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CARDIOVASCULAR

Attentuation of Cardiac Stunning by Losartan in a Cellular Model of Ischemia and Reperfusion Is Accompanied by Increased Sarcoplasmic Reticulum Ca²⁺ Stores and Prevention of Cytosolic Ca²⁺ Elevation

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Received June 14, 2004; accepted August 9, 2004.

	Abstract

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This study investigates whether protective effects of an angiotensin II type 1 receptor antagonist (losartan) in ischemia and reperfusion are mediated by actions on Ca²⁺ cycling. Effects of exposure to losartan (10 µM) in ischemia were evaluated in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion at 37°C. Field-stimulated myocytes were exposed to 30 min of simulated ischemia (hypoxia, acidosis, lactate, hyperkalemia, and glucose-free) and reperfusion with Tyrode's solution for 40 min. Cell shortening was measured with a video edge detector, and Ca²⁺ concentration was measured with fura-2. Field-stimulated myocytes exhibited stunning in reperfusion, which was abolished in cells exposed to losartan. In microelectrode studies, losartan did not alter the responses of resting potentials or action potentials to ischemia and reperfusion. In the absence of losartan, diastolic Ca²⁺ increased in ischemia, and Ca²⁺ transients exhibited a rebound overshoot in early reperfusion. Losartan did not affect amplitudes of Ca²⁺ transients in ischemia but prevented elevations in diastolic Ca²⁺ in ischemia. Furthermore, losartan prevented the overshoot of Ca²⁺ transients in early reperfusion and increased the magnitude of Ca²⁺ transients in late reperfusion. Sarcoplasmic reticulum (SR) Ca²⁺ stores, determined as Ca²⁺ released by rapid application of 10 mM caffeine, were not altered in ischemia and reperfusion. However, losartan increased SR Ca²⁺ stores in late reperfusion, even in cells that were not exposed to simulated ischemia. We conclude that losartan abolishes stunning in reperfusion by preserving normal diastolic Ca²⁺ in ischemia and by increasing Ca²⁺ transients through elevation of releasable SR Ca²⁺.

A number of studies have demonstrated that blockade of angiotensin II receptors with selective angiotensin II type 1 (AT₁) receptor antagonists also is protective in myocardial ischemia and reperfusion (for review, see Schulz and Heusch, 2003). Pretreatment with the AT₁ receptor antagonists losartan or candesartan improves recovery of function and inhibits arrhythmias in Langendorff-perfused hearts exposed to global ischemia (Lee et al., 1997; Paz et al., 1998; Wang and Sjoquist, 1999). Similar protective effects of AT₁ antagonists have been reported in in vivo models subjected to transient coronary artery ligation (Harada et al., 1998; Dorge et al., 1999; Zhu et al., 2000; Yahiro et al., 2003). Other studies, however, in intact hearts have reported that AT₁ receptor antagonists actually have deleterious effects on recovery of contractile function in reperfusion (Ford et al., 1996, 1998; So et al., 1998). Thus, whether AT₁ receptor antagonists exert protective effects in intact hearts exposed to ischemia and reperfusion remains controversial.

We recently reported that losartan attenuates stunning and inhibits the arrhythmogenic transient inward current in voltage-clamp studies of isolated ventricular myocytes exposed to simulated ischemia and reperfusion (Louch et al., 2000). Interestingly, losartan exerted these protective effects both in the absence and presence of exogenous angiotensin II, which suggests that losartan has effects on cardiac myocytes that are independent of AT₁ receptor blockade (Louch et al., 2000). Elevations in intracellular free Ca²⁺ are believed to promote stunning (for review, see Bolli and Marban, 1999) and induce transient inward current (Lederer and Tsien, 1976). Therefore, it is possible that the protective effects of losartan in ischemia and reperfusion are mediated by alterations in Ca²⁺ cycling and a reduction in intracellular free Ca²⁺ levels.

Our previous investigation of losartan in ischemia and reperfusion was conducted under voltage-clamp conditions to correlate effects on L-type Ca²⁺ current and contraction (Louch et al., 2000). However, changes in action potential configuration in response to ischemia and reperfusion would be predicted to contribute to alterations in Ca²⁺ cycling and contractile responses in ischemia and reperfusion. Therefore, the goal of the present study was to examine the effects of losartan on Ca²⁺ cycling and stunning in field or intracellularly stimulated cardiac myocytes where changes in action potential configuration are not eliminated by voltage clamp. Experiments were conducted in a cellular model of simulated ischemia and reperfusion developed in this laboratory (Cordeiro et al., 1994; Louch et al., 2000, 2002). Isolated myocytes exposed to simulated ischemia exhibit many characteristics of true ischemia such as depolarization, abbreviation of action potential duration, intracellular Ca²⁺ overload, and abolition of contraction (Cordeiro et al., 1994; Louch et al., 2000, 2002). Myocytes also exhibit the arrhythmogenic transient inward current in early reperfusion and stunning in late reperfusion (Cordeiro et al., 1994; Louch et al., 2000, 2002). The specific objectives of this study were to determine whether losartan: 1) attenuates stunning in field-stimulated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion; 2) alters resting or action potentials in myocytes subjected to ischemia and reperfusion; 3) prevents elevation of intracellular free Ca²⁺ levels in myocytes exposed to ischemia and reperfusion; and 4) alters Ca²⁺ transients and/or sarcoplasmic reticulum (SR) Ca²⁺ load in cells exposed to simulated ischemia and reperfusion and under normoxic conditions.

	Materials and Methods

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Myocyte Isolation. All experiments were conducted on freshly isolated guinea pig ventricular myocytes and performed in accordance with guidelines published by the Canadian Council on Animal Care. Albino guinea pigs (250-400 g; Charles River Canada, St. Constant, QC, Canada), of which approximately 90% were male and 10% female, were used in this investigation. Guinea pigs were injected with heparin (3.3 IU/g) and anesthetized with sodium pentobarbital (160 mg/kg). The heart was then rapidly cannulated and perfused retrogradely through the aorta (10-12 ml/min) for 7 to 8 min with oxygenated (100% O₂; 37°C) Ca²⁺-free solution of the following composition: 120 mM NaCl, 3.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 10 mM HEPES, and 11 mM glucose (pH 7.4 with NaOH). Collagenase A (25 mg/50 ml) and protease (4.8 mg/50 ml, Sigma-type XIV) were then included in the perfusate for approximately 5 min. Following dissociation, the ventricles were minced and washed in a substrate-enriched high K⁺ buffer of the following composition: 80 mM KOH, 50 mM glutamic acid, 30 mM KCl, 30 mM KH₂PO₄, 20 mM taurine, 10 mM HEPES, 10 mM glucose, 3 mM MgSO₄, and 0.5 mM EGTA (pH 7.4 with KOH). This isolation procedure provided ventricular myocytes that were rod-shaped and free of membrane blebs. Myocytes were then placed in a 0.75-ml chamber on the stage of an inverted microscope. Cells were allowed to adhere to the bottom of the chamber for 5 to 10 min and then were superfused (3 ml min^-1, 37°C) with normal Tyrode's solution of the following composition: 129 mM NaCl, 20 mM NaHCO₃, 0.9 mM NaH₂PO₄, 4 mM KCl, 0.5 mM MgSO₄, 2.5 mM CaCl₂, and 5.5 mM glucose, pH 7.4, gassed with 95% O₂, 5% CO₂.

General Methods. Cells that served as time controls were exposed to 80 min of superfusion with normal Tyrode's solution. In all other experiments, myocytes were exposed to 10 min of normal Tyrode's solution and were then superfused for 30 min with an "ischemic" Tyrode's solution. The ischemic solution was designed to mimic conditions associated with ischemia including hypoxia, hypercapnia, hyperkalemia, acidosis, lactate accumulation, and substrate deprivation (Ferrier and Guyette, 1991). This solution had the following composition: 123 mM NaCl, 6 mM NaHCO₃, 0.9 mM NaH₂PO₄, 8 mM KCl, 0.5 mM MgSO₄, 2.5 mM CaCl₂, and 20 mM sodium lactate, gassed with 90% N₂/10% CO₂, pH 6.8. A 90% N₂/10% CO₂ gas phase was layered over the superfusion chamber throughout simulated ischemia. Reperfusion was achieved by superfusion with normal Tyrode's solution. Each cell was exposed to only one cycle of ischemia and reperfusion. Cells in the treatment group were exposed to 10 µM losartan only during ischemia. This concentration was chosen on the basis of a previous study that reported that 10 µM but not 1.0 µM losartan exerted protective effects in ischemia and reperfusion and that 10 µM losartan blocked the effects of exogenous angiotensin II in guinea pig ventricular tissue (Thomas et al., 1996).

Measurement of Contractions. In some experiments, myocytes were field-stimulated continuously at 2 Hz through a pair of platinum electrodes. Myocytes were visualized with a video camera and TV monitor. Cell length was recorded with a video edge detector (Crescent Electronics, Windsor, ON, Canada). Contraction was measured as the maximum difference between diastolic and systolic cell length. Cell length was recorded at 5-min intervals throughout the experiment, except during the first 5 min of reperfusion when recordings were made every minute. Three responses were averaged for each recording period.

Measurement of Action Potentials. In other experiments, action potentials were measured with conventional microelectrode techniques. Cells were impaled with high-resistance electrodes (18-25 M) to minimize dialysis and avoid buffering intracellular Ca²⁺ levels. Electrodes were filled with 2.7 M KCl, and a 2.7 M KCl-agar bridge was used as a bath ground. Myocytes were stimulated by 3.5-ms current pulses delivered through the recording electrode. Action potentials (averages of five) were recorded every 5 min. Cells were activated regularly throughout the experiments. Recordings were made with an Axoclamp 2B amplifier (Axon Instruments, Inc., Union City, CA). Resting membrane potential (RMP) was measured just prior to action potential trains. Action potential duration (APD) was measured at 80% repolarization with respect to the action potential amplitude.

Intracellular Ca²⁺ Measurements. Intracellular Ca²⁺ was measured by whole cell photometry (DeltaRam; Photon Technology International, Monmouth Junction, NJ). Myocytes were loaded with fura-2 by incubation with fura-2/acetoxymethyl ester (0.1 µM) for 20 min in the dark at room temperature. The ratio of emission at 510 nm, during alternate excitation at 340 and 380 nm, was used to determine intracellular Ca²⁺ concentrations. The fluorescence ratio was converted to Ca²⁺ concentration with a calibration curve determined experimentally at pH 7.2 as described previously (Louch et al., 2002). Ca²⁺ concentration measured with this calibration curve is expected to slightly underestimate actual intracellular Ca²⁺ during ischemia since intracellular acidosis increases the dissociation constant of fura-2 (Martinez-Zaguilan et al., 1991). However, this is not likely to be a problem in reperfusion as intracellular pH recovers rapidly to preischemic levels upon reperfusion (Kitakaze et al., 1988). Fluorescence was recorded and measured with Felix software (version 1.4; Photon Technology International).

Ca²⁺ transients were recorded in both field-stimulated myocytes and in cells stimulated through the microelectrode. Recordings were background-subtracted, and the magnitude of the Ca²⁺ transient was calculated as the difference between diastolic and peak systolic Ca²⁺ concentrations. Ca²⁺ transients were recorded at 5-min intervals throughout the experiment, except for the first 5 min of reperfusion when recordings were made every minute. Three responses were averaged for each recording period.

Estimation of SR Ca²⁺ Stores. Amplitudes of caffeine-elicited Ca²⁺ transients were used as a measure of SR Ca²⁺ content. In these experiments, cells were stimulated with current pulses delivered through the microelectrode at 2 Hz. At 10, 40, 55, 70, and 80 min during the experiment, stimulation was briefly interrupted, and 10 mM caffeine was applied to cells for 1 s at 37°C with a rapid solution switcher. The rapid solution switcher was triggered by the voltage-clamp protocol and changed the solution bathing the cell within 300 ms.

Analyses. Differences between experimental groups were tested for statistical significance with a two-way repeated measures analysis of variance. All other data were analyzed relative to preischemic values with a one-way repeated measures analysis of variance. Post hoc comparisons were made with a Bonferroni test. Statistical analyses were performed with Sigma Stat (version 2.0; SPSS Science, Chicago, IL). Data are presented as means ± S.E.M. The value of "n" represents the number of myocytes sampled (no more than 3 myocytes from a single heart were used).

Compounds. Losartan was a gift from Merck Frosst Canada Inc. (Kirkland, QC, Canada). Collagenase A was obtained from Roche Diagnostics (Laval, QC, Canada). Fura-2/acetoxymethyl ester was purchased from Invitrogen Canada, Inc. (Burlington, ON, Canada). All other drugs and chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada).

	Results

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Effects of Losartan on Contractions throughout Ischemia and Reperfusion. Figure 1 shows representative recordings of cell length at selected time points during ischemia and reperfusion. Control recordings in the absence of drug are shown in Fig. 1A, and recordings obtained in a cell exposed to 10 µM losartan in ischemia are shown in Fig. 1B. In the absence of losartan, contractions were markedly reduced in ischemia, and diastolic length increased slightly (Fig. 1A). After 1 min of reperfusion, contractions recovered and exceeded preischemic levels, and diastolic length decreased (Fig. 1A). After 10 min of reperfusion, diastolic cell length recovered slightly, but contractions were markedly depressed relative to preischemic levels (Fig. 1A). Similar results were obtained in ischemia and early reperfusion in the cell exposed to losartan (Fig. 1B); however, losartan improved the recovery of contractions at 10 min of reperfusion (Fig. 1B).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Losartan treatment during ischemia attenuated contractile depression in late reperfusion. A, representative recordings of cell length from a myocyte exposed to ischemia and reperfusion. Contraction amplitudes were markedly reduced during ischemia. Upon reperfusion, diastolic length decreased and contraction amplitudes increased. With continued reperfusion, diastolic length partially recovered, but contractions became depressed. B, in losartan-treated myocytes, changes in diastolic length and contraction amplitudes during ischemia and early reperfusion were similar to those observed in untreated cells. However, contractile depression in late reperfusion was attenuated in losartan-treated myocytes.

Figure 2A shows mean measurements of cell shortening during time-control experiments and during exposure to simulated ischemia and reperfusion. In time-control experiments, myocytes exhibited only a slight reduction in cell shortening during 80 min of recording. During ischemia, myocytes exhibited a significant decrease in contractions relative to time controls (Fig. 2A). In early reperfusion, contractions were briefly but significantly increased from time-control values. However, after the first few minutes of reperfusion, myocytes exhibited a significant reduction in cell shortening for the remainder of the protocol (Fig. 2A). Losartan-treated cells were similar to untreated cells during ischemia and early reperfusion (Fig. 2B); however, postischemic contractile depression was attenuated following losartan treatment (Fig. 2B). Indeed, cell shortening in losartan-treated cells did not differ significantly from time-control values at any point in reperfusion (Fig. 2B). Thus, field-stimulated myocytes exhibited stunning in reperfusion, which was attenuated by losartan treatment during ischemia.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Losartan protects against stunning in isolated cardiac myocytes. A, mean data demonstrate that ischemia caused a significant decrease in the amplitudes of contractions relative to time controls in the absence of losartan. Upon reperfusion, contractions initially increased; however, significant contractile depression (stunning) was observed with continued reperfusion. B, losartan-treated myocytes also exhibited a significant reduction in the magnitude of contractions in ischemia, followed by an increase in early reperfusion. However, stunning was attenuated in losartan-treated myocytes. Cell shortening was normalized as a percent of preischemic values *, p < 0.05 denotes significant difference from time controls; n = 15 myocytes from 10 hearts in the ischemia/reperfusion group; n = 18 myocytes from nine hearts in the losartan group; n = 12 myocytes from nine hearts in the time-control group.

Effects of Losartan on Resting Membrane Potential and Action Potential Duration during Ischemia and Reperfusion. Contractions in field-stimulated myocytes are initiated by action potentials. Thus, it is possible that losartan might have improved recovery of contractions in reperfusion by effects on RMP or APD. To determine whether losartan treatment altered electrical properties of cells, cells were impaled with microelectrodes. Action potentials were elicited by a 3.5-ms current stimulus delivered through the electrode. Figure 3A shows a representative recording of action potentials in a control cell at selected time points during ischemia and reperfusion. The cell depolarized, and action potentials abbreviated in ischemia; these changes recovered gradually in reperfusion (Fig. 3A). Similar results are shown in representative recordings from a cell exposed to losartan in ischemia (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Changes in APD and RMP during ischemia and reperfusion were similar in the presence and absence of losartan in ischemia. A, representative recordings show action potentials at selected time points in ischemia and reperfusion. In the absence of losartan, action potentials abbreviated, and cell membranes depolarized during ischemia. Both action potential configuration and RMP recovered in reperfusion. B, similar changes in action potential configuration and RMP occurred during ischemia and reperfusion when cells were exposed to losartan in ischemia.

Figure 4 shows mean measurements of RMP (panel A) and APD (panel B) during ischemia and reperfusion. In ischemia, control and losartan-treated cells depolarized to approximately -73 mV from preischemic values near -90 mV (Fig. 4A). RMP recovered rapidly upon reperfusion (Fig. 4A). Figure 4B shows that APD declined gradually during ischemia in both control and losartan-treated cells and recovered gradually in reperfusion in both groups. Thus, losartan treatment did not alter the changes in either RMP or APD during ischemia and reperfusion.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Losartan did not alter mean responses of RMP and APD to ischemia and reperfusion. A, in both untreated (top) and losartan-treated (bottom) cells, ischemia was associated with significant membrane depolarization. RMP recovered rapidly in reperfusion in both groups. B, APD declined gradually during ischemia and recovered slowly in reperfusion in the absence (top) and presence (bottom) of losartan. *, p < 0.05 denotes significantly different from preischemic values; n = 21 myocytes from 20 hearts in the ischemia/reperfusion group; n = 18 myocytes from 15 hearts in the losartan group.

Effects of Losartan Treatment on Diastolic Ca²⁺ and Ca²⁺ Transients. In the next series of experiments, we determined whether losartan altered intracellular Ca²⁺ levels and Ca²⁺ transients in field-stimulated ventricular myocytes paced at 2 Hz. First, we examined intracellular Ca²⁺ levels and Ca²⁺ transients in time-control experiments where cells were not exposed to ischemia and reperfusion. Figure 5 shows representative recordings (panel A) and mean intracellular Ca²⁺ measurements (panel B) for time-control experiments. Ca²⁺ transient magnitudes were relatively stable for the duration of the protocol (80 min), although diastolic Ca²⁺ increased gradually with time (Fig. 5, A and B).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Amplitudes of Ca2+ transients did not change during time-control experiments. Representative recordings (A) and mean data (B, shaded region) from cells that served as time controls show that the magnitude of Ca2+ transients decreased only slightly with time. Diastolic Ca2+ gradually increased with time, but the increase was not statistically significant (n = 5 myocytes from five hearts). Representative times shown in A represent elapsed time in this time-control experiment.

Next, we examined Ca²⁺ transients at selected time points throughout ischemia and reperfusion in the absence of losartan (Fig. 6, A and B). The representative example in Fig. 6A shows that diastolic Ca²⁺ increased in ischemia, but the magnitude of Ca²⁺ transients was unchanged relative to preischemic conditions. At 1 min of reperfusion, diastolic Ca²⁺ levels declined toward preischemic levels, and Ca²⁺ transients increased in magnitude (Fig. 6A). After 30 min of reperfusion, Ca²⁺ transients had recovered, and diastolic Ca²⁺ levels were increased (Fig. 6A). Figure 6B shows mean intracellular Ca²⁺ measurements at intervals throughout ischemia and reperfusion. Diastolic Ca²⁺ increased significantly during ischemia, recovered rapidly in early reperfusion, and increased in late reperfusion (Fig. 6B). Ca²⁺ transient amplitudes (shaded area) also increased in early reperfusion (Fig. 6B).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Losartan treatment during ischemia altered the effects of ischemia and reperfusion on Ca2+ homeostasis. A, representative recordings of Ca2+ transients from a cell exposed to simulated ischemia and reperfusion and mean data are presented in B. Diastolic Ca2+ increased in ischemia with little change in the amplitude of Ca2+ transients (shaded region). Ca2+ transients temporarily exceeded preischemic levels in early reperfusion. Diastolic Ca2+ levels recovered in early reperfusion but rose again in late reperfusion. Representative recordings (C) and mean data (D) in cells treated with losartan show that diastolic Ca2+ increased during ischemia with no change in the magnitude of Ca2+ transients. However, exposure to losartan in ischemia prevented the increase in amplitudes of Ca2+ transients in early reperfusion and increased the magnitude of Ca2+ transients in late reperfusion. Diastolic Ca2+ was significantly elevated in late reperfusion in losartan-treated cells. *, p < 0.05 denotes significantly different from preischemic values, n = 15 myocytes from 12 hearts in the absence of losartan and 11 myocytes from nine hearts in the presence of losartan.

Figure 6, C and D, also shows Ca²⁺ transients at selected time points throughout ischemia and reperfusion in the presence of losartan in ischemia. The representative example in Fig. 6C shows that diastolic Ca²⁺ increased slightly relative to preischemic levels during ischemia and reperfusion. Ca²⁺ transients did not increase in magnitude in early reperfusion but appeared larger after 30 min of reperfusion (Fig. 6C). Figure 6D shows that in the presence of losartan, mean intracellular Ca²⁺ levels increased significantly during ischemia and in late reperfusion when compared with preischemic levels. Ca²⁺ transients did not increase in early reperfusion but did increase in late reperfusion (Fig. 6D).

The preceding results describe changes in diastolic and systolic Ca²⁺ levels in treated and untreated myocytes relative to respective preischemic values. To evaluate the effects of losartan on the effects of ischemia and reperfusion, we compared responses relative to time controls. Figure 7A compares mean diastolic Ca²⁺ levels recorded at selected times in ischemia and reperfusion to diastolic Ca²⁺ levels at corresponding times in time controls. Ischemia caused a significant increase in diastolic Ca²⁺ levels relative to time controls. This increase recovered rapidly in reperfusion (Fig. 7A). In contrast, when myocytes were exposed to losartan in ischemia, the increase in diastolic Ca²⁺ during ischemia was prevented (Fig. 7B). Figure 8A compares mean amplitudes of Ca²⁺ transients at different time points in time controls and cells exposed to simulated ischemia and reperfusion. Throughout most of the experiment, Ca²⁺ transient amplitudes were similar in time controls and in cells exposed to ischemia and reperfusion (Fig. 8A). However, Ca²⁺ transients were significantly increased in amplitude in early reperfusion (Fig. 8A). Interestingly, exposure to losartan in ischemia prevented the increase in amplitudes of Ca²⁺ transients in early reperfusion but increased the magnitude of Ca²⁺ transients late in reperfusion (Fig. 8B). These comparisons demonstrate that losartan attenuates the increase in diastolic Ca²⁺ levels during ischemia, prevents the overshoot of Ca²⁺ transients in early reperfusion, and increases the magnitude of Ca²⁺ transients in late reperfusion.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Losartan treatment attenuated elevations in diastolic [Ca2+]i during ischemia. A, diastolic Ca2+ levels rose significantly in myocytes exposed to ischemia relative to time controls and recovered rapidly in reperfusion. B, in cells exposed to losartan in ischemia, a significant elevation of diastolic Ca2+ was not observed. Amplitudes of Ca2+ transients were normalized to preischemic values. *, p < 0.05 indicates that the effect of ischemia on diastolic Ca2+ is significantly different from time controls; n = 15 myocytes from 12 hearts in the ischemia/reperfusion group; n = 11 myocytes from nine hearts in the losartan group.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Losartan prevented the increase in amplitudes of Ca2+ transients in early reperfusion and increased the magnitude of Ca2+ transients in late reperfusion. A, cells exposed to simulated ischemia and reperfusion exhibited a significant increase in the magnitude of Ca2+ transients in early reperfusion relative to time controls. Otherwise, Ca2+ transients changed little throughout ischemia and reperfusion compared with time controls. B, the increase in amplitudes of Ca2+ transients in early reperfusion was abolished in losartan-treated cells. In addition, the amplitudes of Ca2+ transients increased with further reperfusion. Amplitudes of Ca2+ transients were normalized to preischemic values. *, p < 0.05 denotes significantly different from time controls; n = 15 myocytes from 12 hearts in the ischemia/reperfusion group; n = 11 myocytes from nine hearts in the losartan group.

Effects of Losartan on SR Ca²⁺ Stores during Ischemia and Reperfusion. We next conducted experiments to determine whether losartan alters SR Ca²⁺ stores in ischemia and reperfusion. SR Ca²⁺ stores were assessed by rapid application of 10 mM caffeine with a rapid solution switcher. Representative recordings of caffeine-elicited Ca²⁺ transients in the absence and presence of losartan in ischemia are shown in Fig. 9, A and B. Caffeine-elicited Ca²⁺ transients appeared to decline gradually with time in cells exposed to ischemia and reperfusion (Fig. 9A). However, the magnitude of caffeine transients increased throughout ischemia and reperfusion in myocytes exposed to losartan in ischemia (Fig. 9B).

View larger version (19K): [in this window] [in a new window]   Fig. 9. Losartan increased SR Ca2+ stores in ischemia and reperfusion. Representative examples of caffeine-induced Ca2+ transients in cells exposed to ischemia and reperfusion in the absence (A) and presence (B) of losartan in ischemia. Caffeine-elicited Ca2+ transients were unchanged during ischemia and reperfusion in the absence of losartan (A). In contrast, caffeine-induced Ca2+ transients continued to increase throughout ischemia and reperfusion in losartan-treated cells (B). C, mean caffeine-induced Ca2+ transients were similar in time controls and in cells exposed to ischemia and reperfusion. In cells exposed to losartan in ischemia, caffeine transients continued to increase throughout ischemia and reperfusion. Data have been normalized as a percentage of the first caffeine-induced Ca2+ transient prior to treatment. *, p < 0.05 indicates that the effect of losartan on SR Ca2+ stores is significantly different from time controls; n = 6 myocytes from six hearts in the time-control group; n = 13 myocytes from 12 hearts in the ischemia/reperfusion group; n = 15 myocytes from 12 hearts in the losartan group.

Figure 9C shows mean measurements of the magnitude of caffeine-elicited Ca²⁺ transients at selected intervals during an experiment. Caffeine-induced Ca²⁺ transients declined slightly with time in both time controls and in cells exposed to simulated ischemia and reperfusion (Fig. 9C). However, caffeine transients increased with time when cells were exposed to losartan during ischemia and this increase was significant after 30 min of reperfusion (Fig. 9C). These observations suggest that exposure to losartan in ischemia may increase SR Ca²⁺ stores during reperfusion.

Effects of Losartan on SR Ca²⁺ Stores and Intracellular Ca²⁺ Concentrations under Normoxic Conditions. Exposure to losartan in ischemia appeared to markedly increase SR Ca²⁺ stores in late reperfusion. Therefore, we investigated whether losartan also might increase SR Ca²⁺ stores when cells are exposed to drug under normoxic conditions. In these experiments, myocytes were exposed to 10 µM losartan for 30 min, followed by 40 min of washout. Caffeine was applied at regular intervals to evaluate SR Ca²⁺ stores. Figure 10A shows mean data which demonstrate that losartan increased the magnitude of caffeine-elicited Ca²⁺ transients even in the absence of ischemia and reperfusion. SR Ca²⁺ stores continued to increase, even after the drug was washed out. Therefore, losartan caused a persistent increase in SR Ca²⁺ stores under normoxic conditions. Despite this increase in SR Ca²⁺ stores, signs of Ca²⁺ overload were not observed.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Losartan treatment increases Ca2+ transients and SR Ca2+ stores in the absence of ischemia and reperfusion. Cells were exposed to losartan for 30 min in the absence of ischemia and reperfusion. A, caffeine-elicited Ca2+ transients increased during exposure to losartan and continued to increase following up to 40 min of washout. Caffeine-elicited Ca2+ transients were not altered in time-control experiments. Data have been normalized as a percentage of the first caffeine-induced Ca2+ transient prior to treatment. B, Ca2+ transients increased with time in cells exposed to losartan in the absence of ischemia and reperfusion. In contrast, time controls exhibited a gradual decrease in the magnitude of Ca2+ transients during 80 min of recording time. C, diastolic Ca2+ levels declined gradually in cells that served as time controls but did not decline in cells exposed to losartan. Data have been normalized as a percentage of the first response prior to treatment. *, p < 0.05 denotes significantly different from time controls; n = 6 myocytes from six hearts in the time-control group; n = 7 myocytes from five hearts in the losartan group.

We next determined whether a 30-min exposure to losartan under normoxic conditions altered Ca²⁺ transients and diastolic Ca²⁺ levels. Figure 10B shows mean amplitudes of Ca²⁺ transients plotted as a function of time in time controls and in losartan-treated myocytes. Exposure to losartan increased the amplitudes of Ca²⁺ transients after washout of the drug when compared with time controls. Figure 10C shows mean diastolic Ca²⁺ levels plotted as a function of time. Diastolic Ca²⁺ levels were not significantly different in losartan treated myocytes compared with time controls. Thus, increased SR Ca²⁺ stores in myocytes exposed to losartan under normoxic conditions were accompanied by increased amplitudes of Ca²⁺ transients without elevation of diastolic Ca²⁺ levels.

	Discussion

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The objectives of this study were to determine whether losartan: 1) attenuates stunning in field-stimulated guinea pig myocytes exposed to simulated ischemia and reperfusion, 2) alters resting or action potentials in ischemia and reperfusion, 3) prevents elevation of intracellular free Ca²⁺ levels in cells exposed to ischemia and reperfusion, and 4) alters Ca²⁺ transients and/or SR Ca²⁺ load in ischemia and reperfusion and under normoxic conditions. Our results show that losartan improved recovery of contractile function in field-stimulated myocytes following exposure to ischemia and reperfusion. Losartan did not alter the effects of ischemia or reperfusion on RMP and APD. Losartan prevented the elevation of diastolic Ca²⁺ levels in ischemia and abolished the overshoot of Ca²⁺ transients in early reperfusion. In addition, losartan increased the magnitude of Ca²⁺ transients and SR Ca²⁺ stores in late reperfusion. Interestingly, losartan increased SR Ca²⁺ stores even in cells that were not exposed to ischemia and reperfusion. Thus, losartan may improve contractile function in reperfusion by increasing SR Ca²⁺ stores and thereby SR Ca²⁺ release.

In the present study, we found that losartan reduced postischemic contractile depression in field-stimulated ventricular myocytes. In field-stimulated myocytes, contractions are activated by action potentials with configurations that change with ischemia and reperfusion (Cordeiro et al., 1994). However, we found that losartan did not alter changes in APD and RMP occurring in response to ischemia and reperfusion. Therefore, attenuation of stunning by losartan must occur through an action that does not involve alterations in APD or RMP. This interpretation is supported by our previous observation that exposure to losartan in ischemia improved recovery of contractile function in reperfusion when the magnitude and duration of depolarization was controlled with voltage clamp (Louch et al., 2000).

We also found that exposure to losartan during ischemia prevented elevation of diastolic Ca²⁺ levels which typically occurs in response to ischemia. Prevention of elevated intracellular free Ca²⁺ in ischemia may contribute to the protective effects of losartan in ischemia and reperfusion. Ca²⁺ overload with elevation of cytosolic free Ca²⁺ is believed to promote myocardial stunning by triggering Ca²⁺-dependent proteolysis of myofilaments, which reduces myofilament responsiveness to Ca²⁺ (Gao et al., 1996; Bolli and Marban, 1999). Thus, a reduction in intracellular free Ca²⁺ by losartan in ischemia might preserve myofilament sensitivity and contribute to the attenuation of stunning observed in reperfusion.

Previous studies have shown that losartan decreases the incidence of ischemia and reperfusion-induced arrhythmias in in vivo mouse and rat models (Lee et al., 1997; Harada et al., 1998; Zhu et al., 2000). Furthermore, we previously have demonstrated an antiarrhythmic effect of losartan in early reperfusion in isolated cardiac ventricular muscle preparations exposed to simulated ischemia and reperfusion (Thomas et al., 1996). A reduction in intracellular Ca²⁺ overload also could contribute to the antiarrhythmic actions of losartan reported in these studies. Intracellular Ca²⁺ overload is believed to play an important role in the generation of reentrant arrhythmias (Thomas et al., 1996). In addition, Ca²⁺ overload promotes the arrhythmogenic transient inward current, which is believed to cause triggered arrhythmias (Ferrier, 1977). Indeed, losartan has been shown to inhibit the arrhythmogenic transient inward current in early reperfusion (Louch et al., 2000). Thus, our observation that losartan attenuates the rise in intracellular free Ca²⁺ levels in ischemia may, in part, explain the antiarrhythmic effects of losartan reported in previous studies.

In theory, there are a number of possible mechanisms by which losartan could attenuate the rise in intracellular free Ca²⁺ levels in ischemia. For example, interventions that attenuate acidosis, Na⁺ loading, or Ca²⁺ influx all may protect against elevations in intracellular Ca²⁺ (Kitakaze et al., 1988; Przyklenk et al., 1987; Tani and Neely, 1989, 1990). Interestingly, angiotensin II has been shown to stimulate Na⁺-H⁺ exchange in ischemic hearts and thereby cause intracellular Na⁺ accumulation (Grace et al., 1996). Intracellular Na⁺ accumulation leads to intracellular Ca²⁺ accumulation via the Na⁺-Ca²⁺ exchanger (Tani and Neely, 1989). Thus, it is possible that losartan inhibits Na⁺-loading in ischemia by blockade of the actions of angiotensin II. However, as our study was conducted in isolated myocytes in the absence of exogenous angiotensin II, it would be necessary to postulate that locally produced angiotensin II is responsible for Na⁺ and Ca²⁺ accumulation.

We also demonstrated that losartan abolished the rebound increase in Ca²⁺ transients in early reperfusion. The mechanism by which losartan inhibits this rebound increase has not yet been established. An increase in the activity of the Na⁺-Ca²⁺ exchanger may promote Ca²⁺ entry in early reperfusion. Indeed, studies have shown that oxidative stress and intracellular Ca²⁺ overload in ischemia increase the activity of the Na⁺-Ca²⁺ exchange in early reperfusion (Goldhaber and Liu, 1994; Goldhaber, 1996). Losartan decreases oxidative stress (Franco Mdo et al., 2003; Chamorro et al., 2004) and reduces intracellular Ca²⁺ overload during ischemia. Thus, losartan may decrease the overshoot in Ca²⁺ transients in early reperfusion by reducing oxidative stress and intracellular Ca²⁺ overload in ischemia.

We also found that exposure to losartan gradually increased SR Ca²⁺ stores and increased the magnitude of Ca²⁺ transients, even in cells that were not exposed to ischemia and reperfusion. These observations suggest that losartan may improve contractile function in reperfusion by increasing SR Ca²⁺ stores and therefore SR Ca²⁺ release. Interestingly, signs of Ca²⁺ overload, such as aftercontractions, afterpotentials, or spontaneous beats, did not accompany this increase in SR Ca²⁺. The mechanism by which losartan increases SR Ca²⁺ stores and Ca²⁺ transients may involve increased SR Ca²⁺ uptake. Interestingly, Jaiswal et al. (1991) showed that losartan stimulates release of prostacyclin in vascular smooth muscle and neuronal cells by an action independent of angiotensin II receptor blockade. Prostacyclin stimulates SR Ca²⁺ uptake by increasing phosphorylation of phospholamban, which regulates the SR Ca²⁺ pump (Doni et al., 1994; Karczewski et al., 1998). Thus, losartan may increase SR Ca²⁺ stores in cardiac myocytes by stimulation of prostacyclin release, which would increase the activity of the SR Ca²⁺ ATPase. Alternatively, losartan might increase SR Ca²⁺ stores by AT₁ receptor blockade. Angiotensin II binding activates phospholipase C, which releases diacyl glycerol and inositol triphosphate (Griendling et al., 1987). Inositol triphosphate releases Ca²⁺ from the SR in cardiac myocytes (Fukuta et al., 1998). Thus, locally produced angiotensin II could reduce SR Ca²⁺ stores, and this action would be inhibited by the AT₁ antagonist, losartan. Interestingly, the effects of losartan on SR Ca²⁺ stores and Ca²⁺ transients developed slowly and continued even after losartan was removed. This contrasted with the immediate effect of losartan on diastolic free Ca²⁺ observed during ischemia. The reason for this delay is not clear; however, it is possible that the delay occurs if losartan acts through a signaling pathway in which the levels of intermediates do not return to basal levels quickly. Alternatively, the slowly developing effects may represent accumulation of an active metabolite of losartan, for example, the noncompetitive and potent AT₁ receptor antagonist EXP3174 (Israili, 2000).

To determine whether the protective effects of losartan and its effects on SR Ca²⁺ load are mediated by AT₁-receptor blockade or by non-AT₁ receptor mediated actions, one might compare the effects of losartan to other AT₁ receptor antagonists. If the effects of losartan are mediated by AT₁-receptor blockade, then all AT₁ antagonists would be expected to exert the same effects. However, if the protective effects of losartan represent non-AT₁ receptor actions, only chemically related compounds may exert these effects. For example, the anti-inflammatory and anti-aggregatory effects of losartan, which are not mediated by AT₁-receptor blockade, are shared by losartan's metabolite, EXP3174, and by irbesartan, which are chemically similar to losartan (Sadoshima, 2002). In contrast, these actions are absent with candesartan and valsartan, which are chemically dissimilar to losartan (Sadoshima, 2002).

In summary, we found that exposure of field-stimulated cardiac myocytes to losartan during ischemia markedly improved recovery of contractile function in reperfusion. This improvement in contractile function in reperfusion was not attributable to changes in RMP or APD in ischemia and reperfusion. However, losartan prevented the increase in free intracellular Ca²⁺ levels observed in ischemia, abolished the overshoot in Ca²⁺ transients in early reperfusion, and increased the magnitude of Ca²⁺ transients in late reperfusion. This increase in magnitude of Ca²⁺ transients in late reperfusion was accompanied by an increase in SR Ca²⁺ stores in losartan-treated myocytes. This increase in Ca²⁺ transients and SR Ca²⁺ stores also was observed in cells exposed to losartan in the absence of ischemia and reperfusion. Thus, losartan has important effects on Ca²⁺ homeostasis under normoxic conditions, and these effects are preserved in cells exposed to ischemia and reperfusion. The beneficial effects of losartan on contractile activity in reperfusion may be related both to prevention of ischemia-induced elevation of diastolic Ca²⁺ and to increased SR Ca²⁺ stores. The present study suggests that losartan may exert direct protective effects on the myocardium in patients with ischemic heart disease, in addition to its well established hemodynamic effects.

	Acknowledgements

 
We thank Peter Nicholl, Cindy Mapplebeck, and Steve Foster for excellent technical assistance.

	Footnotes

 
This study was supported by the Heart and Stroke Foundation of Nova Scotia and the Canadian Institutes of Health Research. W.L. was supported by a Scholarship from the Medical Research Council of Canada.

doi:10.1124/jpet.104.072769.

ABBREVIATIONS: ACE, angiotensin-converting enzyme; AT₁ receptor, angiotensin II type 1 receptor; EXP3174, 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl]imidazole-5-carboxylic acid; SR, sarcoplasmic reticulum; APD, action potential duration; RMP, resting membrane potential.

Address correspondence to: Dr. G. R. Ferrier, Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: gregory.ferrier{at}dal.ca

	References

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