Figure 1.
Changes of plasma glucose and body weight in diabetic rats.
Bar graph of plasma glucose (A) and body weight (B). Values were expressed as mean±SEM (n = 8). **P<0.01 compared with control group.
Figure 2.
CaSR protein expression determined by western blot analysis in diabetic rat hearts.
(A) Representative bands of CaSR in diabetic rat hearts. a, control; b, two months. (B) The data represent the mean±SEM of eight independent experiments. The intensity of each band was quantified using densitometry, and the data were normalized to band intensity of GAPDH used as internal control. *P<0.05 compared with control group.
Figure 3.
Effects of CaSR on hemodynamics in diabetic rat hearts.
The hemodynamic parameters, LVSP (A), LVEDP (B), +dp/dtmax and -dp/dtmax (C) were measured throughout the experiment. Data were represented by mean±SEM (n = 8 per group). *P<0.05 compared with control group; @P<0.05 compared with model group; #P<0.05 compared with model+GdCl3 group; &P<0.05 compared with control group.
Figure 4.
Representative illustration of TUNEL staining in cardiomyocytes apoptosis.
(A) Nuclei with brown staining were TUNEL-positive cell, which was defined as apoptotic cell. Magnification at 400 ×. (B) Statistical analysis of cardiomyocytes apoptosis (n = 8). *P<0.05 compared with control group; @P<0.05 compared with model group; #P<0.05 compared with model+GdCl3 group; &P<0.05 compared with control group.
Figure 5.
Flow cytometric analysis of apoptosis.
A, 10,000 cells of each experiment were collected by flow cytometry and analyzed using Cell Quest software. Early apoptotic cells (annexin-V+ and PI-) were displayed in the lower right quadrant, and late apoptotic cells (annexin-V+ and PI+) were shown in the upper right quadrant. (A) Plots of sorted apoptotic cells. Apoptosis were evaluated after treating neonatal rat cardiomyocytes with 25.5 mM glucose, and staining with annexin-V and PI. Flow cytometry profile represents annexin-V-FITC staining in x axis and PI in y axis. The number represents the percentage of apoptotic cells in each condition. (B) Bar graph of cell apoptotic rate. All data were expressed as mean±SEM (n = 6). *P<0.05 compared with control group.
Figure 6.
Detection of apoptosis in neonatal rat cardiomyocytes by flow cytometry.
Bar graph of early (A and B) and late (C and D) apoptotic rate. All data were expressed as mean±SEM (n = 6). *P<0.05 compared with control group; @P<0.05 compared with model group; #P<0.05 compared with model+GdCl3 group; &P<0.05 compared with control group. Groups in A and C were control, model, model+GdCl3, model+NPS-2390+GdCl3, model+NPS-2390, and control+GdCl3. Groups in B and D were control, model, model+GdCl3, siRNA+model+GdCl3, nRNA+model, and siRNA+model. Glucose: 25.5 mM; GdCl3: 300 µM; NPS-2390: 10 µM.
Figure 7.
Measurement of [Ca2+]i in neonatal rat cardiomyocytes.
Fluorescent intensity in [Ca2+]i was recorded by laser scanning confocal microscope in different treatment. (A) Representative images of cardiomyocytes. (B and C) Bar graph of fluorescent intensity. All data were expressed as mean±SEM (n = 6). *P<0.05 compared with control group; @P<0.05 compared with model group; #P<0.05 compared with model+GdCl3 group; &P<0.05 compared with control group. Groups in B were control, model, model+GdCl3, model+NPS-2390+GdCl3, model+NPS-2390, and control+GdCl3. Groups in C were control, model, model+GdCl3, siRNA+model+GdCl3, nRNA+model, and siRNA+model. Glucose: 25.5 mM; GdCl3: 300 µM; NPS-2390: 10 µM.
Figure 8.
Detection of Bcl-2 and Bax protein expression in cardiomyocytes by western blot analysis.
(A and B) Western blot assay for Bcl-2 expression in neonatal rat cardiomyocytes. (C and D) Western blot assay for Bax expression in neonatal rat cardiomyocytes. Average data were represented by mean±SEM (n = 6). *P<0.05 compared with control group; @P<0.05 compared with model group; #P<0.05 compared with model+GdCl3 group; &P<0.05 compared with control group. Groups in A and C were control, model, model+GdCl3, model+NPS-2390+GdCl3, model+NPS-2390, and control+GdCl3. Groups in B and D were control, model, model+GdCl3, siRNA+model+GdCl3, nRNA+model, and siRNA+model. Glucose: 25.5 mM; GdCl3: 300 µM; NPS-2390: 10 µM.
Figure 9.
ERK1/2, JNK, and p38 phosphorylation in neonatal rat myocytes in different groups.
Western blotting for phosphorylated ERK1/2 (A and B), JNK (C and D), p38 (E and F), as well as total ERK1/2, JNK, p38 of cardiomyocytes. All the data were expressed as mean±SEM (n = 6). *P<0.05 compared with control group; @P<0.05 compared with model group; #P<0.05 compared with model+GdCl3 group; &P<0.05 compared with control group. Groups in A, C and E were control, model, model+GdCl3, model+NPS-2390+GdCl3, model+NPS-2390, and control+GdCl3. Groups in B, D and F were control, model, model+GdCl3, siRNA+model+GdCl3, nRNA+model, and siRNA+model. Glucose: 25.5 mM; GdCl3: 300 µM; NPS-2390: 10 µM.