P300-Targeted Acetylome Reveals a Role for HMGB1 Translocation in Central Cardiac Sympathetic Activation Post Myocardial Infarction

Figures

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  Figure 1. Expression profile of the p300 in PVN post-MI. (A) A summary heatmap of quantitative RT-PCR analysis of acetyltransferase. n = 3 per group. (B and C) Western blot showing the expression profiles of p300 (300 kD) in PVN at 0 hr, 1-, 3-, 5-, and 7-days post‐MI. P300 was quantified relative to the β-tubulin (55 kD) levels. n = 4 per group and per time‐point. (D) Double‐immunostaining for Iba1 (green) and p300 (red) in the vehicle‐MI group shows a limited distribution of p300 on Iba1 in the PVN. Bar = 30 μm. (E and F) Qualification of Iba1 positive and p300 positive Iba1 cells in PVN, respectively n = 6 per group. T test and two-way analysis of variance (ANOVA) were applied for two groups and four groups analysis respectively. Each column with a bar represents the mean ± standard deviation. *P < 0.05 and **P < 0.01 vs 0 hr.

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  Figure 2. Targeted p300 knockdown in PVN. (A) Sketch map of targeting virus injection into PVN using stereotaxic apparatus. (B) Image confirming PVN targeted virus transfection into microglia. (C) Representative expression of p300 at 7 days post‐MI as determined via Western blotting. (D) P300 expression was quantified relative to the β-tubulin (55 kD) levels. (E) Representative original color images of Masson’s trichrome staining of the infarcted area: (i) sham group, (ii) sham + shCtrl, (iii) MI + shp300 and (iv) MI + shCtrl. (F) Infarction size calculation by Image J. (G) Representative images of cardiac function recorded by M-mode echocardiography. Ejection Fraction (EF) calculation (H) and fractional shortening (FS) calculation (I). n = 6 per group. Two-way ANOVA was applied for statistical analysis. Each column with a bar represents the mean ± SD. *P < 0.05, **P < 0.01 vs sham + shCtrl group; # P < 0.05 vs MI + shCtrl group.

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  Figure 3. Effects of p300 on sympathetic nerve activity and programmed electrical stimulation at 7 days post-MI. (A) Representative immunohistochemical images of central sympathetic nerve activity marked using Fos family proteins, indicated by brown puncta. Nuclei (blue) were stained with hematoxylin: (i) sham + shCtrl, (ii) sham + shp300, (iii) MI + shCtrl, and (iv) MI + shp300 groups (magnification 200×). Bar = 50 μm. (B) Typical recordings from the left RSNA and integrated RSNA at 7 days post-MI in (i) sham + shCtrl, (ii) sham + shp300, (iii) MI + shCtrl, and (iv) MI + shp300 groups. Bar = 1s. (C) Typical inducible ventricular arrhythmias. (D) Quantitation of Fos‐positive cells. (E) Quantification of baseline of RSNA. (F) Quantification of ventricular arrhythmia scores. (F) Comparisons of the arrhythmia scores across four groups. The vertical bar = 1mV, abscissa bar = 0.5s. (G) Myocardial NE levels detected by ELISA. n = 6 per group. Two-way ANOVA was applied for statistical analysis. Data are expressed as mean ± S.D. **P < 0.01 and *P < 0.05 vs the sham group. #P < 0.05 vs the MI + shCtrl groups.

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  Figure 4. Identification of p300-dependent acetylome. (A) (upper panel); (lower panel) Ice logo shows analysis of the frequency of amino acids surrounding the acetylated lysines targeted by p300. Overall, we analyzed 159 distinct “Ks” derived from a list of 135 proteins, with 10 aa upstream (−10 on the x-axis) and 10 aa downstream (+10 on the x-axis). The overall height of the stack indicates the sequence conservation at that position, while the symbol height within the stack indicates the relative frequency of each amino acid at that position. (B) Go enrichment analysis of proteins acetylated by p300 in biological process, cellular component and molecular function. (C) Based on GO enrichment analysis, the cnetplot of proteins acetylated by p300 suggests HMGB1 is involved in the protein transport process. (D) Motif logo map depicting p300 binding protein in NC and KD groups.

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  Figure 5. The effect of p300 shRNA intervention on HMGB1 levels, intracellular distribution, and acetylation in the PVN of MI rats. (A) Immunofluorescence costaining for HMGB1 (cyan) with Iba1 (green) and p300 (red) of PVN 7 days after MI. (B) Representative Western blot illustrating cytoplasm/nucleus/acetylation HMGB1 protein. (C) Qualification of acylation level of HMGB1. (D and E) Qualification of p300 positive cells and nucleus/cytoplasm HMGB1 percentage in PVN, respectively. (F and G) Qualification of cytoplasm and nucleus HMGB1 level, respectively. Bar = 30 μm. n = 6 per group. Two-way ANOVA was applied for statistical analysis. Values are expressed as the mean ± SD. *P < 0.05 and **P < 0.01 vs the sham group; # #P < 0.01, #P < 0.05 vs the MI + shCtrl groups.

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  Figure 6. P300 acetylates HMGB1 at its K43 residue. (A) Co-transfection of Myc/Flag-tagged p300 into HEK293T cells with Flag-tagged HMGB1-A box or Flag-tagged HMGB1-B box. Immunoprecipitation was performed using an anti-Myc antibody, and Western blots were analyzed using the indicated antibodies. Cell lysates were immunoprecipitated with an anti-Myc antibody and subsequently analyzed using Western blotting. (B) Co-transfection of Myc-tagged p300 with HMGB1 truncation mutants or wild-type HMGB1 into HEK293T cells in the presence of HDAC inhibitors. Immunoprecipitation was performed using an anti-myc antibody and then analyzed using the specified antibodies. (C) Immunostaining for HMGB1 (green) and DAPI (blue) in the p300 /HMGB1 coinfection group (upper) and the p300/HMGB1 K43R mutant coinfection group (lower). Bar = 10 μm. (D) Representative Western blot illustrating cytoplasm/nucleus HMGB1 protein. (E and F) Qualification of cytoplasm and nucleus HMGB1 level, respectively. (G) ELISA for detecting HMGB1 content in cell culture supernatant in the p300 /HMGB1 coinfection group (upper panel) and p300/HMGB1 K43R mutant coinfection group. n = 4 per group. T test was applied for statistical…

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  Figure 7. P300 knockdown diminished the increase in NMDA-mEPSCs post-MI. (A) Western blot showing the expression profiles of GluN1 (105kD) and GLuN2B (166kD) in PVN at day-7 post‐MI. GluN1 and GluN2B expression were quantified relative to the β-tubulin (55 kD) levels. (B and C) Statistical bar graph of relative GluN1 and GLuN2B expression in Sham+shCtrl, Sham+shP300, MI+shCtrl, and MI+shP300 groups. (D) Representative single mEPSC currents of brain slices from Sham+shCtrl, Sham+shP300, MI+shCtrl, and MI+shP300 groups. (E) Statistical bar graph of normalized NMDAR-mEPSC current amplitude in the four groups. (F and G) Statistical bar graph of normalized NMDAR-mEPSC current frequency and interevent intervals in the four groups. n = 5 per group. *P < 0.05 vs the Sham+shCtrl group; # P <0.05 vs the MI+shCtrl group.

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  Figure 8. HMGB1 promotes NMDA-mEPSCs in PVN neurons. (A) Double‐immunostaining for HMGB1 (green) and NMDAR1 (red) in the vehicle‐MI group. (B) Quantification of HMGB1 positive NMDAR1 cells in the PVN. Bar = 50 μm. n = 6 per group. **P < 0.05 vs sham group. (C) The structural diagram illustrates the optimal docking between the HMGB1 ligand (purple) and NMDAR receptor molecule (yellow band:GluN1, the other is GluN2B) is as follows: (a) The enlarged docking site of the optimal combination; (b) Receptor residues bound in the optimal structural diagram (overall, 43 amino acid residues); (c) Surface interaction in the optimal docking structure diagram, with blue representing positively charged surface areas, red indicating negatively charged surface areas, and green representing hydrophobic surface areas. (D) Recordings of representative spontaneous mEPSCs from PVN neurons before and after HMGB1 protein application via voltage-clamp in whole-cell recording mode, with a membrane potential held at −70 mV. (E) The statistical bar graph shows that HMGB1 significantly increased the amplitude and current numbers of mEPSCs without affecting the half-width of the mEPSCs in PVN neurons. (F)…