Kir6.1 improves cardiac dysfunction in diabetic cardiomyopathy via the AKT‐FoxO1 signalling pathway

Figures

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  FIGURE 1. Cardiac and mitochondrial function are decreased in DCM mice and in insulin‐resistant NRVMs. A, The cardiac function of mice was determined by echocardiography. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. Values are expressed as the mean ± SEM. N = 6 mice in each group. P‐values from unpaired Student's t‐test: **P < .01 vs. control. B, Expression of brain natriuretic peptide (BNP) in the serum was analysed by ELISA. Values are expressed as the mean ± SEM. N = 8 mice. P‐values from unpaired Student's t‐test: **P < .01 vs. control. C, Cardiac histology. Representative transverse section from ten mice of left ventricle stained with haematoxylin and eosin (Scale bar = 50 μm/20 μm). For quantification, cell area measurements were performed on five similar sections (Scale bar = 50 μm), and 100 nucleated cells were randomly selected for measuring the mean cell area. Values are expressed as the mean ± SEM. N = 10 mice. P‐values from unpaired Student's t‐test: ***P < .001 vs. control. D, Apoptosis was detected by the TUNEL assay. Representative images from six animals for each group. The number of apoptotic cells in similar sections were counted…

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  FIGURE 2. Kir6.1 in HFD&STZ‐induced type 2 diabetic mice and in chronic insulin‐resistant NRVMs determined by qRT‐PCR and Western blotting. A, B, Kir6.1 in mouse cardiac tissue. N = 6 mice in each group. C, D, Kir6.1 in NRVMs. N = 6 independent experiments in each group. Values are expressed as mean ± SEM P‐values from unpaired Student's t‐test: **P < .01 vs. control, ***P < .001 vs. control

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  FIGURE 3. Kir6.1 overexpression improves cardiac dysfunction in DCM mice and in insulin‐resistant NRVMs. A, Kir6. 1 expression in mice was confirmed by Western blotting. Values are expressed as the mean ± SEM. N = 6 mice in each group. P‐values from unpaired Student's t‐test: ***P < .001 vs. control. B, Kir6.1 expression in NRVMs was confirmed by Western blotting. Values are expressed as the mean ± SEM. N = 6 independent experiments in each group. P‐values from unpaired Student's t‐test: ***P < .001 vs. control. C, Cardiac function in mice was determined by echocardiography. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. Values are expressed as the mean ± SEM. N = 6 mice. P‐values from ANOVA: **P < .01 vs. control, #P < .05 vs. DCM group. D, Brain natriuretic peptide (BNP) expression in the serum was analysed by ELISA. Values are expressed as the mean ± SEM. N = 6 mice. P‐values from ANOVA: ***P < .001 vs. control, ###P < .001 vs. DCM group. E, Cardiac histology. Representative transverse section from ten mice of left ventricle stained with haematoxylin and eosin (Scale bar = 20 μm). For quantification, cell area measurements were performed on five…

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  FIGURE 4. Cardiac‐specific Kir6.1 knockout aggravates cardiac dysfunction in diabetic mice. A, The Kir6.1 mRNA expression in the heart of mice was examined by qRT‐PCR. Values are expressed as the mean ± SEM. N = 6 mice in each group. P‐values from unpaired Student's t‐test: ***P < .001 vs. control. B, The Kir6.1 protein expression in the heart of mice was examined by Western blot analysis. Values are expressed as the mean ± SEM. N = 6 mice. P‐values from unpaired Student's t‐test: ***P < .001 vs. control. C, Mouse cardiac function was determined by echocardiography. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. Values are expressed as the mean ± SEM. N = 6 mice. P‐values from ANOVA: **P < .01 vs. control, ##P < .01 vs. DCM group. D, Brain natriuretic peptide (BNP) expression in the serum was analysed by ELISA. Values are expressed as the mean ± SEM. N = 8 mice. P‐values from ANOVA: ***P < .001 vs. control, ##P < .01 vs. DCM group. E, Cardiac histology. Representative transverse section from ten mice of left ventricle stained with haematoxylin and eosin (Scale bar = 50 μm/20 μm). For quantification, cell area measurements were performed on five…

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  FIGURE 5. Effect of Kir6.1 on the AKT‐FoxO1 signalling pathway in DCM. A, Western blotting was performed to quantify the expression levels of Kir6.1, p‐AKT and p‐FoxO1 in the heart of mice. Values are expressed as the mean ± SEM. N = 5 mice in each group. P‐values from ANOVA: ***P < .001 vs. control, ###P < .001 vs. DCM group. B, Western blotting was performed to quantify the expression levels of Kir6.1, p‐AKT and p‐FoxO1 in the heart of mice. Values are expressed as the mean ± SEM. N = 5 mice. P‐values from ANOVA: ***P < .001 vs. control, ###P < .001 vs. DCM group. C, Western blotting was performed to quantify the expression levels of Kir6.1, p‐AKT and p‐FoxO1 in NRVMs. Values are expressed as the mean ± SEM. N = 5 independent experiments in each group. P‐values from ANOVA: ***P < .001 vs. control, ##P < .01 vs. Insulin group, ###P < .001 vs. Insulin group

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  FIGURE 6. Effect of the AKT‐FoxO1 signalling pathway in regulating Kir6.1 expression and mitochondrial function in cardiomyocytes. A, Western blotting was performed to quantify the expression levels of Kir6.1, p‐AKT and p‐FoxO1 in cardiomyocytes. Values are expressed as the mean ± SEM. N = 5 independent experiments in each group. P‐values from ANOVA: ***P < .001 vs. control, ###P < .001 vs. MK‐2206&INS group. B, Determination of ΔΨm in the three groups of cardiomyocytes by the fluorescent dye, JC‑1 (Scale bar = 200 μm). C, Comparison of the ΔΨm among the three groups of cardiomyocytes. For quantification, 20 cells were randomly selected to calculate the ΔΨm levels by comparing red fluorescent intensity to green fluorescent intensity. Values are expressed as the mean ± SEM. N = 5 independent experiments. P‐values from ANOVA: ***P < .001 vs. control, ###P < .001 vs. MK‐2206&INS group. D, Potential FoxO1‐binding sites are located 2.0 kb upstream of the Kir6.1 gene's transcription initiation site. E, Binding of endogenous FoxO1 from the hearts of mice to the potential FoxO1‐binding site was confirmed by the chromatin immunoprecipitation assay. Representative results from three independent…