Inflammatory Serine Proteases Play a Critical Role in the Early Pathogenesis of Diabetic Cardiomyopathy

Figures

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  Fig. 1.. STZ treatment increases leukocyte infiltration, DPPI expression and activation. LV sections from animals treated with citrate buffer or STZ for 4–20 weeks were assessed for anti-DPPI immunostaining (400X magnification with scale bars 50 μm) (A) and quatification (B), DPPI activity as determined by specific fluorogenic substrates (C), and DPPI immunoblot analysis (D). Double immunistaining with DPPI and NIMP-R14 (neutrophil), chymase (mast cells), or granzyme B (cytotoxic T cells) antibodies (400X magnification with scale bars 50 μm) (E) and quantification (F), respectively. n=5 for each group *=p< 0.05 vs control. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups. Two-way ANOVA and subsequent Tukey test were performed to compare groups with different time points.

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  Fig. 2.. DPPI deletion reduces ISP activity and inflammation after diabetes induction. DPPI (A), cathespin G (CG) (B), chymase (C), and granzyme B (D) activity levels in the left ventricular heart as determined by enzymatic activity assay (n=5 for each group). (E) Representative NIMP-R14, chymase, and granzyme B immunstaining of paraffin-embedded heart sections of WT and DPPI subjected to diabetes induction (400X magnification with scale bars 50 μm). (F) Quantification of NIMP-R14, chymase, and granzyme B-positive cells (n=5 for each groups). Real-time qPCR analysis of IL-6 (G) and TNF-α (H) in controls and after diabetes induction in WT and DPPI-KO mice heart (n=5 for each groups). The data were normalized to GAPDH and were expressed as mean ± SEM. *P<0.05 vs. control, #P<0.05 vs. STZ-treated WT. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups.

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  Fig. 3.. DPPI deletion improves the metabolic profile of diabetic mice. (A) Blood glucose levels. (B) Hemoglobulin A1c. Data are expressed as means ± SEM (n=10–15; only 5 animals are shown in each group).*=p< 0.05 vs control and #=p<0.05 vs STZ-treated WT. Oneway ANOVA followed by the Tukey post hoc test was used to compare multiple groups.

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  Fig. 4.. DPPI deletion improve cardiac function and remodeling induced by T1DM after STZ injection independently of hyperglycemia. (A-F) Echocardiography measurement of left ventricular (LV) ejection fraction (A), fractional shortening (B), E/A ratio (C), LV end diastolic (D) and systolic (E) dimension, and LV end diastolic dimension to wall thickness ration (LVEDD/PWT) ratio (F) in control and STZ-induced diabetic animals. (G-I) Expression of cardiac remodeling markers, atrial natriuretic peptide (ANP), B-type natriuretic peptite (BNP) hypertophic markers, and beta myosin heavy chain (β-MHC). (J) Representative images of WGA staining in the controls and diabetic hearts (400X magnification with scale bars 50 μm). (HK) Quantitative analysis of myocyte cross-sectional area. Data are expressed as means ± SEM (n=10–15; only 5 animals are shown in each group).*=p< 0.05 vs control and #=p<0.05 vs STZ-treated WT. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups.

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  Fig. 5.. DPPI deletion attenuated fibrosis induced by T1DM. (A) Histological sections of left ventricular cardiac staining by picrosirius red and semiquantitative analysis was done by a computer imaging system (400X magnification with scale bars 50 μm). (B-G) cardiac mRNA expression of fibronectin (B), collagen I (C), collagen IV (D), TGF-β (E), connective tissue growth factor (CTGF) (F), and MMP9 (G). Data are presented as mean ± SEM. n=5 for each group. *=p< 0.05 vs control and #=p<0.05 vs STZ-treated WT. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups.

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  Fig. 6.. Myocardial cell apoptosis assessed by TUNEL staining and cardiac caspase-3 protein expression before and after diabetes induction. (A) Left ventricular (LV) tissue sections were assessed for apoptosis with the use of the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (green), tropomyosin (red), and DAPI (4′,6-diamidino-2-phenylindole; blue) staining and the number of TUNELpositive myocytes was expressed as a percentage of total nuclei detected by DAPI staining (600X magnification with scale bars 40 μm). (B) Quantification of caspase-3 activity in LV with the use of caspase-3–specific fluorogenic substrate. RFU indicates relative fluorescence units. Immunoblot analysis and quantification of cardiac expression of Bax (C & D), Bcl-2 (C & E) and cleaved caspase-3 (C & F). (G) Bax/Bcl-2 ratio. Data are presented as mean ± SEM. n=5 for each group. *=p< 0.05 vs control and #=p<0.05 vs STZ-treated WT. Oneway ANOVA followed by the Tukey post hoc test was used to compare multiple groups.

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  Fig. 7.. Signal transduction mechanisms involved in the induction of apoptosis by inflammatory serine protease. (A) Representative Immunoblots of LV lysates from control and diabetic WT or DPPI-KO animals. (A) Top, Immunoblots analysis of phosphor-AKTSer473, (B) phopsho-p38hr180/182, (C) GRP78, and (D) CHOP expression in the LV heart tissue. Botton, Quantification of the experiments represented as fold change compared to WT animals Data are expressed as means ± SEM (n=5).*=p< 0.05 vs control and #=p<0.05 vs STZ-treated WT. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups.