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Am J Physiol Heart Circ Physiol 288: H2260-H2270, 2005. First published January 14, 2005; doi:10.1152/ajpheart.01153.2004
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Defective calcium handling in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion

Harjot K. Saini and Naranjan S. Dhalla

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

Submitted 16 November 2004 ; accepted in final form 4 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although ischemia-reperfusion (I/R) has been shown to affect subcellular organelles that regulate the intracellular Ca2+ concentration ([Ca2+]i), very little information regarding the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts is available. To investigate changes in [Ca2+]i due to I/R, rat hearts in vitro were subjected to 10–30 min of ischemia followed by 5–30 min of reperfusion. Cardiomyocytes from these hearts were isolated and purified; [Ca2+]i was measured by employing fura-2 microfluorometry. Reperfusion for 30 min of the 20-min ischemic hearts showed attenuated cardiac performance, whereas basal [Ca2+]i as well as the KCl-induced increase in [Ca2+]i and isoproterenol (Iso)-induced increase in [Ca2+]i in cardiomyocytes remained unaltered. On the other hand, reperfusion of the 30-min ischemic hearts for different periods revealed marked changes in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i without any alterations in the KCl-induced increase in [Ca2+]i or S(–)-BAY K 8644-induced increase in [Ca2+]i. The I/R-induced alterations in cardiac function, basal [Ca2+]i, and Iso-induced increase in [Ca2+]i in cardiomyocytes were attenuated by an antioxidant mixture containing superoxide dismutase and catalase as well as by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2 for 30 min. These results suggest that abnormalities in basal [Ca2+]i as well as mobilization of [Ca2+]i upon {beta}-adrenoceptor stimulation in cardiomyocytes are dependent on the duration of ischemic injury to the myocardium. Furthermore, Ca2+ handling defects in cardiomyocytes appear to be mediated through oxidative stress in I/R hearts.

Ca2+ mobilization; oxidative stress; ischemic preconditioning

IT IS NOW WELL KNOWN that reperfusion of the ischemic myocardium is essential for preventing the ischemia-induced changes in cardiac performance; however, reperfusion after a certain critical period has been reported to cause deleterious effects on cardiac function (17). In view of the important role played by intracellular Ca2+ in the regulation of heart function, several studies have been carried out to investigate changes in intracellular Ca2+ in hearts subjected to ischemia-reperfusion (I/R). A marked increase in tissue Ca2+ content has been reported in I/R hearts under a wide variety of experimental conditions, and this occurrence of intracellular Ca2+ overload has been suggested to explain I/R injury to the myocardium (7, 11, 17, 22, 26, 41). Likewise, a positive correlation was found to occur between the diastolic intracellular Ca2+ concentration ([Ca2+]i) and left ventricular end-diastolic pressure (LVEDP) in I/R rat hearts (25); however, such a relationship was not evident in postischemic ferret myocardium (20, 24). In fact, some investigators (10) have denied a direct role of intracellular Ca2+ in ischemic diastolic dysfunction in isolated rat and rabbit hearts. Although different studies have examined the Ca2+ handling ability of cardiomyocytes obtained from I/R hearts or undergoing hypoxia-reoxygenation, the results are conflicting (4, 21, 32, 34). Thus no definitive conclusion regarding the status of Ca2+ handling in I/R hearts can be made on the basis of information available in the literature.

It is possible that conflicting reports regarding the characteristics of [Ca2+]i in cardiomyocytes from I/R hearts may be due to the reversible and irreversible nature of the ischemic cell injury. This view is based on the observation that excessive accumulation of intracellular Ca2+ has been shown to occur during irreversible myocardial injury (17), whereas reversible postischemic contractile dysfunction is associated with complete normalization of [Ca2+]i within a few minutes of reperfusion (3, 24). Accordingly, we investigated in detail the Ca2+ handling abilities of nondepolarized and depolarized cardiomyocytes isolated from hearts subjected to different periods of ischemia and reperfusion to test whether changes in [Ca2+]i in cardiomyocytes are related to reversible and irreversible phases of I/R injury. Because catecholamines are known to cause an increase in [Ca2+]i (37), the effect of isoproterenol (Iso), a {beta}1-adrenoceptor agonist (30), on intracellular Ca2+ handling was studied in depolarized cardiomyocytes. In view of the important role of oxidative stress in inducing I/R injury, experiments were carried out to examine whether I/R-induced Ca2+ handling abnormalities in cardiomyocytes are mediated through the generation of oxyradicals. For this purpose, the I/R-induced changes in [Ca2+]i in cardiomyocytes from hearts perfused with a mixture of superoxide dismutase (SOD) plus catalase, which prevents the development of oxidative stress (30), were investigated. Furthermore, this study tested whether the effects of I/R on cardiomyocyte Ca2+ handling are simulated by perfusing the heart with H2O2, a well-known oxidant (7, 28). Because ischemic preconditioning (IP) has been shown to attenuate I/R-induced cardiac dysfunction (23, 42), the status of cardiomyocyte Ca2+ handling was examined in hearts subjected to IP.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolated rat heart preparation. Male Sprague-Dawley rats (250–300 g) were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The hearts were quickly excised, mounted on the Langendorff apparatus, and perfused at 37°C (pH 7.4) with Krebs-Henseleit (K-H) buffer gassed with a mixture of 95% O2-5% CO2 at a constant flow of 10 ml/min. The composition of K-H buffer 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 300 beats/min via a square wave current of 1.5-ms duration throughout the experiment using a Phipps and Bird stimulator (Richmond, VA). The left ventricular systolic pressure (LVSP), LVEDP, rate of change of pressure development (+dP/dt), and rate of change of pressure decay (–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. At the beginning of the experiment, 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 on-line through an analog-to-digital interface (MP-100, Biopac Systems) and stored with a computer program (Acqknowledge 3.5.3) by a Biopac Data-Acquisition System. All hearts were stabilized for a period of 20 min and randomly divided into different experimental groups. In the first set of experiments, control hearts were perfused for 10–60 min after stabilization. Because no significant differences were observed in the cardiac performance and intracellular Ca2+ handling, control values were grouped together. Some hearts were made globally ischemic for 10 or 20 min by stopping the coronary flow, and flow was then restored for 30 min, whereas other hearts in this group were subjected to 30 min of global ischemia followed by 5, 15, and 30 min of reperfusion. In the second set of experiments, isolated hearts were treated with an antioxidant mixture containing SOD (5 x 104 U/l, Sigma-Aldrich; Oakville, Ontario, Canada) and catalase (7.5 x 104 U/l, Fisher Scientific; Nepean, Ontario, Canada) for 10 min before ischemia was induced as well as during the reperfusion period, as described previously (28, 30). 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. In the third set of experiments, IP in hearts was induced by three cycles of 5-min ischemia followed by 5-min reperfusion before hearts were subjected to I/R as described by Temsah et al. (42). All protocols were approved by the University of Manitoba Animal Care Committee in accordance with guidelines of the Canadian Council on Animal Care.

Isolation of cadiomyocytes. Ventricular myocytes were isolated using a method described previously (45). In brief, hearts from different groups were perfused at 37°C 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 and gassed with a mixture of 95% O2-5% CO2. 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 (except for the hearts treated with H2O2, which were perfused for 15 min), the hearts were removed from the cannula. 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 a mixture of 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+ concentrations (250, 500, and 750 µM) to a final concentration of 1 mM. The cell viability in all experimental groups was evaluated using the trypan blue (Sigma-Aldrich) exclusion method. For this purpose, an aliquot of the cardiomyocyte suspension was added to an equal amount of 0.3% trypan blue in normal saline and incubated for 3–5 min. The unstained, stained, and total numbers of cells were counted in the Neubauer chamber. After the cell viability and yield were determined, the preparation was washed two times by centrifugation at 100 g for 1 min to minimize the contamination from dead cells. All preparations were further purified by subjecting them to an isotonic Percoll (Sigma-Aldrich) gradient (pH 7.3) before fura-2 AM loading. In some experiments, cardiomyocytes from control and 30-min I/R hearts were examined under a light microscope to observe any differences in size and shape. The final cell suspension after purification had 90–95% viable cardiomyocytes; 3–5% of cardiomyocytes were observed to beat spontaneously. Because the isolation of cardiomyocytes involved 5 min of Ca2+-free perfusion and 25 min of collagenase perfusion with oxygenated medium, it is understood that cardiomyocytes obtained from hearts subjected to ischemia or I/R underwent additional reperfusion for 30 min.

Measurement of [Ca2+]i. Isolated cardiomyocytes were incubated with 5 µM fura-2 AM for 40 min in 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 for all the experimental groups. Alterations in fluorescence intensity were monitored by a SLM DMX-1100 dual-wavelength spectrofluorometer (SLM Instruments; Urbana, IL) adjusted to an 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 level was calculated according to the following Grynkiewicz equation (15)

where Kd is the effective dissociation constant and was taken as 224 for all the [Ca2+]i measurements. The ratio of the fluorescence signals (R) at 340 and 380 nm was calculated automatically. Rmax and Rmin values were determined by the addition of 20 µl Triton X-100 (10%) and 40 µl EGTA (400 mM), respectively. Sf2 and Sb2 are the fluorescence proportionality coefficients obtained at 380 nm (excitation wavelength) under Rmin and Rmax conditions, respectively. Treatment with different concentrations of Iso was performed by incubating the fura-2 AM-loaded cells in a buffer containing Iso for 5 min at room temperature before the measurement of fluorescence. Unless otherwise indicated in the text, 100 µM Iso, 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. It should be mentioned that Iso was found to have no effect on [Ca2+]i in unstimulated cardiomyocytes; however, this treatment augmented the KCl-induced or S(–)-BAY K 8644-induced increase in [Ca2+]i. The difference between the responses in the presence and absence of Iso treatment was taken as the Iso-induced increase in [Ca2+]i.

Measurement of some biochemical parameters. To gain some information regarding the status of oxidative stress, the lipid peroxidation in control and 30-min I/R heart homogenates was assayed by measuring malondialdehyde (MDA) content (2). In addition, conjugated diene formation was determined in the heart homogenate according to the method of Esterbauer et al. (12). For the determination of binding characteristics of {beta}1-adrenoceptors in control and 30-min I/R hearts, crude membranes were prepared by the method used by Persad et al. (30). Specific binding to {beta}1-adrenoceptors was calculated as the difference between 125I-labeled cyanopindolol binding values in the presence and absence of CGP-20712A, a selective {beta}1-adrenoceptor antagonist. Kd and maximal binding (Bmax) were calculated from the Scatchard plot analysis according to the interactive LIGAND program, as described previously (30)

Statistical analysis. All results are expressed as means ± SE. Statistical analysis was performed using 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
 
Cardiac performance of hearts subjected to different periods of ischemia and reperfusion. For studying the effects of I/R on cardiac function, isolated rat hearts were subjected to global ischemia for 10, 20, and 30 min and then reperfused for 5–30 min. Alterations in LVDP, LVEDP, +dP/dt, and –dP/dt in hearts subjected to 10- and 20-min ischemia followed by 30-min reperfusion are shown in Table 1, whereas those in hearts subjected to 30-min ischemia followed by 5-, 15-, and 30-min reperfusion are shown in Table 2. An increase in LVEDP was observed in hearts subjected to 20- and 30-min ischemia, whereas no such alteration was seen in 10-min ischemic hearts. On the other hand, LVDP, +dP/dt, and –dP/dt were markedly depressed in all ischemic hearts compared with control hearts. These changes in cardiac function in hearts undergoing 10-min ischemia were fully reversible after 30-min reperfusion, whereas a partial improvement in these parameters was observed in hearts exposed to 20-min ischemia followed by 30-min reperfusion (Table 1). The recovery of cardiac performance was markedly impaired in hearts undergoing 5-, 15-, and 30-min reperfusion after 30-min ischemia (Table 2). It should be mentioned that the recovery of cardiac function in hearts subjected to 20-min ischemia and 60-min reperfusion was 90–95%, whereas that in 30-min ischemic and 60-min reperfused hearts was 30–40% of the control values (data not shown). Thus it is evident that functional changes in 10- or 20-min ischemic hearts were reversible, whereas those in 30-min ischemic hearts were irreversible.


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  		Table 1. Cardiac performance, cell viability, and characteristics of cardiomyocyte Ca2+ handling for isolated hearts subjected to 10- and 20-min ischemia followed by 30-min reperfusion

 

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  		Table 2. Cardiac performance and cell viability for isolated hearts subjected to 30-min ischemia followed by different times of reperfusion

 
Effect of I/R on viability and yield of cardiomyocytes. To examine the effect of I/R at the cellular level, cardiomyocytes were isolated from hearts undergoing varying periods of ischemia and reperfusion as shown in Tables 1 and 2. No decrease in cell viability and cardiomyocyte yield was observed in hearts undergoing 10-min ischemia or 10-min ischemia followed by 30-min reperfusion. Likewise, the cell viability and yield of cardiomyocytes did not change significantly in hearts undergoing 20-min ischemia or 20-min ischemia followed by 30-min reperfusion (Table 1). On the other hand, both yield and viability of cardiomyocytes were significantly decreased at different periods (5, 15, and 30 min) of reperfusion in 30-min ischemic hearts (Table 2). However, the size and shape of the viable cardiomyocytes isolated from the 30-min ischemic and 30-min reperfused hearts were not different from the control preparations (Fig. 1). This shows that most of the dead or damaged cardiomyocytes due to I/R were removed during the process of isolation and purification; this fact is evident from the reduced yield of cardiomyocytes obtained from I/R hearts.



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  		Fig. 1. Size and shape of cardiomyocytes isolated from control and ischemic-reperfused (I/R) hearts. A: cardiomyocytes isolated from control perfused hearts (x10). B: cardiomyocytes isolated from hearts subjected to 30-min ischemia (I) followed by 30-min reperfusion (R) (x10). C: cardiomyocytes isolated from hearts subjected to 30-min ischemia followed by 30-min reperfusion (x25). Each micrograph is representative of cardiomyocytes isolated from 4 different preparations from control or I/R groups after application of a Percoll gradient, and the cell pellet was used for taking the micrograph. The number of cardiomyocytes in the preparation from I/R hearts was adjusted equal to that from sham control hearts.

 
Basal [Ca2+]i and KCl-induced increase in [Ca2+]i in cardiomyocytes isolated from I/R hearts. To investigate alterations in Ca2+ handling by I/R, [Ca2+]i was measured in isolated cardiomyocytes using fura-2 microfluorometry. Representative tracings for KCl (30 mM), a known depolarizing agent (33), induced increases in [Ca2+]i in cardiomyocytes isolated from control and I/R hearts, are shown in Fig. 2A. The basal [Ca2+]i was not altered in cardiomyocytes isolated from hearts undergoing 10- and 20-min ischemia (Table 1), whereas a gradual increase in basal [Ca2+]i was observed at different times of reperfusion in cardiomyocytes isolated from 30-min ischemic hearts (Fig. 3A). The data shown in Table 1 indicate that the KCl -induced increase in [Ca2+]i was not altered in cardiomyocytes isolated from hearts subjected to 10- and 20-min ischemia followed by 30-min reperfusion. Similarly, no alterations in the KCl-induced increase in [Ca2+]i were observed in hearts subjected to varying periods of reperfusion (5, 15, and 30 min) after 30-min ischemia (Figs. 2A and 3B). It is pointed out that after we measured the cardiac performance, both ischemic and I/R hearts were further perfused for 5 min with Ca2+-free medium and 25-min perfusion with medium containing collagenase; this 30-min additional reperfusion period should be taken into consideration while interpreting the data in cardiomyocytes isolated from ischemic or I/R hearts.



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  		Fig. 2. KCl-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes with or without isoproterenol (Iso) treatment isolated from hearts subjected to 30-min ischemia followed by 30-min reperfusion. A: KCl-induced increase in [Ca2+]i in untreated cardiomyocytes isolated from control and I/R hearts. B: KCl-induced increase in [Ca2+]i in Iso-treated cardiomyocytes isolated from control and I/R hearts. Upward arrows indicates the time when preparations were exposed to 30 mM KCl. Treatment with 100 µM was carried out for 5 min before intracellular Ca2+ measurements.

 


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  		Fig. 3. Effect of 30-min ischemia and different times of reperfusion on [Ca2+]i in isolated cardiomyocytes. A: effect of I/R on basal [Ca2+]i in isolated cardiomyocytes. B: effect of I/R on the KCl-induced increase in [Ca2+]i in isolated cardiomyocytes. C: effect of I/R on the Iso-induced increase in [Ca2+]i in isolated cardiomyocytes. The basal [Ca2+]i represents [Ca2+]i before the addition of KCl. The increase in [Ca2+]i was calculated as the difference between the peak value and the basal value in each experiment. The Iso-induced increase was calculated by subtracting the values for the KCl-induced increase in [Ca2+]i in untreated cardiomyocytes from those treated with 100 µM Iso. The concentration of KCl was 30 mM. Each point represents the mean ± SE of 4 experiments. *P < 0.05 vs. the control group.

 
Effect of Iso on [Ca2+]i in KCl-depolarized cardiomyocytes isolated from I/R hearts. Because treatment of mainly quiescent cardiomyocytes with Iso, a known {beta}1-adrenoceptor agonist (30), did not affect [Ca2+]i, KCl-depolarized cells were used to examine the effect of 100 µM Iso at the receptor level. Iso was observed to increase [Ca2+]i in KCl-depolarized cardiomyocytes obtained from control hearts (Fig. 2B and Table 1). No alterations in the Iso-induced increase in [Ca2+]i in cardiomyocytes was observed in hearts exposed to 10-min ischemia with or without 30-min reperfusion (Table 1). In contrast, a significant depression in the Iso-induced increase in [Ca2+]i was seen in hearts subjected to 20-min ischemia. However, this alteration in 20-min ischemic hearts was fully reversible after 30-min reperfusion (Table 1). On the other hand, in hearts subjected to 30-min ischemia followed by 5-, 15-, or 30-min reperfusion, a significant depression in the Iso-induced increase in [Ca2+]i was detected at each of these time points (Fig. 3C). It is important to point out that the Iso-induced increase in [Ca2+]i in KCl-depolarized cells was depressed at different concentrations of Iso (10–150 µM) in hearts subjected to 30-min ischemia followed by 30-min reperfusion (Fig. 4). Furthermore, the increase in [Ca2+]i by 100 µM Iso was mediated by {beta}-adrenoceptors because propranolol (50 µM) was found to inhibit this effect completely in cardiomyocytes isolated from both control and I/R hearts.



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  		Fig. 4. Effect of treatments with different concentrations of Iso (10–150 µM) on the KCl-induced increase in [Ca2+]i in cardiomyocytes isolated from hearts subjected to 30-min ischemia followed by 30-min reperfusion. Each point represents the mean ± SE of 4 experiments in each group. *P < 0.05 vs. the control group.

 
Effect of S(–)-BAY K 8644 on [Ca2+]i in cardiomyocytes isolated from I/R hearts. Although no difference in KCl responses was seen in control and I/R cardiomyocytes, the KCl-induced increase in [Ca2+]i in both control and I/R hearts was inhibited up to 70–80% by verapamil (10 µM), a well-known antagonist of L-type Ca2+ channels, but was unaffected by propranolol (50 µM). The involvement of L-type Ca2+ channels in I/R-induced changes in Ca2+ handling was further verified by using S(–)-BAY K 8644 (2 µM), a specific dihydropyridine (DHP) receptor agonist (16). No change in the BAY K 8644-induced increase in [Ca2+]i was observed after 30-min ischemia or after 30-min ischemia followed by 30-min reperfusion. On the other hand, the Iso-induced increase in [Ca2+]i in the presence of BAY K 8644 was significantly depressed in all these groups (Fig. 5). It should be mentioned that the BAY K 8644-induced increase in [Ca2+]i in both control and I/R hearts was 90–95% inhibited by 10 µM verapamil but was not altered by 50 µM propranolol.



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  		Fig. 5. Effect of 30-min ischemia and 30-min reperfusion (I/R) on [Ca2+]i in isolated cardiomyocytes. A: representative tracings showing the S(–)-BAY K 8644 (BAY)-induced increase in [Ca2+]i in cardiomyocytes isolated from control and I/R hearts. B: effect of I/R on the BAY-induced increase in [Ca2+]i in isolated cardiomyocytes. C: effect of I/R on the Iso-induced increase in [Ca2+]i in isolated cardiomyocytes. The basal [Ca2+]i represents [Ca2+]i before the addition of BAY (2 µM). The Iso-induced increase was calculated by subtracting the values for the BAY-induced increase in [Ca2+]i in the untreated cardiomyocytes from those for the 100 µM Iso-treated cardiomyocytes. Each point represents the mean ± SE of 4 experiments in each group. Upward arrows indicate the times when the cardiomyocytes were exposed to 2 µM BAY. *P < 0.05 vs. control.

 
Status of oxidative stress and {beta}1-adrenoceptors in I/R hearts. To gain some information regarding the status of oxidative stress in I/R hearts under the experimental conditions used in the present study, we measured the level of MDA and the formation of conjugated dienes. The data shown in Table 3 reveal an increase in the level of MDA and conjugated dienes formation in I/R hearts compared with control. Because the responsiveness of I/R cardiomyocytes to Iso was attenuated, the status of {beta}-adrenoceptors in control and I/R hearts was also determined. The binding characteristics of {beta}1-adrenoceptors indicated that both the density (Bmax) and affinity (1/Kd) of {beta}1-adrenoceptors were depressed in I/R hearts (Table 3).


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  		Table 3. Alterations in some parameters of oxidative stress and binding characteristics of {beta}1-adrenoceptors in hearts subjected to 30-min ischemia and 30-min reperfusion

 
Effect of SOD and catalase on intracellular Ca2+ handling in isolated cardiomyocytes. To investigate the involvement of oxyradicals in I/R-induced alteration in intracellular Ca2+ handling, isolated hearts were treated with an antioxidant mixture containing SOD and catalase. Treatment of hearts with SOD and catalase did not exert any effect on LVDP, LVEDP, +dP/dt, –dP/dt, cell viability, or cardiomyocyte yield in control and 30-min ischemic hearts (Table 4). On the other hand, the increase in basal [Ca2+]i and decrease in the Iso-induced increase in [Ca2+]i in 30-min ischemic cardiomyocytes, unlike control preparations, were attenuated by SOD and catalase. From the data shown in Table 4, it can be seen that treatment of hearts with SOD and catalase attenuated the I/R-induced alterations in cardiac function, cell viability, and cardiomyocyte yield as well as basal [Ca2+]i and the Iso-induced increase in [Ca2+]i. Although SOD and catalase treatment was observed to increase the KCl-induced increase in [Ca2+]i in both control and 30-min ischemic as well as I/R cardiomyocytes (Table 4), no attempt was made to understand the mechanisms of such an effect.


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  		Table 4. Cardiac performance, cell viability, and characteristics of cardiomyocyte Ca2+ handling for isolated rat hearts treated with SOD and Cat 10 min before ischemia and during the reperfusion period

 
Effect of H2O2 on intracellular Ca2+ handling in isolated cardiomyocytes. To mimic the effects of oxidative stress during I/R, isolated hearts were treated with 50 µM H2O2 for 30 min. H2O2 caused a depression in LVDP, +dP/dt, and –dP/dt with an elevation in LVEDP, as shown in Table 5. The selection of this concentration and time period of perfusion with H2O2 was based on a comparable depression of cardiac performance in 30-min I/R hearts in the present study. A marked decrease in cell viability and number of cardiomyocytes and an increase in basal [Ca2+]i were observed after H2O2 treatment, whereas the Iso-induced increase in [Ca2+]i was significantly depressed in H2O2-treated hearts (Table 5). Although I/R and H2O2 are considered to produce oxidative stress, it is pointed out that unlike I/R, H2O2 was able to augment the KCl-induced increase in [Ca2+]i in isolated cardiomyocytes (Table 5). This effect is in accordance with a previous study (44) and may be due to the activation of sarcoplasmic reticulum (SR) Ca2+ release channels and inhibition of SR Ca2+ pump activity in cardiomyocytes (44). It is also possible that I/R-induced injury may not produce a sufficient amount of H2O2 as employed in our study. Furthermore, the concentration of H2O2 as employed in the present study may be affecting some other sites in addition to those affected by I/R. However, no experiments were carried out to establish the mechanisms of the observed differences in cardiomyocytes isolated from H2O2 and I/R hearts with respect to the KCl-induced increase in [Ca2+]i.


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  		Table 5. Cardiac performance, cell viability, and characteristics of cardiomyocyte Ca2+ handling for isolated rat hearts treated with 50 µM H2O2 for 30 min

 
Effect of IP on intracellular Ca2+ handling in isolated cardiomyocytes. To determine the significance of I/R-mediated changes in intracellular Ca2+ handling at the level of isolated cardiomyocytes, we examined the effect of IP, which is known to prevent I/R-induced changes in cardiac function (23, 42). Although IP depressed the cardiac performance (LVDP, LVEDP, +dP/dt, and –dP/dt) in control hearts, alterations in cardiac function in I/R hearts, unlike ischemic hearts, were attenuated (Table 6). Both cell viability and yield of cardiomyocytes in I/R hearts, unlike control hearts, were improved by IP. On the other hand, cell viability in ischemic hearts was improved by IP without any changes in cardiomyocyte yield (Table 6). Attenuation of basal [Ca2+]i and a marked improvement of the Iso-induced increase in [Ca2+]i were observed in cardiomyocytes isolated from both ischemic and I/R hearts subjected to IP (Table 6). The KCl-induced increase in [Ca2+]i in control, ischemic, and I/R groups was not affected by IP (Table 6). It is also pointed out that a strong correlation (r2 = 0.9814, P < 0.001) was observed between I/R-induced changes in LVEDP and LVDP in IP hearts. A similar correlation (r2 = 0.9179, P < 0.001) was also found between I/R-induced changes in LVDP and cell viability in cardiomyocytes isolated from IP hearts. Such relationships suggest that cardiomyocyte preparations obtained from I/R hearts under different experimental conditions including IP may show a corresponding magnitude of I/R injury.


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  		Table 6. Cardiac performance, cell viability, and characteristics of cardiomyocyte Ca2+ handling for isolated hearts subjected to IP before the induction of 30-min ischemia and 30-min reperfusion

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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In the present study, basal [Ca2+]i was increased in purified cardiomyocytes isolated from hearts subjected to 30-min global ischemia followed by different times of reperfusion, whereas no change in basal [Ca2+]i was observed in hearts undergoing 10- or 20-min ischemia and subsequent reperfusion. By employing whole heart preparations, Seki et al. (36) have also shown an increase in diastolic Ca2+ in hearts undergoing 30-min low-flow ischemia followed by reperfusion, whereas no difference was observed in hearts subjected to reperfusion after 10-min ischemia. Although Meissner and Morgan (25) have shown a significant increase in diastolic [Ca2+]i in isolated hearts exposed to 30-min global ischemia followed by 60-min reperfusion, these investigators were also able to detect an increase in basal [Ca2+]i in 10- or 20-min ischemic hearts upon reperfusion. Similarly, in isolated rat cardiomyocytes, hypoxia-reoxygenation has been shown to cause persistent elevation in cytosolic Ca2+ concentration, which is associated with cell injury (32). An increase in [Ca2+]i has been observed in rat cardiomocytes after 45-min anoxia followed by 10-min reperfusion (34). On the other hand, Chandrashekhar et al. (4) have reported that [Ca2+]i does not change in cardiomyocytes isolated from rat hearts subjected to 20-min global ischemia and 20-min reperfusion and have suggested that impairment of cardiac function in I/R is not linked with abnormal Ca2+ handling in cardiomyocytes. Furthermore, Kim et al. (21) have shown that impaired Ca2+ handling in I/R hearts is species dependent because no defect was evident in cardiomyocytes isolated from rat I/R hearts, whereas a significant impairment in Ca2+ handling was seen in cardiomyocytes isolated from swine I/R hearts. It is pointed out that Dhalla et al. (7) have reported an increase in total tissue Ca2+ in isolated hearts after 30-min I/R. Likewise, Tani and Neely (41) observed an increase in 45Ca2+ uptake in hearts undergoing 30-min zero-flow global ischemia with 30-min aerobic perfusion. Although accumulation of [Ca2+]i during the ischemic period appears to be a consistent finding, it is important to point out that the gradual increase in [Ca2+]i during the reperfusion period depends on the time of the ischemic insult. Different studies subjecting isolated hearts or trabeculae muscles to ischemia for <20 min have shown that the increased [Ca2+]i during ischemia normalizes to preischemic values in the first few minutes of reperfusion (3, 14, 24). Thus it appears that the ability of cardiomyocytes isolated from the I/R heart to exhibit increased levels of basal [Ca2+]i may be related to the irreversible nature of the ischemic injury. In this regard, it should be pointed out that cardiac dysfunction in hearts subjected to 10-min ischemia was fully reversible upon reperfusion for 30 min and that in hearts subjected to 20-min ischemia, unlike 30-min ischemic hearts, was reversible upon reperfusion for 60 min. No changes in cell viability and cardiomyocyte yield in hearts subjected to 10- or 20-min ischemia followed by reperfusion, unlike in 30-min I/R hearts, provide further evidence that the ischemic insult for a period of 10–20 min may produce reversible alterations, whereas irreversible changes are induced by 30-min global ischemia. This view is consistent with observations by other investigators (27, 43) showing that 20–40 min of ischemia in rat and mouse hearts is responsible for the transition from reversible to irreversible injury associated with cardiac dysfunction.

It is now well known that [Ca2+]i is regulated by coordinated functions of sarcolemma (SL), SR, and mitochondria (26, 32, 41, 42). Accordingly, the observed increase in basal [Ca2+]i in cardiomyocytes isolated from I/R hearts may be due to defects in one or more of these subcellular mechanisms. In this regard, it should be noted that both I/R and hypoxia/reoxygenation have been shown to produce marked changes in SL Na+-K+-ATPase, Na+/Ca2+ exchange, Ca2+ pump, and Ca2+ channels (8, 9, 19, 31, 41). Furthermore, alterations in SR Ca2+ uptake and Ca2+ release activities have been observed in I/R hearts (42). Excessive amounts of Ca2+ have also been shown to accumulate in mitochondria during reperfusion of the ischemic myocardium (26). Thus abnormalities in different subcellular organelles and particularly in the SL Na+/Ca2+ exchange system due to I/R can be seen to produce defects in Ca2+ handling by cardiomyocytes and explain the observed increase in basal [Ca2+]i. Nonetheless, an increase in basal [Ca2+]i reflects the occurrence of intracellular Ca2+ overload and is likely to explain the increased LVEDP and depressed ability of I/R hearts to generate contractile force (11, 39). In fact, increases in LVEDP in I/R hearts in all untreated and treated groups were found to be linearly related to increases in basal [Ca2+]i as well as decreases in LVDP, cardiomyocyte yield, and viability (Fig. 6). With the use of a different experimental design, other investigators (25) have also reported a correlation between diastolic Ca2+ and LVEDP in I/R hearts. In view of the role of intracellular Ca2+ overload in inducing cell injury (39), the increased level of basal [Ca2+]i in cardiomyocytes from I/R hearts may also explain the observed decrease in cell viability and cardiomyocyte yield from I/R hearts. In addition, rapid normalization of tissue pH and osmolarity at the time of reperfusion also causes severe mechanical stress on cardiomyocytes leading to their loss in I/R hearts (31).



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  		Fig. 6. Correlation of left ventricular end-diastolic pressure (LVEDP) of isolated hearts with left ventricular developed pressure (LVDP), cell yield, cell viability, basal [Ca2+]i, and the Iso-induced increase in [Ca2+]i. A: correlation of LVDP and LVEDP of isolated hearts. B: correlation of cardiomyocyte yield and LVEDP of isolated hearts. C: correlation of cardiomyocyte viability and LVEDP of isolated hearts. D: correlation of basal [Ca2+]i in cardiomyocytes and LVEDP of isolated hearts. E: correlation of the Iso-induced increase in [Ca2+]i in cardiomyocytes and LVEDP of isolated hearts. The data are taken from the original points of I/R, superoxide dismutase (SOD) + catalase-treated, and ischemia preconditioned (IP) hearts (Tables 1, 2, 4, and 5) as shown in this study. {blacksquare}, Control group; {bullet}, I/R group; {blacktriangleup}, SOD + catalase-treated group; {blacktriangledown}, IP group. P values and the values for the correlation coefficient (r2) are shown.

 
The results in this study indicate an augmentation of the KCl-induced increase in [Ca2+]i upon treatment of cardiomyocytes with Iso. Because such an augmentation of the KCl-induced increase in [Ca2+]i by Iso was prevented by propranolol, a {beta}-adrenoceptor blocker, it is evident that the Iso-induced increase in [Ca2+]i is mediated through activation of {beta}1-adrenoceptors in cardiomyocytes. These results are consistent with earlier observations showing an increase in [Ca2+]i by Iso in KCl-depolarized as well as electrically stimulated cardiomyocytes (37, 40). The data described here show that the Iso-induced increase in [Ca2+]i in KCl-depolarized cells is attenuated in I/R hearts. A highly significant linear relationship between LVEDP and the decrease in the Iso-induced increase in [Ca2+]i was also evident in I/R cardiomyocytes from all untreated and treated groups used in this study (Fig. 6). Because attenuation of the Iso-induced increase in [Ca2+]i was seen in cardiomyocytes stimulated by BAY K 8644, an activator of L-type Ca2+ channels in the SL membrane, it is likely that such an effect of I/R on Ca2+ influx may be mediated through changes in L-type Ca2+ channels. In this regard, note that the Iso-induced increase in [Ca2+]i in control and I/R preparations was markedly prevented by verapamil (10 µM), a Ca2+ channel antagonist (data not shown), without having any effect on basal [Ca2+]i. Although a depression in the density of L-type Ca2+ channels has been reported to occur in I/R hearts (16), Ca2+ channel antagonists have been shown to prevent the I/R-induced increase in [Ca2+]i (22). Because the KCl-induced or BAY K 8644-induced increase in [Ca2+]i in cardiomyocytes was not altered by I/R, it is unlikely that the observed decrease in the Iso-induced increase [Ca2+]i is due to any direct defect in L-type Ca2+ channels. Because verapamil in high concentrations (10 µM), as used in the present study, has been shown to inhibit the Na+/Ca2+ exchanger as well as the increase in [Ca2+]i in KCl-depolarized cardiomyocytes (33), the role of the Na+/Ca2+ exchanger in causing Ca2+ handling abnormalities in I/R hearts cannot be ruled out. On the other hand, the attenuation of the Iso-induced increase in [Ca2+]i may be related to changes in {beta}-adrenoceptors. In fact, both the affinity and density of {beta}1-adrenoceptors were reduced in I/R hearts. Similar alterations in binding characteristics of {beta}1-adrenoceptors in I/R hearts have been shown previously (30). Thus the observed changes in Ca2+ mobilization by Iso, reflecting Ca2+ handling abnormalities in I/R hearts, may be due to defects at the level of {beta}-adrenoceptors. However, in view of the dramatic changes in Ca2+ release and Ca2+ uptake activities in the SR from I/R hearts (42), the contribution of attenuated Ca2+-induced Ca2+ release from the SR cannot be ruled out from the observed depression of Iso-induced increase in [Ca2+]i in cardiomyocytes obtained from I/R hearts.

There is a good possibility that changes in cardiac function as well as basal and Iso-induced increase in [Ca2+]i in I/R hearts may be due to oxidative stress. This view is supported by the observations that the concentration of MDA and conjugated diene formation were increased in I/R hearts. In fact, oxidative stress has been known to produce cardiac dysfunction, alterations in {beta}1-adrenoceptor-mediated signal transduction mechanisms, and changes in subcellular functions in I/R hearts (6, 7, 29). The participation of oxidative stress in inducing changes in Ca2+ handling by cardiomyocytes as well as cardiac function is further evident from the finding that these alterations in I/R hearts were attenuated by treatment with a mixture of SOD and catalase, which is known to scavenge the oxyradicals (28, 30). By employing a similar experimental model, SOD and catalase treatment has also been shown to cause a reduction in MDA and conjugated diene content in I/R hearts (7). In addition, the changes observed in I/R hearts and cardiomyocytes were simulated by treatment with H2O2, a well-known oxidant (29, 44). Note that treatment of I/R hearts with SOD and catalase improved the cell viability, whereas H2O2 produced a reduction in cell viability. The results described in this study also indicate that IP, which is known to upregulate the antioxidant enzymes in I/R hearts (6), was found to prevent changes in cardiomyocyte Ca2+ handling. An IP-mediated reduction in cytosolic Ca2+ has been previously observed in isolated hearts (1). It is important to point out that the changes in Ca2+ handling by I/R are fully reversible by IP, whereas these parameters remained attenuated in SOD + catalase-treated I/R hearts. Although Persad et al. (30) demonstrated that the binding characteristics of {beta}1-adrenoceptors (Kd and Bmax) were completely normalized to control values in I/R hearts after treatment with SOD and catalase under the same experimental conditions used in this study, a partial improvement was observed for I/R-induced changes in adenylyl cyclase activity in the presence of Iso. The poor recovery of cell viability by the antioxidant mixture may also be responsible for the incomplete restoration of the Iso response in cardiomyocytes isolated from SOD + catalase-treated I/R hearts. In addition, the present study suggests that mechanisms other than increasing the antioxidant reserve may be involved in the improvement of defective Ca2+ handling in I/R hearts by IP. Synchronization of SR Ca2+ pump and release channels (42) and translocation and activation of specific PKC isoforms (23) followed by phosphorylation of myofilament regulatory proteins (18) and a reduction of H+ accumulation during ischemia (13) may be associated with improved Ca2+ handling by IP.

In conclusion, the findings presented in this study demonstrated that Ca2+ handling abnormalities occur in cardiomyocytes isolated from I/R hearts and that this alteration is dependent on the period of ischemic insult. The method employed for assessing changes in [Ca2+]i in cardiomyocytes can be seen to have advantage over that used for the measurement of [Ca2+]i in intact heart (10, 20, 24, 25). This view is based on the observations that diastolic Ca2+ measurements in the intact heart are associated with methodological problems including motion artifacts of beating hearts, absorbance of light by chromatic molecules, contribution of changes in signal due to the presence of other types of cells, and the effect of temperature/pH on the intracellular Ca2+-induced signal emission (38). Because a collective response was taken from a large number of cells, the possibility of biasing associated with selection of single cells is eliminated. Although the preparation employed in this study has been used previously to examine the effect of ATP (5, 35), low Na+ (33), and phosphatidic acid (45) on the intracellular levels of Ca2+, some caution should be exercised while interpreting the data presented here. Because the hearts in all experimental groups were subjected to 5- and 25-min perfusion with Ca2+-free and collagenase-containing solution, the effect of ischemia alone cannot be assessed on the basis of the experimental design used in this study. Furthermore, cardiac performance was measured in isolated hearts, and Ca2+ handling studies were performed in isolated cardiomyocytes. Therefore, the correlation between these parameters is of an indirect nature. Although we removed the dead cells from the preparation by applying a Percoll gradient, contamination by some trypan blue-permeant cells cannot be ruled out. The low yield of cardiomyocytes isolated from I/R hearts may be a consequence of decreased cell viability due to ischemic insult as well as increased susceptibility to stress during the isolation procedures used in this study. In addition, the data are representative of viable cardiomyocytes and exclude a large population of cells, which were lost during the I/R insult and isolation procedure. Thus it can be argued that the remaining viable cells from I/R hearts used in this study are I/R resistant. However, it is important to point out that the remaining viable cells exhibited a rod-shaped structure and showed no difference in KCl- or BAY K 8644-induced increases in [Ca2+]i, whereas these cells had increased basal levels of [Ca2+]i and depressed Iso-induced increases in [Ca2+]i. These observations clearly indicate that there occurs a Ca2+ handling defect at the level of cardiomyocytes in I/R hearts. Such an abnormality can be seen to produce intracellular Ca2+ overload and decrease the cell viability leading to cell damage. Furthermore, the experiments described here indicate that oxidative stress may be an important mechanism for causing changes in [Ca2+]i handling in I/R hearts. It seems that there is a functional relationship between cardiac function of isolated hearts and intracellular Ca2+ handling by cardiomyocytes, because IP, which prevents changes in cardiac function, also prevents the alterations in intracellular Ca2+ handling.


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This study 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, Manitoba, 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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 Am. J. Physiol. Heart Circ. Physiol.Home page
H. K. Saini, O. N. Tripathi, S. Zhang, V. Elimban, and N. S. Dhalla
Involvement of Na+/Ca2+ exchanger in catecholamine-induced increase in intracellular calcium in cardiomyocytes
Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H373 - H380.
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 J. Appl. Physiol.Home page
S. Takeda, S. Mochizuki, H. K. Saini, V. Elimban, and N. S. Dhalla
Modification of alterations in cardiac function and sarcoplasmic reticulum by vanadate in ischemic-reperfused rat hearts
J Appl Physiol, September 1, 2005; 99(3): 999 - 1005.
[Abstract] [Full Text] [PDF]

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 Am. J. Physiol. Heart Circ. Physiol.Home page
H. K. Saini, V. Elimban, and N. S. Dhalla
Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion
Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H614 - H623.
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