Paracrine signal emanating from stressed cardiomyocytes aggravates inflammatory microenvironment in diabetic cardiomyopathy

Figures

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  Figure 1. Time-course analyses exhibit cardiac dysfunction associated with maladaptive ER stress response following high-fat high-sucrose diet (HFHSD) (A) Representative images and quantification graphs (protein/Gβ ratio, AU) from densitometry analysis of PAK2 expression and phosphorylation (pPAK2) and ER stress markers from heart lysates of human subjects. ER stress markers include GRP94, GRP78, phosphorylated IRE1, IRE1, spliced XBP1 (XBP1s), unspliced XBP1 (XBP1u), ATF6, phosphorylated PERK (pPERK), PERK, ATF4, and CHOP. (B–D) Data graphs showing (B) serum insulin, (C) fasted blood glucose, and (D) intraperitoneal glucose tolerance test after 8–24 weeks of feeding in C57BL/6J mice and bar graph displaying AUC. (E) Graphs depicting isovolumetric relaxation time (IVRT) and E/A mitral valve flow from pulse-wave Doppler imaging indicating diastolic function. (F) From M-mode echocardiography: fractional shortening (FS%) and ejection fraction (EF%) indicating systolic function. (G) Relative mRNA expression of Nppb (chow normalized to 1, AU, arbitrary unit). (H) Immunoblots and quantification graphs (protein/Gβ ratio, AU) from densitometry analysis of key ER stress transcription factors from…

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  Figure 2. Perturbed ER homeostasis exacerbates myocardial inflammatory reaction in the diabetic myocardium (A and B) Representative immunofluorescence staining (scale bar: 25 μm) of (A) Mac3- (red) and B CD68- (green)-positive non-cardiomyocytes in heart sections of C57BL/6J mice fed with HFHSD indicating myocardial inflammation. Nuclei are stained with DAPI (blue). Cardiomyocytes are counterstained with α-actinin (green) in (A) and with troponin (red) in (B). (C) Relative mRNA expression (normalized to chow, AU) of pro- and anti-inflammatory markers from C57BL/6J hearts. Inset: key for data points; each data point represents one animal. N = 4–8/group. 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.

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  Figure 3. PAK2 deficiency-induced maladaptive cardiac ER stress-associated myocardial inflammation and cardiac dysfunction in diabetes (A) Correlation between Nppb transcript and PAK2 protein level in C57BL/6J mice hearts (N = 15 pairs). (B and C) Graphs depicting (B) IVRT and E/A mitral valve flow from pulse-wave Doppler imaging and (C) JT segment from ECG. (D–G) From M-mode echocardiography (D) calculated FS% and EF%, (E) LV-end diastolic diameter (LVEdD), (F) relative wall thickness (RWT), and (G) diastolic posterior wall (dPW) thickness; N = 5–9/group in (B–G). (H) Immunoblot images of ER stress markers from heart lysates of Pak2fl/fl and Pak2cKO mice. Images are representative of N = 5–8/group. Changes of XBP1s and CHOP (N = 5–11/group) displaying maladaptive ER stress response. Gβ is loading control. (I and J) Representative immunofluorescence staining (scale bar: 25 μm) of (I) Mac3- (red) and (J) CD68- (green)-positive non-cardiomyocytes in heart sections of Pak2fl/fl and Pak2cKO mice fed with HFHSD indicating myocardial inflammation. Nuclei are stained with DAPI (blue). Cardiomyocytes are counterstained with α-actinin (green) in (I) and with troponin (red) in (J); (N = 6/group).…

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  Figure 4. 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…

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  Figure 5. 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 =…

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  Figure 6. 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 α-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). Gβ is…

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  Figure 7. 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 α-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…