AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 289: H614-H623, 2005. First published April 8, 2005; doi:10.1152/ajpheart.00101.2005
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Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Harjot K. Saini, Vijayan Elimban, and Naranjan S. Dhalla

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

Submitted 2 February 2005 ; accepted in final form 1 April 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular ATP is known to augment cardiac contractility by increasing intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes; however, the status of ATP-mediated Ca2+ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subjected to 10–30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca2+]i was measured in purified cardiomyocytes by fura-2 microfluorometry. Reperfusion for 30 min of 20-min ischemic hearts, unlike 10-min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca2+]i; no changes in basal [Ca2+]i were evident in 10- or 20-min I/R preparations. On the other hand, reperfusion of 30-min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca2+]i and a dramatic increase in basal [Ca2+]i. The positive inotropic effect of extracellular ATP was attenuated, and the maximal binding characteristics of 35S-labeled adenosine 5'-[{gamma}-thio]triphosphate with crude membranes from hearts undergoing I/R was decreased. ATP-induced increase in [Ca2+]i in cardiomyocytes was depressed by verapamil and Cibacron Blue in both control and I/R hearts; however, this response in I/R hearts, unlike control hearts, was not affected by ryanodine. I/R-induced alterations in cardiac function and ATP-induced increase in [Ca2+]i were attenuated by treatment with an antioxidant mixture and by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2. The results suggest an impairment of extracellular ATP-induced Ca2+ mobilization in I/R hearts, and this defect appears to be mediated through oxidative stress.

isolated cardiomyocytes; oxidative stress; ischemic preconditioning

ALTHOUGH ATP IS an essential source of energy for a wide variety of cellular processes (7), extracellular ATP has also been recognized as a local regulator of physiological functions in the cardiovascular system (37). Such a role of extracellular ATP has been ascribed because it produces a vasodilator effect due to its action on the endothelium, a vasoconstrictor effect by acting on the smooth muscle (5), and positive inotropic action due to its effect on the ventricular muscle (32). In fact, cardiomyocytes are known to express purinergic ATP receptors including P2X (ligand-gated ion channels) and P2Y (G protein-coupled receptors) (17), and the positive inotropic effect of ATP has been shown to be mediated through the activation of purinergic receptors and a subsequent increase in the intracellular concentration of Ca2+ ([Ca2+]i) (32, 39). Several investigators have demonstrated that influx of Ca2+ through L-type Ca2+ channels in the sarcolemmal (SL) membrane and inositol triphosphate-mediated mobilization of intracellular Ca2+ from the sarcoplasmic reticulum (SR) are the major pathways of ATP-induced increase in [Ca2+]i in isolated cardiomyocytes (9, 10). Furthermore, it has been reported that the ATP-induced increase in [Ca2+]i is augmented by norepinephrine as a consequence of enhanced inward Ca2+ current (42). It should be pointed out that extracellular ATP is released from the sympathetic nerve terminals as a cotransmitter with norepinephrine (5, 6). The level of extracellular ATP has been shown to be increased in the interstitial fluid during ischemia-reperfusion (I/R) (18). In addition, a substantial amount of ATP is released from hypoxic cardiomyocytes and activated platelets, as well as from different types of cells such as erythrocytes, secretory cells, vascular endothelial cells, and smooth muscle cells (37). Thus extracellular ATP can be seen to affect the myocardium under both physiological and pathophysiological conditions; however, very little information regarding the responsiveness of the heart as well as cardiomyocytes to ATP under pathological conditions is available in the literature.

Recent studies from our laboratory (31) have shown attenuation of the positive inotropic effect of extracellular ATP in heart failure as well as depression of the ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from failing hearts. On the other hand, no such defect in ATP responses was observed in cardiomyocytes isolated from diabetic hearts (41). Because the effects of exogenous ATP on cardiac performance in I/R hearts as well as intracellular Ca2+ mobilization in cardiomyocytes isolated from I/R hearts have not been investigated previously, the present study was undertaken to test the hypothesis that the positive inotropic effects of extracellular ATP and ATP-induced increase in [Ca2+]i are depressed in I/R hearts. Because oxidative stress has been shown to play an important role in I/R-induced cardiac dysfunction (11, 25), this study also examined whether the ATP-induced changes in [Ca2+]i are mediated through oxidative stress. For this purpose, ATP-induced alterations in [Ca2+]i mobilization were investigated in cardiomyocytes isolated from I/R hearts treated with an antioxidant mixture containing SOD and catalase, which is known to prevent the development of oxidative stress (13). The present study also investigated whether the effects of I/R on ATP-induced changes in [Ca2+]i handling are simulated by perfusing the hearts with H2O2, a well-known oxidant (40). In addition, because ischemic preconditioning (IP) is known to prevent I/R-induced changes in cardiac function (36), this study tested whether the I/R-mediated alterations in ATP-induced increase in [Ca2+]i in cardiomyocytes are prevented by IP.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All experimental protocols were approved by the University of Manitoba Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care.

Isolated rat heart preparation. Male Sprague-Dawley rats weighing 250–300 g were anesthetized by intraperitoneal injection of a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The hearts were rapidly excised, mounted on Langendorff apparatus, and perfused with Krebs-Henseleit (K-H) solution gassed with 95% O2-5% CO2 at 37°C and pH 7.4. The composition of K-H solution was (in mM) 120 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 1.25 CaCl2, and 11 glucose. The hearts were electrically stimulated at a rate of 300 beats/min via a square-wave current of 1.5-ms duration throughout the experiment with a Phipps and Bird stimulator (Richmond, VA), and the perfusion rate was maintained at 10 ml/min. Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), rate of change of contraction (+dP/dt), and rate of change of relaxation (–dP/dt) were measured via a transducer (model 1050 BP, Biopac Systems, Goleta, CA), which was connected with a water-filled latex balloon inserted into the left ventricle (LV). At the beginning of the experiment, the LVEDP was adjusted to ~10 mmHg by inflating the balloon, and the left ventricular developed pressure (LVDP) was taken as the difference between LVSP and LVEDP. The data were recorded online through an analog-digital interface (MP-100, Biopac Systems) and stored in a computer program (Acqknowledge 3.5.3) with a Biopac data acquisition system (Biopac Systems). All hearts were stabilized for a period of 20 min and were randomly divided into experimental groups.

In the first series of experiments, control hearts were perfused for 10–60 min after stabilization. As no significant differences were observed in the cardiac performance and [Ca2+]i handling during 10- to 60-min perfusion, the control values were grouped together. Some hearts were made globally ischemic for 10 or 20 min by stopping the coronary flow, after which the flow was restored for 30 min, whereas other hearts were subjected to 30 min of global ischemia followed by 5, 15, or 30 min of reperfusion. In the second series of experiments, isolated hearts were treated with an antioxidant mixture containing SOD (5 x 104 U/l; Sigma-Aldrich, Oakville, ON, Canada) and catalase (7.5 x 104 U/l; Fisher Scientific, Nepean, ON, Canada) for 10 min before ischemia was induced as well as during the reperfusion period (26). It should be pointed out that the antioxidant mixture was not washed out before ischemia. Furthermore, to test whether the effects of I/R are simulated by oxidative stress, hearts were perfused with H2O2 (50 µM) for 30 min after the stabilization period as described previously (30). In the third series of experiments, IP in hearts was induced by three cycles of 5-min ischemia followed by 5-min reperfusion before subjecting the hearts to I/R as described by Temsah et al. (36). In some experiments, ATP (50 µM) was infused into the perfusion stream to the heart subjected to 30-min ischemia followed by 30-min reperfusion to study the effect of I/R on the positive inotropic effect of ATP.

Isolation of cardiomyocytes. Ventricular myocytes were isolated with methods described previously (28). In brief, hearts from different groups were perfused for 5 min with Ca2+-free buffer (pH 7.4) containing (in mM) 90 NaCl, 10 KCl, 1.2 KH2PO4, 5 MgSO4, 15 NaHCO3, 30 taurine, and 20 glucose, gassed with 95% O2-5% CO2 at 37°C. These hearts were then switched to the same perfusion medium containing 0.04% collagenase, 0.1% BSA, and 50 µM CaCl2. At the end of a 25-min recirculation period, the hearts were removed from the cannula; the hearts treated with H2O2 were perfused for 15 min. These times of perfusion for I/R hearts or H2O2-perfused hearts with medium containing collagenase were found to yield optimal results for cardiomyocyte isolation. The ventricles were cut into small pieces and subjected to another 15 min of digestion in a fresh collagenase solution in the presence of 1% BSA gassed with 95% O2-5% CO2 in a shaking water bath at 37°C. The ventricular fragments were triturated gently (twice per minute) with a plastic pipette. The cells from three to four harvests were combined and filtered through a 200-µm nylon mesh. The myocytes were resuspended for 5 min in buffers containing gradually increasing extracellular Ca2+ concentration (250, 500, 750 µM) to a final concentration of 1 mM. Cell viability was determined by the Trypan blue (Sigma-Aldrich) exclusion method, and a Neubauer chamber was used to count the viable cells and total number of cells. After the isolation 83–87% of the viable cardiomyocytes in all groups were quiescent. To minimize contamination from dead cells, cardiomyocyte preparations from all experimental groups, after cell viability and yield were determined, were washed two times by centrifugation at 100 g for 1 min and subjected to an isotonic Percoll (Sigma-Aldrich) gradient (pH 7.3) before loading with fura-2 AM. The final cell suspension after purification had 90–95% viable cardiomyocytes, and 3–5% of these cells beat spontaneously. This purified cell preparation was found to be stable for 150 min for studying Ca2+-handling characteristics and thus was used within 120 min of the isolation. Because the isolation of cardiomyocytes involved 5-min Ca2+-free perfusion and 25-min collagenase perfusion with oxygenated medium, it is important to point out that cardiomyocytes obtained from hearts subjected to ischemia or I/R underwent additional reperfusion for 30 min. In some experiments, isolated cardiomyocytes from control hearts were subjected to hypoxia-reoxygenation. For this purpose, cardiomyocytes were suspended in K-H solution without glucose and gassed with 95% N2-5% CO2, at room temperature and pH 7.4, for 30 min. Glucose was replaced with Tris·HCl to maintain osmolarity. This cell suspension was then reoxygenated with K-H solution containing 11 mM glucose and gassed with 95% O2-5% CO2, at room temperature and pH 7.4, for 30 min.

Measurement of [Ca2+]i in purified cardiomyocytes. Freshly isolated cardiomyocytes were incubated with 5 µM fura-2 AM for 40 min in the buffer (pH 7.4) containing 1 mM Ca2+ and washed twice to remove any extracellular dye. The final cell number in the cuvette was adjusted to 0.3 million cells/ml. The alterations in fluorescence intensity were monitored by a SLM DMX-1100 dual-wavelength spectrofluorometer (SLM Instruments, Urbana, IL) adjusted to excitation wavelength of 340/380 nm, emission wavelength of 510 nm, integration time of 0.95 s, and resolution time of 1.0 s. The [Ca2+]i levels were calculated as described previously (28, 31). Treatment with 100 µM isoproterenol (Iso), which produces an optimal response (30), was performed by incubating the fura-2 AM-loaded cells in a buffer containing Iso for 5 min at room temperature before the addition of ATP. It must be noted that Iso was found to have no effect on [Ca2+]i in nonstimulated cardiomyocytes; however, this treatment augmented the ATP- or KCl (30 mM)-induced increase in [Ca2+]i. The difference between the responses in the presence and absence of Iso treatment above the basal [Ca2+]i was taken as the Iso-induced increase in [Ca2+]i. Furthermore, cardiomyocytes were incubated at room temperature for 10 min in a buffer containing the desired concentration of pharmacological agent including verapamil, Cibacron Blue, and ryanodine before the fluorescence intensity measurements. The selection of different concentrations of these agents used in the present study was based on our previous observations (28, 31). Unless otherwise indicated in the text, 50 µM ATP, which produced a maximal effect, was used in this study. The increase in [Ca2+]i at peak [Ca2+]i was calculated as the net increase above the basal value in each experiment. Because a collective response was measured in a large number of cells, the possibility of biasing associated with selection of single cells was eliminated. Furthermore, this preparation was used previously to study the effect of ATP (10, 31, 39), low Na+ (28), and phosphatidic acid (41) on [Ca2+]i in isolated cardiomyocytes.

Preparation of crude membranes and [35S]adenosine 5'-[{gamma}-thio]triphosphate binding. At the end of 30-min ischemia or 30-min ischemia followed by 30-min reperfusion, hearts were removed from the cannula and crude membranes were prepared as described previously (26). In brief, the ventricular tissue was minced and then homogenized in 50 mM Tris·HCl, pH 7.4 (15 ml/g tissue) with a PT3000 Polytron (Brinkman Instruments, Westbury, NY) twice for 20 s each at 15,000 rpm. The resulting homogenate was centrifuged at 1,000 g for 10 min, and the pellet was discarded. The supernatant was centrifuged at 48,000 g for 25 min. The pellet was resuspended and centrifuged twice in the same buffer at the same speed. After the protein content was determined by Lowry's method, these membranes were suspended in 0.2 mM sucrose and 10 mM histidine (pH 7.4) at a concentration of 3–5 mg/ml, stored at –80°C, and used within 2–3 wk without any loss of activity. The purification of isolated membranes was determined by measuring Na+-K+-ATPase (a SL marker enzyme) activity in the presence of 1 mM ouabain and comparing it with that detected in the heart homogenate as described previously (12). However, the presence of other subcellular fractions including mitochondria in the crude membrane fraction cannot be ruled out. The status of purinergic receptors in the membrane was determined by studying the binding characteristics of a slowly hydrolyzable analog of ATP, [35S]adenosine 5'-[{gamma}-thio]triphosphate (ATP{gamma}S) (39). Thirty to fifty micrograms of membrane protein was incubated in 0.5 ml of medium containing various concentrations (0.1–10 nM for the high-affinity site and 1–10 µM for the low-affinity site) of [35S]ATP{gamma}S (65 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA) and 50 mM Tris·HCl (pH 7.5) at 37°C for 30 min as described previously (39). The reaction was terminated by vacuum filtration over wet Whatman filters (GF/B). The filters were washed three times with 6 ml of ice-cold distilled water, and radioactivity was counted with a Beckman scintillation counter. Binding was determined in the absence (total) and presence (nonspecific) of 4 mM ATP (Tris salt; Sigma-Aldrich); specific binding was calculated by subtracting the nonspecific binding from the total binding. To avoid possible artifacts, the binding of radioligand GF/B filters was checked in the absence of membrane proteins. The values for Kd and maximum receptor density (Bmax) were calculated by Scatchard plot analysis with Graph Pad Prism 4 for Windows (version 4.02; GraphPad Software, San Diego, CA).

Statistical analysis. All results are expressed as means ± SE. Statistical analysis was performed with Microcal Origin version 6 (Microcal Software, Northampton, MA). The differences between two groups were evaluated by Student's t-test. The data from more than two groups were evaluated by one-way ANOVA followed by the Newman-Keuls test. Values showing P < 0.05 were considered statistically significant unless otherwise indicated in the text.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of I/R on ATP-induced increase in [Ca2+]i. To examine the effect of I/R on ATP-induced increase in [Ca2+]i in cardiomyocytes, isolated hearts were subjected to 10, 20, or 30 min of global ischemia followed by 30 min of reperfusion. Representative tracings showing the effect of I/R on cardiac function are given in Fig. 1. An increase in LVEDP was observed in hearts undergoing 20 or 30 min of ischemia, whereas no such alteration was seen in 10-min ischemic hearts (Table 1). On the other hand, LVDP and dP/dt were depressed in 10-, 20-, or 30-min ischemic hearts (Fig. 1, Table 1). The depression in cardiac function in 10-min ischemic hearts was fully reversible, whereas a partial recovery was observed in 20-min ischemic hearts after 30 min of reperfusion (Fig. 1, Table 1). Data given in Table 1 also indicate that 30-min ischemia followed by 5, 15, or 30 min of reperfusion caused a marked depression in LVDP and a marked elevation in LVEDP. The depression in cardiac function in 30-min I/R hearts is also evident from the fact that the values for +dP/dt in control and experimental hearts were 6,170 ± 395 and 1,608 ± 423 mmHg/s, respectively, whereas those for –dP/dt were 4,138 ± 270 and 750 ± 78 mmHg/s, respectively. The yield was decreased in cardiomyocytes isolated from hearts subjected to 30 min of ischemia and 30 min of reperfusion (2.6 ± 0.4 vs. 8.7 ± 1.9 million cells/heart in control). Furthermore, it can be seen from the information in Table 1 that the cell viability of cardiomyocytes (before purification) from 30-min I/R hearts was decreased. These changes are in agreement with our earlier report (30).



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  		Fig. 1. Representative tracings showing the left ventricular (LV) pressure and change of pressure development (dP/dt) in isolated hearts. A: isolated hearts subjected to 10 min of ischemia (I) followed by 30 min of reperfusion (R). B: isolated hearts subjected to 20-min ischemia followed by 30 min of reperfusion. C: isolated hearts subjected to 30 min of ischemia followed by 30 min of reperfusion.

 

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  		Table 1. Parameters in isolated hearts subjected to 10, 20, and 30 min of ischemia followed by varying times of reperfusion

 
Measurement of [Ca2+]i in purified preparations revealed an increase in basal [Ca2+]i in cardiomyocytes isolated from 30-min I/R hearts (Fig. 2, Table 1). Extracellular ATP (50 µM), a purinergic receptor agonist (37), caused a significant increase in [Ca2+]i in cardiomyocytes isolated from control hearts; this is in agreement with previous studies (31, 39). However, the increase in [Ca2+]i by ATP was significantly depressed in purified cardiomyocytes isolated from 30-min I/R hearts (Table 1, Fig. 2). The depression in ATP-induced increase in [Ca2+]i in I/R hearts may not be due to an elevated level of basal [Ca2+]i because the increase in [Ca2+]i caused by KCl (30 mM), a known depolarizing agent (28), was not altered in 30-min I/R hearts (110 ± 5.5 vs. 112 ± 4.0 nM in control) (Fig. 2). It was also observed that the ATP-induced increase in [Ca2+]i was depressed at different concentrations of ATP (10–100 µM) in cardiomyocytes isolated from hearts undergoing 30 min of ischemia followed by 30 min of reperfusion (Fig. 3).



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  		Fig. 2. Representative tracings showing the increase in intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes isolated from hearts subjected to 30-min ischemia and 30 min of reperfusion. A: ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from control and ischemia-reperfusion (I/R) hearts. B: KCl-induced increase in [Ca2+]i in cardiomyocytes isolated from control and I/R hearts. The increase in [Ca2+]i was calculated as the difference between the peak value and the basal value in each experiment. Arrows indicate time at which the preparation was exposed to 50 µM ATP or 30 mM KCl.

 


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  		Fig. 3. Effect of different concentrations of ATP (10–100 µM) on the increase in [Ca2+]i in cardiomyocytes isolated from control and 30-min ischemic, 30-min reperfused hearts. Each point represents the mean ± SE of 4 experiments in each group. *P < 0.05 vs. control group.

 
To gain further information regarding the effects of I/R on Ca2+ handling by cardiomyocytes, [Ca2+]i measurements were carried out in preparations isolated from hearts subjected to 10- or 20-min ischemia followed by 30 min of reperfusion as well as 30-min ischemia followed by 5- or 15-min reperfusion. The data in Table 1 indicate that basal [Ca2+]i was not altered in cardiomyocytes isolated from hearts undergoing 10 or 20 min of ischemia or 10- or 20-min ischemia followed by 30-min reperfusion. On the other hand, in cardiomyocytes isolated from hearts undergoing 30-min ischemia or 30-min ischemia followed by 5 and 15 min of reperfusion, a significant increase in basal [Ca2+]i was observed (Table 1). ATP-induced increase in [Ca2+]i was depressed in cardiomyocytes isolated from hearts undergoing 20 or 30 min of ischemia, unlike the 10-min ischemic hearts. This attenuation in ATP-induced increase in [Ca2+]i in 20-min ischemic hearts was partially reversed by 30 min of reperfusion, whereas ATP responsiveness remained markedly depressed in 30-min ischemic hearts undergoing 5 or 15 min of reperfusion (Table 1). Analysis of data from 10-, 20-, and 30-min ischemic-reperfused hearts (Table 1) revealed that changes in LVEDP were linearly related to alterations in ATP-induced changes in [Ca2+]i (r2 = 0.8737; P < 0.0001) as well as basal [Ca2+]i (r2 = 0.8033; P < 0.0001). However, it must be emphasized that after cardiac performance was measured, both ischemic and I/R hearts were perfused for 5 min further with Ca2+-free solution and 25 min with collagenase-containing solution for the isolation of cardiomyocytes. Therefore, the 30 min of additional reperfusion time should be taken into consideration when interpreting the data in cardiomyocytes isolated from ischemic or I/R hearts.

Effect of Iso on ATP-induced increase in [Ca2+]i in cardiomyocytes. To examine the effect of I/R on catecholamine-mediated potentiation of ATP response, cardiomyocytes isolated from I/R hearts were treated with Iso (100 µM), a known {beta}1-receptor agonist (30). As shown in Fig. 4, Iso treatment caused an augmentation in ATP-induced increase in [Ca2+]i; this is in agreement with previous findings (42). On the other hand, a significant depression in Iso-induced increase in [Ca2+]i was observed in cardiomyocytes isolated from hearts undergoing 30 min of ischemia followed by 30 min of reperfusion (Fig. 4). It should be mentioned that Iso treatment did not have any effect on basal [Ca2+]i in cardiomyocytes isolated from control and I/R hearts.



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  		Fig. 4. ATP-induced increase in [Ca2+]i in cardiomyocytes, with or without isoproterenol (Iso) treatment, isolated from hearts subjected to 30-min ischemia followed by 30 min of reperfusion. A: representative tracings showing ATP- and Iso-induced increase in [Ca2+]i in cardiomyocytes isolated from control hearts. B: representative tracings showing ATP- and Iso-induced increase in [Ca2+]i in cardiomyocytes isolated from I/R hearts. C: effect of I/R on Iso-induced increase in [Ca2+]i. The increase in [Ca2+]i was calculated as the difference between the peak value and the basal value in each experiment. Iso-induced increase was calculated by subtracting the values for ATP-induced increase in [Ca2+]i in untreated cardiomyocytes from those treated with 100 µM Iso. Arrows indicate time at which the preparation was exposed to 50 µM ATP. Treatment with 100 µM Iso was carried out for 5 min before intracellular Ca2+ measurements. Values are means ± SE of 4 experiments in each group. *P < 0.05 vs. ATP; #P < 0.05 vs. control group.

 
Effect of SOD and catalase on ATP responsiveness in cardiomyocytes. To investigate the involvement of oxidative stress in I/R-mediated alterations in ATP-induced increase in [Ca2+]i, isolated hearts were treated with an antioxidant mixture containing SOD and catalase. The SOD and catalase treatment caused a significant improvement in heart function in terms of increase in LVDP and decrease in LVEDP in hearts undergoing 30 min of ischemia followed by 30 min of reperfusion (Table 2). Similar improvement in +dP/dt and –dP/dt was observed in I/R hearts with SOD and catalase treatment (data not shown). In addition, I/R-induced increase in basal [Ca2+]i was significantly reduced by SOD and catalase treatment. Furthermore, the depression of ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from I/R hearts was markedly attenuated by the antioxidant mixture (Table 2). From the data in Table 2, it can be seen that treatment with SOD and catalase itself had no effect on LVDP, LVEDP, or basal [Ca2+]i. Although SOD and catalase treatment was observed to augment the ATP-induced increase in [Ca2+]i in control and I/R cardiomyocytes (Table 2), no attempt was made to understand the mechanisms of such an effect.


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  		Table 2. Parameters in isolated rat hearts treated with SOD and catalase 10 min before ischemia and during reperfusion period

 
Effect of H2O2 on ATP-induced increase in [Ca2+]i in cardiomyocytes. To test whether the effects of I/R are mimicked by oxidative stress, isolated hearts were perfused with 50 µM H2O2 for 30 min. H2O2 caused a depression in LVDP, +dP/dt, and –dP/dt as well as an elevation in LVEDP (Table 3). The selection of this concentration and time period of H2O2 perfusion was based on our previous study (30). A marked increase in basal [Ca2+]i was observed, whereas ATP-induced increase in [Ca2+]i was significantly depressed in cardiomyocytes isolated from H2O2-treated hearts (Table 3). Similarly, the Iso-induced increase in [Ca2+]i was also depressed in H2O2-treated hearts.


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  		Table 3. Parameters in isolated rat hearts treated with H2O2 for 30 min

 
Effect of IP on ATP-induced increase in [Ca2+]i in cardiomyocytes. To determine the significance of I/R-mediated changes in ATP-induced increase in [Ca2+]i at the level of isolated cardiomyocytes, we examined the effect of IP, which is known to prevent the I/R-induced changes in cardiac performance (36). Although IP caused a partial depression in cardiac performance in control hearts (Table 4), the I/R-induced alterations in cardiac function were improved by IP (Table 4). Attenuation of basal [Ca2+]i and a marked improvement in ATP-induced increase in [Ca2+]i were observed in cardiomyocytes isolated from I/R hearts subjected to IP (Table 4). Basal [Ca2+]i and the ATP-induced increase in [Ca2+]i did not change in cardiomyocytes isolated from IP hearts compared with control hearts (Table 4).


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  		Table 4. Parameters in isolated hearts subjected to ischemic preconditioning before induction of ischemia and reperfusion

 
Inotropic effect of ATP in I/R hearts. To investigate the relationship between reduced responsiveness of ATP in cardiomyocytes isolated from I/R hearts and the response of ATP in I/R hearts, ATP (50 µM) was infused in hearts subjected to 30 min of ischemia followed by 30 min of reperfusion. Representative tracings showing the positive inotropic effect of ATP in control and I/R hearts are given in Fig. 5. The positive inotropic effect of ATP in control hearts in terms of increase in LVDP, +dP/dt, and –dP/dt was 33.4 ± 1.2%, 36.7 ± 1.9%, and 38.1 ± 2.1% of the respective basal values, respectively. On the other hand, in hearts undergoing 30-min ischemia followed by 30-min reperfusion, the ATP-induced increase in these parameters (LVDP 11.5 ± 0.9%, +dP/dt 8.7 ± 1.4%, and –dP/dt 9.2 ± 1.7% of the respective basal values) was significantly depressed compared with control hearts. No significant difference was observed in LVEDP after ATP infusion in both groups (3.1 ± 1.7% and 2.4 ± 1.3% for control and I/R hearts, respectively).



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  		Fig. 5. Representative tracings showing the effect of ATP on LV pressure and dP/dt in isolated hearts. A: effect of ATP on cardiac function in control perfused hearts. B: effect of ATP on cardiac function in hearts subjected to 30-min ischemia and 30- min reperfusion. Arrow indicates time at which the hearts were perfused with 50 µM ATP.

 
Mechanisms of I/R-induced alterations in cardiomyocytes. To assess whether the attenuation of ATP-induced increase in [Ca2+]i in I/R cardiomyocytes was due to a defect at the purinergic receptor level, the specific binding of [35S]ATP{gamma}S, a slowly hydrolyzable analog of ATP (39), to cardiac membranes isolated from ischemic and I/R hearts was determined. As reported previously (39), ATP was found to bind cardiac membranes at high-affinity and low-affinity binding sites. Although the affinity (1/Kd) of purinergic receptors to ATP at both high-affinity and low-affinity binding sites was increased in 30-min ischemic as well as 30-min ischemic, 30-min reperfused hearts, a significant depression in maximal number of purinergic receptors (Bmax) at both sites was observed in these hearts (Table 5).


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  		Table 5. Changes in ATP binding characteristics in crude membrane isolated from hearts undergoing 30-min ischemia and 30-min ischemia followed by 30-min reperfusion

 
To determine the mechanism of depression of ATP responsiveness at the cellular level, cardiomyocytes isolated from control and 30-min ischemic, 30-min reperfused hearts were incubated with verapamil, an L-type Ca2+ channel blocker in the SL membrane (28), Cibacron Blue, an ATP receptor blocker (31), or ryanodine, an agent that prevents the release of Ca2+ from the SR by opening the ryanodine receptor channel to subconductance level or by locking the channel (3, 29). Treatments with different concentrations of verapamil (1 and 10 µM) and Cibacron Blue (50 and 100 µM) resulted in a significant reduction of ATP-induced increase in [Ca2+]i in both control and I/R hearts (Table 6). On the other hand, ryanodine (2 and 10 µM) treatment caused a significant decrease in ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from control perfused hearts but did not affect the ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from I/R hearts (Table 6). None of these drugs altered basal [Ca2+]i in cardiomyocytes isolated from either control or I/R hearts.


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  		Table 6. Effect of various pharmacological agents on ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from control and I/R hearts

 
Effect of hypoxia-reoxygenation on ATP-induced increase in [Ca2+]i. To eliminate the possibility of any artifact caused by cell damage during the isolation process, cardiomyocytes isolated from control hearts were subjected to 30-min hypoxia (in the absence of glucose) followed by 30-min reoxygenation. The results in Table 7 show that the yield and viability of cardiomyocytes were decreased after hypoxia-reoxygenation; however, an increase in basal [Ca2+]i was observed in cardiomyocytes after hypoxia-reoxygenation. Extracellular ATP (50 µM) caused a significant increase in [Ca2+]i in cardiomyocytes isolated from control hearts; this increase in [Ca2+]i by ATP was significantly depressed in purified hypoxic-reoxygenated cardiomyocytes (Table 7). The depression in ATP-induced increase in [Ca2+]i in hypoxic-reoxygenated cardiomyocytes may not be due to an elevated level of basal [Ca2+]i because the increase in [Ca2+]i by KCl (30 mM), a known depolarizing agent (28), was not altered under similar conditions. On the other hand, the Iso-induced increase in [Ca2+]i was significantly depressed in hypoxic-reoxygenated cardiomyocytes (Table 7).


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  		Table 7. Parameters in control cardiomyocytes subjected to 30-min hypoxia followed by 30 min of reoxygenation at room temperature

 

   DISCUSSION
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In the present study, we have shown that basal [Ca2+]i is increased in purified cardiomyocytes isolated from hearts undergoing 30 min of ischemia followed by 30 min of reperfusion. This finding is in agreement with our previous observations (30) indicating a defect at the cellular level in I/R hearts. The increased basal [Ca2+]i was associated with depressed cardiac function in I/R hearts in terms of elevated LVEDP. Although some investigators have shown a similar relationship between [Ca2+]i and LVEDP in I/R hearts (21, 35), others have denied the defect in Ca2+ handling in cardiomyocytes isolated from I/R hearts (8, 16). In this regard, it is important to point out that accumulation of intracellular Ca2+ during the ischemic phase appears to be a consistent finding in most of the studies (14, 19) and the gradual increase in [Ca2+]i during reperfusion phase depends on the time of ischemic insult. In the present study, we have shown that cardiac dysfunction is fully reversible on reperfusion for 30 min in hearts undergoing 10 min of ischemia and partially reversed on 30 min of reperfusion in hearts subjected to 20 min of ischemia. On the other hand, attenuated recovery in cardiac function was observed after 30 min of ischemia followed by different periods of reperfusion. This view is consistent with the observations by other investigators (24, 38) that 20- to 40-min ischemia is required for the transition from reversible to irreversible injury in isolated rat and mouse hearts. Basal [Ca2+]i was not increased after 10- or 20-min ischemia followed by 30 min of reperfusion, whereas a gradual increase in [Ca2+]i was observed after 30 min of ischemia followed by 5, 15, and 30 min of reperfusion. These observations provide the evidence that the alterations in basal [Ca2+]i are linked with reversible (10 and 20 min of ischemia) and irreversible (30 min of ischemia) phases of I/R injury. In fact, [Ca2+]i is regulated by coordinated function of different ion channels in the SL membrane and SR, where marked changes in these ion channels including SL L-type Ca2+ channels, Na+-K+-ATPase, and Na+/Ca2+ exchanger as well as SR Ca2+ pump and release channels in I/R hearts (4, 33, 35, 44) may be responsible for the occurrence of intracellular Ca2+ overload.

The results of this study indicate that infusion of exogenous ATP caused a significant increase in LV function in control hearts and produced an increase in [Ca2+]i in control cardiomyocytes; these observations are in agreement with our previous studies (31, 39). On the other hand, the positive inotropic effect and increase in [Ca2+]i in cardiomyocytes by extracellular ATP were attenuated in hearts subjected to 30 min of ischemia followed by 30 min of reperfusion. This depression in ATP-induced increase in [Ca2+]i after 30 min of ischemia was evident even after 5 and 15 min of reperfusion, whereas no depression in ATP responsiveness was observed in hearts undergoing 10 min of ischemia and 30 min of reperfusion. Although a depression in ATP-mediated increase in [Ca2+]i was observed after 20-min ischemia, a partial recovery was observed after 30 min of reperfusion. A highly significant relation between depression in ATP-induced increase in [Ca2+]i and LVEDP as well as basal [Ca2+]i was observed in I/R hearts. In view of the fact that previous studies have demonstrated that the positive inotropic effect of ATP in control hearts is mediated by its action on purinergic receptors in cardiomyocytes and subsequent increase in [Ca2+]i (17, 39, 43), it is possible that the attenuated response to ATP in 30-min I/R hearts may be due to alterations in purinergic receptors or defects in associated signal transduction mechanisms. Because the affinity (1/Kd) of ATP, as indicated by the specific binding of [35S]ATP{gamma}S, to purinergic receptors was increased and Bmax was decreased after 30 min of ischemia as well as after 30-min ischemia followed by 30 min of reperfusion, it appears that I/R-induced alterations at the purinergic receptor level are of a complex nature. On the other hand, the depression in ATP-induced increase in [Ca2+]i in cardiomyocytes isolated from 30-min I/R hearts by Cibacron Blue, a well-known ATP receptor antagonist, rules out the possibility of any major defect at the receptor level. It is possible that the decreased density of purinergic receptors may explain the attenuated responses of I/R hearts to ATP whereas the increased affinity to [35S]ATP{gamma}S may serve as a compensatory mechanism to the decreased receptor density for maintaining the purinergic receptor function in I/R hearts. Therefore, postreceptor defects may be responsible for the altered ATP response in I/R hearts and cardiomyocytes isolated from these hearts. In view of the involvement of PLC in the signal transduction of ATP-induced increase in [Ca2+]i (27) and marked alterations in PLC isoforms during I/R (2), the participation of altered PLC-mediated signal transduction mechanisms in depressed ATP responsiveness during I/R seems likely. On the other hand, the depression in Iso (known {beta}1-adrenoceptor agonist; Ref. 30)-mediated increase in [Ca2+]i in I/R hearts may be related to defects in both affinity and density of {beta}1-adrenoceptors in I/R hearts (26). Nonetheless, propranolol (50 µM), a known {beta}-adrenoceptor antagonist, was found to cause a significant depression in Iso-induced increase in [Ca2+]i in cardiomyocytes isolated from both control and I/R hearts (data not shown).

Previous studies have shown the involvement of SL L-type Ca2+ channels in ATP-induced increase in [Ca2+]i (9), which further triggers the mobilization of Ca2+ from the SR (43). Because verapamil, a known Ca2+ channel antagonist, caused a depression in ATP-induced increase in [Ca2+]i in both control and I/R hearts, it is likely that influx through L-type Ca2+ channels is preserved during I/R. It should be noted that Ca2+ channel antagonists are known to prevent the I/R-induced increase in [Ca2+]i (23); however, some investigators have shown the downregulation of SL L-type Ca2+ channels (15), whereas others have shown no alterations in these receptors after I/R (45). Furthermore, the lack of difference in KCl (known depolarizing agent)- as well as BAY K 8644 (dihydropyridine receptor agonist)-mediated increase in [Ca2+]i in control and I/R hearts in our previous study (30) rules out the possibility of any defect in L-type Ca2+ channels. On the other hand, ATP-induced increase in [Ca2+]i in I/R cardiomyocytes, unlike control preparations, was not altered by ryanodine, an agent that depresses Ca2+ release by depleting SR Ca2+ stores as well as by blocking Ca2+ channels (28). Accordingly, it is suggested that abnormal Ca2+ handling at the level of the SR may be responsible for the depressed ATP-induced increase in [Ca2+]i in I/R cardiomyocytes. In this regard, decreased protein content and gene expression of ryanodine receptors have been demonstrated in I/R hearts (36). Furthermore, Zucchi et al. (46) showed a significant reduction in the density of high-affinity and low-affinity [3H]ryanodine binding sites in I/R hearts. In addition, protein kinase C, which is known to be activated during I/R (34) and causes decrease in ATP-induced as well as catecholamine-induced increase in [Ca2+]i (43), may be involved in the depressed ATP and Iso responsiveness in I/R hearts.

There is a possibility that the depressed responsiveness to ATP in I/R hearts may be due to oxidative stress. This view is supported by the observation that the attenuated ATP-induced increase in [Ca2+]i in I/R hearts was prevented by an antioxidant mixture containing SOD and catalase. In fact, oxidative stress has been shown to cause cardiac dysfunction during I/R, which was prevented by SOD and catalase (30). In addition, I/R-induced increase in basal [Ca2+]i was also attenuated by the antioxidant mixture. Furthermore, changes observed in I/R hearts and cardiomyocytes isolated from these hearts were simulated by H2O2, because, like cardiomyocytes from I/R hearts, an increase in basal [Ca2+]i and a depression in ATP-mediated increase in [Ca2+]i were observed in cardiomyocytes isolated from hearts treated with H2O2. On the other hand, Wang et al. (40) showed that H2O2 at high concentrations caused an augmentation of ATP-induced increase in [Ca2+]i in isolated cardiomyocytes. Although H2O2 at the concentration used in the present study has been shown to cause an increase in [35S]ATP{gamma}S binding in purified SL membranes (22), defects in SR Ca2+ handling may be the major reason for the depression in ATP-induced increase in [Ca2+]i. In addition, H2O2 has been known to cause a depression in PLC activity in SL membranes (20), which is associated with ATP-induced increase in [Ca2+]i. The results described in this study also indicate that IP, which is known to upregulate the antioxidant enzymes in I/R hearts (11), prevented the attenuation of ATP-induced increase in [Ca2+]i in I/R hearts. It may be noted that IP caused a depression in basal [Ca2+]i in I/R hearts, and this is in agreement with previous studies (1, 30). Furthermore, the participation of IP-mediated synchronization of SR Ca2+ pump and release channels (36, 44) may be involved in the restoration of ATP-induced increase in [Ca2+]i in I/R hearts.

In conclusion, the present study has shown that 30 min of ischemia followed by 30 min of reperfusion causes a marked increase in basal [Ca2+]i as well as a significant depression in ATP-induced increase in [Ca2+]i; these changes may be associated with defective Ca2+ handling at the level of the SR. Because the data are representative of viable cardiomyocytes and exclude a large population of cells that were lost during the isolation process, it can be argued that the viable cardiomyocytes used in this study are I/R resistant. However, this may not be the case, as cardiomyocytes isolated from control hearts and undergoing hypoxia-reoxygenation have shown a similar depression in ATP- and Iso-induced increase in [Ca2+]i as well as a significant increase in basal [Ca2+]i without any change in KCl response. These alterations in [Ca2+]i in hypoxic-reoxygenated cardiomyocytes are similar to those observed in cardiomyocytes isolated from I/R hearts. Furthermore, the results of the present study indicate that the depression in ATP-induced increase in [Ca2+]i may explain the attenuated positive inotropic effect of ATP in I/R hearts. Development of oxidative stress due to I/R appears to be an important mediator in causing this decrease in ATP response in I/R cardiomyocytes.


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The work reported in this article was supported by a grant from the Canadian Institutes of Health Research. H. K. Saini is a predoctoral fellow of the Heart and Stroke Foundation of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6 (E-mail: nsdhalla{at}sbrc.ca)

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.


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 ABSTRACT
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