Fig 1: Cardiomyocyte-secreted HMGB1 participates in macrophage polarization toward M1 phenotype under diabetic condition(A) Correlation graph of relative Hmgb1 expression to Nppb (N = 23 pairs) in C576L/BJ mouse hearts.(B and C) HMGB1 lysine acetylation from C57BL/6J mice (B) immunoprecipitation from hearts, and (C) serum immunoblot with quantification (N = 3–5/group, AU).(D) Immunoblots from hearts and serum from normal subjects and diabetic patients (N = 3–4/group, AU).(E and F) Immunoblot and quantification (E) tissue lysate and culture medium from cultured human hearts; (n = 3, technical repeat); and (F) isolated adult rat cardiomyocytes (ARCMs) (N = 4), stimulated with high fatty acid (500 µM) and high glucose (33 mM) (HFHG).(G) Experimental overview of macrophage differentiation from BM progenitors from C57BL/6J mice. The BM-derived macrophages (BMM) were selected as (Ly6G-F4/80+) and polarization markers include CD206 (M2) and CD86 (M1); gating in Figure S9A).(H) Data showing calculated M1/M2 ratio (normalized to chow, AU) of basal BMM from chow- and HFHSD-fed mice.(I–K) (I) 3D bar plot representing mean %CD206+ and %CD86+ macrophages from HFHSD-fed mice, stimulated with rHMGB1 (500 ng/mL) with and without HFHG (boxed, mean M1/M2 ratio, AU) (N = 3/group). %CD206+ and %CD86+ macrophages from chow-fed mice following (J) IL4 (20 ng/mL), IFN? (50 ng/mL), lipopolysaccharide (LPS) (100 ng/mL), rHMGB1 (500 ng/mL) with and without HFHG stimulation, and (K) culture medium of HFHG stimulated H9C2 with and without glycyrrhizin (Gly).Boxed: Data showing mean M1/M2 ratio (AU) from each experiment (normalized to control, AU). All IP experiments (N = 1/group/cohort). Each data point represents one animal/experiment. All data are presented as mean ± SEM. p values (shown in each panel) versus corresponding controls determined by two-tailed Student's t test in (C–E) and by ANOVA with Tukey's/Sidak's post hoc tests in (B, F, and H–K). Pearson R coefficient analysis in (A).
Fig 2: A retained ER function is a requisite for anti-inflammatory action of vildagliptin under diabetes(A) Representative immunoblot images and quantification of PAK2 activation and CHOP expression from isolated ARCM in the fact of vildagliptin (Vil) (20 µM) with diabetes-mimicking stress (N = 4).(B) Strategy for Vildagliptin administration (5 mg/kg/day in drinking water) with or without application of AAV9-Pak2 in Pak2cKO mice fed with HFHSD.(C and D) (C) BW (D) intraperitoneal glucose tolerance test.(E and F) (E) IVRT from pulse-wave Doppler analysis and (F) calculated FS from M-mode echocardiography (N = 6 in C–F).(G) Representative immunofluorescence staining (scale bar: 25 µm) and quantification of left: Mac3- (red) and right: CD68- (green)-positive non-cardiomyocytes in heart sections. Cardiomyocytes are counterstained with a-actinin (green) and troponin (red). Nuclei are stained with DAPI (blue) (N = 4/group).(H) Relative mRNA expression (normalized to chow, AU) of pro- and anti-inflammatory markers in Pak2cKO hearts (N = 5–6/group).(I) Immunoprecipitation of acetylated HMGB1 from protein lysate. Immunoblot images and quantification of CHOP and HMGB1 (N = 4/group). Gß is loading control.Inset: key for data points; each data point represents one animal/experiment. All data are presented as mean ± SEM. p values (shown in each panel) versus corresponding controls, determined by ANOVA with Tukey's post hoc tests in (A and C–I).
Fig 3: Glycyrrhizin prevents cardiac inflammation and decelerates DCM development by antagonizing extracellular HMGB1(A) Experimental overview of treating with (+Gly) and without (-Gly) glycyrrhizin (150 mg/kg/day in drinking water).(B) Fasted blood glucose and intraperitoneal glucose tolerance test, and bar graph representing AUC (N = 8/group).(C) IVRT (left) and E/A ratio (right) (N = 8/group).(D and E) (D) FS (left) and EF (right), (E) dPW, LVEsD, and relative wall thickness (N = 8/group).(F) Relative mRNA expression of Nppb (-Gly normalized to 1, AU) (N = 5–7/group).(G) Representative immunofluorescence staining (scale bar: 25 µm) and quantification of Mac3- (red) and CD68- (green)-positive non-cardiomyocytes in heart sections. Cardiomyocytes are counterstained with a-actinin (green) and troponin (red). Nuclei are stained with DAPI (blue) (N = 5–6/group).(H) Relative mRNA expression (-Gly normalized to 1, AU) of pro- and anti-inflammatory markers (N = 5–7/group).(I) Representative flow cytometry gating (Figure S14A) for myocardial macrophages (CD45+Ly6G-F4/80+) and histogram displaying CD206 and CD86 macrophage subtypes. %CD206+(left) and %CD86+ (right) macrophages from CD45+Ly6G-F4/80+ cells. Representative histogram displaying derived parameter (M1/M2) vs. unit area (AU), calculated using derive parameter function on FlowJo from corresponding fluorescent channels of M1 (CD86) and M2 (CD206) (N = 4–5/group).Each data point represents one animal. All data are presented as mean ± SEM. p values (shown in each panel) determined by two-tailed Student's t test.
Fig 4: ER dysfunction facilitates cardiac HMGB1 expression and subsequent release(A) Correlation graph of relative Hmgb1 expression to PAK2 (N = 16 pairs) expression in C576L/BJ hearts. (B-C) From Pak2fl/fl and Pak2cKO mice, (B) immunoprecipitation from hearts (N = 5–8/group, AU) for HMGB1 acetylation.(C) serum analyzed by ELISA (N = 4–6/group) and immunoblot (N = 4/group).(D) Representative immunohistochemical images (scale bar: 45 µm) (black square scale bar: 25 µm) of CHOP-stained nuclei (black arrowheads), with quantification graph depicting nuclear H-score from 10 images from PAK2 mice hearts.(E) Immunofluorescence staining (scale bar: 50 µm) showing cellular localization of CHOP (red) and HMGB1 (green) following HFHG in H9C2. Lysosomes and nuclei are stained with LysoTracker (red) and DAPI (blue), respectively, and yellow puncta denote HMGB1 and lysosome colocalization. Relative CHOP nuclear intensity (AU) from 100 nuclei/data point (N = 4).(F) Schematic figure showing CHOP-binding sites in the promoter region (-8000 bp from transcription start site, TSS) in rat, human, and mouse Hmgb1. ChIP performed on H9C2s using anti-CHOP antibody (normalized to the input chromatin, N = 4). Primer pair 1 product sequence is highlighted gray; forward (F) and reverse (R) primer sequences flanking the corresponding region are annotated as Red = Primer one; Orange = Primer 2 and Green = Primer 3.(G) Strategy for macrophages stimulation using the conditional medium culturing H9C2s.(H–L) Immunoblot from H9C2s and conditional medium from (H and I) CHOP overexpression (N = 5) and (K and L) HFHG stimulation following PAK2 knockdown using siPAK2 (N = 3-5 experiments). Conditioned medium was analyzed for release of acetyl-HMGB1. Gß and Coomassie stain are loading controls.(M and N) Representative flow contour plots and quantification of %CD206+ and %CD86+ macrophages with boxed mean M1/M2 ratio for BMM (chow-fed mice) treated with culture medium of H9C2 with (J) CHOP overexpression, (M) siScramble/PAK2 with HFHG (8 h) stimulation, and (N) Ad-Control/Ad-PAK2 with HFHG (12 h) stimulation. All M1/M2 ratios are calculated from mean %CD206+ and %CD86+ cells gated from parent (Ly6G-F4/80+) macrophages. Each data point represents one animal/experiment. All IP experiments (N = 1/group/cohort).All data are presented as mean ± SEM. p values (shown in each panel) versus corresponding controls determined by two-tailed Student's t test in (C, D, F, H, and I) and by ANOVA with Tukey's/Sidak's post hoc tests in (B, C, E, and J–N). Pearson R coefficient analysis in (A).
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