Rhynchophylline Regulates Calcium Homeostasis by Antagonizing Ryanodine Receptor 2 Phosphorylation to Improve Diabetic Cardiomyopathy

Figures

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  FIGURE 1. Chemical structure of rhynchophylline.

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  FIGURE 2. Effect of rhynchophylline on blood glucose. (A) The bar graph showed the fasting blood glucose levels of mice in the CON group, T2DM mice, DTL group and Rhy group before administration. (B) Blood glucose levels measured 2 months after administration. CON:control group mice; T2DM: Type 2 diabetes model group mice; DTL: T2DM mice treated with Dantrolene (DTL) for 8 weeks; Rhy: T2DM mice treated with rhynchophylline for 8 weeks. Values are presented as mean ± standard error of the mean. **p < 0.01 vs. control group (n = 9–10, 1-way analysis of variance, Holm–Sidak post-hoc test).

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  FIGURE 3. The effects of rhynchophylline on the echocardiographic parameters. Representative M-mode images of left ventricular diameters and wall thicknesses.

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  FIGURE 4. Effect of rhynchophylline on cardiac appearance and morphology. (A) Representative heart photographs of each group. (B) The left ventricular weight index (LVWI) and cardiac weight index (CWI). (C) Representative sections (magnification, ×400) of cardiac tissue morphology stained by hematoxylin and eosin in each group. (D) Pathology scores; (E) Representative sections (magnification, ×400) of cardiac tissue morphology stained by Masson’s trichrome stain in each group; (F) Positive area rate of MASSON staining. Values are presented as mean ± standard error of the mean. **p < 0.01 vs. control group; #p < 0.05 vs. type 2 diabetes mellitus group.

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  FIGURE 5. Effect of rhynchophylline on cardiac enzymes. (A) The levels of creatine kinase (CK). (B) The levels of lactate dehydrogenase (LDH). (C) The levels of aspartate aminotransferase (AST). Values are presented as mean ± standard error of the mean. **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. type 2 diabetes mellitus group.

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  FIGURE 6. Effect of rhynchophylline on the degree of apoptosis in cardiac tissue. (A) Representative sections of apoptosis in cardiac tissues of each group (magnification, ×200). (B) The degree of apoptosis in cardiac tissue. (C–H) Caspase-3, Bax, and Bcl-2 protein expressions in heart tissue were determined by western blot analysis. Relative intensities of caspase-3 (D), Bax (F), and Bcl-2 (H) were calculated by normalization to glyceraldehyde-3-phosphate dehydrogenase in each group; Values are presented as mean ± standard error of the mean. *p < 0.05, **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. type 2 diabetes mellitus group (n = 3, 1-way analysis of variance, Holm–Sidak post-hoc test).

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  FIGURE 7. Effect of rhynchophylline on cardiomyocyte contraction and calcium transient. (A) Representative real-time monitoring of ventricular myocyte contraction and calcium transience images. (B) Representative monitoring of ventricular myocyte contraction in each group. (C) The amplitude of cardiomyocyte contraction. (D) The maximum rate of cell contraction. (E) The time for cells to reach the maximum rate of contraction. (F) Representative monitoring of ventricular myocyte calcium transience in each group. (G) The magnitude of calcium transience (△F/F0). (H) The tau value(τ) of calcium transience. Values are presented as mean ± standard error of the mean. **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. type 2 diabetes mellitus group. Abbreviations: N, mouse; n, cell. n = 3 each group, n ≥ 10 each group, 1-way analysis of variance, Holm–Sidak post-hoc test.

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  FIGURE 8. Effect of rhynchophylline on the phosphorylation levels of ryanodine receptor 2 (RyR2) and PLB protein in cardiomyocytes. (A) The phosphorylation protein-expression levels of RyR2-S2808 and PLB-S16 were detected by Western blot analysis. (B) The phosphorylation levels of RyR2. (C) The phosphorylation levels of PLB. Values are presented as mean ± standard error of the mean. **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. type 2 diabetes mellitus group.

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  FIGURE 9. Effect of rhynchophylline on the microstructure and mitochondria morphology of cardiomyocytes. (A) Representative sections of mouse cardiomyocytes observed under a transmission electron microscope. (B) Representative mitochondrial membrane potential observed under a fluorescence microscope (magnification, ×200). (C) The dynamic change in ΔΨm represented by the ratio of fluorescence intensity of 527 and 590 nm. (D) Adenosine triphosphate content in myocardial tissue. Values are presented as mean ± standard error of the mean. **p < 0.01 vs. control group; #p < 0.05, ##p < 0.01 vs. type 2 diabetes mellitus group.

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  FIGURE 10. Mechanism of rhynchophylline to improve diabetic cardiomyopathy.