28 days post-infarct.

B. A diagram showing the K874R mutation site and N-terminal, SWIRM, SANT and C-terminal functional domains is shown.

C. in BAF170-K874R mice, heart tissue lysates were immunoprecipitated using anti-BAF 170 antibodies and western immunoblotted with anti-ubiquitin (Ub) antibodies.

EF% and FS% in D-F, WT and BAF170-K874R mice.

HW/BW and HW/TL of G-H, WT and BAF170-K874R mice.

Western blot analysis of I-J, BAF170 expression levels

K. Heart tissue was stained using DHE (ethidium bromide) detection kit (red). Scale bar = 20 μm.

L-M, western blot analysis to assess the expression levels of 33-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1) and SOD2 (superoxide dismutase 2).

Western blot analysis of N-O, BCL2 expression levels.

P, activity of mitochondrial oxidative phosphorylation components was assessed using Oroboros O K system.

Q, representative image of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

R-S, western blot analysis to assess expression levels of Cleaved-PARP1 and Cleaved-Aspase 3.

T-V, hematoxylin, eosin (H & E), wheat Germ Agglutinin (WGA) and Masson staining assays to assess the extent of myocardial hypertrophy and fibrosis. Above, scale = 800 μm, below scale = 20 μm.

E-F, O-V, for normal distribution and isovariational data, two-way anova with Bonferroni test was used to compare groups. If the normal or homogeneity of variance is not met, the treatment differences are assessed by the Kruskal-Wallis test and Dunn multiple comparison test (n=7 mice per group). The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance (G-H, J, K, M, O, P, S, U, V, 6 adjusted values) was assessed using a two-way anova with Bonferroni multiple comparison test; kruskal-Wallis with Dunn multiple comparison test.

Figure 6.Bfh772 significantly reduced myocardial cell injury induced by myocardial infarction.

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

B-D, EF% and FS% in mice treated with various concentrations of BFH772 (including 0mg/kg, 20mg/kg, 30mg/kg, 40 mg/kg).

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

F-G, western blot analysis to assess the expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1) and SOD2 (superoxide dismutase 2).

Western blot analysis of H-I, BCL2 expression levels.

The J-K, oroboros O K system was used to evaluate mitochondrial oxidative phosphorylation component activity.

L, representative image of cardiomyocyte mitochondria captured by transmission electron microscopy. Red arrows indicate mitochondria. Scale bar = 1 μm.

M-N, western blot analysis to assess expression levels of Cleaved-PARP1 and Cleaved-Aspase 3.

O-Q, hematoxylin, eosin (H & E), wheat Germ Agglutinin (WGA) and Masson staining were used to detect myocardial hypertrophy and fibrosis. Above, scale = 800 μm, below scale = 20 μm.

C-E, G, I-K, N, P-Q. For data with normalization and isovariabilities, the groups were compared using a one-way anova with Tukey multiple comparison test (n=7 mice per group). The quantitative data are mean ± standard deviation. Statistical significance was assessed by one-way anova with Tukey multiple comparison test (C-E, J-K, P-Q,6 comparison of adjusted values; G, I, N,15 comparison of adjusted values).

Figure 7 quality control results of wwp2 ubiquitin proteomics analysis.

A. principal Component Analysis (PCA) shows the first two principal components of protein intensity, with samples connected by centroid, depending on sample type.

B. Pearson correlation analysis, each value representing a correlation coefficient between two samples.

C. tolerance profile of peptide mass

D. A box plot based on Relative Standard Deviation (RSD), each point represents one RSD value.

E. And (5) a mass spectrum result histogram.

F. Protein molecular weight statistics, each bar height represents the number of proteins.

G. density profile.

H. intensity value distribution bar graph.

I. Box plots based on intensity values (WWP 2-cKO: myh6-Cre+; wwp f/f; WWP2-WT: wwp f/f; WWP2-TG: R26-LSL-Wwp2+/+; myh 6-Creer).

Figure 8 wwp2 alleviates ischemia and hypoxia induced cardiomyocyte injury.

A. Lysates of H9C2-shWWP cell lines treated with H/SD or without H/SD for 12 hours were IP treated with anti-BAF 170 antibodies and Western blots with anti-WWP 2 antibodies.

B. Lysates of H9C2-shWWP cell lines treated with H/SD or without H/SD for 12 hours were IP treated with anti-BAF 170 antibodies and Western blots with anti-ubiquitinated (Ub) antibodies.

C. Cell lysates over-expressing HA-tagged WWP2 treated with H/SD or without H/SD for 12 hours were IP treated with anti-BAF 170 antibody and Western blots with anti-WWP 2 antibody.

D. Cell lysates over-expressing HA-tagged WWP2 treated with H/SD or without H/SD for 12 hours were IP treated with anti-BAF 170 antibodies and Western blots with anti-Ub antibodies.

E and F, HA-WWP2 NTm re-expression in H9C2-shWWP2 cell lines after 12 hours of treatment with or without H/SD, western blot analysis of expression levels of 3-Nitrotyrosine, 8-oxo-dG, SOD1 (superoxide dismutase 1) and SOD2 (superoxide dismutase 2) (independent experiment with n=3).

G-K, mitochondrial ROS levels were assessed using Mitosox Red and CellROX Green staining in H9C2-shWWP2 cells expressing HA-WWP2-NTm after 12 hours of treatment with or without H/SD, while apoptotic cells were quantified using Hoechst33342 (blue). (independent experiments with n=3).

L and M immunoblot analysis of BCL2 protein expression was performed in H9C2-shWWP cells transfected with HA-WWP2 NTm, with or without 12 hours of hypoxia-deficient serum (H/SD) treatment. (independent experiments with n=3).

N and O, JC-1 staining to assess changes in mitochondrial membrane potential in H9C2-shWWP2 cells expressing HA-WWP2-NTm after 12 hours of H/SD treatment and without. (independent experiments with n=3).

Western blot analysis of Cleaved-PARP1 and Cleaved-Aspase expression in H9C2-shWWP2 cells after P and Q, HA-WWP2 NTm re-expression, 12 hours with and without H/SD treatment. (independent experiments with n=3).

F. I-K, M, O, Q three independent experiments were performed. The data is considered normally distributed according to the central limit theorem. The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance was assessed using one-way anova with Tukey multiple comparison test (F, O, Q, 3 comparisons of P values) and two-way anova with Bonferroni multiple comparison test (M, I-K, 15 comparisons of P values). NC, normal control group, no treatment, H/SD, oxygen glucose deprivation, IB, immunoblotting.

FIG. 9.BAF170-K874R inhibits BCL2 transcription and promotes Casp3 transcription by binding to an enhancer.

A. BAF170 binding density was analyzed using a depth tool to generate a heat map showing the distribution of CUT and Tag tags over the binding peaks. Samples of WT and K874R heterozygous mutant mice were compared in microglial and total heart tissue, with peaks arranged by signal intensity.

B. Statistical analysis of differentially expressed genes.

C. Statistical analysis of differential Gene distribution in KEGG pathway

Visualization of D and E, BCL2 and Casp3 genomic loci CUT and Tag signal trajectories, revealed an enrichment pattern.

FIG. 10 BAF170-K905R exacerbates ischemia hypoxia-induced cardiomyocyte injury

A. Alignment of sequences around K874 in BAF170 homologs from different species. Ubiquitin lysine residues at BAF170-K874 are highlighted (bold and red).

B and C, BAF170 expression levels were assessed in HEK293T cells using western blot analysis after treatment with three different short hairpin RNA constructs (shBAF 170121254, 121255 and 121256) compared to the normal control group. (independent experiments with n=3).

D and E, western blot analysis to assess expression of oxidative stress markers (3-Nitrotyrosine, 8-oxo-dG) and antioxidant enzymes (SOD 1, SOD 2) in H9C2-shBAF170 cells expressing WT-BAF170 NTm or K905R-BAF170 NTm with or without 12 hours of H/SD treatment. (independent experiments with n=3).

F-J, mitochondrial ROS levels were assessed using Mitosox Red and CellROX Green staining, whereas apoptotic cells were quantified using Hoechst33342 (blue) after re-expression of WT-BAF170 NTm or K905R-BAF170 NT in H9C2-shBAF170 cells, whether or not H/SD treated for 12 hours. (independent experiments with n=3).

K and L, western blot analysis to assess BCL2 expression in H9C2-shBAF170 cells with and without 12 hours H/SD treatment after reintroduction of either WT-BAF170 NTm or K905R-BAF170 NTm. (independent experiments with n=3).

M and N, JC-1 staining to assess changes in mitochondrial membrane potential in H9C2-shBAF170 cells expressing WT-BAF170 NTm or K905R-BAF170 NTm in hypoxia/serum deprivation (H/SD) therapy and in the absence of hypoxia/serum blockage (H/SD, 12 hours). (independent experiments with n=3).

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 12H H/SD treatment. (independent experiments with n=3).

C. E, H-J, L, N, P, three independent experiments were performed. The data is considered normally distributed according to the central limit theorem. The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance was assessed by one-way anova with Tukey multiple comparison test (C, P values adjusted by 6 comparisons; E, N, P values adjusted by 3 comparisons), and by two-way anova with Bonferroni multiple comparison test (H-J, L, P values adjusted by 15 comparisons). H/SD indicates hypoxia ischemia, IB, immunoblotting, NS has no statistical significance.

FIG. 11 purification of WWP2 protein and truncated BAF170 peptide fragment

A. the sensor pattern shows the concentration-dependent binding kinetics of BAF170 truncation mutants (1-647) to immobilized WWP2 protein in the range of 7.8-125 nM.

B. The sensor pattern shows binding kinetics between BAF170 truncations (1-595) and immobilized WWP2, measured in the 15.63-250nM range.

C-F, sensor maps of binding of BAF170 peptide fragments (1-423, 424-1214, 569-1214, and 648-1214) to WWP2 protein. Peptide concentrations ranged from 0.06 to 1 μm for evaluation of binding interactions by microarray.

Fig. 12. Virtual screening involving small molecule compound BFH 772.

A. surface Plasmon Resonance (SPR) analysis to determine the binding affinity between ten candidate small molecules and BAF170-WWP2 immobilized on the chip surface.

B. WWP2 is fixed on the chip surface. SPR was used to assess the binding interactions of BAF170 and WWP2 in the presence of 10 candidate small molecule compounds or controls.

C. SPR analysis to assess interactions between immobilized WWP2 and BAF170 in the presence or absence of the small molecule inhibitor BFH 772. The molecular structure of BFH772 is shown.

D. three-dimensional structure of small molecule BFH 772.

E. the binding of BFH772 small molecules to BAF170 protein was visualized as a surface representation. The key interactions are highlighted in red, showing the interaction of residue K874 (yellow) with BFH772 (cyan).

F. the binding state of BFH772 to BAF170 is shown.

G. three-dimensional interaction map of BFH772 and BAF 170.

H. two-dimensional interaction diagram of BFH772 and BAF 170.

Fig. 13. Small molecule compound BFH772 reduces ischemia and hypoxia induced cardiomyocyte injury.

A-B, western blot analysis to assess protein expression levels of BAF170, WWP2 and apoptosis markers (Cleaved-PARP 1 and Cleaved-Aspase) after 48 hours exposure to BFH772 (0-100. Mu.M). (independent experiments with n=3).

C. the WWP2 overexpression of the HA-tag was 48h after induction with or without 1 μm BFH 772. Lysates were immunoprecipitated with anti-HA antibodies and analyzed by western blot using anti-BAF 170.

D. cells were co-transfected with HA-tagged WWP2 or vector control and HA-Ub, followed by induction for 48h with or without 1 μm BFH 772. BAF170 ubiquitination was assessed by anti-HA immunoprecipitation.

E-F, H C2 cells were pretreated with BFH772 (36 hours) and then H/SD co-treated (12 hours). Western blot analysis was performed to assess the expression of 3-Nitrotyrosine, 8-oxo-dG and SOD 1. (independent experiments with n=3).

G-H, H C2 cardiomyocytes were treated with BFH772 for 36 hours and then H/SD for 12 hours. Western blot analysis was performed to assess protein levels of BAF170, WWP2, BCL2 and apoptosis markers (Cleaved-PARP 1, cleaved-Aspase 3) (independent experiments with n=3).

B. F, H three independent experiments were performed. The data is considered normally distributed according to the central limit theorem. The relative protein levels were calculated as fold changes compared to the first group. The quantitative data are mean ± standard deviation. Statistical significance was assessed by one-way anova using Tukey multiple comparison test (15 comparisons of B, P values) and two-way anova using Bonferroni test (F, H, 6 comparisons of P values). Ub represents ubiquitin antibody, IB, immunoblotting.

Detailed Description

The technical scheme of the invention is further described below with reference to the specific embodiments.

The non-standard abbreviations and acronyms used in the present invention are as follows:

8-oxo-dG 8-oxo-dG

SOD1 superoxide dismutase 1

SOD2 superoxide dismutase 2

H & E hematoxylin, eosin

WGA wheat germ lectin

CM myocardial cells

Congenital heart disease of CHD

CHX cycloheximide

H/SD hypoxia and serum deprivation

Reactive Oxygen Species (ROS)

EF% ejection fraction

FS% reduction score

HW/BW heart weight/body weight

HW/TL heart weight/tibia length

TEM transmission electron microscope

Parp1 poly ADP ribose polymerase family member 1

Examples

1. Raw materials

Antibodies and reagents used in the following methods are shown in table 1 below:

TABLE 1 antibodies and reagents

2. Experimental method

Proteomics and general proteomics of biotinylation

Protein extraction

The samples were ground with liquid nitrogen to cell powder and then transferred to a 5mL centrifuge tube. Next, four volumes of lysis buffer (8M urea, 1% protease inhibitor cocktail) were added to the cell powder, followed by three sonications on ice using a high intensity sonicator (Scientz). The remaining chips were centrifuged at 12,000g for 10 minutes at 4 ℃. Finally, the supernatant was collected and protein concentration was determined using BCA kit according to the instructions.

Trypsin digestion

Upon digestion, the protein solution was reduced with 5mM dithiothreitol at 56℃for 30 minutes, and then alkylated with 11mM iodoacetamide for 15 minutes in a dark environment at room temperature. The protein sample was then diluted by adding 100mM TEAB and the urea concentration was reduced to below 2M. Finally, trypsin was added at a trypsin to protein mass ratio of 1:50 for a first overnight digestion and 1:100 trypsin to protein mass ratio for a second 4 hours digestion.

Enrichment of post-translationally modified peptide fragments

The peptide fragments were dissolved in Immunoprecipitation (IP) buffer (100mMNaCl,1mM EDTA,50mM Tris-HCl,0.5% NP-40, pH 8.0) and mixed with pre-washed anti-lysine ubiquitination (PTM-1104, jing Jie biological laboratories, inc.) residual antibody resin and incubated overnight with gentle shaking at 4 ℃. The antibody resin was washed with IP buffer and deionized water. Finally, the peptide was enriched by eluting with 0.1% trifluoroacetic acid three times and purified with C18 ZipTips.

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

The trypsin digested peptide fragments were dissolved in liquid chromatography mobile phase a and separated in NanoElute ultra high performance liquid system. Mobile phases a and B were aqueous solutions containing 0.1% formic acid and 2% acetonitrile, and acetonitrile solutions containing 0.1% formic acid, respectively. The gradient of elution of the peptide fragment was set to 0-72 min, 7% -24% B, 72-84 min, 24% -32% B, 84-87 min, 32% -80% B, 87-90 min, 80% B, and the flow rate was constant at 450 nL/min. After separation of the peptide fragments on a capillary column (inner diameter, particle size), ionization was performed by injection into a capillary ion source and analysis was performed by TIMS-TOF Pro mass spectrometer (ion source voltage, 1.6kV; scan range, 100-1700 Da). The parallel cumulative serial fragmentation (PASEF) mode is enabled upon data acquisition. Precursor ions of charge states 0 to 5 were selected for fragmentation, 10 PASEF MS/MS scans were acquired per cycle. The dynamic exclusion time for MS/MS scanning was 30 seconds to avoid multiple scans of the same parent ion.

Database retrieval

The mass spectrum raw data were retrieved by MaxQuant (v1.6.15.0) in the SwissProt protein sequence database (mus_museulus_10090_sp_20210721. Fasta), containing reverse bait entries and common contaminating proteins. trypsin/P digestion allows a maximum of 2 deletion cleavage sites, requiring at least 7 amino acids per peptide stretch. The precursor ion mass error tolerance was 10ppm and the product ion mass error tolerance was 20ppm. Cysteine alkylation (carbamoylmethyl [ C ]) is set as the immobilization modification. Variable modifications include methionine oxidation and N-terminal ubiquitination. Ubiquitination of lysine and diglycine on lysine were also set as variable modifications for the corresponding modification enrichment analysis. The FDR identified for both protein and PSM was 1%.

CUT & Tag experimental method

CUT & Tag experiments were performed using HYPERACTIVE UNIVERSAL CUT & TAG ASSAY KIT for Illumina (Vazyme, TD 903-01) according to the instructions. Briefly, cells were collected and bound to concanavalin A-coated magnetic beads, permeabilized with digitonin and then incubated with BAF170 antibodies (ab 243634, 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 tagging and analyzed on IlluminaNovaSeq 150PE platform.

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

H9C2 cells were purchased from American type culture Collection (ATCC, USA) and cultured in Dulbecco's modified Eagle's Medium (DMEM, hyClone, logan, UT, USA). HEK293T cells were purchased from the Shanghai cell bank of the national academy of sciences and cultured in high sugar Dulbecco's modified Eagle medium. Cells were cultured in a humidified environment of 37℃and 5% CO2, the medium containing 10% fetal bovine serum (FBS, hyClone, logan, UT, USA), penicillin (100U) and streptomycin (100. Mu.g/ml). Cells were cultured in an environment of 1% o2, 5% co2 and 94% n2 under hypoxic and serum deprived conditions.

Plasmid construction and transfection

Table 2 below lists various plasmids and small hairpin RNAs (shRNAs). Full length human BAF170 carrying the K874R/K704R/K694R mutation was cloned into a 3XFlag GV712 vector and six truncated BAF170 plasmids containing different domains, the Flag tagged BAF 170N-terminus and SWIRM and SANT domains, the Flag tagged BAF 170N-terminus and SWIRM domains, the Flag tagged BAF 170N-terminus domain, the Flag tagged BAF 170C-terminus and SANT domains, the Flag tagged BAF 170C-terminus and SANT and SWIRM domains were constructed. HA-WWP2 and HA-Ub were purchased from Biotechnology (Shanghai) Inc. Plasmid transfection was performed using Lipofectamine 3000 according to the instructions, and cells were collected 48 hours after transfection.

TABLE 2 plasmids and small hairpin RNAs (shRNAs) useful in the present invention

Lentivirus production

BAF170 and WWP2 shRNA lentiviruses were purchased from gekka gene. Lentiviruses were collected from HEK293T cells as described. Lentiviral particles were mixed with 5 XPEG-itTM solution. Cells in 6-well plates were infected with lentivirus. Stable cell lines were screened with puromycin (10. Mu.g/ml) for 7 days. Finally, western blotting is used to determine the infection efficiency of the target cells.

Western blot and immunoprecipitation

Cell lysates were incubated with anti-Flag beads overnight at 4 ℃, or with appropriate antibodies for 3 hours at 4 ℃, followed by protein a/G immunoprecipitation beads for 12 hours at 4 ℃. The protein-antibody complex was washed three times with pre-chilled lysis buffer at 4 ℃ and eluted by boiling with SDS loading buffer for 10 minutes.

BAF170 ubiquitination assay

Mouse myocardial tissue samples and 48 hours transfected cells were lysed with 200. Mu.L of 1% SDS buffer (Tris pH 7.5, containing 0.5mM EDTA and 1mM DTT), boiled for 10 minutes, and then diluted with 800. Mu.L Tris-HCl (pH 8.0). Endogenous proteins were immunoprecipitated using anti-BAF170 antibody (1. Mu.g/mg cell lysate) for 2-3 hours at 4℃and then either protein A/G immunoprecipitated beads were incubated for 12 hours at 4℃or lysates were incubated with anti-Flag (B26302; biotool) immunoprecipitated beads for 12 hours at 4 ℃. Ubiquitinated BAF170 was detected with anti-HA antibodies.

JC-1

Mitochondrial membrane potential (Δψm) of H9C2 cells was measured with 5,5', 6' -tetrachloro-1, 1', 3' -tetraethyl-iodinated imidocarbonocyanine (JC-1 fluorescent probe) according to the specification, 1×105H 9C2 cells were cultured per well and placed in 24-well microwell plates overnight, after about 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 for 20 minutes at 37 ℃ in the absence of light after washing 3 times with PBS DMEM was added and fluorescence was observed with fluorescent microscopy JC-1 polymers (red) were converted to monomers (green) when Δψm was changed, indicating that the mitochondrial membrane of H9C2 cells was damaged.

Hoechst 33342

After H9C2 cells were treated on the slide, they were incubated with Hoechst 33342 dye solution for 15min at 37 ℃. After three washes with PBS, the cells were incubated on ice for 15 minutes with PI dye solution in the dark. After washing three more times with PBS, the slides were blocked.

Oxidative stress staining of cells

To detect intracellular ROS levels, cells were washed three times with PBS and incubated with 5 μ M CellROX Green or 5 μM Mitosox Red at 37℃for 30 minutes in the absence of light. After washing sufficiently, the fluorescence intensity in the cells was observed by a fluorescence microscope.

Wwp2 knockout, transgenic and BAF170-K874R mutant mice were modeled

Myh6 Cre+ Wwp f/f mice, WWP2-TG mice, BAF170-K874R mutant mice and corresponding control mice were all established by Shanghai Nannon model Biotechnology Co. BAF170-K874R mutant mice were derived from fourth generation mice in which lysine 874 (AAG) was mutated to arginine (CGC). All animals were kept under pathogen-free conditions. All experiments were performed using mice 8-10 weeks old. And at a proper age, a permanent myocardial infarction model is established by ligating the left anterior descending coronary artery of the mouse, and the modeling effect is stable. During modeling, each group of mice was subjected to cardiac ultrasound examination at days 0, 7, 14 and 28 to verify modeling success. All animal handling was in compliance with the university of chinese medical science animal welfare regulations and animal research protocols were approved by the institutional animal care committee of chinese medical science (CMU 20241520, CMU20241518, CMU20241909, CMU 20251150).

BFH772 pretreatment

BFH772 (6- ((6- (hydroxymethyl) pyrimidin-4-yl) oxy) -N- (3- (trifluoromethyl) phenyl) -1-naphthamide, APE, china) is a potent oral VEGFR2 inhibitor with an IC50 value of 3nM (PMID: 26629594) for the targeted VEGFR2 kinase. In an in vivo experiment, BFH772 powder was diluted with dimethyl sulfoxide and corn oil and 80 μl of each mouse was administered at a ratio of dimethyl sulfoxide to corn oil=1:9. The mice were dosed at 0, 20, 30 and 40mg/kg, respectively. Administration was started 48 hours after myocardial infarction, once daily, for 28 days. In vitro experiments, BFH772 powder was diluted with DMSO to final concentrations of 0.01, 0.1, 1, 10 and 100 μm and drug action time was 48 hours.

Myocardial infarction molding

Target gene expression was induced by intraperitoneal injection of tamoxifen (75 mg/kg body weight, once every other day, 5 times total). Myocardial infarction molding was performed 30 days after injection. Permanent myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD) of C57BL/6J mice. Mice were anesthetized using isoflurane administration systems by inhalation of 1.5-2% isoflurane. The left ventricle is exposed after cutting the left chest cavity between the third and fourth intercostal spaces. The left ventricular aorta was found, sutured and ligated at about 3mm from its origin. When the anterior wall of the left ventricle became lighter, it was confirmed that myocardial ischemia was successfully induced. Immediately after ligation, the heart was returned to the chest.

Echocardiography and left ventricular function assessment

The mice were subjected to echocardiographic examination at baseline (0 day), 7 days, 14 days, and 28 days, respectively. Cardiac function was assessed in each group of mice using Visual Sonics Vevo 2100 real-time high-resolution in vivo microscopic imaging system (Visualsonic, canada; VINNO6 Lab, china). Mice were anesthetized with 1.5% isoflurane and cardiac function analysis was performed using a 40MHz sensor with continuous oxygen supply. Left ventricular function was detected by two-dimensional M-mode recording. The cardiac function measurements are based on inter-chamber thickness (IVSd), left ventricular back wall thickness (PWTd), systolic left ventricular inner diameter (LVDs), diastolic left ventricular inner diameter (LVDd), and left ventricular mass measurements. In addition, left ventricular ejection fraction (EF%) and short axis shortening (FS%) were also measured. After 28 days, mice were euthanized and their Body Weight (BW) and Tibia Length (TL) were measured. The tibial length is the measured distance from the medial articular surface to the most distal projection of the medial malleolus [29915560]. The heart was removed, washed with PBS and Heart Weight (HW) measured.

Histopathological evaluation

After the myocardial tissue samples were fixed with 4% fixative solution for 48 hours, they were embedded in paraffin and cut into 4 μm-sized sections. After dewaxing with xylene and rehydration with gradient ethanol, staining was performed with hematoxylin-eosin (H & E) and Masson trichromatic staining (G1340; solarbio, china). Cardiomyocyte cross-sectional areas were assessed by 5 μm wheat germ lectin (WGA) (Genetex, usa) staining images.

Electron microscope

The morphology of cardiac mitochondria was observed with a Transmission Electron Microscope (TEM). Heart tissue was pre-fixed with EM grade 2.5% glutaraldehyde in 0.1mol/l sodium dimethyl arsenate buffer. The fixed tissues were incubated with 1% osmium tetroxide in 0.1mol/l sodium dimethyl arsenate buffer for 2 hours. The fixed tissue is then dehydrated stepwise for embedding. Heart tissue sections were stained with uranium acetate. Changes in mitochondria in myocardial tissue were observed and photographed using a Hitachi H-7650 transmission electron microscope, and 15 to 24 images were randomly photographed per group of mice.

Animal myocardial tissue oxidative stress staining

In vitro experiments, ROS production was detected by DHE staining. Paraffin-embedded heart sections were mixed with DHE (dihydroethylidine, 5 μmol/L) and incubated for 1 hour at 37 ℃ protected from light. Images were taken by fluorescence microscopy blinding, fluorescence intensity was calculated using ImageJ software and the results were expressed as fold change over the corresponding control group.

Mitochondrial extraction for high resolution respiratory assay system O2k

The mouse heart was extracted and cut into small pieces quickly within 4 minutes. Incubation was performed with mitochondrial isolation buffer (SIGMA ALDRICH, P8038) containing 0.1mg/ml protease for 2 min at 4 ℃. The mitochondrial isolation buffer composition was 50mM Tris×HCl (pH 7.4), 100mM KCl, 100mM sucrose, 1mM KH2PO4, 0.1mM EGTA and 0.2% bovine serum albumin. Tissue was gently homogenized 6 times with a glass homogenizer. After centrifugation at various speeds, the resulting pellet was resuspended in 10mM buffer, which was 10mM Tris XHCl (pH 7.4), 225mM mannitol, 75mM sucrose and 0.1mM EDTA. All experimental steps were performed at 4 ℃.

Mitochondrial respiratory activity measurement

Mitochondrial respiration assessment was performed using a high resolution respirometry system O2k (Oroboros Instruments, innsbruck, austria) at 37 ℃ with a reaction chamber volume of 2mL. Oxygen was calibrated with respiratory medium MIR05 (110 mM sucrose, 60mM potassium lactobionate, 0.5mM EGTA, 1g/L fatty acid free BSA, 3mM MgCl2, 20mM taurine, 10mM KH2PO4 and 20mM HEPES;pH 7.1,37 ℃) prior to the experiment. The medium was equilibrated with air in the oxygen meter chamber and stirred at 750rpm for 20 minutes until the signal stabilized. 70 μg of mitochondrial extract was added per chamber and substrate-uncoupler-inhibitor titration (SUIT) was performed in the order pyruvic acid/malic acid/glutamic acid (PMG) → MgCl2/ADP (D) → succinic acid (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) is calculated by the negative time derivative of oxygen concentration. Data acquisition and analysis useSoftware version 7.4.0.4 (Oroboros Instruments) is complete.

Reagent(s)

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

Protein purification-WWP 2

The recombinant plasmid containing WWP2 gene is transferred into competent cells of escherichia coli BL21 (DE 3), cultured and induced to express a large amount of protein. Cells were collected by centrifugation. For affinity purification, cells were lysed with buffer (50 mM Tris, 300mM NaCl, 0.1% Triton X-100, 0.2mM PMSF,pH 8.0), sonicated and the supernatant was collected by centrifugation to give crude protein. Then, 5ml of Ni-NTA column was equilibrated with five volumes of binding buffer (PBS-NaCl, pH 7.4). The crude protein was incubated with the column packing for 1 hour and the effluent was collected. The column was then washed with binding buffer, followed by washing with washing buffer (PBS-NaCl, 20mM imidazole, pH 7.4) and the effluent was collected. The proteins were eluted with elution buffer (PBS-NaCl, 500mM imidazole, pH 7.4) and the eluate was collected. The eluted samples were further purified by ion exchange chromatography (Q column) and analyzed by SDS-PAGE. The purified eluted sample was subjected to gel filtration chromatography (Superdex 200) using buffer (PBS, 0.12%SKL,pH 7.4). The purified fractions were dialyzed into protein storage buffer (PBS, 0.12%SKL,pH 7.4), concentrated, filtered and sterilized. The purified protein was aliquoted and stored at a temperature of-80 ℃.

Protein purification-BAF 170

A series of recombinant expression vectors encoding different domains of SMARCC2 were constructed by molecular cloning, SMARCC2 (1-647, 1-423, 1-595, 648-1214, 424-1214, and 596-1214). And (3) carrying out recombination and amplification preparation after the recombinant plasmid is correctly constructed through restriction enzyme analysis verification. The plasmid was transfected into 293 cells and the protein purified. First, cells were lysed with buffer C, and after sonication, the supernatant was collected by centrifugation. After 5ml of Ni-NTA column was equilibrated with 5 times the bed volume of binding buffer, the crude protein was incubated with the equilibrated column packing for 1 hour, and the effluent was collected. The equilibrated column was washed with binding buffer, then washing buffer (PBS-NaCl, 20mM imidazole, pH 7.4). Proteins were eluted with elution buffer (PBS-NaCl, 500mM imidazole, pH 7.4). The eluate samples were purified by ion exchange chromatography (Q column) and analyzed by SDS-PAGE. The high purity eluted sample was purified by gel filtration chromatography (Superdex 200) (PBS, 0.12%SKL,pH7.4), the purified fraction was dialyzed into protein storage buffer (PBS, 0.12%SKL,pH 7.4), concentrated, filtered and sterilized, and then sub-packaged for storage at-80 ℃. In SDS-PAGE analysis, protein samples were processed and prepared using 12% separation gel and 5% concentration gel, and molecular weight was determined.

Preparation of compound library

The SMILES form of the selected compounds (15000 molecules) was obtained from PubChem (https:// pubchem. Ncbi. Nl. Gov /), and these compounds were normalized and converted by RDKit (2023.09.1) to generate a library of compounds. All selected compounds are converted to the desired structure (SDF format).

Virtual screening

The binding site of k874 was confirmed by AlphaFold prediction of SMARCC2 protein structural model. The central coordinate is fixed as The size of the grid frame is fixed asDocking of flexible ligand and rigid receptor was performed by AutoDockTools 1.5.6 and Vina, 1.1.2, energy range was set to 4kcal/mol, and degree of exhaustion was set to 12. The best binding mode was found using Genetic Algorithm (GA) and Particle Swarm Optimization (PSO), and the affinity of each binding mode was calculated by a scoring function according to an empirical formula. The binding pattern of each compound to the target protein was ordered according to affinity and the results visualized by pymol2.2.0.

Surface Plasmon Resonance (SPR)

Different domain truncated BAF170 binding assay to WWP2

SPR experiments were performed on BAF170 (1-426), BAF170 (1-595), BAF170 (1-647), BAF170 (424-1214), BAF170 (569-1214) and BAF170 (648-1214) on a Biacore T200 system (Cytiva), at 25℃and flow rates of 30. Mu.l/min. Purified wild-type WWP2 protein was immobilized on S CM5 series sensor chip (Cytiva) by amine coupling chemistry. The different domain truncated BAF170 proteins were serially diluted with running buffer to multiple concentrations and flowed through the chip at a constant flow rate from low to high concentration for 4-6 minutes. The data were analyzed by Biacore T200 evaluation software 3.0 and affinity constants were calculated.

SPR analysis of BFH772 effect on BAF170 binding to WWP2

Small molecule compounds such as colpitan 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, citrulline specific probe, BFH772, BMS195614, LIMKi 3, N-methyl protoporphyrin IX, L-689,560, information of which are shown in table 3, temperature 25 ℃ and flow rate 30 μl/min were subjected to SPR experiments on Biacore T200 system (Cytiva). Purified wild-type WWP2 protein was immobilized on S CM5 series sensor chip (Cytiva) by amine coupling chemistry. 50. Mu.M of different small molecule compound complexes were each flowed through the chip as binding substrates at a constant flow rate, with their equilibrium state as signal baseline, and then injected into BAF170. Through a ternary interaction system, the binding curve and signal change of the WWP2 and the BAF170 before and after the small molecule compound is added are visually compared, and the specific influence of the small molecule compound is determined.

TABLE 3 information on small molecule Compounds

Relative value of

Immunoblots images were analyzed by ImageJ software. The intensity of the internal reference band was used as a reference for the protein loading. First, in an independent experiment, the relative expression amount of the target protein was calculated by dividing the gray value of each band by the gray value of the internal reference. The relative expression level of each band was then divided by its average to give a ratio. This calculation process was repeated in other independent experiments. Next, the ratio of the first group (control group) is averaged, and each ratio in the first group is divided by the average to obtain a relative value. Finally, the ratios of the other groups are divided by this average to obtain the final value for statistical analysis. The fluorescence intensities of DHE (oxidative stress marker), cellROX-Green and Mitosox-Red represent oxidative stress levels, and relative fluorescence units were calculated by imageJ software. The average value of the fluorescence unit values of the first group (control group) is calculated first, and then each value in the first group is divided by the average value to obtain the relative fluorescence intensity. Finally, the values of the other groups are divided by the average value to obtain fluorescence intensity values for statistical analysis.

Statistical analysis

Data are expressed as mean ± Standard Deviation (SD). In vitro experiments, if the cell number is large enough (hundreds of thousands of cells), the ratio (the mean index of detection) is calculated as the protein expression level divided by the internal reference. According to the central limit theorem, the detection average index approximately obeys the normal distribution, and the average index of three independent repeated experiments obeys the normal distribution. In vivo experiments (sample size > 7) were tested for normalization by Shapiro-Wilk, F test (two sets of comparisons) or Bartlett test (two or more sets of comparisons) for variance alignment, with a threshold P value of 0.05. For normally distributed and well-variational data, two sets of studies used unpaired t-test (two-tailed Student-t-test), and multiple sets of comparisons used one-way anova and Tukey multiple comparison test. Two-way anova and Bonferroni multiple comparison test were used when two conditions were considered between the two groups. Data that did not pass the normalization or variance alignment test were compared to two sets of data using the Mann-Whitney test, and the multiple sets of comparisons were tested using the Kruskal-Wallis test and the Dunn multiple comparison test. Multiple comparisons adjust for class I false expansion using Bonferroni correction and adjust for P values when applicable. Each legend contains detailed methods of significance assessment and the number of biological replicates for each set of experiments. The exact P-values for significant variation are indicated in the legend. Statistical analysis was done using SPSS22.0 software (SPSS, usa) and GraphPadPrism 10.0 software (GraphPad, bethesda, MD, usa), P <0.05 indicating statistical significance. Representative images reflecting the average results of each experiment were selected from the figure. "N" or "N" in the legend indicates biological repetition.

3. Results

The experimental results are shown in FIGS. 1-13 and Table 4.

Increased BAF170 protein expression during MI

To explore which proteins and functions in the heart were altered during Myocardial Infarction (MI), we created a mouse model of MI by ligating the left anterior descending coronary artery. Subsequently, we performed high-depth proteomic analysis of heart tissue and compared with control mice heart tissue. The results showed that 1079 proteins were up-regulated (FC >1.5, p value < 0.05) and 225 proteins were down-regulated (FC <0.667, p value < 0.05) among the differentially expressed proteins (fig. 1A). GO functional enrichment analysis showed that SWI/SNF superfamily complexes were most scored among the cellular components, except for cellular structural proteins (FIG. 1B). Among the SWI/SNF chromatin remodeling complex family members identified in the differentially expressed proteins, SMARCC2 (also known as BAF 170) had the lowest p-value (FIG. 1C). This suggests that BAF170 may play an important role in the heart during myocardial infarction.

Hypoxia and serum deprivation (H/SD) are a widely accepted in vitro model for mimicking ischemic hypoxia conditions. The H/SD model has been widely used to study cardiomyocyte responses to ischemic hypoxia. We studied the role of BAF170 under these conditions by modeling H/SD of H9C2 cells, and found that there was a direct correlation between H/SD exposure time and apoptosis rate and BAF170 expression level (FIGS. 1D and 1E). Using Hoechst33342, a commonly used nuclear stain, we observed a gradual increase in the number of apoptotic cells with prolonged H/SD treatment time, which was manifested as blue fluorescence enhancement (FIG. 1E).

To explore the mechanism of elevated BAF170 expression, we performed Immunoprecipitation (IP) using BAF170 antibodies to enrich for interacting proteins from heart tissue in control and Myocardial Infarction (MI) mice. By mass spectrometry we detected changes in downstream protein interactions. The results show that BAF170 binding to WWP2 was significantly reduced in the myocardial infarction model (table 4). Furthermore, in the H/SD model, the expression of WWP2 gradually decreased with the extension of the induction time (FIG. 1F).

After myocardial infarction, BAF170 has reduced interaction with WWP2, indicating a regulatory relationship between the two. Endogenous immunoprecipitation experiments showed a clear interaction between WWP2 and BAF170 (fig. 1g,1 h), which was further confirmed by exogenous immunoprecipitation studies (fig. 1I). To determine the specific region of interaction, researchers performed binding experiments using endogenous WWP2 and various tag-tagged BAF170 constructs, including full-length and truncated versions. The results show that WWP2 specifically interacts with three regions of BAF170, the SANT domain, SWIRM domain and the N-terminal region (fig. 1j,1 k).

WWP2 regulates BAF170 degradation through K874 ubiquitination

To investigate the potential role of WWP2 in regulating BAF170 levels, we studied whether decreasing WWP2 would affect BAF170 expression levels. To this end, we generated a stable WWP2 knockdown (shWWP 2) H9C2 cell line using three different shRNA fragments (fig. 2A). Of these three fragments, 74453 fragment showed the highest knockdown efficiency and was therefore selected for subsequent experiments. Consistent with WWP2 playing a role in regulating BAF170 levels, silencing of WWP2 results in increased BAF170 levels.

To further verify this relationship, we performed gradient over-expression experiments of WWP2 in HEK293T and H9C2 cells. The results showed that BAF170 protein levels decreased in a dose-dependent manner with increasing WWP2 expression (fig. 2B). These findings strongly suggest that WWP2 acts as a negative regulator of BAF170 protein levels.

WWP2 may reduce BAF170 protein levels by promoting its degradation (fig. 2C-2H). To verify this hypothesis, we used Cycloheximide (CHX) as a translational inhibitor for time-gradient treatment, which prevents the synthesis of new proteins, thus enabling monitoring of the stable levels of the protein at different time points. Experiments were performed in normal control and shWWP cells. After the cycloheximide addition, the control group had a faster decline in BAF170 levels, while the shWWP group remained at higher levels (fig. 2c,2 g). This suggests that a decrease 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 under cycloheximide treatment (fig. 2d,2 h).

Next, we tested whether BAF170 was incorporated into the ubiquitin proteasome mediated degradation pathway. To verify this, we performed time-course experiments using the proteasome inhibitor MG132 (fig. 2E-2J). The BAF170 protein levels were significantly elevated in shWWP2 transfected cells compared to the control group (fig. 2e,2 i). Furthermore, after MG132 treatment, the accumulation of BAF170 in cells expressing HA-WWP2 increased significantly, whereas the control group expressing HA-Vector did not (FIGS. 2F, 2J). These results support the conclusion that WWP2 regulates BAF170 protein levels via the proteasome pathway. Likewise, WWP2 overexpression significantly increased the ubiquitination level of BAF170 (fig. 2K), while WWP2 knockdown significantly reduced the ubiquitination level of BAF170 (fig. 2L). Taken together, these findings suggest that WWP2 promotes degradation of BAF170 by proteasome-dependent degradation following ubiquitination.

To determine WWP 2-targeted BAF170 ubiquitination modification sites, we performed comprehensive analysis using a variety of mouse models and advanced proteomic techniques. Two key experimental groups were established in the study, a conditional myocardial-specific WWP2 knockout mouse (WWP 2-cKO: myh6-cre+; wwp2 ^f/f) and its corresponding WWP2-WT (Wwp 2 ^f/f) control group, and a transgenic Rosa26-WwP2-Flag mouse (WWP 2-TG) and its corresponding control group. The specific signaling pathway regulated by WWP2 in the response to myocardial infarction was investigated by complex quantitative proteomic and ubiquitinated modified genomic analysis (fig. 2M). 7020 ubiquitinated sites and 2202 ubiquitinated proteins were identified by four-dimensional unlabeled high-depth proteomics, with 4091 sites and 1082 proteins showing quantitative differences.

Subsequently, we integrated the differential and ubiquitinated proteins using a nine-quadrant plot, focusing specifically on two key populations, ubiquitinated down-regulated and highly expressed proteins in WWP2 knockdown versus wild-type control, and ubiquitinated up-regulated and low-expressed proteins in WWP2 transgene versus wild-type control (fig. 2N). By expression pattern cluster analysis, we found that both proteins (fig. 2O) and ubiquitination sites (fig. 2P) showed significant trends in the different experimental groups. It is particularly notable that as WWP2 levels increase, the expression level of certain proteins decreases (cluster 3), while the degree of modification of certain ubiquitination sites increases (cluster 6). Systematic analysis showed that there was a significant negative correlation between BAF170 protein expression level and WWP2 expression level, accompanied by evidence of ubiquitination modification.

The role of WWP2 in Myocardial Infarction (MI) was studied by biofunction enrichment analysis. In the WWP2 transgenic/wild-type model, upregulated ubiquitinated proteins were analyzed and found to be involved in cardiac contractile regulation, muscle silk slipping and myocardial function (fig. 2Q). Whereas in the WWP2 conditional knockdown/wild-type model, the down-regulated ubiquitinated proteins were analyzed, these proteins were found to be involved in nicotinamide nucleotide metabolism, NADP metabolism and ADP metabolic processes (fig. 2R).

We have paid particular attention to the role of WWP2 in BAF170 ubiquitination regulation. By mass spectrometry data analysis we found six ubiquitination sites on BAF170, K694, K704, K872, K874, K897 and K902. Of these sites, only K694, K704 and K874 were directly related to the expression level of WWP2, i.e., the degree of ubiquitination of these sites was increased when the expression level of WWP2 was increased, and the degree of ubiquitination of these sites was decreased when the expression level of WWP2 was decreased (FIGS. 2U-2W). These findings indicate that K694, K704 and K874 are specific sites for WWP2 mediated BAF170 ubiquitination.

To explore these potential ubiquitination sites further, we analyzed the K694, K704 and K874 sites of BAF170 by creating lysine to arginine (K-R) mutations. We observed changes in BAF170 ubiquitination levels after exogenous overexpression of HA-tagged vectors or HA-tagged WWP2 variants, and when HA-UB was co-transfected in the presence of MG 132. Wild-type BAF170 and mutants thereof (K694R, K R or BAF 170-K874R) were over-expressed, respectively. Co-immunoprecipitation experiments showed that overexpression of BAF170-K874R alone significantly reduced the ubiquitination level of BAF170 compared to the wild-type (FIGS. 2U-2W). These findings indicate that WWP2 site-specifically mediates polyubiquitination of BAF170 through K874, leading to degradation of BAF170 through the proteasome pathway.

Myocardial specific WWP2 deletion results in increased BAF170 expression and exacerbates myocardial cell injury

The specific role of WWP2 in regulating BAF170 during Myocardial Infarction (MI) is not yet clear. To further investigate this relationship we constructed a mouse model of MI by ligating the left anterior descending coronary artery for 28 days using Wwp, ^f/f and Myh6-cre+; wwp, ^f/f mice. Westernblot analysis confirmed that exon 3 of WWP2 was deleted in Myh6-cre+; wwp2 ^f/f mice (FIGS. 3A-B). WWP2 expression was significantly reduced in Myh6-cre+; wwp2 ^f/f mice compared to Wwp2 ^f/f control group, and this reduction was more pronounced in MI (FIGS. 3J-3K).

We confirmed the regulatory effect of WWP2 on BAF170 by analyzing interactions in heart tissue in the Myocardial Infarction (MI) model. The interaction of BAF170 with WWP2 was significantly reduced after MI in heart tissue of Myh6-Cre +; wwp2 ^f/f mice compared to Wwp2 ^f/f control group (fig. 3C). Furthermore, in MI, myh6-cre+; wwp2 ^f/f mice had significantly reduced BAF170 ubiquitination levels (FIG. 3D). IP analysis in shWWP cell line showed that WWP2 binding to BAF170 was similar whether or not subjected to H/SD treatment. H/SD treatment reduced WWP2 interaction with BAF170 in H9C2-shWWP2 cells (FIG. 8A) and reduced WWP 2-mediated ubiquitination of BAF170 (FIG. 8B).

Mitochondrial dysfunction of cardiomyocytes is considered to be an important causative factor of Myocardial Infarction (MI). Myocardial infarction initiates an acute Reactive Oxygen Species (ROS) burst, resulting in mitochondrial injury and respiratory dysfunction, which in turn triggers cardiomyocyte apoptosis, cardiac fibrosis and cardiac dysfunction, ultimately leading to worsening heart failure. Notably, cardiac dysfunction was significantly aggravated after myocardial infarction in Myh6-cre+; wwp ^f/f mice, compared to Wwp2 ^f/f mice, as evidenced by a decrease in ejection fraction (EF%) and foreshortening fraction (FS%) (FIGS. 3E-3G). Evaluation of cardiac hypertrophy markers showed that Myh6-cre+; wwp2 ^f/f mice had higher cardiac weight to body weight ratio (HW/BW) and cardiac weight to tibial length ratio (HW/TL) than the control group (FIGS. 3H-3I). Simultaneous analysis of oxidative stress markers in heart tissue revealed that Myh6-cre+; wwp2 ^f/f mice had elevated endogenous ROS levels (FIG. 3L), increased levels of 3-Nitrotyrosine and 8-oxo-dG, decreased SOD1 and SOD2 expression, and compared to Wwp2 ^f/f mice (FIGS. 3M-3N).

Next, we studied mitochondrial respiratory function, structural abnormalities, oxidative stress injury, and fibrosis in an established MI mouse model. First, we examined the protein BCL2 associated with mitochondrial apoptosis, as its down-regulation is directly associated with mitochondrial damage and dysfunction. The binding of BAF170 to BCL2 enhancer was found to be reduced in MI tissues compared to control group by CUT & Tag and chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis (fig. 9D). Notably, myh6-cre+; wwp2 ^f/f mice had significantly lower expression of BCL2 in MI than the Wwp2 ^f/f control group, indicating enhanced mitochondrial apoptosis (FIG. 3O-3P).

To assess mitochondrial respiratory function, we isolated mitochondrial tissue of the heart and analyzed oxygen consumption of different mitochondrial complexes using Oroboros O K system. In Wwp, 2, ^f/f and Myh6-cre+; wwp, 2f/f mice, the oxidative phosphorylation of complex I, complex I and II, and ATP production were all reduced, while complex II remained unchanged. Notably, myh6-cre+; group Wwp2 ^f/f was significantly reduced in these three parameters compared to group Wwp2 ^f/f (FIG. 3Q).

Transmission Electron Microscopy (TEM) is widely regarded as a gold standard for assessing mitochondrial content and is capable of measuring the bulk density of mitochondria. In Wwp mice ^f/f of the sham surgery group, mitochondria exhibited dense, tightly packed and well organized ridges. However, mice in the MI treated group showed signs of mitochondrial damage, manifested by cristae becoming loose, swollen, destroyed, and vacuolated. This injury was especially severe in Myh6-cre+; wwp2 ^f/f mice (FIG. 3R).

To investigate whether cardiomyocyte apoptosis is associated with changes in apoptosis-related protein expression, we performed comparative analysis on Wwp2 ^f/f mice and Myh6-cre+; wwp2 ^f/f mice. The results show that levels of Cleaved-PARP1 and Cleaved-Caspase3 are significantly elevated in Myh6-cre+; wwp2 ^f/f mice (FIGS. 3S-3T). Furthermore, histological analysis by hematoxylin and eosin (H & E), wheat germ lectin (WGA) and Pinus massoniana staining showed that Myh6-cre+; wwp2 ^f/f mice had significantly increased cardiac hypertrophy and fibrosis (FIGS. 3U-3W).

Next, we studied the oxidative stress markers and endogenous ROS levels of the H9C2-shWWP2 cell line in the presence or absence of H/SD treatment. First, we analyzed several proteins associated with oxidative stress, including 3-Nitrotyrosine, 8-oxo-dG, SOD1 and SOD2. The results show that the level of oxidative stress related proteins was higher than that of the control group, regardless of the presence or absence of H/SD treated H9C2-shWWP cell line. Notably, the re-expression of HA-WWP2 NTm was effective in reducing these elevated levels (fig. 8E-8F). Subsequently, we detected ROS levels by fluorescence methods. CellRox-Green fluorescent probes detect ROS production in intact tissues by staining nuclei, producing Green fluorescence, while Mitosox-Red fluorescent probes are used to measure ROS in mitochondria, showing Red fluorescence. Quantitative analysis of the staining intensity of CellRox-Green and Mitosox-Red probes showed that the ROS production was significantly higher in the H9C2-shWWP2 cell line, whether or not H/SD treated (FIGS. 8G-8J).

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 show that BCL2 levels were significantly reduced in shWWP cells, either control or H/SD model, indicating increased mitochondrial apoptosis. In contrast, BCL2 levels were significantly elevated when HA-WWP2 NTm was re-expressed in shWWP2 cells, indicating reduced mitochondrial apoptosis (fig. 8L-8M). Changes in mitochondrial membrane potential were detected by JC-1 staining. Whether or not subjected to H/SD treatment, the ratio of green JC-1 monomer to red JC-1 aggregate in the H9C2-shWWP2 cell line was increased, indicating a decrease in mitochondrial membrane potential. Importantly, the re-expression of HA-WWP2 NTm eases this change (FIG. 8N-8O).

Finally, we studied the relationship between apoptosis and changes in apoptosis-related protein expression in H9C2 cells. By comparing the H/SD group with the control group, it was found that Cleaved-PARP1 and Cleaved-Caspase3 levels were significantly elevated in the H9C2-shWWP2 cell line, whether or not subjected to H/SD treatment. Notably, the re-expression of HA-WWP2 NTm can mitigate this increase (fig. 8P-8Q). In addition, hoechst33342 staining showed a significant increase in the number of apoptotic cells in the H9C2-shWWP2 cell line under H/SD and control conditions. This effect was also reduced by re-expression of HA-WWP2 NTm (FIGS. 8G-8H, 8K).

WWP2 overexpression down regulates BAF170, and reduces myocardial cell injury after myocardial infarction

In view of the negative impact of reduced WWP2 expression, we tested whether over-expression of WWP2 could alleviate cardiomyocyte injury. By crossing F0 mice with Myh6-Creer mice we generated Rosa26-WwP2-Flag transgenic mice (WWP 2-TG, R26-LSL-Wwp +/+; myh 6-Creer) (FIG. 4A). Myocardial infarction was then induced in wild type (WWP 2-WT, R26-LSL-Wwp < 2+ >; myh 6-Creer-) and WWP2-TG mice by ligating the left anterior descending coronary artery and monitored for 28 days (FIG. 4B). The analysis confirmed the successful production of WWP2-TG mice whose WWP2 expression levels were significantly lower than WWP2-WT mice and significantly decreased after myocardial infarction (fig. 4J-4K).

To investigate the regulatory effect of WWP2 on BAF170 in this model, we analyzed protein interactions in cardiac tissue. An enhanced interaction between BAF170 and WWP2 was observed in cardiac tissue of WWP2 transgenic (WWP 2-TG) mice under Myocardial Infarction (MI) conditions compared to wild type (WWP 2-WT) mice (fig. 4C). Furthermore, we found that the level of ubiquitination of BAF170 was increased in WWP2 transgenic mice following myocardial infarction (fig. 4D). Similarly, in the H/SD model, after transfection of HA-WWP2, the interaction between WWP2 and BAF170 was enhanced and the level of ubiquitination of BAF170 by WWP2 was also increased compared to the HA vector control group (FIGS. 8C-8D).

Cardiac function of WWP2 transgenic mice was significantly improved in Myocardial Infarction (MI), as demonstrated by measurements of ejection fraction (EF%) and shortening fraction (FS%) (fig. 4E-4G). Furthermore, WWP2 transgenic mice showed reduced cardiac hypertrophy markers in myocardial infarction compared to WWP2 wild type mice, as evidenced by a reduction in cardiac weight to weight ratio (HW/BW) and cardiac weight to tibial length ratio (HW/TL) (fig. 4H-4I). In addition, the oxidative stress markers of WWP2 transgenic mice were also decreased, including a decrease in endogenous reactive oxygen species level (FIG. 4L), and a decrease in 3-Nitrotyrosine and 8-oxo-dG levels, while the expression levels of antioxidant enzymes SOD1 and SOD2 remained higher (FIGS. 4M-4N), than the WWP2 wild-type control group.

Subsequently, we studied the effects of mitochondrial respiratory dysfunction, structural abnormalities, oxidative stress damage and fibrosis observed in established MI mouse models. The results showed that WWP2 transgenic mice showed significantly increased BCL2 expression after MI compared to wild type WWP2 mice, indicating reduced mitochondrial apoptosis (fig. 4O-4P). Through Oroboros O K system, we found that after MI, both wild type WWP2 mice and WWP2 transgenic mice, complex I oxidative phosphorylation, complex I and II oxidative phosphorylation, and ATP production were reduced compared to sham-operated groups. Notably, the oxidative phosphorylation capacity of complex II was unaffected. WWP2 transgenic mice performed better than wild-type WWP2 mice in all three parameters (FIG. 4Q).

Electron microscopy showed that in the sham operated group of WWP2-WT mice, the mitochondrial cristae was dense, compact and well organized. In contrast, MI treated mice exhibited loose, swollen, disturbed mitochondrial ridges and vacuolated appearance. However, this structural damage was significantly reduced in WWP2-TG mice after MI treatment (fig. 4R). Levels of Cleaved-PARP1 and Cleaved-Caspase3 were significantly reduced in WWP2-TG mice compared to WWP2-WT mice (FIGS. 4S-4T). Furthermore, histological analysis by H & E staining, WGA staining and Masson staining showed significant improvement in cardiac hypertrophy and fibrosis in WWP2-TG mice (fig. 4U-4W).

To determine the specific effect of WWP2 on BAF170 ubiquitination in Myocardial Infarction (MI), we established stable BAF170 knockdown cell lines using three different short hairpin RNA (shRNA) fragments. 121254 fragments exhibited the best knockdown efficiency and were therefore used in subsequent experiments (fig. 10B-10C). Subsequently, we expressed wild-type BAF170 or mutant forms thereof in these knockdown cell lines. Sequence analysis showed that K874 was an evolutionarily conserved site from rat to mammal, possibly a BAF170 ubiquitination site regulated by WWP 2. This site corresponds to K874 in mice and K905 in rats (fig. 10A). Thus, we focused on residues K874/K905 in particular to investigate the importance of the BAF170 ubiquitination site in oxidative stress. We analyzed a variety of oxidative stress indicators including 3-Nitrotyrosine, 8-oxo-dG, SOD1 and SOD2. Under hypoxic/serum deprived (H/SD) conditions, cells expressing K905R-BAF170NTm 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-BAF170NTm (FIGS. 10D-10E).

We evaluated mitochondrial Reactive Oxygen Species (ROS) levels using fluorescent probes Mitosox-Red and CellRox-Green, H/SD treated and untreated, respectively, in the experiments. The results showed that the mitochondrial ROS levels of cells expressing K905R-BAF170 NTm were consistently higher than those of cells expressing WT-BAF170 NTm, whether or not H/SD treatment was performed (FIGS. 10F-10I). These results strongly indicate that K905R-BAF170 is capable of exacerbating oxidative stress induced cell damage.

Next, we studied the effect of BAF170 on mitochondrial respiratory function. By analyzing whether the H9C2-shBAF170 cell line received H/SD treatment after re-expression of either WT-BAF170 NTm or K905R-BAF170 NTm, we found that re-expression of K905R-BAF170 NTm significantly reduced BCL2 levels, compared to re-expression of WT-BAF170 NTm, indicating enhanced mitochondrial apoptosis (FIGS. 10K-10L). JC-1 staining results showed that the ratio of green JC-1 monomer to red JC-1 aggregates increased significantly after re-expression of K905R-BAF170 NTm under H/SD conditions (FIGS. 10M-10N). These results indicate that the K905R-BAF170 mutation aggravates mitochondrial dysfunction and impairs cardiomyocyte mitochondrial function under ischemic and hypoxic stimulation.

In agreement with the increased mitochondrial apoptosis, the apoptosis markers Cleaved-PARP1 and Cleaved-Caspase3 showed significantly higher expression after re-expression of K905R-BAF170NTm compared to re-expression of WT-BAF170 NTm in H9C2-shWWP2 cells (FIG. 10O-10P). The Hoechst33342 assay demonstrated a significant increase in apoptotic cells in the H9C2-shBAF170,170 cell line under normal and H/SD conditions compared to the control group, and the K905R-BAF170NTm re-expression further amplified this effect (FIGS. 10F-10G, 10J).

Taken together, our results indicate that mutation of lysine (K) residue 874 to arginine (R) in BAF170 prevents WWP 2-mediated proteasome degradation, resulting in increased mitochondrial dysfunction, increased oxidative stress and increased cardiomyocyte apoptosis.

BAF170-K874R disrupts ubiquitination of BAF170, exacerbating myocardial cell injury

To directly demonstrate the role of BAF170-K874 in myocardial infarction, we constructed a hybrid K874R mutant within the SWIRM domain of mouse Smarcc-yl exon 28. The mutation involved a codon change from AAG (lysine) to CGC (arginine), occurring in functionally critical regions (fig. 5A-5B). BAF170 expression levels were significantly elevated in BAF170-K874R mice compared to wild-type BAF170 control. In addition, the expression level of BAF170 was also significantly increased after myocardial infarction (fig. 5I-5J).

We studied the role of WWP2 in BAF170 regulation using established mouse models of MI point mutations. Analysis showed that heart tissue of BAF170-K874R mice was significantly reduced in BAF170 ubiquitination levels after MI compared to wild-type BAF170 mice (fig. 5C). Importantly, BAF170-K874R mice exhibited significantly worse MI cardiac dysfunction compared to WT-BAF170 mice, as shown by lower measured ejection fraction (EF%) and foreshortening fraction (FS%) (fig. 5D-5F). Evaluation of cardiac hypertrophy markers, including cardiac weight/body weight (HW/BW) and cardiac weight/tibial length (HW/TL) ratios, showed an increase in the values of the MI of BAF170-K874R mice compared to the WT-BAF170 control group (FIGS. 5G-5H).

Simultaneous measurement of endogenous ROS levels and Western blot analysis of oxidative stress markers were performed on heart tissues of WT-BAF170 and BAF170-K874R mice. Markers detected include 3-Nitrotyrosine, 8-oxo-dG, SOD1 and SOD2. The results showed that the endogenous ROS levels in BAF170-K874R mice were significantly elevated (FIG. 5K), as were the levels of 3-Nitrotyrosine and 8-oxo-dG. In contrast, these mice had SOD1 and SOD2 levels lower than that of WT-BAF170 mice (FIGS. 5L-5M). Mitochondrial apoptosis is monitored by detecting the expression level of BCL2 protein. Compared to wild-type BAF170 mice, BAF170-K874R mice showed a significant decrease in BCL2 levels after myocardial infarction, indicating enhanced mitochondrial apoptosis (fig. 5N-5O).

Analysis using the Oroboros O K system showed significant changes in mitochondrial function between experimental groups. In the WT-BAF170 and BAF170-K874R groups, the oxidative phosphorylation of complex I, the oxidative phosphorylation of complexes I and II, and the ATP production were reduced after myocardial infarction compared to the sham-operated group. Notably, the oxidative phosphorylation capacity of complex II was unaffected. The values for the BAF170-K874R group were significantly lower over these three parameters compared to the WT-BAF170 group (FIG. 5P).

Electron microscopy showed that there was a clear morphological difference between the groups. In the sham group of WT-BAF170 mice, mitochondria were aligned, cristae was dense and compact, and exhibited an ordered structure. In contrast, mice in the MI group showed signs of mitochondrial dysfunction, manifested as cristae loosening, swelling, destruction, and concomitant cavitation. These structural abnormalities were particularly pronounced in post-MI BAF170-K874R mice (fig. 5Q).

To investigate whether cardiomyocyte apoptosis is associated with changes in apoptosis-related protein expression, we compared wild-type BAF170 mice with BAF170-K874R mutant mice. The results of the study found that the Cleaved-PARP1 and Cleaved-Caspase3 levels were significantly elevated in BAF170-K874R mutant mice (FIGS. 5R-5S). In addition, these mice exhibited more severe cardiac hypertrophy and fibrosis, as demonstrated by H & E, WGA and Masson staining experiments (fig. 5T-5V). These findings indicate that WWP2 no longer promotes degradation of BAF170 by proteasome ubiquitination when lysine (K) at position 874 of BAF170 protein is replaced by arginine (R). This mutation results in increased mitochondrial dysfunction, increased oxidative stress, and increased myocardial apoptosis.

BFH772 can remarkably relieve myocardial cell injury caused by myocardial infarction

Our findings indicate that the BAF170-WWP2 axis is critical to protect the heart from Myocardial Infarction (MI). To find small molecule compounds that could enhance WWP2 binding to BAF170, potentially alleviating the effects of MI, we screened APE company database of bioactive compounds (L-CO-020). From 30,000 small molecule compounds we screened 10 candidate compounds with significant binding affinity, which showed significant binding affinity for BAF170-K874 (see table 3, fig. 12B).

These candidates were subjected to stringent in vitro validation and tested for their effect on the interaction between WWP2 and SMARCC2 domains. We first expressed the full-length WWP2 protein using E.coli BL21 and purified (FIG. 11A). Subsequently, we generated and purified the different domains of SMARCC2 protein (including 1-647, 1-423, 1-595, 648-1214, 424-1214, and 596-1214) by transfection in HEK293T cells (FIG. 11B). 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 176nM and 58.7nM, respectively (FIGS. 11C-11D). Notably, BAF170 truncated peptides 1-595 had significantly reduced affinity for WWP2 compared to 1-647 peptide, consistent with our binding experimental results (fig. 1K). Other BAF170 truncated peptides had no detectable binding to WWP2 (fig. 11E-11F). SPR analysis consistently demonstrated that WWP2 interacted with SANT, SWIRM and N-terminal domains of BAF 170.

We performed Surface Plasmon Resonance (SPR) experiments evaluating 10 pre-screened small molecule compounds aimed at finding compounds that could enhance WWP2 binding to BAF170 truncated peptide (1-647). By means of the ternary interaction system we immobilized WWP2 on chip, then mixed candidate compounds (and blank) with BAF170 and incubated the mixture with chip (fig. 12A). Compounds 5, 6 and 7 showed enhanced signal transduction, indicating improved WWP2-BAF170 binding (fig. 12B). Notably, compound 6 exhibited the most robust and consistent signal enhancement, making it the primary candidate for our further investigation (fig. 12C). This compound, designated BFH772, is a known VEGF inhibitor that exhibits a significant effect in inhibiting melanoma growth. Currently BH772 is undergoing clinical trials for the treatment of rosacea, showing good anti-inflammatory effects. However, the potential therapeutic applications of BH772 in cardiovascular diseases, particularly Myocardial Infarction (MI), have not been fully explored.

The three-dimensional structure of the small molecule BFH772 (fig. 12D) was subjected to a virtual screening assay that mimics the binding of BFH772 to BAF170 protein, presented in the form of a surface map (fig. 12E) and a cartoon map (fig. 12F). The interaction between BFH772 and BAF170, through 3D and 2D visualization (FIGS. 12G-12H), reveals multiple binding sites, hydrophobic interactions with residues A870, V871, K874 and A877, hydrogen bonding with K874, pi-pi electron interactions with the A873 amide group, and halogen bonding with residues A877 and E881.

To further validate our findings, we studied the effect of BFH772 on H9C2 cells in vitro. We treated cells with BFH772 (0,0.01,0.1,1,10 and 100 μm) at different concentrations for 48 hours. The results showed that the expression levels of apoptosis markers Cleaved-PARP1 and Cleaved-Caspase3 gradually decreased with increasing doses. The expression of WWP2 peaks at 1 μm BFH772, whereas the expression of BAF170 drops significantly at this concentration and remains stable at higher concentrations. Based on these results, we determined 1 μm as the optimal BFH772 concentration for the subsequent experiments (fig. 13A-13B).

Subsequently, we studied the interaction between WWP2 and BAF170 using H9C2 cells transfected with HA-WWP2 (with or without 1 μm BFH772 treatment). After 48 hours, BFH772 treatment significantly enhanced WWP2 interaction with BAF170 and promoted ubiquitination of BAF170 (fig. 13C-13D). To investigate the effect of BFH772, H9C2 cells were first pre-treated with this compound for 36 hours followed by 12 hours co-treatment with H/SD. The results showed that oxidative stress markers (3-Nitrotyrosine and 8-oxo-dG) were significantly reduced in BFH 772-treated cells, while antioxidant enzyme (SOD 1 and SOD 2) levels were significantly increased compared to control (fig. 13E-13F). In addition, BFH772 treatment resulted in decreased expression of apoptosis markers (BAF 170, cleaved-PARP1 and Cleaved-Caspase 3) and increased expression of protective proteins (WWP 2 and BCL 2) (FIGS. 13G-13H). These findings suggest that BFH772 exerts its cardioprotective effects through a variety of mechanisms, which enhance WWP 2-mediated ubiquitination of BAF170 and its subsequent degradation, reduce oxidative stress, and inhibit mitochondrial apoptosis. Taken together, these effects lead to a significant protection against cardiomyocyte apoptosis even in vitro experiments.

To further verify the role of BFH772 in Myocardial Infarction (MI) in vivo, we established a MI mouse model by ligating the left anterior descending coronary artery for 28 days. BFH772 (20, 30 or 40 mg/kg) was administered at various concentrations by intraperitoneal injection 48 hours after surgery (FIG. 6A). Mice receiving 30 and 40mg/kg BFH772 had improved MI-induced cardiac dysfunction compared to the control group receiving DMSO treatment. This improvement was demonstrated by an increase in ejection fraction (EF%) and shortening fraction (FS%) on days 14 and 28 (fig. 6B-6D). However, the 20mg/kg dose did not show significant therapeutic effect. Furthermore, mice receiving only 30mg/kg and 40mg/kg BFH772 treatment showed a significant decrease in the HW/BW and HW/TL ratios (FIG. 6E).

Oxidative stress-related protein analysis showed that mice treated with 30mg/kg and 40mg/kg BFH772 showed lower levels of 3-Nitrotyrosine and 8-oxo-dG, while SOD1 and SOD2 levels were higher compared to DMSO-treated mice (FIGS. 6F-6G). Furthermore, expression of the mouse mitochondrial apoptosis-related protein BCL2 was significantly increased in mice receiving 30 and 40mg/kg BFH772, indicating improved mitochondrial function (fig. 6H-6I).

Using the Oroboros O K system, we observed that mice treated with 30mg/kg and 40mg/kg BFH772 had significantly higher oxidative phosphorylation capacity of complex I, complex I and complex II, as well as ATP production than the DMSO-treated control and 20mg/kg groups. Complex II oxidative phosphorylation remained unchanged (fig. 6J). Notably, none of these parameters showed statistically significant changes in the sham groups at the different BFH772 concentrations (fig. 6K).

Electron microscopy showed that the sham surgery group exhibited a dense, tight and ordered arrangement of mitochondrial cristae at all BFH772 doses. In contrast, the surgical group had a different degree of mitochondrial injury, manifested as loosening, swelling, destruction and vacuolation of the cristae. Notably, the 30mg/kg and 40mg/kg treatment groups showed significant improvement over the DMSO group (fig. 6L).

The therapeutic effect of BFH772 was further demonstrated by the reduced levels of apoptosis-related proteins (Cleaved-PARP 1 and Cleaved-Caspase 3) in mice treated at doses of 30mg/kg and 40mg/kg (FIGS. 6M-6N). Histological analysis, including H & E, WGA and Masson staining, showed significant improvement in myocardial hypertrophy and fibrosis in these high dose groups compared to DMSO-treated control and 20mg/kg groups (fig. 6O-6Q).

Our research results indicate that BFH772 can enhance the binding capacity of WWP2 to BAF170, promote ubiquitination of BAF170, and inhibit oxidative stress and mitochondrial apoptosis. These mechanisms help to alleviate myocardial remodeling following myocardial infarction, thereby effectively alleviating symptoms of myocardial infarction.

BAF170-K874R inhibits BCL 2 transcription and promotes Caspase3 transcription by binding to enhancers

Total CUT & Tag analysis using BAF170 antibody showed significant peak enrichment in hearts from both BAF170-K874R and WT-BAF170 mice (fig. 9A). The differential analysis showed that 1,281 gene fragments up-regulated and 820 gene fragments down-regulated in the K874R mutant group compared to the wild-type control group (fig. 9B). To elucidate the role of BAF170 ubiquitination, we performed KEGG pathway analysis on differentially expressed genes. Notably, several key apoptosis mediators occur in multiple signaling pathways—caspase3 occurs in the MAPK signaling pathway and the apoptotic pathway, while BCL2 occurs in the PI3K-Akt pathway (fig. 9C). These findings indicate that ubiquitination of BAF170 affects the apoptotic program by regulating transcription of the target gene. Indeed, chIP-seq peak analysis showed that BAF170 had a direct binding site in the regulatory regions of BCL2 and Caspase 3. Specifically, the K874R mutation enhanced the binding of BAF170 to the Caspase3 enhancer, while decreasing the binding to the BCL2 enhancer, which resulted in increased and decreased transcription of these genes, respectively (FIGS. 9D-9E).

TABLE 4 protein Mass Spectrometry results with reduced binding to BAF170 in heart tissue of heart failure mice after myocardial infarction

4. Conclusion(s)

Using high depth proteomics techniques, we found that the SWI/SNF chromatin remodeling complex family, particularly the BAF170 subunit, plays a key role in myocardial infarction. We determined that WWP2 is responsible for modulating the physiological E3 ubiquitin ligase of BAF170 by polyubiquitination at the K874 site, which triggers degradation of BAF 170. Notably, inhibition of this polyubiquitination process by point mutations (BAF 170-K874R) or WWP2 knockdown significantly increased BAF170 expression, exacerbating cardiomyocyte injury following myocardial infarction. Through CUT & Tag experiments, we determined that BCL2 and Cleaved-Caspase3 are novel downstream apoptotic proteins regulated by BAF 170. Our studies reveal a new pattern 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, and apoptosis signaling pathways. Furthermore, these findings also reveal how the mechanism of chromatin remodeling affects cardiovascular health and disease progression.

The present study provides new mechanistic insights by linking the regulation of BAF170, a component of the SWI/SNF chromatin remodeling complex, with Myocardial Infarction (MI) induced continuous apoptosis of cardiomyocytes. By demonstrating the role of WWP 2-mediated ubiquitination in BAF170 degradation, we introduced a new axis of how oxidative stress and mitochondrial dysfunction act synergistically to maintain cardiomyocyte apoptosis. Our findings not only reveal the molecular pathways involved, but also suggest potential therapeutic targets to mitigate sustained apoptosis of cardiomyocytes, which provides a promising strategy for combating the progression of heart failure.

Our studies found that BAF170 expression was significantly increased during cardiac remodeling following myocardial infarction. This increase is associated with three negative effects, exacerbation of mitochondrial dysfunction, increased levels of oxidative stress, and exacerbation of cardiomyocyte apoptosis. These findings mark a significant advance in our understanding of the role of BAF170 in cardiovascular disease.

This study further demonstrates that WWP2 acts as a physiological E3 ubiquitin ligase responsible for the polyubiquitination of BAF170-K874 and its subsequent degradation. It was found that WWP2 mediated BAF170-K874 has impaired ubiquitination and degradation functions during cardiac remodeling following myocardial infarction, leading to BAF170 accumulation. This accumulation causes a series of adverse consequences including increased mitochondrial dysfunction, oxidative stress, and increased cardiomyocyte apoptosis. These findings reveal the mechanism of action of ubiquitination in chromatin remodeling and cardiovascular disease progression.

By elucidating the relationship between chromatin dynamics and apoptosis, our findings open up new research directions for studying the potential role of chromatin remodeling in other cell death pathways, particularly in terminally differentiated cells. This view significantly improves our understanding of the mechanisms of regulation of cell death and indicates direction for future studies in this field.

It is exciting that by extensively screening small molecule compounds, we found that BFH772, a highly selective VEGFR-2 inhibitor, is well known for its remarkable effect in cancer treatment, capable of significantly reducing myocardial cell damage caused by heart failure following myocardial infarction. This finding effectively improves mitochondrial dysfunction, reduces oxidative stress, reduces cardiomyocyte apoptosis, and ultimately reduces fibrosis in myocardial infarction and heart failure. Our study underscores the importance of the WWP2-BAF170 axis in protecting cardiomyocytes from post-myocardial infarction and heart failure injury.

In summary, we have discovered a new ubiquitination-dependent mechanism that regulates chromatin remodeling following myocardial infarction. Our studies show that ubiquitination for BAF170-K874 can be a promising strategy for treating cardiac remodeling following myocardial infarction. In addition, we identified a potential small molecule compound BFH772, which laid a solid foundation for developing targeted therapies for cardiac remodeling following myocardial infarction. This finding opens up new approaches for therapeutic intervention and requires further research in preclinical and clinical settings.

Claims (8)

1. A BAF170 mutant is compared with a wild BAF170, wherein the BAF170 mutant is a K874 locus mutation, and the amino acid sequence of the BAF170 mutant is shown as SEQ ID NO. 1.

2. An isolated nucleic acid molecule encoding the BAF170 mutant of claim 1.

3. A vector comprising the isolated nucleic acid molecule of claim 2.

4. A host cell, wherein the host cell comprises the vector of claim 3.

5. A pharmaceutical composition comprising the BAF170 mutant of claim 1.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable diluent, excipient, and/or carrier.

7. Use of a BAF170 mutant or a pharmaceutical composition of claim 1 in the manufacture of a medicament for the prevention, alleviation and/or treatment of a cardiovascular disease.

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