Progressive decay of Ca²⁺ homeostasis in the development of 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 [9]. 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 [10]. 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/dt_max). Myocardial relaxation was evaluated according to the left ventricular end-diastolic pressure (LVEDP) and the maximum descending rate of left ventricular pressure (−dP/dt_max) [11]. 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 [12]. In brief, the hearts were cannulated, and perfused for 10 minutes at 2 mL/min (37°C) with oxygenated Ca²⁺-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, Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ sparks, the criteria was set at greater than 3.8× the standard deviation (SD) of the background noise over the mean background noise, and Ca²⁺ sparks were automatically counted with the Sparkmaster Plug-in for Image [13].

Western blot analyses (n = 6, in each group) were conducted as previously described [14] to assess the expressions of the Ca²⁺-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 (1:10⁴; 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).

Blood glucose levels were significantly higher in all diabetic rats (22.62 ± 5.07 mmol/L) compared with the control rats (7.57 ± 1.69 mmol/L; P = 0.00), but were not significantly different among the diabetic groups (P > 0.05; Table 1). Relative to the body weight of the control rats, that of the streptozotocin-induced diabetic rats was significantly lower, and progressively decreased from 4 weeks (group B) to 12 weeks (group D, P < 0.05). Heart weights also decreased during the 12-week development of diabetes, although the difference was statistically significant only in group D compared with the control group (P = 0.013).

Similar heart rates were observed in all groups (P = 0.627; Table 1). The left ventricular end-diastolic diameters (LVEDD) and left ventricular end-systolic diameters (LVESD) were comparable in all 3 diabetic groups (P > 0.05). With regard to ejection fraction (EF) and fractional shortening (FS), both showed a trend toward gradual decline in the diabetic rats over the 12 weeks, with significant reductions (P < 0.05) appearing at the twelfth week after diabetes was induced (Figure 1). E waves exhibited a downward trend during the progression of diabetes, but there were no significant differences among the diabetic groups (P > 0.05). In group D, there was a slight but substantial decline in E/A ratio compared with that in control group (P = 0.027).

As diabetes progressed over the experimental period, LVPSP decreased gradually from group B (4 weeks) to group D (12 weeks; Figure 2e). Compared with the controls, LVPSP was reduced significantly in group D (P = 0.009), which was consistent with the marked drops in EF and FS. Concurrently, the left ventricular end-diastolic pressure (LVEDP) increased gradually. Relative to the control rats, a significant increase in LVEDP first appeared in group C (8 weeks, P = 0.003, Figure 2f), which was earlier than that of the significant difference in LVPSP. Both + dP/dt_max and –dP/dt_max began to decrease significantly in group C, and were sustained in group D, which suggested marked impairments of both systolic and diastolic capabilities of the diabetic myocardium (Figure 2g and h).

Over the 12-week progression of diabetes, there were alterations in the properties of Ca²⁺ sparks (Figure 3). The frequency of Ca²⁺ sparks showed a significant decline, beginning at 4 weeks (group B), and was sustained in groups C (8 weeks) and D (12 weeks; Figure 3b). Concurrently, relative to the control group the Ca²⁺ spark peak amplitude (ΔF/F) was unchanged in group B, but significantly increased from group C to group D (Figure 3c). The Ca²⁺ spark decay time constant (Tau) increased significantly and progressively from group B to group D (Figure 3f).

In addition, caffeine stimulation caused a sudden and transient Ca²⁺ release from the SR and an increase in intracellular Ca²⁺ concentration, which represented the SR Ca²⁺ load. The SR Ca²⁺ load showed a marked decline, which was first observed in group B, and declined further in groups C to D (Figure 3g). However, neither the Ca²⁺ spark rise times nor the full width at half-maximum amplitude (FWHM) were significantly different in the different stages of diabetes from that of the control rats (Figure 3d and e).

RyR2, SERCA, and NCX1 are the major channel proteins for Ca²⁺ flux, and their expressions were analyzed by western blot (Figures 4 and 5). Levels of RyR2 proteins were significantly lower in group B, relative to those of the control, with progressively less in groups C and D (Figure 4b). Expressions of FKBP12.6 protein, a regulatory protein of RyR2, were similar in the control rats and group B, but significantly less in group C, and reduced further in group D (Figure 4c). Both SERCA and NCX1 showed a trend of gradual decline over the 12 weeks in diabetic rats: significantly less in group B relative to the controls, and further declines from group C to group D (Figure 5b, 5c). In addition, expressions of PLB protein, a regulatory protein of SERCA, in group B were similar to those in the control, but significantly and progressively higher levels were detected from group C to group D (Figure 6b). Concurrently, expressions of PLB-Thr17 and PLB-Ser16 proteins were also significantly less in group B relative to the controls, and further declines were detected from group C to group D (Figure 6c, d).