AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 292: H912-H920, 2007. First published September 29, 2006; doi:10.1152/ajpheart.00824.2006 Free Article
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Restoration of diabetes-induced abnormal local Ca2+ release in cardiomyocytes by angiotensin II receptor blockade

Nazmi Yaras,1 Ayca Bilginoglu,1 Guy Vassort,2 and Belma Turan1

1Department of Biophysics, School of Medicine, Ankara University, Ankara, Turkey; and 2Institut National de la Santé et de la Recherche Médicale, Physiopathologie Cardiovasculaire, Centre Hospitalier Universitaire Arnaud de Villeneuve, Montpellier, France

Submitted 1 August 2006 ; accepted in final form 19 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Stimulation of local renin-angiotensin system and increased levels of oxidants characterize the diabetic heart. Downregulation of ANG II type 1 receptors (AT1) and enhancement in PKC activity in the heart point out the role of AT1 blockers in diabetes. The purpose of this study was to evaluate a potential role of an AT1 blocker, candesartan, on abnormal Ca2+ release mechanisms and its relationship with PKC in the cardiomyocytes from streptozotocin-induced diabetic rats. Cardiomyocytes were isolated enzymatically and then incubated with either candesartan or a nonspecific PKC inhibitor bisindolylmaleimide I (BIM) for 6–8 h at 37°C. Both candesartan and BIM applied on diabetic cardiomyocytes significantly restored the altered kinetic parameters of Ca2+ transients, as well as depressed Ca2+ loading of sarcoplasmic reticulum, basal Ca2+ level, and spatiotemporal properties of the Ca2+ sparks. In addition, candesartan and BIM significantly antagonized the hyperphosphorylation of cardiac ryanodine receptor (RyR2) and restored the depleted protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6). Furthermore, candesartan and BIM also reduced the increased PKC levels and oxidized protein thiol level in membrane fraction of diabetic rat cardiomyocytes. Taken together, these data demonstrate that AT1 receptor blockade protects cardiomyocytes from development of cellular alterations typically associated with Ca2+ release mechanisms in diabetes mellitus. Prevention of these alterations by candesartan may present a useful pharmacological strategy for the treatment of diabetic cardiomyopathy.

heart; candesartan; Type 1 diabetes; thiol oxidation

CHRONIC DIABETES ALTERS the structure and function of the human heart, and individuals with diabetes mellitus usually develop a specific cardiac dysfunction known as diabetic cardiomyopathy (37). Several mechanisms involved in the development of cardiomyopathy have been postulated, including alterations in intracellular ion homeostasis and glucose metabolism and enhanced oxidative stress. Although alteration of Ca2+ signaling via changes in critical processes that regulate intracellular Ca2+ has become a hallmark of this type of cardiomyopathy, controversies, currently going on, relate to specific alterations in Ca2+ signaling pathways contributing to the cardiac defects in diabetes (7, 20). Recently, we reported that these defects result partially from altered local Ca2+ signaling due to a dysfunction of cardiac PKA-mediated ryanodine receptor Ca2+ release channel (RyR2) (51).

Several mechanisms have been proposed to explain how all of the pathologies involved in the progression of diabetic cardiomyopathy might result from hyperglycemia. Increased PKC isoform expression and increased polyol pathway flux are two main hypotheses presented to describe how hyperglycemia might cause all of the diabetic complications (6). Furthermore, it has been demonstrated that hyperglycemia activates the local renin-angiotensin system (RAS) and enhanced RAS activity in diabetes (33, 40). In addition, it is known that ANG II has direct effects on cardiomyocytes through the ANG II type 1 (AT1) receptor (9–11). Thus, stimulation of the AT1 receptor generates oxygen-derived free radicals, which have detrimental effects on the cardiovascular system (28). The AT1 receptor has been shown to be coupled to several postreceptor signaling pathways, including NADPH oxidase (33). Furthermore, there is a significant amount of evidence demonstrating that increased PKC activity contributes to cardiovascular complications associated with diabetes and that elevated PKC activity has been found in diabetic hearts (25). PKC is known to phosphorylate a number of proteins that are directly involved in cardiac excitation-contraction coupling (8, 24, 25). Therefore, upregulation of cardiac RAS and increased PKC activity in diabetic animals suggest the importance of these pathways in the development of cardiac complications.

Defects in cardiac intracellular Ca2+ have been proposed as one possible contributory factor to the depressed myocardial contractile activity in diabetic cardiomyopathy (1, 32). Most alterations were attributed to anomalous sarcoplasmic reticulum (SR) pump activity (15), SR-Ca2+ storage (5), and partly to alterations in RyR2 properties (3, 27, 35, 49, 51). Recently, it has been shown that some cardioprotective agents correct the defective FK506 binding protein 12.6 (FKBP12.6)-mediated stabilization of RyR2, leading to an improvement in cardiac function of heart failure (29, 50). Recently, Gassanov et al. (16) described marked increases in the ANG II-induced depressed parameters of Ca2+ sparks with candesartan. We and others have shown that chronic administration of ANG II receptor blockers could protect the heart from the development of cellular alterations typically related with diabetes (18, 29, 30, 33, 34). Although ANG II antagonism leads to an attenuation of the depressed SR-Ca2+-ATPase (SERCA) and to an improvement in intracellular Ca2+ handling in heart failure (16, 29), it is not known whether this antagonism can correct the defective interaction of RyR2 and FKBP12.6 that occurs in the SR during the development of diabetes-induced cardiomyopathy (51).

To get further insight and to evaluate a potential direct role of ANG II activation in diabetic heart dysfunction, we examined direct effects of an AT1 receptor blocker, candesartan, on cardiac intracellular Ca2+ metabolism in terms of global Ca2+ release (electrically stimulated Ca2+ transients) and local Ca2+ release by RyR2 modulated by PKA (Ca2+ sparks) and also their possible relation with activated PKC in cardiomyocytes isolated from streptozotocin (STZ)-induced diabetic rats. Our results demonstrated that ANG II antagonism significantly restores the defective interaction of FKBP12.6 and RyR2 in addition to its restoring effects on activated PKC and oxidized protein thiols in these diseased cardiomyocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental groups. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg body wt and dissolved in 0.1 M citrate buffer, pH 4.5) in adult male Wistar rats (200–250 g), whereas rats for the control group received citrate buffer alone. A week after injection of STZ, blood glucose levels were measured and only the rats having blood glucose levels at least three times higher than the preinjection levels were used later on. All rats had free access to standard rat chow and water. All animals were used 5 wk after STZ or citrate buffer injections. All experiments were approved by the Ankara University School of Medicine and performed in accordance with its ethics guidelines for the care and use of laboratory animals.

Cell isolation. Cell isolation was performed as described previously (51). Hearts were rapidly removed following anesthetization of the rats with pentobarbital sodium (30 mg/kg body wt). The hearts were cannulated on a Langendorff apparatus and perfused through the coronary arteries with a Ca2+-free solution containing (in mM) 145 NaCl, 5 KCl, 1.2 MgSO4, 1.4 Na2HPO4, 0.4 NaH2PO4, 5 HEPES, and 10 glucose at pH 7.4, bubbled with O2 at 37°C. Hearts were perfused for 3–5 min to remove the remaining blood, and this was followed by perfusion with the same solution containing 1 mg/ml collagenase (collagenase A, Boehringer) for 30–35 min. Ventricles were then removed and minced into small pieces and gently massaged through a nylon mesh, and dissociated cardiomyocytes were washed with the collagenase-free solution. Subsequently, Ca2+ was increased in a graded manner to a concentration of 1 mM with three steps. Cells were kept in this solution at 37°C until used the same day.

Cytosolic Ca2+ sparks measurement. Ca2+ sparks measurement was performed as described previously (51). Cardiomyocytes were placed into the recording chamber setting over the stage of an inverted microscope equipped with a laser scanning confocal microscope (200-M, LSM-Pascal, Zeiss, Germany). In the experiments, x40 (numerical aperature = 1.3) oil immersion objectives were used for imaging cardiomyocytes located over cover glass base of the recording chamber. A 488-nm laser line from an argon laser (25 mW) was used to excite the Ca2+-sensitive dye, fluo-3, and emitted fluorescence collected at 505 nm. Changes in intracellular free Ca2+ were recorded in line-scan mode [spatial (x) vs. temporal (t), 1.9 ms per line]. To prevent photobleaching and cell damage, the laser line was kept at 4–6% of maximal intensity.

Image analysis was performed by using LSM Image Examiner. The F value for fluorescence intensity of image was calculated by averaging pixels except potential spark areas. Then {Delta}F/F image was created by using this F value. Ca2+ sparks were manually detected and converted to temporal lines by averaging fluorescence intensity of two to three pixels, aligning the peak of fluorescence intensity over time. The signals were filtered using a Butterworth digital filter. The temporal profiles were then fitted to gamma function to analyze time to peak (TP), peak amplitude of fluorescence intensity (PA), and decrease time to half-maximum (DT50).

To assess the effects of candesartan (dissolvable form of candesartan cilexetil), an AT1 receptor blocker, or of the nonspesific PKC inhibitor bisindolylmaleimide I (BIM) (30) on the parameters of Ca2+ sparks in isolated cardiomyocytes, a fraction of diabetic and control cardiomyocytes were incubated with either candesartan (1 µM) or BIM (100 nM) or buffer solution for 6–8 h at 37°C. BIM was dissolved in DMSO; however, final dilution of DMSO in experimental solutions was <0.01%.

Measurement of cytosolic Ca2+ transients. Intracellular Ca2+ transients were measured by fura-2 fluorescence at room temperature (21 ± 2°C) as described previously (51). The cells were incubated at 37°C for 50 min with 4 µM fura-2 AM for loading and then washed with fresh buffer. Fluorescence was recorded using a PTI Ratiomaster microspectrophotometer and FELIX software (Photon Technology International). Cells were excited at 340/380 nm, and emission was measured at 510 nm. The fluorescence ratio of the emitted lights on excitation at 340 and 380 nm (F340/380) was calculated and used as an indicator of intracellular Ca2+ concentration ([Ca2+]i). F340/380 was measured at a frequency of 10 Hz. The composition of the bath solution used in [Ca2+]i measurement was as follows: (in mM) 130 NaCl, 4.8 KCl, 1.2 MgSO4, 1.5 CaCl2, 1.2 KH2PO4, 10 HEPES, and 10 glucose (pH 7.4). Following the monitoring of [Ca2+]i for 20 s at rest, field-stimulation pulses of 20–30 V with 10-ms duration were applied at 0.2-Hz frequency. PA (difference between basal and peak F340/380 ratios), TP, and DT50 were determined from Ca2+ transients evoked by field stimulation. Background fluorescence measured from a cell-free field was substracted from all recordings before calculation of ratios.

Caffeine-induced Ca2+ transients were induced by bath application of 10 mM caffeine on cardiomyocytes 30 s after stopping field stimulation of the cells with electrical pulses (for 2 min at 0.2-Hz stimulation frequency) to ensure stable SR-Ca2+ load. Caffeine responses were measured in buffer and in both candesartan (1 µM)- and BIM (100 nM)-incubated cardiomyocytes from diabetic and control group rats for 6–8 h at 37°C.

Western blot analysis. Isolated cardiomyocytes were homogenized with a motor-driven Teflon-to-glass homogenizer in cold Tris·HCl buffer containing (in mM) 50 Tris·HCl (pH 7.4), 200 NaCl, 20 NaF, 1.0 Na3VO4, 1 dithiothreitol, and protease inhibitors (complete tablet from Roche) and centrifuged at 1,000 g for 30 min at 4°C. Supernatant was centrifuged at 30,000 g for 30 min, and the pellet was then washed out via centrifugation at 30,000 g for 1 h at 4°C. Pellet was suspended with the homogenization buffer, and protein amount was measured by Bradford method as described previously (51).

The samples (100 µg protein) were subjected to 5% SDS-PAGE for RyR2 assay and 10% SDS-PAGE for both FKBP12.6 and actin assays and then transferred electrophoretically to nitrocellulose membrane. Immunoblotting was performed by using antibodies against RyR2 and phosphorylated RyR2 (kind gift of Drs. A. Marks and X. Wehrens; dilutions, 1:5,000 and 1:3,000, respectively) and FKBP12.6 and actin (Santa Cruz), and then enhanced chemiluminescence (ECL) was performed. The samples (30 µg protein) were subjected to 10% SDS-PAGE for PKC assays and then transferred electrophoretically to nitrocellulose membrane. Immunoblotting was performed by using antibodies against PKC (Santa Cruz), and then ECL was performed as mentioned previously (51). Briefly, nitrocellulose membranes were incubated 1 h at 4°C in PBS (20 mM NaH2PO4-Na2HPO4, pH 7.6, containing 154 mM NaCl, 3% bovine serum albumin, and 8% nonfatty dry milk). Blots were washed several times with PBS containing 0.1% Tween and were then incubated with antiserum at room temperature for 1–2 h by shaking. Blots were then washed several times with PBS, incubated with horseradish peroxidase-labeled anti-rabbit IgG (Santa Cruz, CA) (dilution, 1:10,000) for 1 h at room temperature. Blots were washed several times with PBS and then incubated with ECL Western blotting reagent (Amersham, Vienna, Austria) for 1 min and exposed to X-ray film for 45–90 s.

Determination of sulfhydryl groups in isolated cardiomyocytes. Cardiomyocytes from both control and diabetic groups were first washed with a HEPES-buffered solution containing (in mM) 123 NaCl, 5.4 KCl, 1.7 MgCl2, 1.8 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). The cells were then divided into two groups: untreated group (cells kept in the HEPES-buffered solution) and treated group [cells incubated in the same solution supplemented with either candesartan (1 µM) or BIM (100 nM) under the same conditions]. After 6- to 8-h incubation at 37°C, the cells from each group were washed with the HEPES-buffered solution and used for estimation of total and acid-soluble sulfhydryl (SH) groups. These estimations were done with Ellman’s reagent as described elsewhere (44). The cells were thawed and lysed in 0.2 M Tris·HCl buffer (pH 8.1) containing 2% sodium dodecyl sulfate. For the determination of total SH groups, 0.05-ml aliquots of cell lysates were mixed with 0.8-ml distilled water and 0.1 ml of 2 mM 5,5'-dithiobis-(2-nitrobenzoic acid), waiting 20 min for color development. Absorbance of the supernatants was read at 412 nm. After correction of the absorbance with sample and reagent blanks, concentrations of SH groups in each sample were calculated by employing an extinction coefficient of 1.31 mM–1·mm–1. To determine the acid-soluble SH groups, 0.7 ml aliquots of cell lysates were mixed with 0.35 ml of 20% trichloroacetic acid and then centrifuged at 13,000 g for 10 min. The precipitates were then washed with 0.2 ml of 20% trichloroacetic acid in a similar manner. Later, the supernatants were adjusted to pH 8 with NaOH. The SH content of the supernatants was measured as described for total SH measurements.

Chemicals and statistical evaluation of data. All chemicals used were purchased from Sigma (Sigma-Aldrich Chemie, Steinheim, Germany) except for fura-2 AM and fluo-3, which were purchased from Molecular Probes (Eugene, OR), and collagenase A, which was purchased from Boehringer-Mannheim (Roche Diagnostics, Manheim, Germany). Candesartan was a gift from AstraZeneca (AstraZeneca R&D Mölndal, Sweden).

Groups were tested and compared using ANOVA and Tukey post hoc test. P values < 0.05 were taken as significant, and significance levels are given in the text. Data are presented as means ± SE throughout the text.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General effects. Diabetic animals had significantly high glucose levels (~4-fold) compared with control animals at the end of the 5-wk experimental period (105 ± 3 and 421 ± 5 mg/dl control vs. diabetics). During this period following STZ injection, they stopped gaining weight (219.2 ± 9.2 vs. 196.8 ± 7.9 g), whereas control animals continued to gain weight (211.4 ± 6.9 vs. 240.2 ± 7.8 g). Besides the measurement of heart weight of each animal, we also estimated cell capacitance of the isolated cardiomyocytes and compared these values between the groups. Mean cell capacitances of the control and diabetic rats were similar as previously reported by us (189.9 ± 12.2 and 180.6 ± 13.6 pF, respectively) (30).

Candesartan restores diabetes-induced altered parameters of Ca2+ sparks of cardiomyocytes. Since Ca2+ sparks represent SR-Ca2+ release from clusters of RyR2, we examined the effects of AT1 receptor blocker candesartan (+Can) on the characteristics of spontaneously arising Ca2+ sparks in cardiomyocytes isolated from STZ-induced diabetic (DM) and control (Con) rat hearts. Representative line-scan images of the candesartan- or buffer-incubated cardiomyocytes from diabetic rats and buffer-incubated control rats (individual Ca2+ spark image x vs. t) are displayed in Fig. 1. Representative temporal profiles of the Ca2+ sparks are shown in Fig. 1, A–D. The spatial (x) and (t) time courses were extracted as the mean of all recorded spatial or temporal pixels centered at the peak of the {Delta}F/F of the Ca2+ sparks. These transients were then fitted by a gamma distribution function to obtain objective kinetic and spatial values of the events (30).


Figure 1
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  		Fig. 1. Line-scan images with spontaneously arising Ca2+ sparks recorded in representative cardiomyocytes. Left: representative line-scan images recorded from untreated cardiomyocytes from control (Con; A) and diabetic (DM; B) rats and from treated cardiomyocytes from DM rats incubated with candesartan (+Can; 1 µM; C) or bisindolylmaleimide I (+BIM; 100 nM; D). Right: line plots (derived from line-scan images at the location along the scan line indicated by the arrows from each scan) demonstrate the fluorescence as a function of time. Color bar shows the intensity of fluorescence signals. F/F0, ratio of fluorescence intensity to basal fluorescence intensity.

 
Quantitative data for Ca2+ spark characteristics are summarized in Fig. 2. The Ca2+ spark amplitude (PA; as indicated by the ratio of peak fluorescence over baseline fluorescence) was not affected by candesartan (0.56 ± 0.02 for Con; 0.54 ± 0.02 for Con + Can; 0.51 ± 0.02 for DM; and 0.53 ± 0.02 for DM + Can) (Fig. 2A, right). On the other hand, TP Ca2+ fluorescence (rise time) and half-decay time (DT50) were 10.35 ± 0.43 and 25.36 ± 1.05 ms in candesartan-incubated diabetic cells compared with 12.88 ± 0.50 (P < 0.05) and 32.90 ± 1.29 ms (P < 0.05) in buffer-incubated diabetic cells and compared with 9.08 ± 0.30 (P < 0.05) and 20.78 ± 0.87 ms (P < 0.05) in buffer-incubated control cells (Fig. 2A, left). Consistent with these findings, significant differences were obtained for the mean frequency of Ca2+ sparks width at half maximum (FWHM). The spatial spread (FWHM) was significantly prolonged in the diabetic group with respect to the control group (3.16 ± 0.20 µm vs. 2.43 ± 0.07 µm), and incubation of these diabetic cardiomyocytes with candesartan could restore significantly (2.62 ± 0.12 µm) (Fig. 2B), whereas there was no significant effect on the controls (2.35 ± 0.10 vs. 2.43 ± 0.07 µm).


Figure 2
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  		Fig. 2. Can or BIM restores diabetes-induced altered parameters of Ca2+ sparks of the cardiomyocytes. A: averaged spatial and temporal values of Ca2+ sparks. Time-to-peak amplitude (TP), half-decay time (DT50), and peak amplitude (PA) of Ca2+ sparks are measured in cardiomyocytes from Con and DM groups (nrat = 4, ncell = 30, and nspark = 152 vs. nrat = 4, ncell = 25, and nspark = 145 in DM vs. Con), and Can (1 µM)- or BIM (100 nM)-incubated cardiomyocytes from DM group (nrat = 5, ncell = 44, and nspark = 185 vs. nrat = 4, ncell = 25, and nspark = 133 in Can vs. BIM). B: spatial distribution of the sparks, Ca2+ sparks width at half their maximal amplitude (FWHM). C: comparison of Ca2+ sparks frequency (10 vs. 12 cardiomyocytes from Con vs. DM groups). *P < 0.05 vs. Con; {dagger}P < 0.05 vs. DM.

 
Spontaneous Ca2+ sparks frequency was significantly higher in diabetic cardiomyocytes (0.051 ± 0.006 s–1·µm–1) than in the control cells (0.032 ± 0.003 s–1·µm–1), whereas they were significantly reduced after candesartan incubation (0.042 ± 0.005 s–1·µm–1) (Fig. 2C). No significant difference for spark frequency was present between incubated and nonincubated cardiomyocytes from the respective control groups (0.035 ± 0.004 s–1·µm–1 vs. 0.032 ± 0.003 s–1·µm–1).

BIM restores diabetes-induced altered parameters of Ca2+ sparks. It has been demonstrated that incubation of cardiomyocytes with a PKC inhibitor could restore depressed potassium currents in diabetic rats, suggesting a role of increases in ANG II, AT1 receptors, and PKC activity in diabetic rats (41). Therefore, we incubated isolated diabetic cardiomyocytes with BIM for 6–8 h to assess the role of cardiac PKC (a known downstream mediator of AT1 receptor action) in the altered parameters of the Ca2+ sparks obtained in the cardiomyocytes from the diabetic rats. Incubation of diabetic cardiomyocytes with BIM, similar to candesartan incubation, significantly restored the prolonged TP and DT50 values, whereas it had no significant effect on the diminished amplitude of Ca2+ sparks (Fig. 2A). BIM incubation of these diabetic cardiomyocytes could significantly restore spontaneous Ca2+ sparks frequency (0.039 ± 0.005 s–1·µm–1) and the FWHM (2.59 ± 0.13 s–1 · µm–1) with respect to the controls as well (Fig. 2, B and C). Moreover, incubation of the cardiomyocytes with BIM from the control group did not affect these parameters significantly (data not shown).

Candesartan restores global Ca2+ release in diabetic rat heart. Fig. 3A shows original recordings of Ca2+ transients elicited in the control and the diabetic rat cardiomyocytes (incubated with buffer or candesartan). The averaged PA of {Delta}F340/380 was significantly recovered in candesartan-incubated cardiomyocytes from diabetic rats compared with buffer-incubated diabetic cells (0.24 ± 0.01 vs. 0.25 ± 0.01, respectively; Fig. 3B). The TP amplitude of Ca2+ transients of candesartan-incubated diabetic cells (0.183 ± 0.004 s) was significantly restored compared with buffer-incubated diabetics (0.249 ± 0.006 s), whereas the DT50 was significantly shortened in candesartan-incubated diabetic group with respect to buffer-incubated diabetic group (0.62 ± 0.02 vs. 0.68 ± 0.02 s) (Fig. 3B). Candesartan incubation of the cells from the control group could significantly affect neither PA nor TP and DT50. The averaged diastolic value of {Delta}F340/380 was 0.436 ± 0.006 in candesartan-incubated diabetic cells, and this value was significantly smaller than in the buffer-incubated diabetic cells (0.46 ± 0.01). This is indicative that the basal [Ca2+]i level is larger in the diabetic groups with respect to the controls (0.42 ± 0.01), and candesartan incubation could restore this value to the levels of the control. Candesartan incubation of the cells from control group did not change the diastolic value of {Delta}F340/380 (0.43 ± 0.01 vs. 0.42 ± 0.01) (Fig. 3C).


Figure 3
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  		Fig. 3. Effect of Can incubation on the parameters of intracellular Ca2+ transients from DM rats. A: intracellular Ca2+ transients recorded from different groups of cells are given with different colors as indicated. B: changes in PA of the fluorescence Ca2+ transients ({Delta}F340/380 = F340/380 peak – F340/380 basal), TP, and DT50 of the DM (nrat = 5, and ncell = 35) and the Con groups (nrat = 5, and ncell = 44). Traces were obtained from cardiomyocytes of Con, DM, and Can (1 µM)- or BIM (100 nM)-incubated cells (4 h). Ca2+ transients were induced by field stimulation (20–30 V with 10-ms duration at 0.2 Hz). C: changes of basal intracellular [Ca2+] in the DM and Con groups with or without Can or BIM incubations. Values are expressed as means ± SE of 48–60 cells from at least 5 animals. D: caffeine-induced peak intracellular Ca2+ transients elicited in cardiomyocytes. Bar graph {Delta}F340/380 values of the 2 groups are expressed as means ± SE of 20–27 cells from at least 4 animals. *P < 0.05 vs. Con; {dagger}P < 0.05 vs. DM.

 
Effects of BIM on the altered parameters of the Ca2+ transients seen in diabetes were also tested in the present study. We observed that incubation of diabetic cardiomyocytes with BIM for 6–8 h significantly restored the prolongation in both TP and DT50 values of the diabetic cardiomyocytes. Additionally, BIM incubation caused a significant recovery of the diminished Ca2+ transient amplitude in the diabetic cardiomyocytes (Fig. 3, A–C).

Effect of candesartan on SR-Ca2+ content of diabetic rat cardiomyocytes. It is well known that caffeine application causes a sudden and transient increase in intracellular Ca2+ in cardiomyocytes due to a Ca2+ release from SR. The size of the caffeine-induced Ca2+ release has been used to assess the SR-Ca2+ load of the diabetic cardiomyocytes. We observed that caffeine-induced peak of intracellular Ca2+ transient was smaller in the diabetic cardiomyocytes compared with control cells without any significant change in the kinetic parameters. Candesartan or BIM incubation of the cardiomyocytes could counteract the reduction in caffeine responses (in terms of the amplitude of the transients) of Ca2+ release from SR in the diabetic rats (Fig. 3D), whereas no significant effect was observed in the controls.

Candesartan inhibits PKC activation in diabetic rat cardiomyocytes. To confirm that the increased PKC activity has an important role in the pathogenesis of the heart complications in diabetes, we estimated, using Western blot analysis, total PKC protein content in the cytosolic and membrane fractions of the cardiomyocytes from both control and diabetic rats before and after candesartan incubation. Total PKC content in the membrane increased markedly in diabetic hearts, whereas it decreased markedly in the cytosolic part (Fig. 4, A and B). Nonetheless, candesartan significantly attenuated these changes in both cytosolic and particulate fractions. The ratio of the protein content of total PKC in the membrane to that in the cytosolic fraction was increased very markedly in the diabetic hearts; candesartan incubation caused significant decrease in this value compared with control values (Fig. 4C). Western blot analysis was also performed with BIM incubation. As can be seen from Fig. 4, we obtained significant recovery in the parameters of PKC similar to the data with candesartan. Neither candesartan nor BIM incubations of the cardiomyocytes from control rats have any significant effect in the parameters of the controls (data not shown).


Figure 4
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  		Fig. 4. Effect of Can or BIM incubation on the protein content of total PKC in cardiomyocytes from DM rat heart. A: Western blot bands showing the total PKC protein content in the cytosolic fraction of the hearts from the Con, DM, and Can (1 µM)- or BIM (100 nM)-incubated (6–8 h) cells. The histographic representation of quantified data (in arbitrary units) of the total PKC content is given in the upper part of each band. B: total PKC content in the membrane fraction of the hearts from the same groups. C: ratio of membrane to cytosolic fractions (means ± SE; 4 rats with double assays in each sample from each group for each type of measurement). *P < 0.05 vs. Con; {dagger}P < 0.05 vs. DM.

 
Effects of candesartan or BIM on RyR2 in diabetic rat cardiomyocytes. We previously hypothesized and showed that the alterations in Ca2+-release behavior observed at the global and local spark level in STZ-induced diabetic rat cardiomyocytes were due to a defect in RyR2 function following its hyperphosphorylation and release of FKBP12.6 (51). The phosphorylation level of RyR2 in candesartan- or BIM-incubated (6–8 h, at 37°C) diabetic cardiomyocytes was evaluated by using specific antibodies directed against RyR2 and phosphorylated RyR2. Total RyR2 in the diabetic group was significantly recovered following candesartan or BIM incubation as estimated from the Western blot bands, whereas no effect on control cells was observed (data not given) (Fig. 5A). The significant phosphorylation of RyR2 in diabetic rat heart was significantly preserved with treatment with both candesartan and BIM at a level of about 50% (Fig. 5B). The amount of FKBP12.6, which was decreased by about 40% in the diabetic rat heart compared with control rat heart, was significantly preserved by treatment of cardiomyocytes with either candesartan or BIM (Fig. 5C).


Figure 5
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  		Fig. 5. Effect of Can or BIM incubation on ryanodine receptor (RyR2) in DM rat cardiomyocytes. Representative Western blots and the average changes of RyR2 (A), phosphorylated RyR2 (RyR2-P; B), and FK506 binding protein 12.6 (FKBP12.6; C) measured in cardiomyocytes from the Con, DM, and Can or BIM-incubated cells; +BIM groups are given as bar graphs (4 animals with double assays). *P < 0.05 vs. Con; {dagger}P < 0.05 vs. DM.

 
AT1 receptor blockage effects occur through reduction of oxidized protein thiols. It is well known that metabolic shifts brought about by diabetes increase production of reactive oxygen species (ROS) and nitrogen species (see review in Ref. 47). In addition, in our previous study we, like others, have shown that oxidative stress is involved in the etiology of diabetes-induced downregulation of transient outward K+ currents. Moreover, Shimoni et al. (43) have recently shown that the attenuation of transient outward K+ currents with diabetes-related oxidative stress through generated ANG II increased superoxide ion levels and their recovery following ACE inhibition through a reduction in oxidative stress. In this regard, our results indicated that the effects of AT1 receptor blocker occurred mainly through the alteration in the oxidative state of protein thiols (Table 1). Total and acid-soluble SH concentrations were measured in the isolated cardiomyocytes. The results, summarized in Table 1, demonstrate that both total and soluble SH levels of diabetic cardiomyocytes were significantly decreased with respect to the controls. Both candesartan and BIM incubation of these diabetic cardiomyocytes could markedly preserve these values to the levels of the controls.


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  		Table 1. Effects of Can and BIM on sulfhydryl contents of cardiomyocytes isolated from Con and DM rat hearts

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major novel result of our study is that candesartan applied on cardiomyocytes isolated from STZ-induced diabetic rats exerts a protective action against anomalous Ca2+ homeostasis that is attributable to reduced RyR2 phosphorylation level and recovery of protein levels of both RyR2 and FKBP12.6. Furthermore, candesartan downregulates PKC translocation to the membranes. Similar recoveries are also obtained by treating the diabetic cells with a PKC inhibitor, BIM. In this regard, the present data demonstrate that activation of AT1 receptor plays a major role in inducing functional changes of heart under diabetic conditions via a defective interaction of FKBP12.6 with RyR2, which is triggered by the hyperphosphorylation of RyR2 via modulation with PKA, as well as an upregulation of the PKC and increase in ROS generation, although our study did not clarify all the intermediate steps.

All components of the RAS are present in the cardiac tissue, and ANG II is generated in the heart. However, ANG II generation depends largely, if not completely, on kidney-derived renin. The significant effects of application of candesartan, an AT1 antagonist, on isolated cardiomyocytes imply that the AT1 receptors are activated. This could seem surprising at first, since the cardiomyocytes were enzymatically dissociated at least 1 h before the application of the antagonist. However, diabetic cardiomyocytes produce ANG II (14, 43). Most probably, ANG II could be released and will act in an autocrine fashion, similar to a manner in neonatal cardiomyocytes in which ANG II was shown to be released 100-fold under stretching (ref. 38; see review in Ref. 7a). Also, one cannot exclude the fact that in the diabetic heart, AT1 receptors may exhibit a relatively stable binding to ANG II or would be constitutively active.

Enhanced RAS action in tissues and related signaling pathways have been consistently demonstrated to contribute to cardiovascular disease in diabetes, including diabetic cardiomyopathy (12, 34, 39). We previously reported that, in a rat model of STZ-induced diabetes, a prominent abnormal Ca2+ leak occurs through the RyR2, owing to a partial loss of RyR2-bound FKBP12.6 and PKA-mediated hyperphosphorylation of RyR2 (51). Both protein kinases, PKA and PKC, are expressed in cardiac muscle (22), and they strongly controlled cardiac inotropic activity (17, 19, 45). PKA and PKC play a central role in transducing signal and potentiating intracellular cross talk by phosphorylating diverse substrates, including receptors, enzymes, and transcription factors. They control the cardiac pump function and might be activated by various mediators including ANG II (13). The marked recovery in the characteristics of Ca2+ sparks with candesartan may be attributable in part due to an indirect effect of ANG II on PKA activation (34). These findings suggest that, in diabetic rat hearts, a hyperadrenergic signal is transmitted into the cell, resulting in a hyperphosphorylation of RyR2, which triggers a defective interaction of FKBP12.6 with RyR2. Hence, candesartan, by acting on AT1, would reduce RyR2 hyperphosphorylation. In a very recent review article, Yano et al. (50) discussed the stabilization of RyR2 as a new therapeutic paradigm in heart failure due to the effects of ANG II receptor blockers that are suppressing the hyperadrenergic state, thereby reversing PKA-mediated hyperphosphorylation of RyR2, restoring the stoichiometry of its macromolecular complex, and inhibiting the Ca2+ leak from SR. This hypothesis is further supported by our data. We have shown that with candesartan incubation, the cardiomyocytes could prevent diabetes-induced hyperphosphorylation of RyR2 and defect in the interaction of FKBP12.6 with RyR2. This prevention can further cause a recovery in abnormal Ca2+ leak from SR and a significant recovery in intracellular Ca2+ overload.

It is known that stimulation of AT1 receptors in diabetic heart results in a marked activation in PKC activity (8, 14, 26). Our competitive studies with an AT1 receptor blocker candesartan and a nonspecific inhibitor BIM revealed that there is a close relationship between increased levels of ANG II receptors, enhanced PKC activity, and abnormal Ca2+ release in diabetic heart. Furthermore, AT1 receptor stimulation induces the generation of oxygen-derived free radicals, which can have detrimental effects on heart function (8, 25, 28). Recently published data support this idea (23, 31). They reported the role of ROS in the amplification of PKC signaling and ANG II activation in high glucose-induced cell dysfunction. With these findings in mind, it can be suggested that PKC can play important roles in the alterations of both local and global Ca2+ releases in cardiomyocytes from diabetic rat hearts.

The heart, like many other organs, has a local RAS (9–11, 14), and the activation of RAS is shown to contribute to diabetic cardiomyopathy (12). Supporting this idea, Gassanov et al. (16) have recently demonstrated that ANG II could induce marked changes in the characteristics of Ca2+ sparks in human atrial myocytes and that candesartan (1 µM) could cause significant recovery in these parameters within 12 h of incubation. Moreover, other studies with both AT1 receptor blockers and antioxidants showed significant augmentation in diabetes-induced cardiac cell dysfunctions (41–43). It is important to note that hyperglycaemia can lead to oxidative stress by a wide variety of mechanisms, including activation of RAS, and activation of PKC also leads to oxidative stress (25, 26). In the present study, we have demonstrated that both total and soluble SH levels in diabetic cardiomyocytes were significantly decreased with respect to control. Both candesartan and BIM incubations of these diabetic cardiomyocytes could markedly preserve these values to the levels of control. With these findings kept in mind and the data related to recovery in the hyperphosphorylation level of RyR2 with both candesartan and BIM, it can be suggested that oxidative stress can play important roles in the alterations of both local and global Ca2+ releases in cardiomyocytes from diabetic rat hearts. This is strongly supported by the study of Bidasee et al. (4), which demonstrated significant increased disulfide bond formation and alterations in RyR2 functions in STZ-induced diabetic heart (4). The role of diabetes in the modification of SH groups of heart preparations has been reported earlier. In that study (32), ATPase activities of myofibrils and myofibrillar SH reactivity to 5,5'-dithiolbis(2-nitrobenzoic acid) of isolated cardiomyocytes from diabetic rat hearts were measured. Both parameters were depressed and responded differentially to two different types of specific SH group modifiers. Therefore, the mechanism for the effect of candesartan on altered Ca2+ release mechanisms in heart during diabetes could involve, partially, marked recovery in modified SH groups into the cells. It is also well known that metabolic shifts brought about by diabetes increase production of ROS and nitrogen species (see review in Ref. 47). These species are also capable of reacting with several amino acid residues on proteins. Like glycation, modifications of amino acid residues by these species can alter the tertiary structure, and these changes, in turn, could alter the function of RyR2 (4). In addition, in our previous study we, like others, have shown that oxidative stress is involved in the etiology of diabetes-induced downregulation of cardiac function and that there is a close relationship between insulin deficiency, or impaired insulin signaling, and alterations in the cardiac function via depressed endogenous defense mechanisms (1, 41, 48).

Another possible mechanism to account for an AT1 receptor blockade-induced reversal of slowed relaxation of the intracellular Ca2+ transients in diabetic cardiomyocytes is restoration of the function of SERCA, which is diminished in STZ-induced diabetes (7, 25, 30). Activation of PKC has also been shown to decrease SR-Ca2+ uptake and to have a negative inotropic effect in rat cardiomyocytes (36). Decreased caffeine-induced intracellular Ca2+ transient in diabetic cardiomyocytes has been interpreted as decreased SR-Ca2+ load due to the depressed SERCA activity (7). In the light of these published data, our findings indicated that the decreased SR-Ca2+ load could also be restored via restoration of SERCA activity by candesartan treatment, besides its possible effect on RyR2 hyperphosphorylation. This hypothesis is supported by the study of Hanatani et al. (18) in which candesartan treatment prevented left ventricular dysfunction via normalization of the SERCA and Na+-Ca2+ exchanger levels.

Our present data demonstrate that both PKA and PKC, either by separate ways or in a common pathway, can play important roles in the diabetes-induced alterations in Ca2+ homeostasis as well as ANG II-induced cell dysfunction in diabetes. We have shown that both candesartan and BIM have similar protective effects on these altered parameters related with Ca2+ homeostasis and finally with heart function from male rats. Our data are supported with several studies performed with different diseased heart models. Recently, Watanuki et al. (46) investigated alterations of expression and site-specific phosphorylation of phospholamban, and SERCA in STZ-induced diabetes, and showed that PKA-dependent phosphorylation might incompletely counteract the function of phospholamban as an inhibitor of SERCA activity in diabetes in which PKC expression and activity were enhanced. Alltogether, our data point out the roles of PKA and PKC, either together or individually, via the activation of ANG II and oxidant stress in diabetes-induced heart dysfunction.

Viewed together, our observations are consistent with the concept that ANG II is upregulated in the heart during diabetes, and this causes several major alterations in the Ca2+ homeostasis of cardiomyocytes. This research extends our previous observations that chronic ANG II blockade protects the heart from the development of cellular alterations typically related with diabetes, such as prolonged action potentials or reduced Ca2+ transient and contraction (30). Both PKA and PKC are involved in these alterations either individually or together in the same pathway. It is clear that activities of both kinases are closely related to diabetes and also to oxidative stress status in the cell. Our results should help to identify a better basis to understand the molecular mechanisms of diabetes-induced heart dysfunction, the common clinical observations that use ANG II receptor blockers to improve prognoses among patients with diabetes, and other treatments. These agents appear to prevent severe disease complications from occurring, partially by ameliorating the defective FKBP12.6-mediated stabilization of the RyR2 as well as SERCA function. This new therapeutic strategy, targeting the intracellular Ca2+ homeostasis, should be promising for the treatment of diabetic cardiomyopathy.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Ankara University Biotechnology Institute Project Grant 2001K-120-240 and projects of TUBITAK-SBAG-PIA-10 (105S149) and TUBITAK-SBAG-3056 (104S591).


   ACKNOWLEDGMENTS
 
We thank Drs. S. Ozdemir, N. Purali, H. Gurdal, and M. Ugur for technical contributions and helpful discussions and AstraZeneca (Istanbul, Turkey and Mölndal, Sweden) for a generous gift of candesartan. We thank Drs. A. M. Marks and X. Wehrens for a generous gift of RyR2 and phosphorylated RyR2 antibodies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Turan, Ankara Univ., School of Medicine, Dept. of Biophysics, Sihhiye, 06100, Ankara, Turkey (e-mail: belma.turan{at}medicine.ankara.edu.tr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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