PDCD4 deficiency ameliorates left ventricular remodeling and insulin resistance in a rat model of type 2 diabetic cardiomyopathy

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  Figure 1. Programmed cell death 4 (PDCD4) was involved in the development of diabetic cardiomyopathy (DCM) and its deficiency improves abnormal lipid metabolism and insulin resistance in type 2 diabetic rats. (A, B) Representative images and quantitative analysis of PDCD4 protein expression in the myocardium of control and diabetic rats. (C–D) Representative images and quantitative analysis of PDCD4 protein expression changes in H9c2 cells accompanied by time-dependent high-glucose (HG) stimulation. (E–F) Quantitative analysis of PDCD4 mRNA expression in the myocardium of control and diabetic rats. (G–K) Quantitative analysis of serum total cholesterol (TC) levels, serum triglyceride (TG) levels, serum free fatty acid (FFA) levels, serum fasting blood glucose (FBG) levels, and serum fasting insulin (FNS) levels in four groups of rats. (L) Quantitative analysis of serum insulin sensitive index (ISI) levels in four groups of rats. (M–N) Quantitative analysis of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) between wild-type (WT) and PDCD4-/- rats after ingesting a high-fat diet for 8 weeks. (O–P) Quantitative analysis of IPGTT and the area…

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  Figure 2. Programmed cell death 4 (PDCD4) deficiency can improve left ventricular remodeling and function in type 2 diabetic rats and affects the morphological changes of cardiac myocytes. (A) Quantitative analysis of B-type natriuretic peptide (BNP) mRNA expression between four groups of rats. (B) Representative echocardiographic images of four groups of rats. (B-1) Two-dimensional echocardiogram showing left ventricular long-axis view; (B-2) M-mode echocardiogram showing left ventricular dimensions; (B-3) Pulse-wave Doppler echocardiogram depicting diastolic mitral flow; (B-4) Tissue Doppler echocardiogram displaying mitral annular velocities. (C) Quantitative analysis of left ventricular ejection fraction (LVEF) in four groups of rats. (D) Quantitative analysis of left ventricular fractional shortening (LVFS) in four groups of rats. (E) Quantitative analysis of left ventricular end-diastolic diameter (LVEDD) in four groups of rats. (F) Quantitative analysis of left ventricular end-systolic diameter (LVESD) in four groups of rats. (G) Quantitative analysis of the ratio of early to late diastolic mitral flow velocities (E/A) in four groups of rats. (H) Quantitative analysis of the ratio…

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  Figure 3. Programmed cell death 4 (PDCD4) deficiency attenuates myocardial fibrosis and inflammation in diabetic rats. (A, I-1) Representative images and quantitative analysis of Sirius Red staining of myocardial fibers (scale bar: 100 µm). (B, I-2) Representative images and quantitative analysis of Masson’s trichrome staining of myocardial fibers (scale bar: 100 µm). (B1) Representative images of Masson’s trichrome staining of myocardial matrix fibers (scale bar: 100 µm). (B2) Representative images of Masson’s trichrome staining of perivascular fibers (scale bar: 100 µm). (C–E, I-3–I-5) Representative immunohistochemical staining and quantitative analysis of Collagen I, Collagen III, and transforming growth factor (TGF)-β1 protein expression in the myocardium of four groups of rats (scale bar: 100 µm). (J) Quantitative analysis of Collagen I, Collagen III, and TGF-β1 mRNA expression in the myocardium of four groups of rats. (F–H, I-6–I-8) Representative immunohistochemical staining and quantitative analysis of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 protein expression in the myocardium of four groups of rats (scale bar: 100 µm). (K) Quantitative analysis of TNF-α,…

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  Figure 4. Programmed cell death 4 (PDCD4) expression intervention affects proliferation, transformation, and oxidative stress of cardiac fibroblasts stimulated by HG in vitro. (A) Immunofluorescence analysis of six groups of cells expressing Ki67 (red) and nuclei stained with DAPI (blue) (scale bar: 50 µm). (C-1) Quantification of Ki67-positive cells. (B) Immunofluorescence analysis of six groups of cells expressing S100A4 (red), α-SMA (blue), and nuclei stained with DAPI (blue) (scale bar: 50 µm). (C-2) Quantification of α-SMA and S100A4, both positive cells. (D) Representative dihydroethidium (DHE) staining of apoptosis in six groups of cells (scale bar: 50 µm). (E) Quantification of DHE-positive cells in six groups of cells. Data were expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. DAPI, 4',6-diamidino-2-phenylindole; HG, high glucose; MOCK, mock-vehicle transfection group; NG, normal glucose; OC, osmotic control; SiP, SiRNA-PDCD4; siRNA, small interfer RNA; α-SMA, alpha-smooth muscle actin.

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  Figure 5. Programmed cell death 4 (PDCD4) regulated myocardial apoptosis and PI3K-AKT pathway in vivo and in vitro. (A) Representative TUNEL staining of apoptosis in the myocardium of four groups of rats (scale bar: 50 µm). (B) Quantification analysis of TUNEL-positive cells in four groups of rats. (C) Quantification analysis of flow cytometry results of apoptosis in six groups of cells. (D) Representative western blot of PDCD4, pPI3K, pAKT, PI3K, and AKT expression in six groups of cells. (E) Quantification analysis of PDCD4, pPI3K, and pAKT protein expression in six groups of cells. (F) Representative western blot of pPI3K, PI3K, pAKT, and AKT expression in the myocardium of four groups of rats. (G) Quantification analysis of PDCD4, pPI3K, and pAKT protein expression in the myocardium of four groups of rats. Data were expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. AKT, protein kinase B; DAPI, 4',6-diamidino-2-phenylindole; DM, diabetes mellitus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HG, high glucose; KO, knockout; MOCK, mock-vehicle transfection group; ND, normal diet; NG, normal glucose; OC, osmoticcontrol; PI3K, phosphatidylinositol 3-kinase; SiP, SiRNA-PDCD4; SiRNA,…