Decay of Ca2+ Homeostasis in Diabetic Cardiomyopathy
The Beijing Friendship Hospital Animal Care Committee approved all animal handling protocols. The diabetic rat model was induced with a single intraperitoneal injection of streptozotocin (60 mg/kg diluted in 0.1 M citrate buffer, pH = 4.4; Sigma, USA) in male rats (Sprague–Dawley; 200 ± 20 g). Rats in the control group were given an injection of a matched volume of citrate buffer (0.1 M). Subsequently on day three and day five after the injection, random blood glucose concentrations were measured. Only rats with blood glucose levels ≥16.7 mmol/L on both days were defined as diabetic and used in the study.
Four experimental groups (n = 12, each) consisted of the control group (group A) and the diabetic model rats, examined at 4, 8, or 12 weeks after injection (groups B, C and D, respectively). All the rats were housed (three per cage) in a controlled environment at 20 ± 2°C, 30–70% humidity, and a 12:12 h light–dark cycle, and given standard chow and water ad libitum. In accordance with the protocol, all diabetic model rats were euthanized prior to the experiments at the assigned time points. Control rats were euthanized at the twelfth week after an injection with citrate buffer.
Echocardiography was performed to evaluate cardiac structure and function in all animals involved in the study (n = 12, in each group). The rats were weighed and anesthetized with 10% chloral hydrate at 0.3 mL/100 g body weight. Two-dimensional and M-mode echocardiographic measurements were carried out with a VEVO 770 high-resolution in vivo imaging system (VisualSonics, Toronto, Canada). The transthoracic echocardiography images were obtained via long- and short-axis views using standard echocardiography techniques. Cardiac structure was principally evaluated by the left ventricular end diastolic and systolic diameters. The left ventricular systolic function was assessed according to ejection fraction and fractional shortening, and the left ventricular diastolic function was determined by Doppler waveforms of mitral inflows, which were obtained from an apical four-chamber. The variables included the peak early diastolic filling velocity (E wave), the peak late diastolic filling velocity (A wave), and the ratio of the peak early-to late-filling velocity (E/A).
To assess the hemodynamic condition, an ultra-miniature catheter connected to a polygraph instrument (BL-420, TaiMeng, ChengDu, China) was inserted into the carotid artery, and then to the left ventricle of the anesthetized animals (n = 6, in each group). After a 5-min period of stabilization, the parameters were acquired and recorded. The myocardial contractility was assessed according to the left ventricular systolic peak pressure (LVPSP) and the maximum rate of ascending pressure change in the left ventricle (+dP/dtmax). Myocardial relaxation was evaluated according to the left ventricular end-diastolic pressure (LVEDP) and the maximum descending rate of left ventricular pressure (−dP/dtmax). The hearts were then removed and stored at −80°C for further study.
Ventricular myocytes were isolated from the rats (n = 6, in each group) as described previously. In brief, the hearts were cannulated, and perfused for 10 minutes at 2 mL/min (37°C) with oxygenated Ca-free buffer, containing 145 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.4 mM Na2HPO4, 0.4 mM NaH2PO4, 5 mM HEPES, and 10 mM glucose; pH 7.4, adjusted with NaOH. Then the hearts were perfused with an enzyme solution, containing 0.6 mg/mL collagenase type II (Invitrogen, USA) for about 15 min. The ventricular muscle tissue was cut into small pieces, and the ventricular myocytes were released by agitating the cell/tissue suspension. After filtration through a nylon mesh, Ca was gradually reintroduced up to 1.8 mM in the cell suspension. The ventricular myocytes were then ready to be used in further studies.
For the Ca spark imaging, cardiomyocytes were incubated with 10 μM Fluo-4 AM (Invitrogen, USA) in a normal Tyrode's solution for 5 min at 37°C, and transferred into a recording chamber. Cells were perfused with the Tyrode's solution for 20 min for de-esterification. SR Ca load was estimated by a rapid application of 10 mM caffeine via a nearby pipette. To control the rest potentiation, experiments were performed after about 30 minutes, upon reintroduction of Ca to a concentration of 1.8 mM in each group. Confocal line-scan imaging was performed using a Leica SP5 confocal microscope (Germany, 40 ×, 1.25 NA) with an excitation at 488 nm. Line-scan images were acquired at a sampling rate of 1.43 ms per line, along the longitudinal axis of the cell. Digital image processing was performed using MATLAB 7.1 (Math Works). For a minimal detection of Ca sparks, the criteria was set at greater than 3.8× the standard deviation (SD) of the background noise over the mean background noise, and Ca sparks were automatically counted with the Sparkmaster Plug-in for Image.
Western blot analyses (n = 6, in each group) were conducted as previously described to assess the expressions of the Ca-associated proteins RyR2, SERCA, NCX1, FKBP12.6, and phospholamban (PLB), and the phosphorylation status of PLB at serine-16 (PLB-Ser16) and threonine-17 (PLB-Thr17). In brief, the heart tissue was homogenized in a cold Tris–HCl buffer (120 mM NaCl, 1.0%, Triton X-100, 20 mM Tris–HCl, pH 7.5, 10% glycerol, 2 mM EDTA, protease inhibitor cocktail). Total protein was quantified using a BCA protein assay kit as required (CW Bio Tech, 02912E, China). Equal amounts of protein (40 μg) were separated by SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane and blocked with Tris buffered saline containing 5% skim milk powder and 0.05% Tween 20. Then the corresponding primary antibodies, RyR2 (1:1000, Abcam, USA), SERCA (1:1000, Abcam, USA), NCX1 (1:300, Santa Cruz, USA), PLB (1:1000, Abcam, USA), PLB-Thr17 (1:300, Santa Cruz, USA), PLB-Ser16 (1:300, Santa Cruz, USA), and FKBP12.6 (1:1000, R&D, USA) were added in series. The membranes were incubated with the diluted antibody preparations overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) antibodies (1:10; Jackson, USA) for 40 min at room temperature. The blots were visualized using an enhanced chemiluminescence detection kit (Millipore, USA). Target proteins were quantified and normalized relative to β-actin (1:1000, Zhongshan, China).
Data were evaluated by one-way ANOVA expressed as the mean ± SD. When a significant difference was identified by ANOVA, a post-hoc analysis was performed using the Student-Newman-Keuls test. Data were analyzed using SPSS 13.0 software, and a P-value < 0.05 was considered significant.
Methods
Animal Models
The Beijing Friendship Hospital Animal Care Committee approved all animal handling protocols. The diabetic rat model was induced with a single intraperitoneal injection of streptozotocin (60 mg/kg diluted in 0.1 M citrate buffer, pH = 4.4; Sigma, USA) in male rats (Sprague–Dawley; 200 ± 20 g). Rats in the control group were given an injection of a matched volume of citrate buffer (0.1 M). Subsequently on day three and day five after the injection, random blood glucose concentrations were measured. Only rats with blood glucose levels ≥16.7 mmol/L on both days were defined as diabetic and used in the study.
Four experimental groups (n = 12, each) consisted of the control group (group A) and the diabetic model rats, examined at 4, 8, or 12 weeks after injection (groups B, C and D, respectively). All the rats were housed (three per cage) in a controlled environment at 20 ± 2°C, 30–70% humidity, and a 12:12 h light–dark cycle, and given standard chow and water ad libitum. In accordance with the protocol, all diabetic model rats were euthanized prior to the experiments at the assigned time points. Control rats were euthanized at the twelfth week after an injection with citrate buffer.
Echocardiographic Measurements
Echocardiography was performed to evaluate cardiac structure and function in all animals involved in the study (n = 12, in each group). The rats were weighed and anesthetized with 10% chloral hydrate at 0.3 mL/100 g body weight. Two-dimensional and M-mode echocardiographic measurements were carried out with a VEVO 770 high-resolution in vivo imaging system (VisualSonics, Toronto, Canada). The transthoracic echocardiography images were obtained via long- and short-axis views using standard echocardiography techniques. Cardiac structure was principally evaluated by the left ventricular end diastolic and systolic diameters. The left ventricular systolic function was assessed according to ejection fraction and fractional shortening, and the left ventricular diastolic function was determined by Doppler waveforms of mitral inflows, which were obtained from an apical four-chamber. The variables included the peak early diastolic filling velocity (E wave), the peak late diastolic filling velocity (A wave), and the ratio of the peak early-to late-filling velocity (E/A).
Hemodynamic Measurements
To assess the hemodynamic condition, an ultra-miniature catheter connected to a polygraph instrument (BL-420, TaiMeng, ChengDu, China) was inserted into the carotid artery, and then to the left ventricle of the anesthetized animals (n = 6, in each group). After a 5-min period of stabilization, the parameters were acquired and recorded. The myocardial contractility was assessed according to the left ventricular systolic peak pressure (LVPSP) and the maximum rate of ascending pressure change in the left ventricle (+dP/dtmax). Myocardial relaxation was evaluated according to the left ventricular end-diastolic pressure (LVEDP) and the maximum descending rate of left ventricular pressure (−dP/dtmax). The hearts were then removed and stored at −80°C for further study.
Examination of Ca Homeostasis in Cardiomyocytes
Ventricular myocytes were isolated from the rats (n = 6, in each group) as described previously. In brief, the hearts were cannulated, and perfused for 10 minutes at 2 mL/min (37°C) with oxygenated Ca-free buffer, containing 145 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.4 mM Na2HPO4, 0.4 mM NaH2PO4, 5 mM HEPES, and 10 mM glucose; pH 7.4, adjusted with NaOH. Then the hearts were perfused with an enzyme solution, containing 0.6 mg/mL collagenase type II (Invitrogen, USA) for about 15 min. The ventricular muscle tissue was cut into small pieces, and the ventricular myocytes were released by agitating the cell/tissue suspension. After filtration through a nylon mesh, Ca was gradually reintroduced up to 1.8 mM in the cell suspension. The ventricular myocytes were then ready to be used in further studies.
For the Ca spark imaging, cardiomyocytes were incubated with 10 μM Fluo-4 AM (Invitrogen, USA) in a normal Tyrode's solution for 5 min at 37°C, and transferred into a recording chamber. Cells were perfused with the Tyrode's solution for 20 min for de-esterification. SR Ca load was estimated by a rapid application of 10 mM caffeine via a nearby pipette. To control the rest potentiation, experiments were performed after about 30 minutes, upon reintroduction of Ca to a concentration of 1.8 mM in each group. Confocal line-scan imaging was performed using a Leica SP5 confocal microscope (Germany, 40 ×, 1.25 NA) with an excitation at 488 nm. Line-scan images were acquired at a sampling rate of 1.43 ms per line, along the longitudinal axis of the cell. Digital image processing was performed using MATLAB 7.1 (Math Works). For a minimal detection of Ca sparks, the criteria was set at greater than 3.8× the standard deviation (SD) of the background noise over the mean background noise, and Ca sparks were automatically counted with the Sparkmaster Plug-in for Image.
Western Blot Analysis
Western blot analyses (n = 6, in each group) were conducted as previously described to assess the expressions of the Ca-associated proteins RyR2, SERCA, NCX1, FKBP12.6, and phospholamban (PLB), and the phosphorylation status of PLB at serine-16 (PLB-Ser16) and threonine-17 (PLB-Thr17). In brief, the heart tissue was homogenized in a cold Tris–HCl buffer (120 mM NaCl, 1.0%, Triton X-100, 20 mM Tris–HCl, pH 7.5, 10% glycerol, 2 mM EDTA, protease inhibitor cocktail). Total protein was quantified using a BCA protein assay kit as required (CW Bio Tech, 02912E, China). Equal amounts of protein (40 μg) were separated by SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane and blocked with Tris buffered saline containing 5% skim milk powder and 0.05% Tween 20. Then the corresponding primary antibodies, RyR2 (1:1000, Abcam, USA), SERCA (1:1000, Abcam, USA), NCX1 (1:300, Santa Cruz, USA), PLB (1:1000, Abcam, USA), PLB-Thr17 (1:300, Santa Cruz, USA), PLB-Ser16 (1:300, Santa Cruz, USA), and FKBP12.6 (1:1000, R&D, USA) were added in series. The membranes were incubated with the diluted antibody preparations overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) antibodies (1:10; Jackson, USA) for 40 min at room temperature. The blots were visualized using an enhanced chemiluminescence detection kit (Millipore, USA). Target proteins were quantified and normalized relative to β-actin (1:1000, Zhongshan, China).
Statistical Analysis
Data were evaluated by one-way ANOVA expressed as the mean ± SD. When a significant difference was identified by ANOVA, a post-hoc analysis was performed using the Student-Newman-Keuls test. Data were analyzed using SPSS 13.0 software, and a P-value < 0.05 was considered significant.