Sulfhydryl Redox State Affects Susceptibility to Ischemia and Sarcoplasmic Reticulum Ca²⁺ Release in Rat Heart

Implications for Ischemic Preconditioning

From the Scuola Superiore St Anna (R.Z.), Dipartimento di Scienze dell'Uomo e dell'Ambiente (G.Y., P.G., G.R., S.R.-T.), and Dipartimento di Cardiologia (M.M.), University of Pisa, Italy.

Correspondence to Riccardo Zucchi, MD, Scuola Superiore St Anna, via Carducci 40, I-56100 Pisa, Italy. E-mail rzucchi{at}bm.med.unipi.it

	Abstract

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Abstract—We investigated the effect of sulfhydryl and disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca²⁺ release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. Necrosis was evaluated on the basis of lactate dehydrogenase release and triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca²⁺ release and [³H]-ryanodine binding were determined before the sustained ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca²⁺ release and reduced [³H]-ryanodine binding. The disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion buffer either before or after the preconditioning procedure. In preconditioned hearts, dithiothreitol increased sarcoplasmic reticulum Ca²⁺ release and ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents 4,4'-dithiodipyridine (25 µmol/L) and N-ethylmaleimide (16 µmol/L) increased the resistance to ischemia and reduced sarcoplasmic reticulum Ca²⁺ release and [³H]-ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and disulfide reagents produced similar effects on Ca²⁺ release and [³H]-ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca²⁺ release.

Key Words: sarcoplasmic reticulum • Ca²⁺ • ischemia • ryanodine receptor • thiol

	Introduction

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Ischemia and ischemia-reperfusion are associated with an oxidative stress, which can potentially produce extensive molecular changes.¹ In particular, protein sulfhydryl groups may be oxidized, leading to the formation of intramolecular or intermolecular disulfide bonds. Although in many experimental models the exposure to oxidative systems leads to cellular injury, it has been suggested that a moderate oxidative stress might increase the resistance to ischemia and contribute to the phenomenon known as ischemic preconditioning. In isolated rabbit hearts, free radical generating systems produced a protection from a subsequent ischemic injury,^{2 3} whereas in open chest rabbits, superoxide dismutase removed the protection provided by ischemic preconditioning.⁴ The involvement of sulfhydryl oxidation was suggested by the observation that thiol compounds such as N-2-mercaptopropionylglycine and N-acetylcysteine produced similar effects.^{2 4 5} However, the effectiveness of superoxide dismutase and N-2-mercaptopropionylglycine was not confirmed in other studies.^{6 7}

In the present work, we investigated whether changes in the redox state of sulfhydryl groups, produced by several kinds of thiol reagents, affected the susceptibility of rat heart to ischemic injury and whether they influenced ischemic preconditioning. Such actions were correlated with effects on the sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor, because we have reported that the SR Ca²⁺ release capability is reduced after ischemia reperfusion and that this phenomenon might be involved in the pathogenesis of ischemic preconditioning.^{8 9}

	Materials and Methods

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Animals and Perfusion Technique
Male Wistar rats (bw, 275 to 300 g), fed with standard diet, were anesthetized with a mixture of ether and air. After injection of 1000 U sodium heparin in the femoral vein, the heart was excised quickly and perfused according to the working heart technique, as described previously.⁸ The preload (height of the atrial chamber) and the afterload (height of the aortic chamber) were set at 20 and 100 cm, respectively. The treatment of animals was approved by the competent authority of the University of Pisa.

Perfusion Protocol
The hearts were perfused in the working mode for a period of 5 minutes and then subjected to three 3-minute periods of global ischemia, each followed by 3 minutes of retrograde reperfusion (aortic pressure, 95 cm H₂O; ie, 70 mm Hg). After this preconditioning procedure, the hearts were perfused aerobically in the working mode for 5 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 protocol, except that the preconditioning procedure was replaced by aerobic perfusion. During working heart perfusion, the buffer was recirculated (volume, 200 mL), whereas during retrograde perfusion, it was not recirculated and was collected on ice to assay lactate dehydrogenase (LDH) activity, as described previously.⁸ During sustained ischemia, the heart chamber was filled with perfusion buffer, and its temperature was monitored closely and kept at 36.8±0.1°C.

In other experiments, substances interacting with thiol or disulfide groups were added to the perfusion buffer. Each compound was added to the recirculating buffer used for working heart perfusion; therefore, it was absent during the final 120 minutes of retrograde reperfusion. The following substances were used: 25 µmol/L 4,4'-dithiodipiridine (DTDP), 16 µmol/L N-ethylmaleimide (NEM), 10 µmol/L thimerosal (merthiolate), 1 mmol/L DTT. DTDP, NEM, and thimerosal are sulfhydryl reagents, which induce the formation of mixed disulfides, alkylated derivatives, and mercaptides, respectively. DTT is a disulfide reducing agent. Contrary to the other reagents, thimerosal is hydrophilic and poorly membrane permeable.^{10 11} After perfusion, tissue injury was measured on the basis of triphenyltetrazolium chloride (TTC) staining, as described previously.⁹

Although sulfhydryl and disulfide reagents were absent during the final 120 minutes of reperfusion, in pilot experiments, we ascertained that DTT, DTDP, and NEM, at the concentration used in the perfusion experiments, had no direct effect on either LDH assay or TTC staining. In parallel experiments, perfusions were interrupted just before sustained ischemia (28th minute), or at the end of the sustained ischemia (58th minute), and the hearts were used to assay tissue thiols, Ca²⁺-induced Ca²⁺ release, or [³H]-ryanodine binding, as detailed below. In some additional experiments, the perfusion apparatus was switched to the working mode after 60 minutes of retrograde reperfusion (118th minute) to estimate functional recovery. A scheme of the perfusion protocol is shown in Figure 1.

View larger version (36K): [in this window] [in a new window]   Figure 1. Scheme of the perfusion protocols used in this study. Arrows delimitate the period during which each reagent was administered. In parallel experiments, perfusion was interrupted before or after sustained ischemia, and [3H]-ryanodine binding or Ca2+-induced Ca2+ release were determined. Final concentrations: DTT, 1 mmol/L; DTDP, 25 µmol/L; NEM, 16 µmol/L; and thimerosal, 10 µmol/L.

Preparation of Cellular Fractions
At the end of perfusion, the ventricles were minced finely and homogenized in 5 volumes 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 in the assays of Ca²⁺-induced Ca²⁺ release and of [³H]-ryanodine binding.

In other experiments, a microsomal fraction enriched in SR was obtained from pooled homogenates derived from 3 or 4 hearts, all subjected to the same perfusion protocol. The preparation procedure has been described in detail elsewhere.⁸ The protein content of each fraction was determined by the Lowry method, using BSA as a standard.

Assay of Tissue Thiols
Protein thiols were determined in the microsomal fraction by Ellman's reagent, ie, 5,5'-dithiobis(2-nitrobenzoic acid), as described by Lesnefsky et al.¹² In a few experiments, total thiols were measured in unfractionated homogenates, as described by Riddles et al.¹³

Assay of SR Ca²⁺ Release
SR Ca²⁺-induced Ca²⁺ release was determined after passive loading with 10 mmol/L ⁴⁵CaCl₂, as described previously.^{9 45}Ca release was induced by washing the loaded vesicles with a release buffer containing (in mmol/L): HEPES potassium 20 (pH 6.8), KCl 100, CaCl₂ 1.01, and EGTA 1 (free Ca²⁺ concentration was 18 µmol/L). A rapid filtration system with time resolution on the order of 10 ms was used,^{14 15} and the rate constant of quick Ca²⁺ release (K_r) was calculated over the first 100 ms by exponential fitting.⁹ To confirm that the quick phase of Ca²⁺ release represented SR Ca²⁺ release, we tested the inhibition of Ca²⁺ release by using 2 types of nonrelease buffers: the first buffer contained (in mmol/L): HEPES potassium 20 (pH 6.8), KCl 100, and CaCl₂ 10; the second buffer contained 20 mmol/L HEPES potassium (pH 6.8), 100 mmol/L KCl, 10 mmol/L MgCl₂, and 10 µmol/L ruthenium red.^{14 15 16}

Assay of [³H]-Ryanodine Binding
High-affinity ryanodine binding was assayed as described previously.^{8 9} Unless otherwise specified, free Ca²⁺ concentration was 18 µmol/L. Binding usually was determined using 6 concentrations of the ligand. Incubations were performed in duplicate, and nonspecific binding was determined in the presence of 10 µmol/L unlabeled ryanodine. The difference between the counts of duplicate samples was <10% in all cases.

Chemical and Radionuclides
Ryanodine was purchased from Calbiochem. EGTA was obtained from Sigma Chemical Co. All other reagents were of analytical grade. Free Ca²⁺ concentration was calculated according to Fabiato and Fabiato.¹⁷ Free Ca²⁺ was also measured with the antipyrylazo III technique.¹⁸ 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]-Ryanodine and⁴⁵CaCl₂ were obtained from New England Nuclear-DuPont.

Statistical Analysis
Results are expressed as mean±SEM. Least squares linear regression analysis was used to calculate binding and release parameters, after appropriate linearizing transformations (Scatchard transformation and logarithmic transformation, respectively). Differences between groups were evaluated as follows. One-way ANOVA was used a global test for differences between means. If between-groups variance was significantly (P<0.05) higher than within-groups variance, individual groups were compared with the control group by Dunnett test, as described by Glantz.¹⁹ The computer software (version 4.02i, Italian edition) enclosed in Glantz's¹⁹ book was used for data processing.

	Results

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Hemodynamic Variables and Myocardial Protection
The hemodynamic variables and the extent of tissue injury are summarized in Tables 1 and 2, respectively. Some examples of TTC staining are shown in Figure 2. Ischemic preconditioning caused minor changes in mechanical performance and was effective in protecting the heart during sustained ischemia, because both LDH release and the extent of TTC-negative tissue were decreased significantly versus the control values (by 25% to 40%). We previously⁹ have shown that ventricular myocardium was completely reperfused (>98%) under our experimental conditions so that the results were not affected by the occurrence of the no-reflow phenomenon. Consistent with this observation, the coronary flow measured over the final 120 minutes of reperfusion did not show any significant difference between the experimental groups (it averaged 879±30 mL/g wet wt).

View larger version (121K): [in this window] [in a new window]   Figure 2. Examples of TTC staining. Hearts subjected to 30 minutes of ischemia and 120 minutes of retrograde reperfusion, which had been preceded by: control aerobic perfusion (C); ischemic preconditioning (P); and ischemic preconditioning in the presence of DTT (P+DTT). Measurements of stained areas were actually performed on both sides of each slice.

The inclusion of 1 mmol/L DTT in the perfusion buffer did not produce significant changes in contractile performance, in either control or preconditioned hearts. The infusion of DTT before the preconditioning procedure abolished the protective effect of ischemic preconditioning (LDH release, 125±10 versus 114±7 U/g; TTC-negative tissue, 22.6±2.4% versus 26.0±2.1%). Similar results were obtained when DTT was infused between the 23rd and 28th minutes of perfusion, ie, after the preconditioning procedure and before the sustained ischemia (LDH release, 110±14 U/g; TTC-negative tissue, 21.8±3.9%).

In the absence of ischemic preconditioning, DTT did not affect the susceptibility to ischemia, because neither TTC staining nor LDH release were significantly different from control. Although TTC-evaluated necrosis was larger in control hearts perfused with DTT than in preconditioned hearts perfused with DTT, LDH release was lower in the former group. We conclude that the susceptibility to ischemia was similar in control and preconditioned hearts when DTT was included in the perfusion buffer.

In control conditions, ie, in the absence of ischemic preconditioning, perfusion with DTDP, NEM and thimerosal caused minor changes in contractile performance. In particular, cardiac output decreased by 15% to 20%, whereas aortic pressure, coronary flow, and heart rate were unchanged. The extent of ischemic injury decreased in the DTDP and NEM groups (LDH release, 79±6 and 76±15 U/g; P<0.05 in both cases; TTC-negative tissue, 16.4±2.0% and 9.7±3.8%; P<0.05 and P<0.01), but thimerosal did not produce any significant protection. The beneficial effects of NEM and of ischemic preconditioning were not additive, because TTC-evaluated tissue necrosis was not further reduced when NEM was infused in preconditioned hearts.

Our investigation was focused on cellular necrosis rather than on functional injury. However, our results suggest that the latter was also affected by thiol reagents. After 30 minutes of ischemia, several hearts developed irreversible ventricular fibrillation. The percentage of hearts recovering sinus rhythm was higher in the preconditioning, DTDP, and NEM groups than in the control group (Table 2), although the difference did not reach statistical significance, because the number of hearts was relatively low for ² analysis. In some additional experiments (Table 3), contractile function was evaluated in these groups by switching the perfusion apparatus to the working mode after 30 minutes of ischemia and 60 minutes of retrograde reperfusion (if the hearts resumed sinus rhythm). No heart was able to sustain an afterload of 95 cm. By reducing the afterload until the heart could produce a stable aortic flow of a few milliliters per minute, we observed that cardiac output was significantly higher in the preconditioning, DTDP, and NEM groups than in the control group.

Tissue Thiols
Ischemic preconditioning was associated with thiol oxidation (Table 4). In the microsomal fraction, the concentration of protein thiols averaged 117±2 nmol/mg protein in the control condition and decreased significantly (99±2 nmol/mg protein; P<0.01) after the preconditioning procedure. As expected, perfusion with sulfhydryl reagents produced a significant decrease in protein sulfhydryl concentration. Perfusion with DTT normalized sulfhydryl concentration in the preconditioned hearts, whereas it produced no significant effect in the control group.

After 30 minutes of ischemia and 120 minutes of reperfusion, thiol concentration decreased also in the control group, but lower values were still observed in the DTDP, NEM and preconditioning groups. In the experiments aimed at determining ryanodine binding (see below), total thiols were assayed in the crude homogenate, and decrease after ischemic preconditioning was confirmed (mean values, 90 versus 112 nmol/mg protein).

Ca²⁺-Induced Ca²⁺ Release
After passive loading, the⁴⁵Ca²⁺ content of control heart homogenate was on the order of 9 to 12 nmol/mg protein. Exposure to the release buffer determined the quick release of 3 to 5 nmol/mg, which was completed over 100 to 120 ms. Thereafter the rate of Ca²⁺ release decreased considerably. 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 it represented Ca²⁺ efflux through the SR release channels (release curves were quite similar to those reported previously).⁹

The extent of ⁴⁵Ca²⁺ loading and the ⁴⁵Ca²⁺ pool subjected to quick release was not significantly different between the experimental groups. K_r values are shown in Figure 3. Under control conditions, K_r averaged 25.1 s^-1, corresponding to a t_1/2 of 30 ms. Ischemic preconditioning was associated with an 25% decrease in K_r (P<0.05), in accordance with our previous observations.⁹ Perfusion with DTT had no effect on SR Ca²⁺ release in the absence of ischemic preconditioning, whereas in preconditioned hearts, it increased the rate of Ca²⁺ release, which was no longer significantly different from the control value. Decreased K_r was observed in hearts perfused with DTDP and NEM in the absence of ischemic preconditioning. The decrease was on the order of 25% and 40%, respectively (P<0.05 and P<0.01, respectively).

View larger version (16K): [in this window] [in a new window]   Figure 3. Rate constant of Ca2+-induced Ca2+ release in crude homogenates obtained from hearts perfused under different experimental conditions. In the preconditioning+DTT group, DTT was infused throughout the preconditioning procedure. Bars represent mean±SEM of 4 to 7 hearts in each group. *P<0.05; **P<0.01 vs the control group.

Similar results were obtained with regard to the actual rate of Ca²⁺ release. The absolute rate of Ca²⁺ release is equal to the product of K_r and the amount of intravesicular Ca²⁺; therefore, it decreases over time. The initial rate of Ca²⁺ release observed in the various groups (at an average Ca²⁺ load of 4 nmol/mg homogenate protein) is shown in Table 5.

In some experiments, we determined SR Ca²⁺ release after 30 minutes of ischemia and 120 minutes of retrograde reperfusion. A significant decrease versus the baseline occurred in the control group (K_r=17.1±2.1 s^-1; P<0.05), and slightly lower values were observed in the preconditioning, DTDP, and NEM groups (average K_r, 16.6±3.7, 16.8±3.5, and 16.4±3.5 s^-1, respectively).

Additional experiments were performed to assess the effect of disulfide and sulfhydryl reagents in vitro (Figure 4). The homogenate obtained from control or preconditioned hearts was incubated in the presence of DTT, DPDT, or NEM before inducing Ca²⁺ release. In the tissue obtained from control hearts, DTT had no effect, whereas in preconditioned hearts, DTT increased K_r up to the control values. On the other hand, DTDP and NEM decreased K_r in control heart homogenate, but no further reduction was observed in preconditioned tissue.

View larger version (17K): [in this window] [in a new window]   Figure 4. Rate constant of Ca2+-induced Ca2+ release in crude homogenates obtained from control or preconditioned hearts, which were incubated in vitro in the presence or in the absence of 1 mmol/L DTT, 25 µmol/L DTDP, or 200 µmol/L NEM. DTT and DTDP were used at the same concentrations as in the perfusion experiments. Bars represent mean±SEM of 4 to 7 hearts in each group. *P<0.05 vs the control group.

Our main findings were confirmed in the microsomal preparation (not shown). In particular, we obtained the following results: K_r decreased by 43% in preconditioned versus control hearts (the initial release rate decreased from 148 to 84 nmol/min per milligram microsomal proteins); in preconditioned hearts, incubation with 1 mmol/L DTT increased K_r by 39% (initial release rate, 118 versus 84 nmol/min per milligram); and in control hearts, incubation with 25 µmol/L DTDP decreased K_r by 27% (initial release rate, 108 versus 148 nmol/min per milligram).

[³H]-Ryanodine Binding
In the control hearts, [³H]-ryanodine binding was characterized by K_d value of 2.0 nmol/L and B_max of 386 fmol/mg protein. In accordance with our previous observations,^{8 9} ischemic preconditioning was associated with reduced ryanodine receptor density, without any change in the affinity for ryanodine (Table 6). The Ca²⁺ dependence of ryanodine binding was not significantly different between control and preconditioned hearts (mean EC₅₀ for Ca²⁺, 0.95 µmol/L; Hill coefficient, 1.58).

Perfusion with sulfhydryl or disulfide reagents did not affect the affinity for ryanodine. Administration of DTDP or NEM determined a decrease in ryanodine receptor density, which was similar to that produced by ischemic preconditioning. Thimerosal produced only a slight effect, which did not achieve statistical significance. DTT had no effect in control hearts, whereas in preconditioned hearts, it increased ryanodine binding. Conversely, the infusion of NEM in preconditioned hearts did not cause any further decrease in ryanodine binding.

Sustained ischemia also decreased ryanodine binding, but at the end of this period, ryanodine binding was still lower in the hearts that previously had been subjected to ischemic preconditioning. After 30 minutes of ischemia and 120 minutes of reperfusion, ryanodine binding was still significantly reduced versus the baseline in all groups, and a greater reduction was observed in the preconditioned hearts and in the hearts perfused with sulfhydryl reagents.

In other experiments (not shown), the effect of DTT was assayed in vitro, because ryanodine binding was measured in the homogenate obtained from control or preconditioned hearts, and 1 mmol/L DTT was included in the binding buffer. Two different [³H]-ryanodine concentrations were used, namely 3.6 nmol/L and 12.5 nmol/L. In preconditioned tissue, [³H]-ryanodine binding increased in the presence of DTT (13% and 16% with 3.6 and 12.5 nmol/L [³H]-ryanodine, respectively), whereas no change was observed in control tissue (1% and 0.5%).

	Discussion

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Sulfhydryl Redox State and Ischemic Preconditioning
Our findings show that changes in thiol redox state affect cardiac susceptibility to ischemia and might be involved in the pathogenesis of ischemic preconditioning. Treatment of rat hearts with sulfhydryl oxidizing or blocking agents such as DTDP and NEM increased the resistance to a subsequent 30-minute period of global ischemia. Although NEM might interact with amino groups, such reaction occurs only at high concentrations and is quite slow¹⁰ ; therefore, it should be negligible under our experimental conditions. The involvement of sulfhydryl groups was confirmed by the effectiveness of DTDP (25 µmol/L), a highly specific thiol reagent. No protection was elicited by 10 µmol/L thimerosal, a hydrophilic thiol reagent that is poorly membrane permeable. Because 10 µmol/L thimerosal was as effective as 50 µmol/L DTDP in modulating sarcolemmal Ca²⁺ channels,¹¹ our results suggest that the protective effect is associated with the oxidation of intracellular thiols.

The disulfide-reducing agent DTT abolished the protective effect of ischemic preconditioning. The presence of DTT during the preconditioning procedure was not required to produce this effect, because similar results were obtained if DTT was added immediately before the sustained ischemia. We conclude that the pathogenetic mechanism of ischemic preconditioning involved the oxidation of intracellular sulfhydryl groups and that DTT removed the protection by reducing such groups. Although DTT may act as a scavenger of free radical species, the effects that we have observed when DTT was infused after the preconditioning procedure cannot be explained on this basis, because the bulk of free radical production occurs at an earlier stage.^{20 21 22}

Under control conditions, the intracellular environment is highly reductive, and intracellular thiols are in the reduced state,²³ which likely is the reason why DTT did not modify ischemic injury in control hearts. Ischemia reperfusion is known to produce an oxidative stress, largely due to the generation of oxygen free radicals.¹ As a consequence, oxidation of intracellular thiols occurs, as observed in the present investigation and in other experimental models of ischemia reperfusion.^{24 25}

Although oxidative stress often has been associated with tissue injury, other studies have suggested that oxidative reactions may play a crucial role in ischemic preconditioning. In rabbit heart, a mild oxidative stress was cardioprotective,^{2 3} and in rat heart, the thiol compound N-acetylcysteine abolished the protective effect of ischemic preconditioning,⁵ whereas controversial results have been obtained with superoxide dismutase^{4 6} and N-2-mercaptopropionylglycine.^{2 4 7}

Because many proteins (eg, ion channels, ATPases, contractile proteins, and proteases) contain sulfhydryl groups that are critical to their function,^{11 23 26 27 28} the response to sulfhydryl reagents is likely to be a complex process. We hypothesize that oxidation of some highly reactive thiol groups increases the resistance to ischemia, whereas oxidation of additional less-reactive thiols may be detrimental. This may be the reason why the protective effects of NEM and ischemic preconditioning were not additive. Consistent with this hypothesis, in preliminary experiments (unpublished observations), we have noticed that high concentrations of DTDP (>50 µmol/L) caused a remarkable impairment in contractile function and increased ischemic injury.

Sulfhydryl Redox State and SR Ca²⁺ Release
It is not easy to identify the molecular effector(s) responsible for the observed protection. The latter does not appear to be accounted for by hemodynamic changes. In fact, only a slight decrease in cardiac output was produced, and in a previous work,⁹ we showed that much greater changes in contractile performance, obtained by reducing the preload, were not associated with any protection.

In the present study, we have examined the effects of sulfhydryl and disulfide reagents on the SR Ca²⁺ channel, also known as ryanodine receptor. The rationale is our recent observation that the Ca²⁺ release capability of the SR was reduced after brief ischemia and reperfusion.⁸ Such a process might play a role in the pathogenesis of ischemic preconditioning by delaying the increase in cytosolic Ca²⁺ concentration produced by a subsequent ischemic insult. In accordance with this hypothesis, in a model of ischemic preconditioning, the time course of the SR channel changes was closely parallel to the time course of myocardial protection.⁹

Perfusion with DTDP or NEM decreased ryanodine binding and the rate constant of SR Ca²⁺ release. The effect was similar to that produced by ischemic preconditioning, and it was not additive with the latter. On the other hand, DTT antagonized the effect of ischemic preconditioning on the SR Ca²⁺ channel. DTDP, NEM, and DTT produced similar actions when added in vitro to control or preconditioned myocardium. Therefore, we suggest that thiol groups in the SR channel may be the target, or at least one of the targets, of the oxidative process involved in the pathogenesis of ischemic preconditioning.

Our study was focused on the effects occurring before the sustained ischemia, because the early phase of ischemia appears to be critical for the development of cytosolic Ca²⁺ overload. However, the observed changes persisted at later stages. As expected, sustained ischemia produced thiol oxidation and SR channel changes also in the control group, but, at the end of the experimental protocol, ryanodine binding and SR Ca²⁺ release were still slightly lower in the preconditioning, DTDP, and NEM groups than in the control group. Therefore, ischemic preconditioning and sulfhydryl reagents produced a better recovery of contractile performance in spite of a decreased rate of SR Ca²⁺ release, which is likely to reflect a reduced injury to other molecular structures (eg, contractile proteins, ion pumps, cytoskeletal elements, and membrane components).

Most of our experiments were performed in the crude homogenate, because the preparation procedure used to purify the SR may select vesicles that are not representative of the entire SR, which might be misleading in the study of ischemic injury.^{8 29 30} SR Ca²⁺ release can be measured reliably in crude preparations, because no other structure can support a Ca²⁺ release that has t_1/2 on the order of a few milliseconds and is inhibited by millimolar Ca²⁺ or Mg²⁺ and ruthenium red.^{9 16 31 32} In any case, we repeated the assay in a microsomal preparation, with similar results.

In our experiments, sulfhydryl oxidation or blockade inhibited Ca²⁺ release. This observation may appear in contrast with previous findings, suggesting that thiol oxidation induced Ca²⁺ release.^{33 34 35 36} In fact, several investigations have reported biphasic or multiphasic responses to sulfhydryl reagents.³⁶ Although the immediate effect was channel opening, prolonged exposure to heavy metals, hydrogen peroxide, thimerosal, or NEM led to channel inactivation.^{37 38 39 40} Consistently, incubation with thimerosal, NEM, or DTDP inhibited ryanodine binding.^{38 40 41} In control myocardium, DTT had no major effect on ryanodine binding and Ca²⁺ release, which is in agreement with previous reports.^{42 43 44} At variance with our results, in skeletal muscle SR, 1 mmol/L DTT reduced the affinity for ryanodine without major changes in receptor density.⁴⁵ Inhibition of the sheep cardiac channel by DTT was observed in bilayer experiments, but the effect was significant only at concentrations 5 mmol/L.⁴⁶

A reasonable conclusion is that the ryanodine receptor contains different classes of sulfhydryl groups, possibly located in different domains, with different functional roles in modulating the gating of the channel and its interaction with contiguous proteins.^{36 40} Ischemia reperfusion appears to be associated with the oxidation of a specific class of thiols, and the functional consequence is channel inhibition and reduced ryanodine binding.

In summary, our results suggest that the protective effect of ischemic preconditioning is associated with reactions involving sulfhydryl oxidation. A possible target of such reactions is the SR Ca²⁺ channel/ryanodine receptor. Although the ryanodine receptor might be attacked directly by oxygen free radicals, the activation of indirect mechanisms cannot be excluded. Many mediators have been implicated in the pathogenesis of ischemic preconditioning, eg, adenosine, protein kinase C, and nitric oxide,^{47 48 49 50} and it is still uncertain whether some of these pathways may be activated by an oxidative stress or produce oxidative changes in the ryanodine receptor.

	Acknowledgments

 
This study was supported by MURST and CNR. G.Y. is supported by a grant from Knoll SpA. We thank Dr Giuseppe Rossi for his help in statistical analysis.

Received January 5, 1998; accepted August 7, 1998.

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