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(Circulation Research. 1995;76:1049-1056.)
© 1995 American Heart Association, Inc.

Articles

Postischemic Changes in Cardiac Sarcoplasmic Reticulum Ca²⁺ Channels

A Possible Mechanism of Ischemic Preconditioning

Riccardo Zucchi, Simonetta Ronca-Testoni, Gongyuan Yu, Paola Galbani, Giovanni Ronca, Mario Mariani

From Scuola Superiore S. Anna (R.Z.), Istituto di Cardiologia (R.Z., M.M.), and Istituto di Chimica Biologica (S.R.-T., G.Y., P.G., G.R.), University of Pisa (Italy).

Correspondence to R. Zucchi, MD, Scuola Superiore S. Anna, via Carducci 40, I-56100 Pisa, Italy.

	Abstract

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Abstract We investigated the modifications of cardiac ryanodine receptors/sarcoplasmic reticulum Ca²⁺ release channels occurring in ischemic preconditioning. In an isolated rat heart model, the injury produced by 30 minutes of global ischemia was reduced by preexposure to three 3-minute periods of global ischemia (preconditioning ischemia). The protection was still present 120 minutes after preconditioning ischemia but disappeared after 240 minutes. Three 1-minute periods of global ischemia did not provide any protection. In the crude homogenate obtained from ventricular myocardium, the density of [³H]ryanodine binding sites averaged 372±18 fmol/mg of protein in the control condition, decreased 5 minutes after preconditioning ischemia (290±15 fmol/mg, P<.01), was still significantly reduced after 120 minutes (298±17 fmol/mg, P<.05), and recovered after 240 minutes (341±21 fmol/mg). Three 1-minute periods of ischemia did not produce any change in ryanodine binding. The K_d for ryanodine (1.5±0.3 nmol/L) was unchanged in all cases. In parallel experiments, the crude homogenate or a microsomal fraction was passively loaded with ⁴⁵Ca, and Ca²⁺-induced Ca²⁺ release was studied by the quick filtration technique. In both preparations, the rate constant of Ca²⁺-induced Ca²⁺ release decreased 5 and 120 minutes after preconditioning ischemia (homogenate values: 19.7±1.4 and 18.9±0.9 s^-1 vs a control value of 25.4±1.7 s^-1, P<.05 in both cases) and recovered after 240 minutes (23.0±1.9 s^-1). The Ca²⁺ dependence of Ca²⁺-induced Ca²⁺ release was not affected by preconditioning ischemia. In conclusion, changes in sarcoplasmic reticulum Ca²⁺-release channels occur after brief ischemia and reperfusion, are closely correlated with the development of myocardial protection versus sustained ischemia, and might play a role in the pathogenesis of ischemic preconditioning.

Key Words: sarcoplasmic reticulum • Ca²⁺ • ischemia • reperfusion

	Introduction

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The development of myocardial ischemic injury is delayed if the tissue has been previously subjected to one or more brief periods of ischemia/reperfusion. This phenomenon is known as ischemic preconditioning,¹ and its mechanisms are not well known.²

A crucial issue in the pathogenesis of ischemic injury is the increase in cytosolic Ca²⁺ concentration, which occurs in the first minutes of ischemia.^{3 4 5} Several mechanisms contribute in determining cytosolic Ca²⁺ overload: reduction in ATP phosphorylation potential⁶ may block Ca²⁺ transport by the sarcoplasmic reticulum (SR) and plasma membrane Ca²⁺-ATPases; Ca²⁺ influx may occur through the Na⁺-Ca²⁺ exchanger, as a consequence of increased intracellular Na⁺ and of membrane depolarization^{7 8} ; and Ca²⁺ may be released from the SR because the SR Ca²⁺-release channels open as soon as cytosolic Ca²⁺ increases.^{9 10} Since overall cellular Ca²⁺ content is unchanged until reperfusion,^{11 12} inversion of Na⁺-Ca²⁺ exchange appears to be a late phenomenon, and it can be assumed that at least in the early phase of ischemia, cytosolic Ca²⁺ overload reflects a redistribution of intracellular Ca²⁺, ie, Ca²⁺ release from the SR.¹³

Steenbergen et al¹⁴ have reported that the development of cytosolic Ca²⁺ overload is delayed in the preconditioned myocardium. We have recently observed¹⁵ that the density of cardiac ryanodine (Ry) receptors, which correspond to active SR Ca²⁺-release channels, decreases after a short period of ischemia. It seems likely that reduced channel density might delay the increase in cytosolic Ca²⁺ during a subsequent ischemic insult. Therefore, in the present study we investigated whether postischemic changes in cardiac SR Ca²⁺-release channels/Ry receptors may be related to ischemic preconditioning. In particular, we determined the time course of the modifications in [³H]Ry binding and in Ca²⁺-induced Ca²⁺ release produced by ischemic preconditioning and compared it with the time course of myocardial protection.

	Materials and Methods

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Animals and Perfusion Technique
Sprague-Dawley rats fed with standard diet (275 to 300 g body weight) were anesthetized with a mixture of ether and air. After injection of 1000 U sodium heparin in the femoral vein, the heart was quickly excised and perfused according to the working heart technique, as described previously.¹⁵ The concentration of CaCl₂ in the perfusion buffer was 1.5 mmol/L. Unless otherwise specified, the preload (height of the atrial chamber) and the afterload (height of the aortic chamber) were set at 20 and 100 cm, respectively.

Experimental Protocols
The hearts were perfused in the working mode for a period of 7 minutes and were then subjected to a preconditioning procedure that included three 3-minute periods of global ischemia, each followed by 3 minutes of retrograde reperfusion (aortic pressure, 80 mm Hg). In some experiments, the preconditioning procedure was modified and included three 1-minute periods of ischemia, each followed by 3 minutes of retrograde reperfusion. After the preconditioning procedure, the hearts were perfused aerobically in the working mode for a period ranging from 5 to 240 minutes, which was followed by 30 minutes of global ischemia (sustained ischemia) and 120 minutes of retrograde reperfusion. Control hearts were subjected to the same protocols, except that the preconditioning procedure was omitted. During working heart perfusion, the buffer was recirculated (approximate volume, 200 mL); during retrograde perfusion, it was not recirculated and was collected on ice to assay creatine kinase (CK) release as described previously.¹⁵ During sustained ischemia, the heart chamber was filled with perfusion buffer, and its temperature was closely monitored and kept at 36.6±0.1°C.

In other experiments, the preconditioning procedure was substituted by preload reduction (9 cm) or by the addition of Ry to the perfusion buffer (final concentration, 5 nmol/L). Low preload perfusion and Ry administration lasted 30 and 45 minutes, respectively; thereafter, the hearts were subjected to 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. An overview of the different experimental protocols is given in Fig 1.

View larger version (26K): [in this window] [in a new window]   Figure 1. Schematic diagram of the perfusion protocols used in the present study. The duration of each period is expressed in minutes. In Tables 1 through 3, "time" represents the time elapsed after the first 25 minutes of perfusion (dotted line in this figure), which corresponds to the end of the preconditioning procedure in the preconditioning groups. In parallel experiments, the perfusion was interrupted before sustained ischemia (arrow), and [3H]ryanodine binding or Ca2+-induced Ca2+ release were determined in the tissue. CK indicates creatine kinase; TTC, triphenyltetrazolium chloride.

After perfusion, tissue injury was measured on the basis of triphenyltetrazolium chloride (TTC) staining. The hearts were removed from the perfusion apparatus and washed in isotonic saline. The ventricles were separated from the atria and great vessels and cut into four transverse slices, which were weighed and incubated at 37°C for 20 minutes in a buffer containing 1% TTC in 50 mmol/L Tris-HCl (pH 7.4). TTC causes living tissue to stain in deep red.^{16 17} Pictures were taken from both sides of each slice, and the area of TTC-negative tissue was measured planimetrically.

In parallel experiments, the perfusion was interrupted just before sustained ischemia, and the hearts were used to assay [³H]Ry binding or Ca²⁺-induced Ca²⁺ release, as detailed below.

In a few experiments, the effectiveness of reperfusion and the possible occurrence of the no-reflow phenomenon¹⁸ were evaluated. Ten milliliters of 0.3% Monastral blue was injected into the aortic root through a side arm of the aortic cannula just before the end of the final reperfusion period. The ventricles were then fixed in 10% formalin and cut into four transverse slices, and tissue staining was measured planimetrically.

Preparation of Cellular Fractions
In the experiments aimed at determining [³H]Ry binding and Ca²⁺-induced Ca²⁺ release, the ventricles were finely minced and homogenized in 5 vol of 300 mmol/L sucrose and 10 mmol/L imidazole (pH 7.0 at 4°C) by 15+15 passes in a Potter-Elvejheim homogenizer set at 800 rpm and kept in a cold room at 4°C. The homogenate was then filtered through one layer of cheesecloth and used for the assay of Ca²⁺-induced Ca²⁺ release and of [³H]Ry binding.

In other experiments, the homogenate was used to prepare purified fractions, namely, a microsomal fraction enriched in SR (obtained as described previously from pooled homogenates derived from three or four hearts, all subjected to the same perfusion protocol)¹⁵ and heavy SR membranes (obtained as described by Holmberg and Williams¹⁹ from pooled homogenates prepared from six hearts, all subjected to the same perfusion protocol). The protein content of each fraction was determined by the Lowry method,²⁰ with bovine serum albumin used as a standard.

Assay of [³H]Ry Binding
High-affinity Ry binding was assayed as described previously.¹⁵ Binding was usually determined by using six concentrations of the ligand. Incubations were performed in duplicate, and nonspecific binding was determined in the presence of 10 µmol/L Ry. The difference between the counts of duplicate samples was <10% in all cases.

Assay of SR Ca²⁺ Release
Vesicles (15 to 20 mg/mL of homogenate protein, 3 to 5 mg/mL of microsomal protein, or 1 to 2 mg/mL of SR protein) were passively loaded for 120 minutes at 23°C in a medium containing (mmol/L) ⁴⁵CaCl₂ 10 (specific activity, 4 Ci/mol; corresponding to 40 µCi/mL), potassium HEPES 20 (pH 6.8), KCl 100, and NaN₃ 5. ⁴⁵Ca release was determined by using a rapid filtration system.^{21 22} The loaded vesicles were diluted 100- to 200-fold (10 or 20 µL to 2 mL) into a buffer (rinse buffer) having the same ionic composition of the loading medium, except that ⁴⁵Ca was replaced with unlabeled Ca²⁺, applied to cellulose nitrate filters with 0.45-µm pores (Sartorius), and washed with 4 mL of rinse buffer. At 30 seconds after vesicle dilution, either a release or a nonrelease buffer was passed through the filters for a preset time by using a rapid filtration apparatus (RFS-4, Bio-Logic). The filtration time ranged from 10 milliseconds to 4 seconds, and five data points were taken within the first 100 milliseconds.

To follow ⁴⁵Ca release for a longer time, in other assays the loaded vesicles were diluted directly into release or nonrelease buffer and filtered under vacuum at 30 to 180 seconds after vesicle dilution.^{9 23}

The filters were shaken overnight in 8 mL of scintillation fluid (Optiphase II, LKB), and radioactivity was then measured at 90% efficiency in an LKB Wallac 1214 scintillation counter. Filtration experiments were always performed in duplicate.

The release buffer contained (mmol/L) potassium HEPES 20 (pH 6.8), KCl 100, CaCl₂ 1.01, and EGTA 1 (free Ca²⁺ concentration was 18 µmol/L). In some experiments, its Ca²⁺ content was changed to obtain free Ca²⁺ concentrations ranging from 1 to 200 µmol/L; in others, the ionophore A23187 was added (final concentration, 10 µmol/L). Two types of nonrelease buffer were used: the first buffer contained (mmol/L) potassium HEPES 20 (pH 6.8), KCl 100, and CaCl₂ 10; the second one contained (mmol/L) potassium HEPES 20 (pH 6.8), KCl 100, and MgCl₂ 10, along with 10 µmol/L ruthenium red.

The rate constant of quick Ca²⁺ release (K_r) was calculated over the first 100 milliseconds. Since quick Ca²⁺ release followed first-order kinetics, the experimental data were fitted by the following equation: ln[(R-R_res)/(Ro-R_res)]=-K_r · t, where t represents time, R represents radioactivity at time t, Ro represents radioactivity at time zero, and R_res represents the radioactivity remaining on the filter after completion of quick Ca²⁺ release.²³ The latter was calculated by back extrapolation to time zero of the late phase of Ca²⁺ release, and in practice it was equal to the residual radioactivity at 1 to 2 seconds (see below). This calculation procedure was validated by comparison with more sophisticated algorithms involving multiple exponential models. In particular, the analysis of representative experiments showed that no improvement in data fitting was obtained with three-component versus two-component models and that the slow component of Ca²⁺ release was responsible for <1% of the release observed in the first 100 milliseconds.

In preliminary experiments, we observed that the inclusion of protease inhibitors (5 µmol/L leupeptin, 2 µmol/L pepstatin, 0.25 µmol/L aprotinin, 200 µmol/L phenylmethylsulfonyl fluoride, 2 mmol/L iodoacetamide, and 2 mmol/L benzamide) in the homogenization and assay buffers produced no difference (<5%) in the rate constant of rapid Ca²⁺ release and in the amount of quickly releasable Ca²⁺, in both control and ischemic hearts. We have previously obtained similar results with regard to Ry binding.¹⁵ For these reasons, protease inhibitors were omitted in most experiments.

Chemical and Radionuclides
Ry was purchased from Calbiochem as a mixture of Ry and didehydroryanodine, and its concentration was checked on the basis of the absorbance at 268 nm, using an extinction coefficient of 1.45x10⁴ (mol/L)^-1, which was calculated by the manufacturer for the specific lot of Ry used in these experiments. EGTA was obtained from Sigma Chemical Co, and its alleged purity was 97%. All other reagents were of analytic grade.

Free Ca²⁺ concentration was calculated according to Fabiato and Fabiato²⁴ by using a computer program that included an empirical correction for the ionic strength of the buffer. The pH of each solution containing Ca²⁺ and EGTA was carefully adjusted. Free Ca²⁺ was also measured with the antipyrylazo III technique.²⁵ The values reported in the text as "free Ca²⁺" represent the results of such measurements and were generally in accordance with the theoretical values.

[³H]Ry and ⁴⁵CaCl₂ were obtained from New England Nuclear-DuPont and diluted to the desired specific activity with cold Ry and with a stock solution of CaCl₂ purchased from BDH, respectively.

Statistical Analysis
The results are expressed as mean±SEM. The binding data were analyzed through the SCAFIT (LIGAND) program.²⁶ Differences between groups were evaluated by ANOVA: Fisher's F test was first used to compare between-group variance and within-group variance; if the former was significantly (P<.05) higher than the latter, individual groups were compared by appropriate techniques for multiple comparisons, as described by Armitage.²⁷

	Results

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Hemodynamic Variables and Myocardial Protection
The hemodynamic variables and the extent of tissue injury are shown in Tables 1 and 2. In control hearts subjected to 30 minutes of sustained ischemia and 120 minutes of reperfusion, CK release averaged 75±7 U/g wet wt. The extent of myocardial necrosis, as evaluated by TTC staining, corresponded to 28±8% of the ventricular mass. These results were not affected by the occurrence of the no-reflow phenomenon, since Monastral blue staining showed that the ventricular myocardium was completely reperfused (>98%) under our experimental conditions. A moderate impairment in contractile performance was observed in hearts perfused for >180 minutes, consistent with previous reports.²⁸ However, prolonged perfusion did not affect the susceptibility to ischemic injury: if ischemia was produced after 240 minutes of aerobic perfusion, CK release and TTC-negative tissue averaged 71±6 U/g and 31±7%, respectively.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Variables

View this table: [in this window] [in a new window]   Table 2. Tissue Injury

Three 3-minute periods of ischemia/reperfusion caused minor changes in mechanical performance, without any leakage of intracellular enzymes (1 U/g for CK). This procedure was effective in protecting the heart during sustained ischemia. As already reported,^{2 29} the time lag between preconditioning ischemia and sustained ischemia was critical: CK release and the extent of TTC-negative tissue decreased significantly (by 40% to 65%) if the lag did not exceed 180 minutes, but the protection was lost after 240 minutes. The duration of preconditioning ischemia was also crucial, since three 1-minute periods of ischemia did not provide any protection. As in control hearts, Monastral blue staining showed the absence of nonreperfused regions in all groups. Prolonged reperfusion (>180 minutes) was associated with a moderate decrease in cardiac output, which was similar to the decrease observed in control hearts subjected to prolonged aerobic perfusion.

Although the present study was not designed to evaluate cardiac arrhythmias, we noticed that 81% of the control hearts versus 48% of the preconditioned hearts developed irreversible ventricular fibrillation after sustained ischemia (P<.05, ² test; all preconditioned hearts were pooled for this comparison, since the incidence of ventricular fibrillation was still as low as 40% at 240 minutes after preconditioning ischemia). Within each group, the occurrence of ventricular fibrillation was not related to differences in tissue injury, [³H]Ry binding, or Ca²⁺-induced Ca²⁺ release. However, it must be acknowledged that our data were not sufficient to draw definite conclusions on these issues.

Treatment with 5 nmol/L Ry was effective in reducing tissue injury. The extent of TTC-negative tissue decreased significantly (12±3% versus 28±8%, P<.01), and CK release was also reduced (52±7 versus 75±7 U/g, P=.08). The addition of Ry to the perfusion buffer produced a progressive decrease in cardiac output and aortic pressure but no significant change in heart rate. Contractile performance stabilized after 40 minutes, when cardiac output and aortic pressure had decreased by 35% and 15%, respectively. To assess whether the protective action of Ry could be attributed to its negative inotropic action per se, some hearts were subjected to preload reduction. This intervention produced similar impairment in mechanical performance but was not associated with any change in CK release and TTC staining.

[³H]Ry Binding
Saturation binding curves for [³H]Ry are shown in Fig 2. Nonspecific binding was <10% at Ry concentrations 1.5 nmol/L, and the binding data were well interpolated by a single–binding site model. The values of the dissociation constant (K_d) and of the binding site density (B_max) are shown in Table 3.

View larger version (19K): [in this window] [in a new window]   Figure 2. Saturation binding curves for [3H]ryanodine in control conditions () and at 5 minutes (), 120 minutes (), and 240 minutes () after ischemic preconditioning. [3H]Ryanodine binding was determined at pCa 4.7 (free Ca2+, 20 µmol/L) in the crude homogenate. Data points represent mean values of six to eight hearts per group. Bars represent SEM. See Table 3 for Kd and Bmax values and for statistical significance.

View this table: [in this window] [in a new window]   Table 3. Ryanodine Binding and Sarcoplasmic Reticulum Channel Function

In the homogenate obtained from control hearts, the K_d and the B_max averaged 1.5±0.3 nmol/L and 372±18 fmol/mg of protein, respectively. The B_max was slightly higher than previously reported by Naudin et al³⁰ and by our group.¹⁵ From our experience, slightly different B_max values were obtained in similar experimental conditions when different batches of Ry were used, and we presume that differences in the purity of commercial Ry preparations may be responsible for these variations. All the results of the present study were obtained with a single batch of Ry.

Prolonged perfusion, preload reduction, or previous Ry infusion did not modify [³H]Ry binding. Our standard preconditioning procedure (three 3-minute periods of ischemia/reperfusion) produced a 20% to 25% reduction in the density of [³H]Ry binding sites, which was statistically significant (P<.01). Such a reduction persisted after 120 minutes of working heart perfusion, whereas recovery occurred after 240 minutes. If the preconditioning procedure was modified, replacing the three 3-minute periods of ischemia with three 1-minute periods, no change in [³H]Ry binding was produced. The K_d was not affected by preconditioning ischemia nor was the Ca²⁺ dependence of [³H]Ry binding (data not shown).

In our previous study,¹⁵ the modifications of [³H]Ry binding observed in the crude homogenate after ischemia and reperfusion were confirmed by using a binding buffer with physiological ionic strength and could be reproduced in the microsomal fraction. For this reason, in the present study, [³H]Ry binding was not determined at low ionic strength or in purified preparations.

Ca²⁺-Induced Ca²⁺ Release
Representative results of Ca²⁺-induced Ca²⁺-release experiments performed in the crude homogenate are shown in Fig 3. In the presence of 1 to 200 µmol/L extravesicular Ca²⁺, Ca²⁺-induced Ca²⁺ release followed a biexponential kinetic. Total tissue ⁴⁵Ca was on the order of 10 to 15 nmol/mg. Approximately 40% was released over 100 to 120 milliseconds; thereafter, the rate of Ca²⁺ release decreased considerably. As reported in similar experimental models, Ca²⁺ release was delayed in the presence of millimolar Ca^{2+21 22} or millimolar Mg²⁺ and ruthenium red.⁹ In particular, the left panel of Fig 3 shows that the quick component of Ca²⁺ release was abolished by 10 mmol/L extravesicular Ca²⁺ and by 10 mmol/L Mg²⁺ plus 10 µmol/L ruthenium red, confirming that this component represented Ca²⁺ efflux through the SR release channels. By repeating the experiments in the presence of the ionophore A23187, we estimated that nonreleasable ⁴⁵Ca accounted for 8% of the total ⁴⁵Ca.

View larger version (14K): [in this window] [in a new window]   Figure 3. Representative results of Ca2+-induced Ca2+ release in the crude homogenate obtained from control hearts. The preparation was passively loaded with 10 mmol/L 45CaCl2 for 120 minutes and then exposed to either release (, 18 µmol/L Ca2+) or nonrelease (, 10 mmol/L Ca2+; , 10 µmol/L ruthenium red plus 10 mmol/L MgCl2) media. Left, Tissue radioactivity is expressed as a function of time in a semilogarithmic plot. Filtration was performed either through an automatized rapid-filtration system (experimental points in the 0.01- to 4-second range; some of these points are omitted here for clarity) or manually (experimental points in the 30- to 120-second range). Zero time values were obtained from parallel experiments, in which quick filtration was omitted, and the filters were counted 30 seconds after vesicle dilution. Back extrapolation to time zero of the late phase of 45Ca release allowed calculation of the fraction of 45Ca subjected to quick release. In this experiment, such a fraction yielded 4.2 nmol/mg of protein, corresponding to 41% of total vesicle Ca2+. Right, The quick phase of 45Ca release is reproduced with a different time scale. The vertical axis shows the logarithm of the ratio of 45Ca to 45Ca at time zero, after subtraction of the amount of 45Ca not involved in quick Ca2+ release. The slope of the regression line represents the rate constant of Ca2+-induced Ca2+ release (29.3 s-1 in this experiment). See "Materials and Methods" and Reference 23 for further details.

The Ca²⁺ dependence of quick Ca²⁺ release is shown in Fig 4. In accordance with previous findings,⁹ channel activation was maximum at pCa 4.75 (18 µmol/L free Ca²⁺), and the maximum value of the rate constant (K_r) was on the order of 30 s^-1, which corresponded to a half-time (t) of 20 milliseconds.

View larger version (15K): [in this window] [in a new window]   Figure 4. Graph showing Ca2+ dependence of Ca2+-induced Ca2+ release. The rate constant of the quick phase of Ca2+ release (Kr) was calculated as shown in Fig 3 in the homogenate obtained from one control heart () and one heart subjected to ischemic preconditioning (). We did not assess quick Ca2+ release at pCa 7, since in this range of concentration chelation of the excess free Ca2+ remaining in the hydrated filters after vesicle dilution may become a rate-limiting step and bias the results obtained in the first 50 to 100 milliseconds.23

In the microsomal fraction and in heavy SR vesicles, Ca²⁺ release had the same characteristics as in the crude homogenate (data not shown). The only difference lay in the extent of the Ca²⁺ pool subjected to quick release, which was on the order of 8 to 9 and 13 to 15 nmol/mg of protein, respectively (in these preparations the B_max for Ry averaged 1.6 and 3.0 pmol/mg, respectively). The K_r values were similar to those obtained in the homogenate (24.7±2.7 s^-1 in the microsomal fraction and 28.8±3.5 s^-1 in heavy SR vesicles versus 25.4±1.7 s^-1 in the homogenate). These findings support the view that the assay of Ca²⁺-induced Ca²⁺ release performed in the crude homogenate provided a reliable measurement of SR Ca²⁺ release.

After our standard preconditioning procedure, the K_r decreased by 25%, while its Ca²⁺ dependence was preserved (Fig 4), and the total amount of ⁴⁵Ca released was unchanged. The K_r values obtained in the crude homogenate at pCa 4.75 (18 µmol/L free Ca²⁺) are shown in Table 3, and release curves are plotted in Fig 5. The effect of preconditioning ischemia was statistically significant (P<.05) and persisted after 120 minutes of reperfusion, whereas recovery occurred after 240 minutes. These results were confirmed in the microsomal fraction. In particular, the K_r had similar values: it averaged 24.7, 15.0, 15.0, and 21.7 s^-1 in the control condition and 5, 120, or 240 minutes after preconditioning ischemia, respectively (in the homogenate the corresponding values were 25.4, 19.7, 18.9, and 23.0 s^-1). Decreased Ca²⁺ release 5 minutes after ischemic preconditioning was observed also in heavy SR vesicles (K_r=20.4 versus 28.8 s^-1), but in this fraction the time course of the postischemic changes in SR function was not evaluated. Preload reduction and previous Ry infusion did not affect SR Ca²⁺-induced Ca²⁺ release.

View larger version (20K): [in this window] [in a new window]   Figure 5. Graph showing Ca2+-induced Ca2+ release measured in the crude homogenate in control conditions () and at 5 minutes (), 120 minutes (), and 240 minutes () after ischemic preconditioning. Ca2+ release was induced with a buffer containing 18 µmol/L free Ca2+. The quick phase of Ca2+ release is plotted as in the right panel of Fig 3. Data points represent mean values of four to six hearts per group. Bars represent SEM. See Table 3 for rate constant values and for statistical significance.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we assessed the effect of ischemic preconditioning on cardiac Ry receptors/SR Ca²⁺-release channels and compared the time course of channel modifications with the time course of myocardial protection. Ry binding site density (B_max) was reduced after ischemic preconditioning, and this was associated with myocardial protection: after 120 minutes reduced B_max and myocardial protection could still be shown, whereas after 240 minutes Ry binding normalized and the protection disappeared. When the preconditioning procedure was modified by reducing the duration of each ischemic period to 1 minute, both Ry binding and the susceptibility to sustained ischemia were not affected. Ry binding and SR Ca²⁺ efflux have been shown to be closely associated in vitro.^{9 19 31 32} This was confirmed in our experimental model: reduced B_max in binding experiments was associated with reduced K_r in Ca²⁺-release experiments.

Since the preparation procedures used to purify the SR may select vesicles that are not representative of the whole SR,^{15 33 34} all our experiments were performed in the crude homogenate. Although Ca²⁺-induced Ca²⁺ release is usually determined in purified fractions, the use of the crude homogenate should not introduce any bias, since no other structure is able to support a Ca²⁺ release that has t on the order of a few milliseconds, is induced by micromolar Ca²⁺, and is inhibited by millimolar Ca²⁺ or by Mg²⁺ and ruthenium red. This conclusion is supported by quantitative considerations. In the homogenate, the amount of quickly releasable Ca²⁺ averaged 5 nmol/mg of homogenate protein, corresponding to 500 nmol/g wet wt, which is close to previous estimates on the basis of the assay of Ca²⁺ uptake,^{35 36} Ca²⁺ flux studies,³⁷ and quantitative morphological analysis.³⁸ However, we repeated the assay of Ca²⁺-induced Ca²⁺ release in purified preparations. The purification of SR activities was rather low, as already reported in similar rat heart preparations,^{15 39} but the results confirmed those obtained in the homogenate.

Some investigators have reported indirect evidence of reduced SR Ca²⁺-release capability in the postischemic myocardium. In skinned cardiomyocytes, the stimulation of SR Ca²⁺ uptake produced by inhibitors of the SR Ca²⁺-release channel was reduced after simulated ischemia and reperfusion⁴⁰ ; in human atrial fibers, caffeine-induced tension development was reduced after surgical ischemia.⁴¹ Darling et al⁴² have reported that SR Ca²⁺ release was not affected by ischemia. However, this investigation was aimed at studying the regulation of SR Ca²⁺ release by cations and nucleotides, which was preserved in ischemic tissue, a finding that is consistent with our results. Quantitative data on the rate constant of Ca²⁺ release were not determined.

There is now evidence that the pathophysiology of ischemic preconditioning is related to the control of Ca²⁺ homeostasis. Cytosolic Ca²⁺ overload is one of the major determinants of ischemic injury. In an isolated rat heart model, Steenbergen et al¹⁴ have observed that cytosolic Ca²⁺ overload was delayed in the preconditioned myocardium. This phenomenon was interpreted as follows: preconditioning determines glycogen depletion, which delays the development of intracellular acidosis, of Na⁺ overload (through Na⁺-H⁺ exchange), and of Ca²⁺ influx through Na⁺-Ca²⁺ exchange. However, total cellular Ca²⁺ content was found to be unchanged during ischemia,^{11 12 13} and interventions able to prevent acidosis did not affect the development of Ca²⁺ overload.⁴³ Therefore, cytosolic Ca²⁺ overload appears to represent a redistribution of intracellular Ca²⁺.¹³ Our hypothesis is that the overload is initiated by the inhibition of SR Ca²⁺-ATPase activity due to reduced ATP phosphorylation potential⁶ and possibly intrinsic protein dysfunction^{44 45} and is amplified by a positive-feedback mechanism, since the opening probability of the SR Ca²⁺-release channel increases as soon as cytosolic Ca²⁺ concentration rises. The channel alterations observed in the present work should reduce the rate of SR Ca²⁺ release and delay the development of cytosolic Ca²⁺ overload. This might be the molecular basis of the protection provided by ischemic preconditioning.

In our model, contractile performance was normal up to 2 hours after the preconditioning procedure. This observation must be interpreted on the basis of the concept that the amount of Ca²⁺ released from the SR depends on its Ca²⁺ content, on the number of active channels, and on their opening probability, which is modulated by changes in the cytosolic milieu, eg, in Ca²⁺, Mg²⁺, ATP, H⁺, and Cl^- concentrations.^{9 10 19 31 32 46} Therefore, the effect of a slight reduction in the number of active SR channels might be compensated by changes in the cytosolic milieu, eg, higher cytosolic Ca²⁺ in diastole.⁴⁷ In addition, in rats contractile proteins appear to be fully activated by the physiological Ca²⁺ release occurring during each cardiac cycle,⁴⁸ so that even if Ca²⁺ release were reduced after ischemia/reperfusion, it might still be sufficient to support a normal contractile function. On the other hand, during sustained ischemia, when the SR channels remain persistently open, changes in the number of active channels are expected to affect the development of cytosolic Ca²⁺ overload.

The results obtained with the administration of nanomolar Ry to perfused hearts support the hypothesis that reduced SR Ca²⁺ release delays the development of ischemic injury. Nanomolar Ry increases the opening probability of the SR channel or, more precisely, locks the channel open, in a state of subnormal conductivity.⁴⁹ The final effect is a progressive depletion of the SR Ca²⁺ pool and of cardiac contractile performance.^{48 50 51} Under these conditions and in accordance with previous findings,⁵¹ the susceptibility to ischemic injury was reduced. This effect cannot be accounted for by reduced mechanical performance, since preload reduction had the same mechanical consequences as Ry infusion but was not protective.

We should point out some limitations in the present study. It is not certain that results obtained in the rat in vitro may apply to in vivo models or to other species. In particular, contractile performance decreased after several hours of isolated heart perfusion. It could be argued that the cause of the increased susceptibility to ischemic injury observed at 240 versus 120 minutes after preconditioning was the functional deterioration of hearts subjected to prolonged perfusion. This interpretation is unlikely, because the susceptibility to ischemia was unchanged after up to 240 minutes of control perfusion, and the duration of the protection provided by preconditioning was in accordance with the results of previous in vivo studies.^{2 29}

Finally, the molecular mechanism(s) responsible for SR channel alterations remains unknown. Possible mechanisms include changes in redox potential, intracellular acidosis, exposure to reactive oxygen species,⁵² proteolysis, phosphorylation or dephosphorylation,^{53 54} and modification of membrane phospholipids.⁵⁵ Whatever the mechanism may be, it appears to be operating in the early phase of ischemia and to require a few minutes for full activation. At present, there is no evidence that any of the mediators thought to be involved in preconditioning (such as adenosine, nitric oxide, or prostacyclin)² may affect the SR Ca²⁺-release channel, but this possibility has not been specifically investigated.

	Acknowledgments

 
This study was supported in part by Ministero dell'Università e della Ricerca Scientifica e Tecnologica. We thank Maria Fantin for English language revision.

Received July 13, 1994; accepted February 9, 1995.

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