MiR-30c/PGC-1β protects against diabetic cardiomyopathy via PPARα

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  Fig. 1. PGC-1β was increased in diabetic heart. a–c KEGG pathway analysis of DEGs between diabetic hearts and normal ones. Genes altered at 3 day (a), 28 day (b), 42 day (c) after diabetes induction were obtained by statistical reanalysis of the GSE4745 data. Bubble diagrams were generated using ggplot2 package. The y-axis shows the name of KEGG pathway; and the enrichment factor in x-axis denotes the ratio of the number of DEGs to the number of all unigenes in each reference pathway. The color of the dot represents p-value, and the size of the dot represents the exact number of DEGs mapped to this pathway. Cardiac tissues used in d–g were from 28-week-old male db/db mice and their C57 littermates. d Western blots and quantitative analysis of PGC-1β in the heart tissues. β-actin was used as an internal control. e Representative images and quantitative analysis of oil red O staining of cardiac lipid deposition (n = 5–8, bar = 50 μm). f Representative images and quantification of DHE staining of cardiac ROS production (n = 5–8, bar = 50 μm). g Representative images and quantification of TUNEL assay determining cell apoptosis in heart tissues (n = 5–8, bar = 100 μm). For d–f, data are…

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  Fig. 2. PGC-1β knockdown relieved high palmitate induced lipotoxicity in vitro. H9c2 cells were transfected with PGC-1β siRNA and then subjected to palmitate (1 mmol/L) stimulation. a Representative Western blots and quantification of PGC-1β, CD36, CPT1B, PDK4, GLUT4 in H9c2 cells with different treatment. β-actin was used as an internal control. b Representative images and quantitative analysis of BODIBY 493/503 fluorescent dye staining of neutral lipid level in cells (bar = 25 μm). c ROS generation in cells was examined by flow cytometry with DHE probe treatment. d ATP content of H9c2 cells with different treatment. e Apoptosis of H9c2 cells was measured by flow cytometry with Annexin V-PE and propidium iodide staining. For all panels, data are representative of three independent experiments and expressed as mean ± SEM, *p < 0.05

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  Fig. 3. PGC-1β expression was reduced by miR-30c. a Venn diagram showing the overlap number of microRNA targeting PGC-1β predicted by TargetScan, PicTar and TarBase websites. b Sequence alignment of miR-30c on the 3′ UTR of PGC-1β from different organisms. c miR-30c expression was determined by RT-PCR in the heart tissues from 28-week old male db/db diabetic mice and their C57 littermates (n = 3). d miR-30c expression was determined by RT-PCR in H9c2 cells with or without palmitate (1 mmol/L) treatment. e Animals (db/db mice and C57 control mice) were injected with the corresponding rAAVs at 8–10 weeks of age and sacrificed at 28 weeks of age (n = 8–10). f H9c2 cells were transfected with miR-30c mimics/inhibitors (or their control) and then subjected to palmitate (1 mmol/L) stimulation. Representative Western blots and quantitative analysis of PGC-1β in the cardiac tissues (e) and H9c2 cells (f). β-actin was used as an internal control. g PGC-1β mRNA level was determined by RT-PCR in the whole RNA and the RNA of the anti-ago2 co-IP complex from H9c2 cells treated with miR-30c mimics or miR-control. h Schematic diagram of luciferase reporter plasmids pMIR PGC-1β 3′ UTR and mutant PGC-1β…

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  Fig. 4. Overexpression of miR-30c alleviated high palmitate induced lipotoxicity in vitro. H9c2 cells were transfected with miR-30c mimics/inhibitors (or their control) and then subjected to palmitate (1 mmol/L) stimulation. a Representative Western blots and quantification of PGC-1β, CD36, CPT1B, PDK4, GLUT4 in H9c2 cells with different treatment. β-actin used as an internal control. b Representative images and quantitative analysis of BODIBY 493/503 fluorescent dye staining of neutral lipid level in cells (bar = 25 μm). c ROS generation in cells was examined by flow cytometry with DHE probe treatment. d ATP content of H9c2 cells with different treatment. e Apoptosis of H9c2 cells was measured by flow cytometry with Annexin V-PE and propidium iodide staining. For all panels, data are representative of three independent experiments and expressed as mean ± SEM, *p < 0.05

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  Fig. 5. Overexpression of miR-30c attenuated cardiac dysfunction in db/db mice. Db/db mice and C57 control mice were injected with the corresponding rAAVs at 8–10 weeks of age and then sacrificed at 28 weeks of age (n = 8–10). a miR-30c expression was determined by RT-PCR in the heart tissues of mice. b Echocardiographic detection of mice. c Hemodynamic analysis measured by Millar cardiac catheter system of mice. d Representative images and quantitative analysis of oil red O staining of cardiac lipid deposition (bar = 50 μm). e Representative images and quantitative analysis of lipid droplets from cardiac tissues analyzed by transmission electron microscopy (bar = 2 μm). f Representative images and quantification of DHE staining of cardiac ROS production (bar = 50 μm). g Representative images and quantification of TUNEL assay determining cell apoptosis in heart tissues (bar = 100 μm). H. Representative Western blots and quantification of CD36, CPT1B, PDK4, GLUT4 in heart tissues with different treatment. β-actin used as an internal control. Data are expressed as mean ± SEM, *p < 0.05. EF ejection fraction, FS fractional shortening, dP/dtmax peak instantaneous rate of left ventricular…

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  Fig. 6. PGC-1β knockdown blocked PPARα transcriptional activity in diabetic heart. a Immunoprecipitation with the anti-PPARα (left) or anti-PGC-1β antibody (right) in H9c2 cells, followed by Western blotting with antibodies for anti-PPARα and anti-PGC-1β. b ChIP PCR for analysis of PGC-1β binding activity on the CD36 and PDK4 promoter in H9c2 cells treated with PPARα siRNA or siRNA control. Primers were designed according to PPARα/RXR binding site. c Effect of PGC-1β or miR-30c on PPARα transcriptional activity was evaluated by luciferase reporter assays in H9c2 cells. Cells were first transfected with PGC-1β or miR-30c, and then transfected with pTK-PPREx3-Luc plasmid. d Effect of miR-30c on PPARα transcriptional activity after PGC-1β inhibition was determined by luciferase reporter assays in H9c2 cells. Cells were transfected with miR-30c after PGC-1β inhibition by siRNA, and then transfected pTK-PPREx3-Luc plasmid. e Schematic representation of the association among miR-30c, PGC1β, and abnormality in diabetic heart. In cardiomyocyte of diabetic heart, miR-30c was decreased due to high fatty acid (FA). The loss of miR-30c resulted in PGC1β activation, which enhanced the PPARα…