Development of diabetes leads to dysfunction of several tissues including heart. Patients with diabetes demonstrate a substantially increased risk of congestive heart failure (1). A number of experimental and clinical observations indicate that structural, functional, and biochemical changes occur in diabetic heart (2). In animal studies among other biochemical changes elevated level of adenosine in diabetic heart have been reported (3). Adenosine is an endogenous nucleoside with potent action on a variety of physiological systems including cardiovascular system. Its action is mediated by cell-surface receptors coupled to G proteins (4). To date four adenosine receptors have been identified namely A1, A2a, A2b, and A3. The affinity for adenosine and signaling mechanism varies among these receptors, therefore, cell possessing more than one type of AR display different responses to changes in adenosine concentration. In heart activation of A1 receptor attenuates b-adrenoceptor stimulation (5), delays ischemic contracture (6) and stimulates anaerobic glycolysis (7). Coupling of adenosine to A2 receptor leads to vasodilation, and cardioprotection during postischemia reperfusion (8). The physiological role of A3 adenosine receptor in heart is currently undefinied. The experimental data from A3–AR knockout mice indicate that this receptor is important for heart performance during ischemic-hypoxic stress (9).

The goal of our study was to evaluate the expression level of adenosine receptors in diabetic heart and in isolated cardiomyocytes and to assess the effect of administration of insulin to diabetic rats on the level of AR in the heart.


MATERIALS AND METHODS
Experimental diabetes

Diabetes was induced in male Wistar rats (200-240 g) by a single injection of streptozotocin (75 mg/kg body weight) into tail vein. Streptozotocin (STZ) was dissolved in 10 mM citrate buffer, pH 4.5. Control rats (hereafter referred to as normal rats) were injected with citrate instead of STZ. On the 1st, 5th, 10th day after STZ injection and on the day of the experiment, blood glucose levels were measured from tail blood. Only rats with the glucose level of 20-30 mM were used for further experiments. One group of rats on day 10 after STZ treatment were injected with insulin (long-acting, 10 units/kg) once a day for 4 days. During the insulin treatment blood glucose levels were measured from tail blood once a day. On day 14 rats (Table 1) were killed by decapitation the heart was removed and the cardiac myocytes were isolated. The experiments on animals were conducted in accordance with the protocol approved by Regional Bioethical Commission at the Medical University of Gdansk (permission- NKEBN/24/2003).

Table 1. Summary of data characterizing the three experimental groups of rats. One group of diabetic rats on day 10 after STZ treatments were injected with insulin (10 U/kg) once a day for 4 days. Values are mean ± S.D. (for normal and STZ treated rats N=11, for diabetic rats treated with insulin N=5). P < 0.001 vs. normal rats.

Isolation of cardiac myocytes

Cardiac myocytes were isolated as described previously (10). Briefly, rats were injected intraperitoneally with heparin (1 U/g body wt) 30 minutes before decapitation. Heart was rapidly removed into ice-cold Tyrode solution, the aorta was cannulated and retrogradely perfused for 5 min with the Tyrode solution containing 0.1 mM EGTA, followed by 10 minutes perfusion with 1 mg/ml of collagenase type II (Gibco) in the same medium. After perfusion, the ventricles were cut from the atria and placed into 5 ml fresh Tyrode solution containing 1 mg/ml collagenase II, disrupted with forceps into small pieces and shaken for 5-10 min. The cell suspension was filtered trough nylon sieve with pore size 250 µm, and allowed to sediment gravitationally (10 min). The settled cells were washed twice with Tyrode solution containing increased concentration of Ca2+ up to 1 mM, and used for experiments. Usually the entire procedure of cardiac myocytes isolation took no more than 60 min. The purity of isolated rod-shaped cells assessed by examination under light microscopy was in the range 75-80%.

RNA extraction and reverse transcription

Total RNA was extracted from cells with the use of Total RNA Prep Plus Kit (A&A Biotechnology), and stored at - 80 °C. RNA was stored as a pellet under ethanol at - 20 °C. Reverse transcription was performed in 20 µl of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM dNTPs, 250 ng oligo(dT), 14 Units of moloney murine leukemia virus reverse transcriptase (Epicentre Technologies), 10 Units of Rnasin (Promega), and 1-5 µg of RNA. Reactions were incubated for 45 min at 42 °C and 5 min at 95 °C.

Real-time PCR analysis

The levels of AR transcripts were analyzed by real-time PCR performed in a Light Cycler 2.0 (Roche, Mannheim, Germany) using the Light Cycler DNA SYBR Green I Kit, and the primers described previously (11). The reaction mixture contained 2 µl Master Mix, 1 pmol of each primer and 1 µl of cDNA. As negative controls water was run with every PCR. In order to control the PCR product melting curve analysis was performed. The ratio of ß-actin/AR was calculated for each sample. Analysis of the data was done using Light Cycler software 4.0.

Western blot analysis

The AR protein levels were examined in whole heart (without ventricles) extract and in cardiac myocytes. The heart extract was prepared by homogenization of minced with razor blade tissue in 3 vol. of buffer A (20 mM Tris-HCl, pH 7.2, 1 mM dithiothreitol, 0.2 mM Pefabloc SC, and 5 µM leupeptin) in a glass homogenizer with a power-driven Teflon pestle. The extract of cardiac myocytes was obtained by sonication (3 x 15 s) of cell suspension in buffer A (1:2). The homogenates after addition of sodium dodecyl sulfate (SDS) to the final concentration of 2% were boiled for 3 minutes, and insoluble debris were removed by centrifugation. An equivalent amount of protein from obtained extracts were separated by 12% SDS-polyacrylamide gel electrophoresis, and electrophoretically transferred to Immobilon polyvinylidene difluoride transfer membrane (Milipore). The membrane was incubated at 4 °C (overnight) with 3% bovine serum albumin in Tris-buffered saline. The membrane was than cut horizontally at the appropriate position (based on positions of prestained molecular mass markers) and incubated with appropriate primary antibodies for 6 h. After being washed with Tris-buffered saline, membrane was incubated with alkaline phosphatase-conjugated secondary antibodies. Membrane-bound antibodies were visualized with 5-bromo-4-chloro-3-indoyl phosphate and nitro blue tetrazolium. The developed bands were quantified by Gel Doc 2000 system, and relative amounts (normalized to reference protein) were compared using the computer program Quantity One. As a reference protein for A1-AR, A2b-AR, and A3-AR ß-actin was used. For A2a-AR blots the p14-3-3 protein (28 kDa) was used. Primary rabbit polyclonal antibodies to A1-AR (A-268), and blocking peptide for A-268 were from Sigma-Aldrich Sp. z o.o. Rabbit polyclonal antibody to A2b-AR (AB1589P) and blocking peptide were from Chemicon International. Goat polyclonal antibodies to ß-actin (I-19), p14-3-3 (K-19), A2a-AR (R-18), A3-AR (C-17), and corresponding blocking peptides were from Santa Cruz Biotechnology.

Statistical analysis

The statistical analysis was carried out using the STATISTICA 5PL statistical package (StatSoft). Statistical significance was determined using the t-test. P values below 0.05 were considered as significant.


RESULTS
Expression level of adenosine receptors in diabetic heart

In order to evaluate the impact of STZ-induced diabetes on expression level of adenosine receptors in rat heart, we examined the AR mRNA and protein levels in heart of normal and diabetic rats. The changes in AR mRNA were evaluated based on results from real-time PCR performed on cDNA transcribed from RNA isolated from heart and cardiac myocytes.

Examination of ARs expression level in rat heart revealed the presence of detectable amounts of mRNA for A1, A2a, A2b, and A3 adenosine receptor. The expression levels of ARs in heart of diabetic rat 14 days after STZ administration were significantly altered (Fig. 1). The mRNA level for A2a-AR and A3-AR was increased by 40% and 60%, respectively whereas, there were no significant changes in A1 and A2b receptor mRNA level. In order to discriminate the impact of diabetic milieu on ARs expression level from the possible direct effect of STZ on day 10 after STZ treatment the rats were injected with insulin (long-acting, 10 units/kg) once a day for 4 days. Determination of ARs mRNA level in heart of diabetic rats treated with insulin indicated that the expression level of ARs returned to the level observed in heart of normal rats (Fig. 1). Since the protein level not always follows the change in mRNA we evaluated the heart ARs protein level by Western blot. The identity of stained protein bands and specificity of used antibodies were examined by using blocking peptides (immunogens). The antibody to A1-AR, A2b-AR, A3-AR recognized in the rat heart extracts protein bands of 37, 35, and 36 kDa, respectively. In the presence of adequate blocking peptide there was no staining of these bands (not shown); therefore, we assumed that these protein bands represent the respective ARs. The antibody to A2a-AR reacted with two proteins bands migrating as proteins of 50 and 45 kDa (Fig. 2). The reactivity of these protein bands with A2a-antibody was blocked by a peptide corresponding to C-terminal part of A2a-AR. We assumed that the 50 kDa protein band represents the glycosylated form of the receptor (12). Quantitation of normalized to ß-actin Western blots indicated that the protein level of A1-AR increased almost 40% in diabetic heart but this change was not statistically significant due to high variability among blots performed on protein extracts from different hearts (Fig. 2). On the other hand in diabetic heart significant increase in A3-AR protein level was detected. Treatment of diabetic rats with insulin for four days resulted in returning of the A3-AR protein to the level observed in heart of normal rat. A slight decrease (~30%) of A2b-AR protein in diabetic heart was also observed, but it did not reach statistical significance. Despite significantly increased A2a-AR mRNA level, we did not observe any significant changes in the A2a-AR protein level in extracts of whole diabetic hearts.

Fig. 1. The adenosine receptors mRNA levels in heart of diabetic rat. Total RNA was extracted from heart of normal, diabetic, and diabetic rats receiving insulin for 4 days, and the levels of adenosine receptors mRNA was measured by real-time PCR as described under "Materials and Methods". The results normalized to ß-actin mRNA are presented as percent of AR mRNA measured in normal heart ± SD of at least four experiments. The ß-actin\AR ratio for normal heart (100 %) corresponds to 1.33±0.15, 0.53±0.09, 0.97±0.13, and 0.30±0.05 for A1, A2a, A2b and A3 receptor, respectively. *, P < 0.05 relative to normal heart; #, P < 0.05 relative to diabetic heart.

Fig. 2. Changes in adenosine receptors protein level in diabetic rat heart. The protein extracts of whole heart obtained from normal (C), diabetic (D), and diabetic rats receiving insulin for 4 days (D+I) were prepared as described under "Materials and Methods". The proteins (40 µg) were separated on 12% PAGE-SDS and immunoblotted with appropriate antibodies. The blots were scanned and quantified. The presented blots (A) are representative of those obtained in at least in three independent experiments. The quantified results (B) normalized to appropriate reference protein are presented as percent of AR/(reference protein) measured in normal (control) heart ± SD of at least three experiments. *, P < 0.05 relative to control; #, P < 0.05 relative to diabetic cells.

Expression level of adenosine receptors in cardiac myocytes isolated from diabetic heart

Myocardial tissue consists of several cell types of which cardiac myocytes account only for 30-40% of cell numbers. On the other hand, this cell type occupies ~75% of the organ's total volume (13) and forms the major functional and structural unit of the heart. Therefore, we investigated the AR expression level in isolated cardiac myocytes. Evaluation of ARs mRNA levels in isolated cardiac myocytes indicated that the mRNA level for A1-AR and A2b-AR remained unchanged in diabetic cells (Fig. 3). Comparison of A2a-AR mRNA levels in cardiac myocytes of normal and diabetic rats indicated significantly decreased level of this mRNA in diabetic cells. This is in contrast to increased level of this receptor mRNA in whole heart (Fig. 1) and may indicate that the expression level of A2a-AR in other heart cells exceeds that found in cardiac myocytes. The mRNA level of A3-AR was increased in diabetic cells to the similar extend as that observed in whole heart (Fig. 3).

Fig. 3. The adenosine receptors mRNA levels in cardiac myocytes isolated from diabetic rat heart. Total RNA was extracted from cardiac myocytes isolated from normal, diabetic, and diabetic rats receiving insulin for 4 days, and the levels of adenosine receptors mRNA was measured by real-time PCR as described under "Materials and Methods". The results normalized to ß-actin mRNA are presented as percent of AR mRNA measured in cells isolated from normal heart ± SD of at least four experiments. The ß-actin\AR ratio for cardiac myocytes isolated from normal heart (100 %) corresponds to 0.95±0.16, 0.36±0.07, 2.03±0.32, and 0.85±0.14 for A1, A2a, A2b and A3 receptor, respectively. *, P < 0.05 relative to normal cells; #, P < 0.05 relative to diabetic cells.

Western blot analysis performed on cardiac myocytes protein extracts showed protein bands that reacted with antibodies to A1-AR, A2a-AR, A2b-AR, and A3-AR. We did not observe any significant alteration of A2b-AR, and A2a-AR protein levels in diabetic cardiac myocytes. Evaluation of A1-AR protein level indicated that the amount of this protein increased by 50% in diabetic cells (Fig. 4). A similar increase of the protein level in diabetic cardiac myocytes was visible for A3-AR. Treatment of diabetic rats with insulin resulted in normalization of the AR protein level in cardiac myocytes.

Fig. 4. Changes in adenosine receptors protein level in diabetic cardiac myocytes. The protein extracts of cardiac myocytes isolated from normal (C), diabetic (D), and diabetic rats receiving insulin for 4 days (D+I) were prepared as described under "Materials and Methods". The proteins (30 µg) were separated on 12% PAGE-SDS and immunoblotted with appropriate antibodies. The blots were scanned and quantified. The presented blots (A) are representative of those obtained in at least in three independent experiments. The quantified results (B) normalized to appropriate reference protein are presented as percent of AR/(reference protein) measured in normal (control) cells ± SD of at least three experiments. *, P < 0.05 relative to control; #, P < 0.05 relative to diabetic cells.

DISCUSSION
Adenosine receptors are key elements in mediating cardioprotective action of adenosine. Our data presented in this report indicate that all four subtypes of adenosine receptors are expressed in the rat heart and in cardiac myocytes. This is consistent with previous studies on adenosine receptors in heart although, much of the current evidence on adenosine receptor subtypes in cardiac myocytes originates from pharmacological studies performed on cultured immature cells (reviewed in 14). Here we showed that these receptors are also expressed in adult cells. Nevertheless, the data presented show diabetes-induced changes in heart adenosine receptors occurring at the level of gene expression and receptor's protein content. Some of these alterations as discussed below correspond to abnormalities characteristic of the diabetic cardiomyopathy.

Traditionally, A1 receptor has been considered to be the key player in cardioprotection. Stimulation of A1 receptor in the heart leads to inhibition of cAMP production, suppresses the ß-adrenergic stimulation (5), and improves anaerobic glucose utilization (7). Overall, activation of A1 receptor reduces the cardiac work and myocardial oxygen consumption. A number of experimental and clinical data point to the importance of A1-AR in mediating anti-ischemic actions of adenosine (8). We have demonstrated that in diabetic rat the mRNA level for A1 receptor changed neither in whole heart nor in isolated cardiac myocytes, however, the A1-AR protein level increased significantly in diabetic cardiomyocytes. Given the central role of A1-AR in cardioprotection during ischemia and reperfusion an increase in A1 receptor content would be considered beneficial for diabetic cardiac myocytes. It was demonstrated that overexpression of A1 receptor leads to increased protection against ischemia-induced myocyte injury and enhanced preconditioning effect (15). On the other hand epidemiological studies indicate that diabetic patients are more prone to develop myocardial infarction and postinfarction complications (16). This may be due to decreased ability of diabetic myocardium to ischemic preconditioning, although confounding findings do exist with respect to response of diabetic heart to preconditioning. Some animal studies indicate that diabetic hearts are more resistant to infarction than normal heart and that preconditioning results in additional protection (17) whereas, other have shown that in diabetic heart ischemic preconditioning does not confer cardiac protection (18). These divergent results may be due to some experimental differences because other studies on STZ-induced diabetic rats showed that the diabetic heart is more resistant to ischemia/reperfusion injury in the early phase (up to two weeks) of diabetes, but become more sensitive at late phase of diabetes development (four to six weeks) (19). This is consisted with our results showing increased level of A1 receptor after 14 days from SZT administration. The other lesson coming from our study is that the A1-AR/A2a-AR ratio (both on protein and mRNA level) increases in cardiomyocytes of diabetic rat. This may have important physiological consequences since; both receptors coexist on the same cell and have similar affinity for adenosine (12), but activation of A2a-AR counteracts the anti-adrenergic action of A1-AR (20). Several studies reported A2a-AR-mediated protection of the heart during postischemia reperfusion by modulation the vascular function (21, 22), but the precise physiological role of A2a receptors located on cardiac myocytes remains to be determined. We assume that an increase in A1-AR/A2a-AR ratio may alter the physiological balance between pro- and antiadrenergic action of adenosine, which may have important consequences for failing heart. Recently it was reported that stimulation of A2a receptor protected the cardiac cells against apoptotic death by modulating the expression level of anti-apoptotic Bcl2 and pro-apoptotic Bax proteins (23). On the other hand, diabetes has been associated with an increased apoptosis and necrosis of cardiac myocytes (24, 25). In our report we demonstrate that expression level of A3-AR increases significantly in diabetic cardiac myocytes. It should be noted that activation or overexpression of adenosine A3 receptor induced apoptosis of several cell types including cardiac myocytes (26, 27), a deleterious effect applicable to diabetic myocardium. Experiments with A3-AR knockout mice demonstrated that the absence of adenosine A3 receptor leads to ischemia-tolerant phenotype, supporting view on detrimental rather than protective function of this receptor in the heart (9).

Concluding, presented evidence indicates that in the rat cardiac myocytes diabetes induces changes in adenosine receptors expression level, and that these changes correspond to some cardiac complication observed in diabetes.

Acknowledgments: This work was supported by the State Committee for Scientific Research (KBN) grant No. 3 P05A 054 24 to TP.


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