INTRODUCTION

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BRIEF EPISODES OF myocardial ischemia and reperfusion have been shown to protect against the adverse effects of subsequent sustained ischemia-reperfusion, a phenomenon termed as "ischemic preconditioning" (IP) (17). In this regard different investigators have reported the protective effects of IP with respect to limiting the infarct size or preventing arrhythmias due to ischemia-reperfusion (17, 19, 21). Several mechanisms have been proposed to explain the beneficial effects of IP; these include alterations in antioxidant defense (2), stimulation of adenosine receptors (15), activation of protein kinase C (25), induction of heat shock proteins (13), and changes in intracellular Ca²⁺ fluxes (22). In fact, IP has been reported to attenuate the detrimental rise in the intracellular concentration of free Ca²⁺ caused by sustained ischemia-reperfusion (22). In view of the central role played by the sarcoplasmic reticulum (SR) in the regulation of intracellular concentration of Ca²⁺ and observed changes in cardiac SR Ca²⁺-uptake and Ca²⁺-release activities due to ischemia-reperfusion (4, 10, 20, 29), there are a few reports indicating protection of SR function in the preconditioned hearts (24, 30). However, these studies do not provide any information as to whether the protective effect of IP on SR function in the ischemic- reperfused heart is associated with prevention of changes in SR Ca²⁺-release channel and Ca²⁺-pump proteins. Furthermore, despite the role of phosphorylation by Ca²⁺/calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA) in regulating SR function (3, 12, 27), previous studies have not examined the effect of IP on changes in SR phosphorylation by these protein kinases. In this study therefore we have investigated the effect of IP on ischemia-reperfusion-induced alterations in cardiac SR Ca²⁺-uptake and Ca²⁺-release activities as well as in Ca²⁺-release channel and Ca²⁺-pump protein contents in the SR membrane. In addition, the status of SR phosphorylation by endogenous CaMK and exogenous PKA in ischemic-reperfused hearts with or without IP was assessed.

	METHODS

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Heart perfusion and experimental protocol. Male Sprague-Dawley rats (300-350 g) were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg) mixture. The hearts were rapidly excised and perfused by the Langendorff technique. The perfusion medium containing (in mM) 120 NaCl, 25 NaHCO₃, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.25 CaCl₂, 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 (Phipps and Bird, Richmond, VA), and the coronary flow rate was maintained at 10 ml/min. For measurement of the left ventricular pressures, 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 of 9-10 mmHg. Values for the left ventricular developed pressure (LVDP), peak rate of contraction (+dP/dt), and peak rate of relaxation (dP/dt) were obtained through the program AcqKnowledge for Windows 3.0 (Biopac Systems, Goleta, CA). These hearts were perfused with oxygenated medium for 30 min for stabilization before being used in the experiments carried out in this study. For the control group, hearts were further perfused for 30 min with oxygenated medium, followed by 30 min of global ischemia and 30 min of reperfusion. For the preconditioning group, hearts were subjected to three cycles of 5-min ischemia and 5-min reperfusion, followed by 30 min of global ischemia and 30 min of reperfusion. Global ischemia was induced by stopping the coronary flow with a clamp, whereas reperfusion was started by releasing the clamp; the hearts were kept in a humidified chamber at 37°C throughout the experiment. This experimental protocol is shown in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Experimental protocol for study. Hearts perfused by Langendorff technique were stabilized for 30 min before experimental protocol was started for control and preconditioning groups. Preconditioning was induced by 3 cycles of 5-min ischemia and 5-min reperfusion, whereas control hearts were perfused for 30 min with oxygenated medium. Global ischemia was induced by clamping coronary flow for 30 min, whereas reperfusion of ischemic hearts was produced by perfusing hearts for 30 min with oxygenated medium. SR, sarcoplasmic reticulum.

Preparation of SR vesicles. The SR vesicles were prepared using a method described elsewhere (1). The heart was placed immediately in ice-cold saline solution, and the major vessels and right ventricle were removed. The left ventricular tissue was weighed, minced, and transferred to a tube containing a solution (10 ml/g tissue) of (in mM) 10 NaHCO₃, 5 NaN₃, and 15 Tris · HCl at pH 6.8. The tissue was homogenized for 45 s on ice with a Polytron homogenizer (Brinkmann, Westbury, NY) at a setting of 5. The homogenate was centrifuged at 9,500 rpm for 20 min to remove cellular debris, and the supernatant was transferred to a new tube and centrifuged at 19,000 rpm for 45 min. This pellet was suspended in 8 ml of 600 mM KCl and 10 mM Tris · HCl, pH 6.8, and centrifuged at 19,000 rpm for 45 min. The pellet was resuspended in 1 ml of 250 mM sucrose and 10 mM histidine buffer. The concentration of the SR proteins was determined by the method of Lowry et al. (16). The activities of glucose-6-phosphatase, rotenone-insensitive NADPH cytochrome c reductase, ouabain-sensitive Na⁺-K⁺-ATPase, and cytochrome-c oxidase in SR preparations were measured to assess cross-contamination according to methods described earlier (1).

Measurement of Ca²⁺ release. Ca²⁺ release from the ⁴⁵Ca²⁺-loaded SR vesicles was studied according to the method of Ganguly et al. (6). The SR preparation (62.5 µg of SR protein) was suspended in 625 µl of a loading buffer containing (in mM) 100 KCl, 5 MgCl₂, 5 potassium oxalate, 5 NaN₃, and 20 Tris · HCl (pH 6.8), and the samples were incubated with 10 µM ⁴⁵CaCl₂ (20 mCi/l) and 5 mM ATP for 45 min at room temperature. To measure EGTA-induced Ca²⁺ release, 1 mM EGTA was added to the medium and the reaction was stopped at 15 s using the Millipore filtration technique. Radioactivity in the filter was counted in 10 ml of the scintillation fluid. Ca²⁺-induced Ca²⁺ release from the Ca²⁺-loaded SR vesicles was measured by using 1 mM EGTA and 1 mM Ca²⁺ to initiate Ca²⁺ release. The Ca²⁺-induced Ca²⁺ release was blocked by 90-95% in the presence of 20 µM ryanodine in all groups and was linear for 20 s under the experimental conditions employed in this study. The status of SR Ca²⁺-release channels was further verified by measuring specific ³H-labeled ryanodine ([³H]ryanodine) binding with SR preparations in the presence of 1, 2, 5, 10, 20 and 40 nM [³H]ryanodine (30) and analyzing the data in terms of values of maximum binding capacity (B_max) and dissociation constant (K_d) from the Scatchard plots.

Measurement of Ca²⁺ uptake. Ca²⁺-uptake activity of the SR vesicles was determined by employing ⁴⁵Ca²⁺ and the Millipore filtration technique as detailed elsewhere (1). The standard incubation medium (total volume 250 µl) contained 50 mM Tris-maleate (pH 6.8), 5 mM NaN₃, 5 mM ATP, 5 mM MgCl₂, 120 mM KCl, 5 mM potassium oxalate, 0.1 mM EGTA, 0.1 mM ⁴⁵CaCl₂ (20 mCi/l), and 25 µM ruthenium red. The ruthenium red in the incubation medium was added to inhibit Ca²⁺-release channel activity under the assay conditions employed in this study. The reaction was initiated by the addition of SR (12 µg protein) to the standard incubation medium. The reaction was terminated after 1 min by filtering 200-µl aliquots of the incubation mixture through the Millipore filter (0.45 µm). The filter was washed with 5 ml cold water and dried at 60°C for 1 h, and then the radioactivity was counted using the standard liquid scintillation counting technique. In some experiments, oxalate-supported Ca²⁺ uptake in the ventricular homogenate was determined according to a method described elsewhere (1). The status of Ca²⁺ pump in the SR membranes was determined by measuring the Ca²⁺-stimulated ATPase activities in all experimental groups according to a method used previously (1).

Western immunoblot analysis. The protein contents of Ca²⁺-release channels, Ca²⁺ pump, and phospholamban in the SR membrane were determined by Western immunoblotting techniques. For immunoassay of Ca²⁺-release channels, Ca²⁺ pump, and phospholamban, SR (20 µg protein/lane) samples were subjected to SDS-PAGE in 6, 10, and 15% gels, respectively. The protein bands from these gels were then transferred electrophoretically to nitrocellulose membrane (for Ca²⁺-release channels) or polyvinylidene difluoride membrane (for Ca²⁺ pump or phospholamban). The membranes were used for incubation with anti-Ca²⁺-release channel (anti-ryanodine receptor; 1:1,400), anti-sarco(endo)plasmic reticulum Ca²⁺ ATPase (anti-SERCA2; 1:1,400), or anti-phospholamban (1:2,000) antibodies. A peroxidase-linked anti-mouse IgG was used for Ca²⁺ pump or phospholamban as the secondary antibody (1:5,000), whereas anti-mouse IgG was used for Ca²⁺-release channels as the secondary antibody (1:2,500) and then incubated with streptavidin-conjugated horseradish peroxidase solution (1:5,000). Protein bands reactive with antibodies were visualized using the enhanced chemiluminescence detection system from Amersham (Amersham, UK). The intensity of each band was scanned using an Imaging Densitometer with the aid of Molecular Analyst Software version 1.3 (Bio-Rad, Hercules, CA).

Phosphorylation of SR by CaMK and PKA. The SR protein phosphorylation due to endogenous CaMK was determined using the procedure described by Netticadan et al. (18). The incubation medium (total volume 50 µl) for phosphorylation by endogenous CaMK contained 50 mM HEPES (pH 7.4), 10 mM MgCl₂, 100 µM CaCl₂, 100 µM EGTA, 2 µM calmodulin, 0.8 mM -³²P-labeled ATP ([-³²P]ATP; specific activity 200-300 cpm/pmol), and SR (30 µg protein). The initial concentration of free Ca²⁺ as determined using the computer program of Fabiato (5) was 3.7 µM. The phosphorylation reaction was initiated by the addition of [-³²P]ATP following preincubation for 3 min at 37°C. PKA-dependent phosphorylation was performed in the Ca²⁺/calmodulin-free incubation medium in the presence of PKA (catalytic subunit; 4 µg/50 µl assay medium). Reactions were terminated after 2 min by adding 15 µl SDS sample buffer, and the samples were subjected to SDS-PAGE in 4-18% gradient slab gels. The gels were stained with Coomassie brilliant blue, dried, and autoradiographed. The intensity of each phosphorylated band was scanned using the Imaging Densitometer (Bio-Rad).

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

	RESULTS

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Left ventricular function. The time course of changes in left ventricular function of the control and preconditioned groups are depicted in Fig. 2. During the preischemic phase, IP decreased the LVDP, +dP/dt, and dP/dt by 24, 48, and 56%, respectively (P < 0.01). Sustained ischemia for 30 min produced a marked depression (95-100%) of cardiac function in both control and preconditioned hearts. After 30 min of reperfusion of the ischemic hearts, the recovery of LVDP, +dP/dt, and dP/dt in each group (control vs. preconditioning) was 15.9 ± 7.2 vs. 40.2 ± 6.7, 11.9 ± 5.6 vs. 31.9 ± 4.9, and 14.6 ± 5.2 vs. 33.0 ± 5.1% (P < 0.05), respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Time course of changes in left ventricular (LV) function due to ischemia-reperfusion in control and preconditioned rat hearts. Values for LV developed pressure (LVDP), peak rate of contraction (+dP/dt), and peak rate of relaxation (dP/dt) are expressed as percentages of respective basal values before experiment was started. Values are means ± SE of 6 hearts in each group. * P < 0.05 vs. control.

SR Ca²⁺-release and Ca²⁺-uptake activities. EGTA-induced Ca²⁺-release and ATP-dependent uptake activities were determined in SR preparations from control and preconditioned hearts, and the results are shown in Fig. 3. IP was found to depress the EGTA-induced Ca²⁺-release and Ca²⁺-uptake activities in SR preparations by 64 and 44% (P < 0.001), respectively, during the preischemic phase. Although SR Ca²⁺-release (1.7 ± 0.1 nmol · mg¹ · 15 s¹) and Ca²⁺-uptake (7.9 ± 0.4 nmol · mg¹ · min¹) activities decreased markedly in the control ischemic hearts, these changes were significantly attenuated in the preconditioned ischemic hearts (Ca²⁺ release: 6.8 ± 1.1 nmol · mg¹ · 15 s¹; Ca²⁺ uptake: 23.7 ± 2.6 nmol · mg¹ · min¹; P < 0.01). Reperfusion of the control ischemic heart did not result in the recovery of changes in SR Ca²⁺-release and Ca²⁺-uptake activities; however, the values for these parameters in the preconditioned hearts were higher than those in the control ischemic-reperfused group (P < 0.05).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   SR Ca2+-release and -uptake activities in preischemic, ischemic, and ischemic-reperfused control and preconditioned rat hearts. A: EGTA-induced Ca2+-release from Ca2+-loaded SR vesicles. B: oxalate-supported Ca2+-uptake in SR vesicles in presence of ruthenium red. Values are means ± SE of 6 different preparations for each time point in each group. * P < 0.05 vs. corresponding control value. # P < 0.05 vs. preischemic group.

Protein contents of SR. Ca²⁺-release channel, Ca²⁺-pump, and phospholamban protein contents in SR membranes were determined using the Western blot analysis (Fig. 4), and the results are shown in Fig. 5. Ca²⁺-release channel protein of the preconditioned hearts decreased by 24% (P < 0.01), whereas Ca²⁺-pump and phospholamban proteins in the preconditioned group did not show any change during the preischemic phase compared with the respective control value. Ca²⁺-pump protein of the control hearts decreased markedly by 47% (P < 0.01), and IP attenuated this change significantly (P < 0.05). No alterations in SR Ca²⁺-release channel and phospholamban proteins were evident in either control or preconditioned ischemic hearts. Although Ca²⁺-release channel and Ca²⁺-pump proteins decreased by 40 (P < 0.01) and 51% (P < 0.001), respectively, after reperfusion of the control ischemic hearts, IP preserved these changes in protein contents. Ischemia-reperfusion produced no alteration in SR phospholamban protein content in either control or preconditioned hearts.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Typical Western immunoblots of SR Ca2+-release channel (A), Ca2+-pump (B), and phospholamban (C) proteins in control and preconditioned rat hearts. Pre Is, preischemic; Post Is, postischemic; RE, ischemic-reperfused.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Relative protein content of SR Ca2+-release channel (ryanodine receptor) (A), Ca2+ pump (SERCA2; B), and phospholamban (C) in control and preconditioned rat hearts. Values are means ± SE of 6 different preparations for each time point in each group and are expressed as percentages of control densitometric unit (preischemia). * P < 0.05 vs. corresponding control value. # P < 0.05 vs. preischemic value in each group.

Phosphorylation of SR proteins by endogenous CaMK. Phosphorylation of SR proteins in control and preconditioned hearts was determined using autoradiography (Fig. 6), and the analysis of these results is shown in Fig. 7. Note from Fig. 6 that CaMK-mediated phosphorylation in SR membrane occurred at Ca²⁺-release channel, Ca²⁺-pump, and phospholamban proteins. The SDS-PAGE protein profiles of SR preparations from hearts subjected to various experimental protocols do not reveal the appearance of any new protein bands in these preparations; this provides further evidence concerning the purity of SR preparations employed in this study. The data in Fig. 7 indicate that IP produced a significant decrease of Ca²⁺-release channel phosphorylation by endogenous CaMK (P < 0.05), whereas phosphorylation of Ca²⁺ pump and phospholamban was not altered during the preischemic phase. Phosphorylation of Ca²⁺-release channel, Ca²⁺ pump, and phospholamban by endogenous CaMK in the control ischemic hearts decreased markedly by 93, 74, and 33%, respectively (P < 0.05); IP preserved the CaMK-mediated phosphorylation of these SR proteins. Phosphorylation of SR Ca²⁺-release channel and Ca²⁺ pump in the preconditioned hearts decreased significantly compared with that in the postischemia group (P < 0.01), whereas SR protein phosphorylation of the control ischemic hearts recovered moderately on reperfusion. Nonetheless, no difference with respect to SR Ca²⁺-release channel, Ca²⁺-pump, and phospholamban phosphorylations was seen between control and preconditioned ischemic-reperfused hearts (Fig. 7). Because the contents of SR protein substrates are altered in control and preconditioned hearts, it was necessary to establish changes in phosphorylation per unit amount of substrate protein. Accordingly, the data in Figs. 5 and 7 were analyzed in terms of the ratios of substrate phosphorylation and the amount of immunoreactive protein, and the values are given in Table 4. Essentially, the alterations in substrate phosphorylation when calculated per unit amount of substrate were similar to the changes described above for phosphorylation of SR proteins in all groups, except that a significant decrease in SR Ca²⁺-pump phosphorylation in the preconditioned ischemic-reperfused heart was evident compared with the control ischemic-reperfused heart (Table 4).

View larger version (60K): [in this window] [in a new window]   Fig. 6.   Typical SDS-PAGE protein profiles of SR preparations (A) as well as typical autoradiogram of SR phosphorylation (B) by endogenous Ca2+/calmodulin-dependent protein kinase (CaMK) in control and preconditioned rat hearts. Phosphorylation reaction was carried out for 2 min in absence () or presence (+) of Ca2+ and calmodulin (CaM) as indicated. PLN(H), high-molecular-weight form of phospholamban; PLN(L), low-molecular-weight form of phospholamban.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Relative phosphorylation by CaMK of SR Ca2+-release channel (A), Ca2+ pump (B), and phospholamban (C) in control and preconditioned rat hearts. Values are means ± SE of 6 different preparations at each time point in each group and are expressed as percentages of control densitometric unit (preischemia). * P < 0.05 vs. corresponding control value. # P < 0.05 vs. preischemic value in each group.

Phosphorylation of SR proteins by exogenous PKA. Under the assay conditions employed, the major SR peptide bands phosphorylated by exogenous PKA were the high- and low-molecular-weight forms of phospholamban (Fig. 8, bottom). There were no differences between the phosphorylation of phospholamban in the control group and the preconditioning group at preischemic, postischemic, and ischemic-reperfusion phases (Fig. 8). Although PKA-mediated phosphorylation in the control ischemic-reperfused SR was significantly higher than the respective control value in the preischemic phase, such differences were not evident between control ischemic and ischemic-reperfused preparations (Fig. 8 and Table 4).

View larger version (58K): [in this window] [in a new window]   Fig. 8.   Phosphorylation of SR phospholamban by exogenous cAMP-dependent protein kinase (PKA) in control and preconditioned rat hearts. Top: relative phosphorylation of phospholamban by exogenous PKA. Values are means ± SE of 6 different preparations at each time point in each group and are expressed as percentages of control densitometric unit (preischemia). #P < 0.05 vs. preischemia. Bottom: typical autoradiogram of SR phosphorylation in gel. Phosphorylation reaction was carried out in absence () or presence (+) of PKA. Major peptide bands phosphorylated by PKA are PLN(H) and PLN(L) forms of phospholamban.

	DISCUSSION

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In this study preconditioning was observed to depress the EGTA-induced Ca²⁺ release, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release, and [³H]ryanodine binding with SR preparations from hearts at the preischemic phase. These observations confirm the findings of other investigators concerning the effect of preconditioning on [³H]ryanodine binding with heart homogenate (30) as well as on SR Ca²⁺-release activity calculated from the difference between Ca²⁺-uptake values in the presence and absence of ryanodine (24). Steenbergen et al. (22) reported that the development of cytosolic Ca²⁺ overload was delayed in the preconditioned heart, so the reduced SR Ca²⁺-release activity in preconditioned hearts at the preischemic phase may delay the development of cytosolic Ca²⁺ overload and subsequent ischemic injury. Although Tani et al. (24) reported that the beneficial effects of IP were associated with a decrease in ryanodine-sensitive SR Ca²⁺ release, these investigators showed that the depression in Ca²⁺ release in the preconditioned hearts persisted after ischemia-reperfusion. On the other hand, in our study the reduced SR Ca²⁺-release activity at the preischemic phase of the preconditioned hearts was recovered significantly after reperfusion. This difference may be due to the different methods used for assessing the SR Ca²⁺-release activity, because we have measured the amount of Ca²⁺ released from the SR through the ryanodine-sensitive and ryanodine-insensitive Ca²⁺-release channels directly. Nonetheless, the recovery of SR Ca²⁺-release activity due to ischemia-reperfusion in the present study correlated well with the recovery of cardiac function in the preconditioned hearts. It should be noted that SR Ca²⁺-uptake activity was also decreased in the preconditioned hearts at the preischemic phase, and this can be seen to induce cytosolic Ca²⁺ overload in sustained ischemia. However, greater reduction in the SR Ca²⁺-release channels compared with the reduction in Ca²⁺ uptake, as observed in the preconditioned hearts at the preischemic phase, may negate the effects of reduced SR Ca²⁺ uptake. Alternatively, a decrease in SR Ca²⁺ uptake at the preischemic phase may reduce the Ca²⁺ stores in SR, and thus less Ca²⁺ may be available for release at the end of preconditioning. Thus the observed changes in both SR Ca²⁺-release channels and Ca²⁺ pump due to IP may play an important role in eliciting beneficial effects on the ischemia-reperfusion-induced alterations in cardiac function.

Some investigators (10, 29) have reported the degradation of SR Ca²⁺-release channel or Ca²⁺-pump protein in the ischemic-reperfused hearts. This degradation of specific proteins may contribute to the functional abnormalities in the SR membrane and subsequent contractile dysfunction in the ischemic-reperfused myocardium. In our study the protein contents of SR Ca²⁺-release channel as well as Ca²⁺ pump were decreased in ischemic-reperfused hearts, and IP was found to prevent these changes in SR proteins. Such alterations in SR membrane seem to be of some specific nature because protein content of phospholamban in SR membranes was unaltered in the ischemic-reperfused hearts. Furthermore, SR Ca²⁺-release channel protein decreased without any change in the SR Ca²⁺-pump protein during the preconditioning phase, whereas SR Ca²⁺-pump protein decreased without any changes in the SR Ca²⁺-release channel protein in the ischemic hearts. Although these changes in SR proteins may partly explain the observed alterations in SR Ca²⁺-release and Ca²⁺-uptake activities during preischemic, ischemic, and ischemic-reperfused phases, the decrease in SR Ca²⁺-uptake activity due to preconditioning was not associated with any reduction in the SR Ca²⁺-pump protein or SR Ca²⁺-stimulated ATPase activity. Thus some other mechanisms involving changes in the regulation of SR Ca²⁺ pump have to be invoked for explaining the depression in SR Ca²⁺-uptake activity in the preischemic phase.

IP was found to depress the endogenous CaMK-mediated phosphorylation of SR Ca²⁺-release channels during the preischemic phase. This observation correlated well with decreased SR Ca²⁺-release activity at the preischemic phase. Witcher et al. (28) and Takasawa et al. (23) have shown that phosphorylation of SR Ca²⁺-release channel by endogenous CaMK may maintain the Ca²⁺-release channel in an open state and thus enhance Ca²⁺ release from SR, so the observed decrease in SR Ca²⁺-release channel phosphorylation may lead to reduced Ca²⁺ release in the ischemic myocardium. On the other hand, Hain et al. (7) have reported that the activation of endogenous CaMK may lead to closure of the SR Ca²⁺-release channels, and thus some caution should be exercised in interpreting the results obtained in this study. In addition, it should be noted that phosphorylation of SR Ca²⁺ pump or phospholamban by endogenous CaMK has been reported to enhance Ca²⁺ uptake in the SR vesicles (26). However, the SR Ca²⁺-uptake activity in the preconditioned hearts was decreased at the preischemic phase, whereas CaMK phosphorylation of the SR Ca²⁺-pump and phospholamban proteins showed no change. Nonetheless, it should be noted that phosphorylation of SR Ca²⁺-release channel, Ca²⁺ pump, and phospholamban by endogenous CaMK in the preconditioned hearts increased at the postischemic phase and correlated well with the SR function. These results suggest that the beneficial effects of IP on SR function due to the regulation of SR phosphorylation by endogenous CaMK may become evident not only at the preischemic phase but also at the postischemic phase. The observed decrease in CaMK-mediated phosphorylation of SR Ca²⁺-release channels and Ca²⁺ pump in the preconditioned ischemic hearts on reperfusion compared with that in the postischemic phase may indicate the reversible nature of this process.

The role of cardiac SR phosphorylation by protein kinases, especially CaMK and PKA, has been well documented under physiological conditions (12, 27); however, the present study is the first to report changes in CaMK-mediated phosphorylation without any effect on PKA-mediated phosphorylation in the SR membrane in ischemic-reperfused hearts. The finding that the major SR peptide band phosphorylated by exogenous PKA under the assay conditions employed was phospholamban is consistent with an earlier study by Hawkins et al. (9), who reported similar results and indicated that the SR Ca²⁺-release channel and Ca²⁺-pump proteins were not phosphorylated to any significant extent by PKA. Furthermore, there was no difference in the phospholamban phosphorylation by exogenous PKA between the control and preconditioning groups before or after ischemia-reperfusion. Lamers et al. (14) reported similar results in the porcine stunned myocardium, indicating that the phosphorylation of phospholamban by PKA was unchanged after two cycles of 10-min ischemia and 30-min reperfusion. As reported by other investigators (11), brief periods of ischemia-reperfusion during IP may evoke an increase in the intracellular concentration of Ca²⁺ and thus can be seen to activate CaMK in the SR membrane (8). Furthermore, modification of the ischemia-reperfusion-induced changes in CaMK-mediated phosphorylation by preconditioning of the heart may support the view that this process may play an important role in regulating the function of SR proteins under pathophysiological situations. It is possible that some of the changes regarding protein phosphorylation may be due to changes in protein phosphatase activity in control and preconditioned hearts; however, extensive studies are needed to appreciate the potential contribution of protein phosphatase activity in the preischemic, ischemic, and ischemic-reperfused hearts.