Ivabradine Ameliorates Cardiac Diastolic Dysfunction in Diabetic Mice Independent of Heart Rate Reduction

Figures

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  FIGURE 1. JNK and p38 MAPK mediate high glucose (HG)-induced fibrogenic protein upregulation in vitro (A) NRCFs were confirmed by vimentin and DAPI staining (original magnification × 400). Scale bars, 50 μm. Data were obtained from three independent experiments (B and C) NRCFs transfected with or without JNK or p38 MAPK siRNA were evaluated for the protein expression of JNK and p38 MAPK by western blotting (D) NRCFs were exposed to HG for 0–48 h and JNK/p38 MAPK phosphorylation and total JNK/p38 MAPK expression were determined by western blotting (E) NRCFs were transfected with or without JNK/p38 MAPK siRNA and then exposed to HG for the indicated periods of time. The protein expression of PCNA, α-SMA, TIMP2, collagen I, collagen III, and MMP2 was determined by western blotting. Data in (B–E) are presented as mean ± SEM (n = 3). p-values in (B–D) were calculated using paired Student’s t-test. p-value in (E) was calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the normal group; # p < 0.05, compared to the HG group.

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  FIGURE 2. JNK and p38 MAPK mediate the diabetes-induced increase in fibrogenic protein expression in vivo (A) Left ventricular fibroblasts isolated from mice were confirmed by vimentin staining. Data was obtained from three independent experiments (B and C) Isolated left ventricular fibroblasts from wild-type mice that were or were not injected with lentivirus via a tail vein were evaluated for JNK and p38 MAPK protein expression after 4 weeks (D) Left ventricular fibroblasts were isolated from mice and JNK and p38 MAPK phosphorylation and total JNK and p38 MAPK expression were determined by western blotting (E) Isolated left ventricular fibroblasts from diabetic mice that had or had not been injected with lentivirus via a tail vein were evaluated for PCNA, α-SMA, TIMP2, collagen I, collagen III, and MMP2 expression after 4 weeks (F) Between 7 and 11 weeks following the tail vein injection of lentivirus, the blood glucose concentration and body mass of the mice were measured. Data in (B–F) are presented as mean ± SEM (n = 6). p-values in (B–D) were calculated using paired Student’s t-test. p-values in (E-F) were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05,…

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  FIGURE 3. Myocardial fibrosis and cardiac diastolic dysfunction (DD) are ameliorated by the knockdown of JNK or p38 MAPK (A) Massonʼs trichrome staining of heart tissue (original magnification ×400). The columns show the differences in collagen accumulation. Scale bars, 50 μm (B) Echo-Doppler traces for transmitral flow (C) Transmitral pulsed-wave Doppler measurements of E/A, E Dec t, and IVRT (D) Hemodynamic parameters: −dP/dt min and the time constant, Tau. The columns show the differences in left ventricular DD and hemodynamic parameters. Data are presented as mean ± SEM (n = 6). p-values were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the db/db mice groups.

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  FIGURE 4. Ivabradine, but not zatebradine, reduces JNK/p38 MAPK activation and the high glucose (HG)-induced increase in fibrogenic protein expression in vitro (A) NRCFs were pretreated with or without ivabradine at the indicated concentrations for 30 min, and then exposed to HG for 48 h. JNK and p38 MAPK phosphorylation and total JNK and p38 MAPK expression were determined by western blotting (B) NRCFs were treated as described in (A) and the protein expression of PCNA, α-SMA, TIMP2, collagen I, collagen III, and MMP2 was determined by western blotting (C and D) NRCFs were pretreated with or without zatebradine at the indicated concentrations for 30 min, and then exposed to HG for 48 h. The proteins described in (A) and (B) were quantified by western blotting (E and F) NRCFs were treated as described in (A) and (C) and the cell proliferation rate was determined using an MTT assay. Data are presented as mean ± SEM (n = 3). p-values were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the normal group; # p < 0.05, compared to the HG group.

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  FIGURE 5. Effects of ivabradine and zatebradine on heart rate (HR) and biometric parameters (A) During treatment with or without ivabradine for 7–11 weeks, the mean HR of mice was recorded by electrocardiography (ECG) (B and C) Mice were treated as described in (A) and their blood glucose concentration and body mass were measured (D) During treatment with ivabradine or zatebradine at the indicated doses for 7–11 weeks, the mean HR of mice was recorded by ECG (E and F) During treatment with 10 mg/kg/day zatebradine for 7–11 weeks, the blood glucose concentration and body mass of the mice were measured. Data are presented as mean ± SEM (n = 8–10). p-values were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the Control group; # p < 0.05, compared to the db/db mice group; & p < 0.05, compared to the db/db + Iva (20 mg/kg/d) group or the db/db + Zate (10 mg/kg/d) group.

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  FIGURE 6. Ivabradine reduces JNK and p38 MAPK activation and inhibits diabetes-induced CF proliferation and activation in vivo (A) Left ventricular fibroblasts isolated from mice treated with ivabradine at the indicated dose. JNK and p38 MAPK phosphorylation and total JNK and p38 MAPK expression were determined by western blotting (B) In CFs from mice treated as described in (A), the expression of PCNA and α-SMA was determined by western blotting (C and D) In left ventricular fibroblasts isolated from mice treated with zatebradine at the indicated dose the expression of the proteins listed in (A) and (B) was measured by western blotting. Data are presented as mean ± SEM (n = 3). p-values were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the Control group; # p < 0.05, compared to the db/db mice group.

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  FIGURE 7. Ivabradine, but not zatebradine, ameliorates the diabetes-induced increases in fibrogenic protein expression and fibrosis (A) Collagen I, collagen III, TIMP2, and MMP2 protein expression was determined by western blotting in left ventricular fibroblasts isolated from mice administered ivabradine at the indicated doses (B) Left ventricular fibroblasts were isolated from mice administered zatebradine at the indicated doses and the expression of the proteins listed in (A) was measured by western blotting (C and D) Mice were treated as described in (A) and (B) and the degree of cardiac fibrosis was determined by Massonʼs trichrome staining and immunohistochemistry. The columns show the differences in collagen accumulation (original magnification ×400). Scale bars, 50 μm. Data in (A–B) (n = 3) and (C–D) (n = 6) are presented as mean ± SEM. p-values were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the Control group; # p < 0.05, compared to the db/db mouse group.

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  FIGURE 8. Ivabradine and zatebradine ameliorate diabetes-induced cardiac diastolic dysfunction (DD) (A) Mice were administered ivabradine or zatebradine at the indicated doses and echo-Doppler traces for the transmitral flow were recorded (B) Mice were treated as described in (A) and transmitral pulsed-wave Doppler measurements of E/A, E Dec t, and IVRT were obtained (C) Mice were treated as described in (A) and the hemodynamic parameters −dP/dt min and Tau were measured. The columns show the differences in left ventricular DD and hemodynamic parameters. Data are presented as mean ± SEM (n = 6). p-values were calculated using one-way ANOVA with Tukey multiple comparison test. *p < 0.05, compared to the Control group; # p < 0.05, compared to the db/db mouse group.