Dapagliflozin Attenuates Pericardial Adipose Tissue–Derived Leptin‐Mediated Myocardial Remodeling in Obese Rats

Figures

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  Figure 1. Dapagliflozin improves myocardial remodeling in obese rats. A, Hematoxylin and eosin and Masson staining of myocardial sections from the 4 experimental groups. B, Quantification of myocardial collagen volume fraction. C–H, Western blotting analysis of collagen I, collagen III, α‐SMA, MMP‐1, and TIMP‐1. I–K, Western blotting analysis of phosphorylated p38MAPK and PCNA. L–P, Western blotting for cytochrome C, cleaved‐caspase3, Bax, and Bcl2. Q, R, Representative TdT‐mediated dUTP nick‐end labeling staining and quantification of apoptotic cardiomyocytes. For B and R, n=5/group; for D–H, J, K, and M–P, n=6/group. Data are presented as mean±SD. DAPA indicates dapagliflozin; HFD, high‐fat diet; MMP‐1, mitochondrial membrane potential‐1; ND, normal diet; p38MAPK, p38 mitogen‐activated protein kinase; PCNA, proliferating cell nuclear antigen; ɑ‐SMA, ɑ‐smooth muscle actin; and TIMP‐1, tissue inhibitor of metalloproteinases‐1.

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  Figure 2. dapagliflozin inhibits the oxidative stress in the hearts of obese mice and regulates the expression of NKA. A, DCFH‐DA fluorescence staining of myocardial sections from the 4 experimental groups. B, Quantitation of DCFH‐DA fluorescence intensity. C–D, Myocardial SOD and malondialdehyde levels. E, NKA activity in cardiac tissue. F, Immunohistochemical staining of NKA α1 and Src in myocardial sections. G, H, Quantitative analysis of immunohistochemical staining expressed as AOD. I–K, Western blotting of NKA α1 and Src. For B, G, and H, n=5/group; For C and D, n=11/group; for E, J, and K, n=6/group. Data are presented as mean±SD. AOD indicates average optical density; DAPA, dapagliflozin; DCFH‐DA, 2′,7′‐dichlorodihydrofluorescein diacetate; HFD, high‐fat diet; MDA, malondialdehyde; ND, normal diet; NKA, Na+‐K+‐ATPase; and SOD, superoxide dismutase.

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  Figure 3. Dapagliflozin reduces and promotes the differentiation of PAT in obese rats. A, Representative images of hearts and PAT from the four experimental groups. B, Representative hematoxylin and eosin staining of PAT sections. C, Quantification of PAT mass. D–E, Quantitative analysis of adipocyte number and adipocyte area in PAT. F–I, Western blotting for FAS, FABP4, and CEBP α of PAT. For C, n=10/group; for D and E, n=5/group; for G–I, n=6/group. Data are presented as mean±SD. CEBP α indicates CCAAT/enhancer‐binding protein α; DAPA, dapagliflozin; FABP4, fatty acid–binding protein 4; FAS, fatty acid synthase; HFD, high‐fat diet; ND, normal diet; and PAT, pericardial adipose tissue.

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  Figure 4. PAT‐derived leptin promotes myocardial injury. A, Representative images of C57BL/6J and ob/ob mice under normal diet and high‐fat diet conditions. B, Representative images of hearts and PAT from C57BL/6J and ob/ob mice. C, Masson staining of myocardial sections from the 4 experimental groups. D, Quantification of myocardial collagen volume fraction. E–H, Western blotting for collagen III, collagen I, α‐SMA in cardiac fibroblasts cultured in PAT‐CM from C57BL/6J and ob/ob mice. I–L, Western blotting for cleaved‐vasepase3, casepase3, Bax, Bcl2, and cytochrome C in cardiomyocytes cultured in PAT‐CM from C57BL/6J and ob/ob mice. PAT indicates pericardial adipose tissue. For D, n=5/group; for F–H and J–L, n=3/group. Data are presented as mean±SD. HFD indicates high‐fat diet; ND, normal diet; PAT, pericardial adipose tissue; PAT‐CM, pericardial adipose tissue conditioned medium; and ɑ‐SMA, ɑ‐smooth muscle actin.

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  Figure 5. Dapagliflozin attenuates myocardial remodeling by reducing PAT‐derived leptin. A–C, Western blotting analysis for p‐p38MAPK, p38MAPK, and PCNA in cardiac fibroblasts following PAT‐CM intervention. D–G, Western blotting analysis for collagen III, collagen I, and α‐SMA in cardiac fibroblasts following PAT‐CM intervention. H and I, Western blotting analysis of NKA α1 in cardiac fibroblasts. J, Activity of NKA α1 in cardiac fibroblasts after PAT‐CM intervention. K, L, Representative DCFH‐DA staining and quantification in cardiac fibroblasts after PAT‐CM intervention. M–P, Western blotting analysis for cytochrome C, cleaved‐caspase3, Bax, and Bcl2 in cardiomyocytes. Q, R, Representative MMP and quantification in cardiomyocytes. S, T, Representative mPTP and quantification in cardiomyocytes after PAT‐CM intervention. U–W, Western blotting analysis of NKA α1 in cardiomyocytes. For L, R, and T, n=5/group; for B, C, E–G, I, J, N–P, V, and W, n=3/group. Data are presented as mean±SD. CM indicates conditioned medium; DAPA, dapagliflozin; DCFH‐DA, 2′,7′‐dichlorodihydrofluorescein diacetate; HFD, high‐fat diet; MMP, mitochondrial membrane potential; mPTP, mitochondrial permeability transition…

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  Figure 6. Dapagliflozin reduces leptin release by activating the PI3K/AKT pathway and inhibiting the ERK1/2 signaling pathway. A–E, Western blotting analysis for p‐PI3K, PI3K, p‐AKT, AKT, p‐ERK1/2, ERK1/2, and leptin after dapagliflozin intervention on pericardial adipocytes. F–O, Western blotting analysis for p‐PI3K, PI3K, p‐AKT, AKT, p‐ERK1/2, ERK1/2, and leptin in different intervention groups. For B–E, G–J, and L–O, n=3/group. Data are presented as mean±SD. AKT indicates protein kinase B; CON, control group; DAPA indicates dapagliflozin; ERK1/2, extracellular signal‐regulated kinase 1/2; IR, insulin resistance; p‐AKT, phosphorylated protein kinase B; p‐ERK1/2, phosphorylated extracellular signal‐regulated kinase 1/2; PI3K, phosphoinositide 3‐kinase; p‐PI3K, phosphorylated phosphoinositide 3‐kinase.

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  Figure 7. Dapagliflozin improves leptin‐induced myocardial fibrosis by blocking the ROS/NKA/Src oxidative stress loop. A–F, Western blotting analysis for collagen I, α‐SMA, JAK2, p‐JAK2, STAT3, p‐STAT3 and NKA α1 in 4 groups of cardial fibroblasts. G, Representative DCFH‐DA staining and quantification in cardiac fibroblasts. H, Representative immunofluorescence staining and quantification of Collagen I and NKA α1 in cardiac fibroblasts. I–M, Western blotting analysis for collagen I, STAT3, p‐STAT3, NOX4, and SOD2 in cardiac fibroblasts treated with dapagliflozin, S3I‐201 and leptin. N–S, Western blotting analysis for collagen I, NOX4, NKA, p‐Src, Src, p‐ERK1/2, and ERK1/2 in cardiac fibroblasts treated with dapagliflozin, N‐acetylcysteine, pNaKtide, PP2, PD98059 and leptin. For G and H, n=5/group; For B–F, J–M and O–S, n=3/group. Data are presented as mean±SD. DAPA indicates dapagliflozin; DCFH‐DA, 2′,7′‐dichlorodihydrofluorescein diacetate; JAK2, Janus kinase 2; NAC, N‐acetylcysteine; NKA, Na+‐K+‐ATPase; PD98059, inhibitor of ERK1/2; p‐JAK2, phosphorylated Janus kinase 2; PP2, inhibitor of Src; p‐STAT3, phosphorylated signal transducer and activator of transcription 3; ROS, reactive…

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  Figure 8. dapagliflozin attenuates leptin‐induced myocardial apoptosis by blocking the ROS/NKA/Src oxidative stress loop and suppressing the STAT3/Bax signaling. A–D, Western blotting analysis of cytochrome C, cleaved‐caspase3, Bax, and Bcl2 in cardiomyocytes treated with dapagliflozin and leptin. E, F, Western blotting analysis of NKA α1 in cardiomyocytes treated with dapagliflozin and leptin. G–I, Western blotting analysis of p‐JAK2, JAK2, p‐STAT3, and STAT3 in cardiomyocytes. J, Representative DCFH‐DA staining in cardiomyocytes treated with dapagliflozin, colivelin, and leptin. K–O, Western blotting analysis of NKA α1, Src, p‐Src, NOX4, and cytochrome C in cardiomyocytes treated with dapagliflozin, N‐acetylcysteine, pNaKtide, PP2, and leptin. P, Q, MMP and mPTP of cardiomyocytes treated with dapagliflozin, colivelin and leptin. R–U, Western blotting analysis of Bax, cytochrome C, cleaved‐caspase3 of cardiomyocytes treated with dapagliflozin, colivelin, and leptin. For G, M, and U–V, n=5/group; For B–D, F, H, I, L–O and S–U, n=3/group. Data are presented as mean±SD. DAPA indicates dapagliflozin; JAK2, Janus kinase 2; MMP, mitochondrial membrane potential; mPTP, mitochondrial permeability…