INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BRIEF PERIODS OF MYOCARDIAL ischemia and reperfusion have been shown to render cardioprotection against a prolonged period of ischemia-reperfusion; this phenomenon has been termed as ischemic preconditioning (IP) (19). It has been previously reported that IP has beneficial effects with respect to myocardial infarct size, arrhythmias, and cellular injury due to ischemia-reperfusion (14, 19, 21, 26). Several endogenous agonists such as adenosine (14), norepinephrine (27), and bradykinin (7), as well as free radicals (5), have been implicated in mediating the beneficial effects of IP. Protein kinase C (PKC), a major signal transduction mechanism, has been shown to play an important role in protecting the ischemic-preconditioned heart (15). Other targets such as tyrosine kinase (6), ATP-sensitive K (K_ATP) channels (23), and phosphorylation by Ca²⁺/calmodulin-dependent protein kinase II (CaMK II) (20) have also been considered as mechanisms underlying the cardioprotective effects of IP. In a recent study, we have shown that IP exerted beneficial effects in hearts subjected to Ca²⁺ paradox (11), a phenomenon where a brief period of Ca²⁺ depletion followed by Ca²⁺ repletion results in marked myocardial cell damage and contractile dysfunction (31). Because both Ca²⁺ paradox and ischemia-reperfusion injury are known to be associated with the occurrence of intracellular Ca²⁺ overload (3, 24), it is possible that IP may elicit beneficial effects on the myocardium by preventing the development of intracellular Ca²⁺ overload. Whereas a great deal of work has been carried out to understand the mechanisms of the cardioprotective effects of IP with respect to alterations due to ischemia-reperfusion, the mechanisms underlying the IP-mediated cardioprotection of changes due to Ca²⁺ paradox are not known. Thus a study concerning the nature of IP effects on the Ca²⁺ paradox-induced alterations in the heart can be seen to provide further information in the area of cardioprotection.

Under physiological conditions, a small amount of Ca²⁺ entering through the L-type Ca²⁺ channels in the sarcolemmal (SL) membrane induces a release of Ca²⁺ from the sarcoplasmic reticulum (SR) stores and thus results in cardiac contraction (8). Cardiac relaxation occurs when the major part of the cytosolic Ca²⁺ is pumped back into the SR via the Ca²⁺-pump ATPase and the rest is extruded by the SL Na⁺/Ca²⁺ exchanger and the SL Ca²⁺-pump ATPase. Because of its remarkable ability to regulate the intracellular concentration of Ca²⁺, SR is known to play a central role in the contraction-relaxation cycle of the cardiac muscle (8). Earlier studies have demonstrated that IP may exert beneficial effects on the ischemia-reperfusion induced alterations in the SR Ca²⁺-uptake and Ca²⁺-release activities in the heart (2, 20, 32) and these may partly explain the cardioprotective effects of IP. Although an impairment in the SR function has been reported to occur in the Ca²⁺ paradox hearts (2), the effects of IP on Ca²⁺ paradox-induced changes in SR have not been investigated. The present study was therefore undertaken to examine the effects of IP on cardiac SR function by employing an isolated rat heart model of Ca²⁺ paradox. Because adenosine, PKC, and CaMK II (14, 15, 20) are considered to be important in mediating the effects of IP on ischemia-reperfusion-induced changes in the heart, we examined the role of these mediators in hearts subjected to Ca²⁺ paradox.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Heart perfusion and experimental protocol. Male Sprague-Dawley rats (300-350 g) were anesthetized by an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg) mixture. The hearts were rapidly excised and perfused by the Langendorff procedure. The perfusion medium containing (in mmol/l) 120 NaCl, 25 NaHCO₃, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.25 CaCl_2, and 11 glucose was gassed with 95% O₂-5% CO₂ and maintained at pH 7.4 at 37°C. The hearts were paced at 300 beats/min by an electrical stimulator (Phipp and Bird, Richmond, VA), and the coronary flow rate was maintained at 10 ml/min. For measuring the left ventricular pressure, the left atrium was removed and a latex balloon connected to a pressure transducer was inserted through the mitral valve into the left ventricle. The balloon was filled with the perfusion medium and adjusted to the left ventricular end-diastolic pressure (LVEDP) of 5-7 mmHg. Values for the left ventricular developed pressure (LVDP), rate of pressure development (+dP/dt), and rate of pressure decay (dP/dt) were obtained through the program AcqKnowledge for Windows 3.0 (Biopac System, Goleta, CA). The balloon technique employed here allowed stable recording of the hemodynamic parameters over a period of more than 60 min. All hearts were stabilized for 30 min by perfusion with the oxygenated medium. The experimental protocols for inducing IP and Ca²⁺ paradox as well as for drug treatments are shown in Fig. 1. For the Ca²⁺ paradox group, the hearts were perfused for 18 min with oxygenated medium followed by 3 or 5 min of Ca²⁺-free perfusion and 30 min of repletion. The durations of Ca²⁺ depletion and repletion were adapted from our previous study (11). For the preconditioning group, the hearts were subjected to three cycles of 3-min global ischemia and 3-min reperfusion, followed by 3 or 5 min of Ca²⁺-free perfusion and 30 min of repletion. For the control group, the hearts were perfused for a comparable period with the oxygenated medium. To test whether the inhibitors of adenosine receptors, CaMK II, or PKC attenuate the protective effect of IP against Ca²⁺ paradox, we used 8-(p-sulfophenyl)-theophylline (8-SPT) (Research Biochemicals International, Natick, MA), KN-93 (Sigma Chemical, St. Louis, MO), or chelerythrine chloride (Sigma Chemical), respectively. The vehicle (perfusion medium) or each inhibitor was delivered by an infusion pump into the perfusion stream directly above the aortic cannula at 1 ml/min for 10 min before starting the preconditioning protocol as well as during the three cycles of intermittent reflow. The final concentrations of 8-SPT, KN-93, and chelerythrine were 10 µM, 1 µM, and 5 µM, respectively; these concentrations were selected on the basis of previous studies (11, 12, 16) showing inhibitory constant (K_i) value for 8-SPT for adenosine receptors (2.63 µM), KN-93 for CaMK II (0.37 µM), and chelerythrine for PKC (0.67 µM).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Experimental protocols. Protocol 1 indicates points at which changes in heart were examined before initiating Ca2+ depletion, at 5 min of Ca2+ depletion, at 10 min of Ca2+ repletion after 5 min of Ca2+ depletion, and at 30 min of Ca2+ repletion after 5 min of Ca2+ depletion. Protocol 2 indicates conditions under which changes in hearts subjected to 5-min Ca2+-free perfusion followed by 30 min of Ca2+ repletion were examined. Drug treatments were carried out 10 min before starting as well as during the three cycles of preconditioning. CP, Ca2+ paradox; IP, ischemic preconditioning; 8-SPT, adenosine antagonist 8-(p-sulfophenyl)-theophylline (10 µM); KN-93, Ca2+/calmodulin-dependent protein kinase (CaMK II) inhibitor (1 µM); CHT, PKC inhibitor chelerythrine chloride (5 µM). All hearts in this protocol were examined at 30 min of Ca2+ repletion.

Preparation of SR vesicles. The SR preparation was isolated by the method of Osada et al. (20) with slight modification. Briefly, the LV tissue was pulverized and homogenized twice for 20 s each at a one-half maximal setting using a Polytron homogenizer (Brinkmann, Westbury, NY). The homogenization buffer contained (in mmol/l) 10 NaHCO₃, 5 NaN₃, 15 Tris · HCl, pH 6.8, and protease inhibitors (in µmol/l): 1 leupeptin, 1 pepstatin, and 100 phenylmethylsulfonyl fluoride. The homogenate was then centrifuged for 20 min at 9,500 rpm (Beckman, JA 20.0), and the supernatant was further centrifuged for 45 min at 19,000 rpm (Beckman, JA 20.0). The pellet obtained was suspended in a buffer containing 0.6 M KCl and 20 mM Tris · HCl, pH 6.8, and recentrifuged at the same speed and duration as indicated in the previous step. The resulting pellet obtained was suspended in a mixture of 250 mM sucrose and 10 mM histidine, pH 7.0. All steps were performed at 0-4°C, and the resulting SR preparation was used for various assays. The purity of the SR preparations was determined by measuring the activities of marker enzymes such as ouabain-sensitive Na⁺-K⁺-ATPase, cytochrome-c oxidase, glucose-6-phosphatase, and rotenone-insensitive NADPH cytochrome-c reductase according to methods described earlier (1). The SR preparations from all groups were found to contain minimal (3-5%) but equal cross contamination by other subcellular organelles.

Measurement of Ca²⁺-uptake activity. Ca²⁺-uptake activity of SR membranes was determined by the procedure described earlier (20) with slight modifications. The standard reaction mixture (total volume 250 µl) contained (in mmol/l) 50 Tris-maleate (pH 6.8), 5 NaN₃, 5 ATP, 5 MgCl₂, 120 KCl, 5 K-oxalate, 0.1 EGTA, 0.1 ⁴⁵CaCl₂ (12,000 counts · min¹ · nmol¹), and 0.025 ruthenium red. The initial free Ca²⁺ in this medium determined by the program of Fabiato (9) was 8.2 µmol/l. Ruthenium red was added to inhibit Ca²⁺-release channel activity under these conditions. The reaction was initiated by the addition of SR membranes to the Ca²⁺-uptake reaction mixture and terminated after 1 min by filtering a 200-µl aliquot of the reaction mixture. The filters were washed, dried, and counted in a beta liquid scintillation counter.

Determination of Ca²⁺-stimulated ATPase activities. The status of the Ca²⁺ pump in the SR membranes was determined by measuring the Ca²⁺-stimulated ATPase activities according to the method used previously (10). Total (Mg²⁺ and Ca²⁺) and basal (Mg²⁺) ATPase activities were determined in the presence or absence of Ca²⁺ in a reaction medium containing (in mmol/l) 100 KCl, 20 Tris · HCl, 5 MgCl₂, and 5 NaN₃, respectively. The reaction was started by the addition of 5 mM Tris · ATP in the presence of 0.05 mg/ml of SR protein and was terminated with 12% cold TCA. Inorganic phosphate liberated during the reaction was estimated in the protein-free filtrate by a spectrophotometric method (10). The Ca²⁺-stimulated, Mg²⁺-dependent ATPase (Ca²⁺-pump ATPase) activity was calculated as the difference between the total and basal ATPase activities.

Measurement of ryanodine-sensitive, Ca²⁺-induced Ca²⁺ release. The Ca²⁺-release activity of SR vesicles was measured by a procedure described previously (20). In brief, the SR fraction (62 µg protein) was suspended in a total volume of 625 µl of loading buffer containing (in mmol/l) 100 KCl, 5 MgCl₂, 5 K-oxalate, 5 NaN₃, and 20 Tris · HCl (pH 6.8). After incubation with 10 µM ⁴⁵CaCl₂ (20 mCi/l) and 5 mM ATP for 45 min at room temperature, Ca²⁺-induced Ca²⁺ release was carried out by adding 1 mM EGTA plus 1 mM CaCl₂ to the reaction mixture. The reaction was terminated at 15 s by filtering a 100-µl aliquot of the reaction mixture. The filters were washed, dried, and counted in a beta scintillation counter. The Ca²⁺-induced Ca²⁺ release was completely prevented (90-97%) by the treatment of SR with 20 µM ryanodine.

Western blot analysis. The protein contents of Ca²⁺-release channel [ryanodine receptor (RyR)], Ca²⁺ pump [sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2)], and phospholamban (PLB) in the SR membrane were determined by Western immunoblotting techniques. For immunoassay of RyR, SERCA2a, and PLB, SR (20 µg protein/lane) samples were subjected to SDS-PAGE in 6%, 10%, and 15% gels, respectively. The quantity of protein loaded was within the linear range (14). The protein bands from these gels were then transferred electrophoretically to nitrocellulose membrane (for RyR) or polyvinylidene difluoride membrane (for SERCA2a and PLB). The membranes were used for incubation with anti-RyR receptor (1:1400), anti-SERCA2 (1:1400), or anti-PLB (1:3500) antibodies. A peroxidase-linked, anti-mouse IgG was used for SERCA2a or PLB as the secondary antibody (1:5000), whereas anti-mouse IgG was used for Ca²⁺-release channel as the secondary antibody (1:2500) and then incubated with streptavidin-conjugated horseradish peroxidase solution (1:5000). Protein bands reactive with antibodies were visualized using the ECL detection system from Amersham (Buckinghamshire, UK). The intensity of each band was scanned by Imaging Densitometer with the aid of Molecular Analyst Software version 1.3 (Bio-Rad, Hercules, CA). After the detection of SR proteins by Western blot analysis, the membranes were stained with Coomassie Blue, and each lane was scanned to normalize the well-to-well variability in protein loading. The total intensity of the bands in each lane was calculated, but no significant changes in total intensity were observed among different lanes.

Statistical analysis. Data are expressed as means ± SE. The statistical difference among different groups was determined by factorial ANOVA (Statview 4.02, Abacus Concepts). All groups were analyzed with post hoc testing by the Scheffé's procedure. P < 0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Left ventricular function and Ca²⁺ uptake. Changes in the left ventricular function and SR Ca²⁺ uptake due to Ca²⁺ paradox with or without IP are shown in Fig. 2. During predepletion phase, IP did not affect the LVDP, LVEDP, and SR Ca²⁺ uptake; there was no change in LVEDP in hearts that did not undergo IP. Ca²⁺-free perfusion for 5 min (depletion phase) produced a marked depression of LVDP and SR Ca²⁺-uptake activity in hearts with or without IP; however, the extent of changes in both conditions was similar. A marked increase in LVEDP and a depression in SR Ca²⁺-uptake activity were seen on perfusing the Ca²⁺-depleted hearts with normal medium for 10 or 30 min without any recovery in LVDP. At the 10-min repletion phase, IP significantly improved the recovery of LVDP and decreased the LVEDP of the Ca²⁺ paradox hearts; however, there was no significant difference in SR Ca²⁺-uptake activities between groups with or without IP. At the 30-min repletion phase, IP significantly improved LVDP as well as SR Ca²⁺ uptake and markedly decreased the LVEDP of the Ca²⁺ paradox hearts (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Left ventricular (LV) function and sarcoplasmic reticulum (SR) Ca2+-uptake activities in CP hearts with or without IP. Top: percentage recovery of LV developed pressure (LVDP). Values for LVDP are expressed as % of basal values (82.6 ± 3.42 mmHg). Middle: LV end-diastolic pressure (LVEDP). Bottom: SR Ca2+-uptake activities. Hearts were examined before Ca2+ depletion, at 5 min after Ca2+ depletion, at 10 min after Ca2+ repletion after 5 min of Ca2+ depletion, and at 30 min after Ca2+ repletion after 5 min of Ca2+ depletion. Each value is a mean ± SE of 6 hearts in each group. *P < 0.05 vs. CP without IP.

SR protein content in IP and Ca²⁺ paradox. The SR content of RyR, SERCA2a, and PLB in control and Ca²⁺ paradox hearts with and without IP were determined by using the Western blot analysis and values are expressed as percentage of control (Fig. 3, B and C). The density of each band from the control heart was considered to be 100%, and the other groups CP, IP, and IP plus CP were compared with this control value. RyR, SERCA2a, and PLB protein levels did not change due to IP when compared with control value. On the other hand, RyR, SERCA2a, and PLB protein levels in the Ca²⁺ paradox hearts were decreased by 94.6%, 96.8%, and 44.7% of the control value, respectively. IP significantly prevented the depression in protein content of RyR and SERCA2a due to Ca²⁺ paradox. Although PLB content in the Ca²⁺ paradox hearts was 17.3% higher on treatment with IP, this change was not significant.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of ischemic preconditioning and CP on SR protein content in rat heart. Western blot autoradiograms and analysis of ryanodine receptor (A), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) (B), and phospholamban (PLB) (C) of SR protein content in control (C), IP, CP, and IP + CP. Each value is a mean ± SE of 4 separate experiments. *P < 0.05 vs. C and  vs. CP.

Modification of changes in cardiac function. The effects of IP on cardiac performance in Ca²⁺ paradox induced by 5-min Ca²⁺ depletion and 30-min Ca²⁺ repletion with or without some inhibitor treatments are shown in Table 1. Hearts subjected to Ca²⁺ paradox under control conditions showed significant depressions in LVDP, +dP/dt, and dP/dt and a marked increase in LVEDP in comparison with the control values. 8-SPT, KN-93, or chelerythrine treatment did not affect the LV function of the Ca²⁺ paradox hearts significantly. On the other hand, IP improved the recovery of LVDP, +dP/dt, as well as dP/dt significantly and markedly reduced the increase in LVEDP in the Ca²⁺ paradox hearts. Treatments with 8-SPT, KN-93, or chelerythrine abolished these beneficial effects of IP.

Modification of changes in SR Ca²⁺ uptake and Ca²⁺ pump ATPase activities. Oxalate-supported SR Ca²⁺-uptake activities were determined in SR preparations in the control and Ca²⁺ paradox hearts (5-min Ca²⁺ depletion and 30-min Ca²⁺ repletion) with or without some inhibitor treatments and the results are shown in Fig. 4. The Ca²⁺-uptake activity was significantly reduced in Ca²⁺ paradox hearts in comparison with control hearts. IP prevented the decrease in SR Ca²⁺ uptake due to Ca²⁺ paradox significantly; this improvement was attenuated with 8-SPT, KN-93, or chelerythrine treatment. To show whether the observed changes in SR Ca²⁺ uptake were due to alterations in the SR Ca²⁺-pump, Ca²⁺-stimulated ATPase activities were determined. The results in Fig. 5 reveal that the SR Ca²⁺-pump ATPase activity in the Ca²⁺ paradox hearts was significantly reduced in comparison to control hearts. IP improved Ca²⁺-pump ATPase activity of the Ca²⁺ paradox hearts; this beneficial effect of IP was lost on treatment with 8-SPT, KN-93, or chelerythrine.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   SR Ca2+-uptake activities in control and CP hearts with and without IP. SR Ca2+ uptake was measured at 30 min of Ca2+ repletion as described in Fig. 1. Each value is a mean ± SE of 5 different preparations in each group. *P < 0.05 vs. control, #P < 0.05 vs. CP + IP.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   SR Ca2+-stimulated ATPase activities in control and CP hearts with and without IP. SR Ca2+-ATPase activities were determined at 30 min of Ca2+ repletion as described in Fig. 1. Each value is a mean ± SE of 5 different preparations in each group. *P < 0.05 vs. control, #P < 0.05 vs. CP with IP.

Modification of changes in SR Ca²⁺-release activity. The Ca²⁺-induced Ca²⁺-release activity was determined in SR preparations from the control and Ca²⁺ paradox hearts (5-min Ca²⁺ depletion and 30-min Ca²⁺ repletion) with or without some inhibitor treatments and results are shown in Fig. 6. Hearts subjected to Ca²⁺ paradox exhibited a significant depression of the SR Ca²⁺-release activity in comparison with control; IP attenuated the reduction in SR Ca²⁺ release in Ca²⁺ paradox hearts. This improvement due to IP was lost when the hearts were treated with 8-SPT, KN-93, or chelerythrine.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   SR Ca2+-release activities in control and CP hearts with and without IP. SR Ca2+-release activities were determined at 30 min of Ca2+ repletion as described in Fig. 1. Each value is a mean ± SE of 5 different preparations in each group. *P < 0.05 vs. control, #P < 0.05 vs. CP with IP.

Modification of changes due to mild Ca²⁺ paradox. Because perfusion for 5 min with Ca²⁺-free medium and reperfusion for 30 min with Ca²⁺-containing medium produced severe changes in the heart, some experiments were carried out to examine the effects of IP on changes in SR function due to a mild Ca²⁺ paradox injury induced by 3-min Ca²⁺ depletion and 30-min Ca²⁺ repletion (Table 2). The mild Ca²⁺ paradox was associated with a significant decrease in LVDP, a marked increase in LVEDP, as well as significant decreases in SR Ca²⁺-uptake, Ca²⁺-pump ATPase, and Ca²⁺-release activities in comparison to the control values. IP prevented the reduction in LVDP and the increase in LVEDP significantly in addition to preventing changes in SR Ca²⁺ uptake, Ca²⁺-pump ATPase, and Ca²⁺-release activities in the Ca²⁺ paradox hearts.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study we have provided evidence for a significant protection by IP against SR dysfunction due to Ca²⁺ paradox induced by perfusing the heart for 5 min with a Ca²⁺-free medium followed by reperfusion with Ca²⁺-containing medium for 30 min. The beneficial effects of IP were also evident under a mild Ca²⁺ paradox condition where the hearts were subjected to 3-min Ca²⁺-free perfusion and 30-min reperfusion with a normal medium. Although several mechanisms have been put forward to explain the beneficial effects of IP against the ischemia-reperfusion-induced changes in the heart, attenuation of cytosolic Ca²⁺ overload appears to be a major factor (24). In fact, the intracellular Ca²⁺ overload is considered to be the hallmark of Ca²⁺ paradox injury (3) and is believed to be due to an alteration in the ability of the myocardium to regulate the level of intracellular Ca²⁺ (17). Such an alteration in the Ca²⁺ paradox heart has been shown to be in part due to a defect in the SR function (2), which is considered to be intimately involved in intracellular Ca²⁺ homeostasis and cardiac excitation-contraction coupling (8). Changes in the SR Ca²⁺ uptake and Ca²⁺-release activities have also been shown to occur during other pathological conditions, resulting in the occurrence of intracellular Ca²⁺ overload and subsequent cardiac dysfunction (1, 10, 22). Thus the observed alterations in SR Ca²⁺-pump and Ca²⁺-release activities as well as SR Ca²⁺-pump and Ca²⁺-release channel proteins in hearts subjected to Ca²⁺ paradox are consistent with impaired cardiac function in this experimental condition. In addition, we have demonstrated that the recovery of cardiac functions in Ca²⁺ paradox hearts by IP was associated with the ability of IP to partially prevent changes in SR Ca²⁺-uptake, Ca²⁺-pump ATPase, and Ca²⁺-release activities as well as SR Ca²⁺-pump and Ca²⁺-release channel proteins. These observations allow us to speculate that IP rendered cardioprotection by reducing the intracellular Ca²⁺ overload via an improved Ca²⁺-handling ability of the SR in Ca²⁺ paradox hearts.

Since Murry et al. (19) observed the phenomenon of IP, many factors have been proposed to explain the beneficial effects of IP (29). In particular, the formation of adenosine and subsequent activation of adenosine receptors in the heart have been suggested to be an important mediator of IP (14). However, the role of adenosine in mediating IP in rat hearts has been questioned because pretreatment with an adenosine receptor blocker failed to prevent the ischemia-reperfusion damage (13). On the other hand, Ashraf et al. (4) reported that adenosine may be involved in Ca²⁺ preconditioning for conferring significant protection against the Ca²⁺ paradox injury in the rat heart (4). Moreover, adenosine acting via A₁ receptors has also been shown to render significant cardioprotection against rat hearts subjected to Ca²⁺ paradox (25). The results in the present study have shown that the beneficial effects of IP on SR function were abolished by pretreatment of the heart with 8-SPT, an adenosine receptor antagonist. Thus it is likely that the formation of adenosine may be one of the mechanisms by which IP protects the heart against Ca²⁺ paradox injury. Although on the basis of the effects of 8-SPT, which is known to act on the adenosine A₁ receptors (14, 25), it appears that A₁ receptors may be involved in the IP-induced cardioprotection against Ca²⁺ paradox, the involvement of other types of adenosine receptors cannot be ruled out at this time.

In view of the fact that the activation of PKC plays a central role in IP (15, 29), Miyawaki et al. (16) demonstrated that a transient increase in intracellular Ca²⁺ concentration during IP represents the trigger for the activation of PKC and subsequent cardioprotection against the ischemic-reperfusion injury. Moreover, Movsesian et al. (18) reported that PKC may regulate Ca²⁺ uptake by cardiac SR via phosphorylation of PLB. In this study, we show that the treatment of hearts with a PKC inhibitor, chelerythrine, attenuated the beneficial effects of IP on SR function in Ca²⁺ paradox hearts. Our results regarding IP are consistent with a recent study demonstrating a role for PKC in the protection against Ca²⁺-paradox injury by Ca²⁺ preconditioning in the rat heart (28). Because Osada et al. (20) reported that IP prevented alterations in the SR CaMK II-mediated phosphorylation of SR Ca²⁺-cycling proteins as well as SR and cardiac functions in the ischemia-reperfused hearts, the SR CaMK II can also be seen to represent an important mechanism underlying the beneficial effects of IP against the ischemic-reperfusion injury in the isolated rat heart. In the present study, we also show that the treatment of hearts with a CaMK II inhibitor, KN-93, attenuated the beneficial effects of IP on the SR function in the Ca²⁺ paradox hearts. Thus it appears that the activation of both PKC and CaMK II in addition to adenosine receptors may represent the mechanisms by which IP protects the hearts against the Ca²⁺ paradox injury. Extensive research is required to establish whether adenosine receptors, PKC, and CaMK II represent three parallel pathways or act sequentially leading to protection of the Ca²⁺ paradox heart by IP.

Although the observed reduction in SR Ca²⁺ uptake and Ca²⁺-release activities due to Ca²⁺ paradox and the protection of SR function due to IP can be explained on the basis of corresponding changes in SERCA2a and RyR protein contents of the SR membrane, the depressed levels of PLB protein in the Ca²⁺ paradox heart can be seen to relieve the inhibitory effect of PLB on the SR Ca²⁺ pump and thus increase the Ca²⁺-uptake activity. However, this effect due to the reduction of PLB content may not be sufficient to compensate for the depression in the SR Ca²⁺-uptake activity due to the loss of SERCA2a, because the reduction in the SERCA2a level was considerably greater than that in the PLB content in the Ca²⁺ paradox heart. The loss of SR proteins in hearts subjected to Ca²⁺ paradox may be due to the activation of proteases because intracellular Ca²⁺ overload under pathological conditions such as ischemia-reperfusion has been reported to result in SR protein degradation due to protease activation (30). It should be pointed out that the loss of proteins may not be a generalized phenomenon in the paradoxic hearts because the loss of PLB in the SR membrane due to Ca²⁺ paradox with or without IP was considerably smaller than that of RyR and SERCA2a proteins. Furthermore, other proteins such as inhibitory guanine binding proteins and ₂-adrenergic receptor proteins were unaltered under similar conditions of Ca²⁺ paradox (unpublished observations). In view of the role of K_ATP channels in the protective effect of IP against the ischemia-reperfusion injury (23), it is possible that K_ATP channels may also mediate the beneficial effects of IP against Ca²⁺ paradox with respect to changes in cardiac contractile activity. However, such a mechanism cannot be invoked to explain the beneficial effects of IP against the Ca²⁺ paradox-induced or ischemic-reperfusion-induced (20) changes in SR function because the relationship between K_ATP channels and the SR Ca²⁺ transport system is not understood at the present time.