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Am J Physiol Heart Circ Physiol 278: H1791-H1798, 2000;
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Vol. 278, Issue 6, H1791-H1798, June 2000

Ischemic preconditioning prevents I/R-induced alterations in SR calcium-calmodulin protein kinase II

Mitsuru Osada1, Thomas Netticadan1, Kenichi Kawabata1, Kohji Tamura2, and Naranjan S. Dhalla1

1 Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6; and 2 Second Department of Internal Medicine, Yamanashi Medical University, Yamanashi, Japan 409-38


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although Ca2+/calmodulin-dependent protein kinase II (CaMK II) is known to modulate the function of cardiac sarcoplasmic reticulum (SR) under physiological conditions, the status of SR CaMK II in ischemic preconditioning (IP) of the heart is not known. IP was induced by subjecting the isolated perfused rat hearts to three cycles of brief ischemia-reperfusion (I/R; 5 min ischemia and 5 min reperfusion), whereas the control hearts were perfused for 30 min with oxygenated medium. Sustained I/R in control and IP groups was induced by 30 min of global ischemia followed by 30 min of reperfusion. The left ventricular developed pressure, rate of the left ventricular pressure, as well as SR Ca2+-uptake activity and SR Ca2+-pump ATPase activity were depressed in the control I/R hearts; these changes were prevented upon subjecting the hearts to IP. The beneficial effects of IP on the I/R-induced changes in contractile activity and SR Ca2+ pump were lost upon treating the hearts with KN-93, a specific CaMK II inhibitor. IP also prevented the I/R-induced depression in Ca2+/calmodulin-dependent SR Ca2+-uptake activity and the I/R-induced decrease in the SR CaMK II activity; these effects of IP were blocked by KN-93. The results indicate that IP may prevent the I/R-induced alterations in SR Ca2+ handling abilities by preserving the SR CaMK II activity, and it is suggested that CaMK II may play a role in mediating the beneficial effects of IP on heart function.

ischemia-reperfusion; cardiac sarcoplasmic reticulum; cardiac sarcoplasmic reticulum calcium pump


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC PRECONDITIONING (IP) is a powerful intervention for preventing the lethal injury in the ischemia-reperfused myocardium (17, 21). Extensive studies (10, 12, 13, 15, 16) have been carried out to examine the mechanisms of IP. Some investigators have suggested the role of adenosine receptors (10, 13) or protein kinase C (PKC) (15, 24) in mediating the effects of IP; however, others have failed to establish the role of these mediators (12, 16) in eliciting the actions of IP. Because the sarcoplasmic reticulum (SR) is known to play a central role in regulating the intracellular concentration of Ca2+ and subsequent cardiac contraction and relaxation processes, improved handling of Ca2+ by SR has been indicated to explain the beneficial effects of IP in the heart (29, 32). We have recently demonstrated that IP may exert beneficial effects on the ischemia-reperfusion (I/R)-induced alterations in SR function by preventing changes in SR protein content and their phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMK II) (20). Accordingly, it appears that the regulation of SR Ca2+ pump is a crucial factor that may determine the protective action of IP on the I/R-induced contractile dysfunction in the heart. In this regard, it is pointed out that phosphorylation of SR Ca2+ pump and phospholamban proteins by CaMK II has been shown to enhance the Ca2+-uptake activity in the SR vesicles (7, 30), and that CaMK II is present in the cardiac SR membranes as a distinct delta -CaMK isozyme (2). Because there is no report showing the involvement of SR CaMK II in protecting SR function in the preconditioned heart, the status of CaMK II in IP was examined in this study. The role of CaMK II in the beneficial effects of IP on SR function in the I/R rat heart was tested by the use of a specific inhibitor of the CaMK II activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Heart perfusion and experimental protocol. Male Sprague-Dawley rats (300-350 g) were anesthetized by an intraperitoneal injection of a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The hearts were rapidly excised and perfused by the Langendorff technique (20). The perfusion medium containing (in mmol/l) 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 mixture and maintained at pH 7.4 (37°C). The hearts were paced at 300 beats/min by an electrical stimulator (Phipps & Bird, Richmond, VA), and the coronary flow rate was maintained at 10 ml/min throughout the experiment. 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 of 9 mmHg. Values for the left ventricular developed pressure (LVDP) were obtained by using the AcqKnowledge program for Windows 3.0 (Biopac Systems, Goleta, CA). All hearts were perfused with oxygenated medium for 30 min for stabilization before the experiment was initiated. Hearts that did not produce >40 mmHg of LVDP or had frequent paroxysmal ventricular contractions during stabilization were discarded.

To test whether the inhibitor of CaMK II attenuates the protective effect of IP against postischemic cardiac dysfunction, we used KN-93 (Sigma Chemical, St. Louis, MO). The vehicle (perfusion medium) or the inhibitor was then delivered by an infusion pump into the perfusion stream directly above the aortic cannula at 1 ml/min for 10 min before the preconditioning cycles were induced; the control group also received similar treatments during the corresponding 10-min period. The final concentration of KN-93 in the perfusion medium was 1 µmol/l; this concentration was selected on the basis of a previous study (11) indicating the inhibition constant (Ki) value of KN-93 for CaMK II as 0.37 µmol/l. For the control group, the hearts were perfused for 30 min with oxygenated medium followed by 30 min of global ischemia and 30 min of reperfusion. For the preconditioned group, the hearts were subjected to three cycles of 5 min of ischemia and 5 min of 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. Preliminary experiments revealed that three cycles of brief I/R episodes produced maximal protection with respect to changes in heart function due to sustained I/R. The hearts were removed for the preparation of SR vesicles just before the inducement of ischemia (preischemia), after 30 min of ischemia (postischemia), and after 30 min of reperfusion in the control or preconditioned groups.

Preparation of SR vesicles. Membrane fraction enriched with SR vesicles (microsomal fraction) was prepared by the method described elsewhere (1). The heart was placed immediately in ice-cold saline solution, and the major vessels were removed. The ventricular tissue was weighed, minced, and transferred to a tube containing a solution (10 ml/g tissue) of 10 mmol/l NaHCO3, 5 mmol/l NaN3, and 15 mmol/l Tris · HCl, pH 6.8. The homogenate buffer also contained protease inhibitors (in µM): 1 leupeptin, 1 pepstatin, and 100 phenylmethylsulfonylfluoride. The tissue was homogenized for 45 s on ice with a Polytron homogenizer (Brinkmann, Westbury, NY) at 12,000 rpm. The homogenate was centrifuged at 10,000 g for 20 min to remove cellular debris; the supernatant was transferred to a new tube and centrifuged at 40,000 g for 45 min. The pellet obtained was suspended in 8 ml of 600 mmol/l KCl and 10 mmol/l Tris · HCl, pH 6.8, and centrifuged at 40,000 g for 45 min. The pellet was resuspended in 750 µl of 250 mmol/l sucrose and 10 mmol/l histidine buffer. The concentration of SR proteins was determined by the method used previously (1). The activities of glucose-6-phosphatase, rotenone-insensitive NADPH cytochrome c reductase, ouabain-sensitive Na+-K+-ATPase and cytochrome-c oxidase in SR preparations (1) of control and preconditioned hearts at each perfusion phase showed that the SR preparations from these hearts were equally but minimally (3-5%) contaminated with other subcellular organelles.

Measurement of SR Ca2+ uptake, Ca2+-pump ATPase, and CaMK II activities. Oxalate-supported Ca2+-uptake activity of the SR vesicles in the I/R hearts was determined by employing 45Ca2+ and Millipore filtration technique as detailed elsewhere (1). The status of the Ca2+ pump in the SR membranes was further determined by measuring the Ca2+-stimulated ATPase activities in all experimental groups according to the method used previously (1). The activity of SR CaMK II was determined by using the CaMK II assay kit (Upstate Biotechnology, Lake Placid, NY). This assay is based on phosphorylation of specific synthetic peptide autocamtide-3 (KKALRRQETVDAL) as indicated by the transfer of [gamma -32P] from [gamma -32P]ATP by CaMK II. The phosphorylated substrate was then separated from the residual [gamma -32P]ATP using P81 phosphocellulose paper and quantitated by using a liquid scintillation counter. The experiments were performed in the presence and absence of the exogenous substrate, autocamtide-3; the CaMK activity was calculated by subtracting the values in the absence from those in the presence of the exogenous substrate.

Measurement of Ca2+/CaM-dependent Ca2+-uptake activity. Ca2+-uptake activity of SR phosphorylated in the presence or absence of Ca2+/CaM was determined by employing 45Ca2+ and Millipore filtration technique as described elsewhere (7) with slight modifications. The standard incubation medium (total volume 50 µl) for Ca2+/CaM-mediated phosphorylation contained 50 mmol/l HEPES (pH 7.4), 10 mmol/l MgCl2, 100 µmol/l CaCl2, 100 µmol/l EGTA, 1 µmol/l calmodulin, and 0.8 mmol/l ATP and SR (10 µg protein). The concentration of free Ca2+ in this assay medium as determined by using the computer program of Fabiato (5) was 3.7 µmol/l. Phosphatase inhibitors microcystin-LR (10 nM) and sodium pyrophosphate (1 mM) were added to the reaction mixture to inhibit any endogenous phosphatase activity. Phosphorylation reaction was initiated by the addition of ATP following preincubation of the rest of the assay components for 3 min at 37°C. The standard incubation medium for SR vesicle phosphorylation in the absence of Ca2+/CaM contained 10 µmol/l W-7 and 1 mmol/l EGTA; W-7 was added to inhibit endogenous CaM activity (28), whereas EGTA was added to chelate Ca2+. The phosphorylated SR vesicles were transferred into the Ca2+-uptake assay medium after 1 min of the reaction.

The Ca2+-uptake assay medium (total volume 250 µl) contained 50 mmol/l Tris-maleate (pH 6.8), 5 mmol/l NaN3, 5 mmol/l ATP, 5 mmol/l MgCl2, 120 mmol/l KCl, 5 mmol/l potassium-oxalate, 0.1 mmol/l EGTA, 0.1 mmol/l 45CaCl2 (20 mCi/l), and 25 µmol/l ruthenium red; the free Ca2+ in this assay medium as determined by the program of Fabiato (5) was 8.2 µmol/l. Ruthenium red in the assay 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 phosphorylated SR (6 µg of protein) to the assay medium. The reaction was terminated after 1 min by filtering a 200-µl aliquot of the incubation mixture through the Millipore filter (0.45 µm). The filter was washed with 3 ml of cold water and dried at 60°C for 1 h, and the radioactivity was counted by using the standard liquid scintillation counting technique. Ca2+/CaM-dependent Ca2+-uptake activities were calculated as the difference in Ca2+-uptake activities of the phosphorylated SR vesicles in the presence and absence of Ca2+/CaM.

Measurement of protein phosphatase activity. To test whether changes in the SR phosphorylation by endogenous CaMK II were associated with changes in SR protein phosphatase activity in control and preconditioned hearts, we evaluated the SR protein phosphatase activities in the preischemic, ischemic, and ischemic-reperfused hearts of the control and preconditioning groups. The activity of SR protein phosphatase was determined by using the Ser/Thr phosphatase assay kit (Upstate Biotechnology). This assay is based on dephosphorylation of synthetic phosphopeptide (KRpTIRR). Inorganic phosphate released during the reaction was detected by the addition of Malachite Green Solution, and the absorbance was measured at 650 nm. The experiments were performed in the presence and absence of the exogenous substrate, and the phosphatase activity was calculated by subtracting the values in the absence from those in the presence of the exogenous substrate.

Measurement of PKC activity. To examine whether the abolition of the beneficial effects of IP by KN-93 was mediated through the inhibition of PKC activity, homogenate obtained from the control hearts was treated with different concentrations of KN-93, and the PKC activity was determined by a method described earlier (31). The ventricular tissue was homogenized in 50 mM Tris · HCl (pH 7.5), 0.25 mM sucrose, 10 mM EGTA, 4 mM EDTA, 20 µg/ml leupeptin, and 200 U/ml aprotinin, with a Polytron homogenizer (Brinkmann, Westbury, NY) at 8,000 rpm twice for 30 s each and sonicated twice for 15 s each. The homogenate was incubated with 1% Triton X-100 on ice for 60 min to solubilize the PKC enzyme. The Triton X-100-treated homogenate was then centrifuged at 100,000 g for 60 min in a Beckman L70 ultracentrifuge (Beckman Instruments); PKC activity in the supernatant was determined by using the PKC assay kit (Upstate Biotechnology). This assay is based on the phosphorylation of a specific substrate peptide (QKRPSQRSKYL) using the transfer of the gamma -phosphate from [gamma 32-P]ATP by PKC. The phosphorylated substrate was then separated from the residual [gamma 32-P]ATP using P81 phosphocellulose paper and quantiated by using a liquid scintillation counter. The experiments were performed in the presence and absence of the exogenous substrate; the PKC activity was calculated by subtracting the values in the absence from those in the presence of the exogenous substrate.

Statistical analysis. Data are expressed as means ± SE. The statistical difference among different groups was determined by factorial analysis of variance (ANOVA) (STATVIEW 4.02, Abacus Concepts) followed by Bonferroni-Dunn's test. A P value of less than 0.05 was considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Left ventricular function. Control and preconditioned hearts perfused in the absence or presence of KN-93 were subjected to 30 min of ischemia and 30 min of reperfusion, and the values for LVDP and LV pressure development over time (dP/dt) at the preischemic, postischemic, and reperfusion phases are shown in Table 1. KN-93 (1 µmol/l) did not significantly (P < 0.05) affect the LV function of the isolated hearts during the 10-min administration in all experimental groups (Table 1). Although IP significantly (P < 0.05) depressed the LV function at the preischemic phase, the recovery rate after 30 min of ischemia followed by 30 min of reperfusion was higher in the preconditioning group when compared with control values, 44.8% vs 14.1%, respectively. Pretreatment with 1 µmol/l KN-93 abolished the beneficial effect of IP, whereas, a 10-fold lower concentration of the inhibitor (0.1 µmol/l KN-93) did not affect the LVDP recovery in the preconditioned hearts (data not shown).

                              
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  		Table 1.   Influence of KN-93 on left ventricular function in preischemic, postischemic, and ischemic-reperfused control and preconditioned rat hearts

To examine the possibility that the inhibitory effect of KN-93 given before inducing IP was not maximal, another set of experiments was carried out. In this case the hearts were not only treated with KN-93 (1 µmol/l) for 10 min before IP was induced but also were infused for 5 min during each of the three cycles of brief I/R. Control hearts were treated similarly with this agent during the corresponding periods of perfusion. When control and preconditioned hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion, the beneficial effects of preconditioning with respect to the LV function were lost upon treatment with KN-93. The results obtained from this protocol were similar to those described in Table 1 and establish the validity of the experimental design of drug treatment employed in this study.

SR Ca2+ uptake and Ca2+-pump ATPase activities. The oxalate-supported SR Ca2+-uptake activity in the I/R hearts was determined in SR preparations from control, preconditioning, and preconditioning plus inhibitor groups. KN-93 attenuated the SR Ca2+-uptake activity in nonischemic preconditioned hearts by 32% (data not shown). The Ca2+-uptake activity was significantly higher in the preconditioning group when compared with that in the control (Table 2); pretreatment with KN-93 attenuated this effect. To show whether the observed changes in SR Ca2+ uptake were associated with changes in the SR Ca2+ pump, the Ca2+-stimulated ATPase activity in SR preparations was determined. The results in Table 2 reveal that the SR Ca2+-pump ATPase activity in the preconditioned hearts was significantly higher than that for the control; this IP effect was prevented by pretreatment with KN-93. It may be noted from Fig. 1 that individual values for SR Ca2+ uptake in each experiment were correlated significantly with those for LVDP in all groups (r = 0.742, P < 0.0001).

                              
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  		Table 2.   SR oxalate-supported Ca2+ uptake, Ca2+-stimulated ATPase activity, and phosphatase activity in control and preconditioned hearts



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  		Fig. 1.   Plot of left ventricular developed pressure (LVDP) and sarcoplasmic reticulum (SR) Ca2+-uptake activity from control (), preconditioning (open circle ), and preconditioning + KN-93 () hearts. Solid line is a linear regression of two parameters (r = 0.742, P < 0.0001).

SR CaMK II activity. The CaMK II activity was determined in SR vesicles from control, preconditioning, and preconditioning plus KN-93 groups, and the results are shown in Fig. 2. Preconditioning did not affect the CaMK activity in the preischemic phase; however, the enzyme activity was decreased when the hearts were preconditioned in the presence of KN-93. The CaMK II activity in the control group decreased after ischemia as well as I/R (P < 0.05 vs. preischemia); however, these changes were prevented upon preconditioning the hearts. With KN-93 treatment, the SR CaMKII activity was partly depressed in the postischemic phase but was completely depressed in the reperfusion phase in comparison to the respective preconditioning groups (P < 0.05). It may be noted from Fig. 3 that the SR CaMK II activity correlated well with the SR Ca2+-uptake activity in the experimental hearts (r = 0.816, P < 0.0001).


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  		Fig. 2.   Enzymatic activity of SR CaMK II in preischemic, postischemic, and ischemia-reperfused control and preconditioned rat hearts. KN-93 (1 µmol/l) was infused into the heart for 10 min before preconditioning cycles were induced in the preconditioning + KN-93 group. Each value is a mean ± SE of 9-11 different preparations at each time point in each group. #P < 0.05 vs. corresponding control value. ¶ P < 0.05 vs. preischemic value. *P < 0.05 vs. corresponding preconditioning value. CaMK II, Ca2+/calmodulin-dependent protein kinase II.



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  		Fig. 3.   Plot of SR Ca2+-uptake activity and SR CaMK II activity from control (), preconditioning (open circle ), and preconditioning + KN-93 () groups in ischemic-reperfused rat hearts. Solid line is a linear regression of two parameters (r = 0.816, P < 0.0001).

Ca2+/CaM-dependent Ca2+-uptake activity. Ca2+-uptake activities of SR vesicles phosphorylated in the absence or presence of Ca2+/CaM were determined in the control, preconditioning, and preconditioning plus KN-93 groups, and the results are shown in Figs. 4-6. Preconditioning did not significantly affect the Ca2+/CaM-dependent Ca2+-uptake activity in the preischemia phase but was depressed when the hearts were preconditioned in the presence of KN-93 (Fig. 4). The low values for Ca2+ uptake in the SR preparations phosphorylated in the absence of Ca2+/CaM (Figs. 4-6) were due to the fact that the endogenous CaM activity was inhibited and Ca2+ was chelated by compound W-7 and EGTA present in the phosphorylation medium, respectively. Although Ca2+-uptake activities of the phosphorylated SR vesicles from the control group decreased markedly at the postischemic and reperfusion phases, these changes were significantly attenuated in the preconditioning group (P < 0.01). This trend was readily evident when Ca2+/CaM-dependent Ca2+-uptake activities were calculated as differences between the Ca2+-uptake activities of phosphorylated SR preparations in the presence and absence of Ca2+/CaM (insets in Figs. 5 and 6). The values at the preischemic, postischemic, and reperfusion phases were 30.7 ± 7.5, 6.9 ± 1.1, and 13.6 ± 1.4 nmol Ca2+ · mg-1 · min-1 in the control group and were 25.7 ± 1.9, 18.4 ± 2.1, and 23.4 ± 3.9 nmol Ca2+ · mg-1 · min-1 in the preconditioning group, respectively. With KN-93 treatment the Ca2+/CaM-dependent Ca2+-uptake activity was partly depressed in the postischemic phase and almost completely depressed in the reperfusion phase in comparison to the preconditioning group.


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  		Fig. 4.   SR Ca2+-uptake activities of hearts in control, preconditioning (PRECON), and preconditioning + KN-93 groups at preischemic phase. Inset, Ca2+/calmodulin (CaM)-dependent SR Ca2+-uptake activities as difference between Ca2+-uptake activities in presence and absence of Ca2+/CaM. Values are means ± SE of 9 different preparations in each group. *P < 0.05 vs. preconditioning.



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  		Fig. 5.   SR Ca2+-uptake activities of hearts in control, preconditioning (PRECON), and preconditioning + KN-93 groups at postischemic phase. Inset, CaM-dependent Ca2+-uptake activities. Values are means ± SE of 9 different preparations in each group. #P < 0.05 vs. control. *P < 0.05 vs. preconditioning.



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  		Fig. 6.   SR Ca2+-uptake activities of hearts in control, preconditioning (PRECON), and preconditioning + KN-93 groups at reperfusion phase. Inset, CaM-dependent Ca2+-uptake activities. Values are means ± SE of 9-11 different preparations in each group. #P < 0.05 vs. control. *P < 0.05 vs. preconditioning.

Protein phosphatase activity. SR protein phosphatase activities were decreased in the ischemic and ischemic-reperfused hearts in both control and preconditioning groups; these alterations were attenuated by IP (Table 2). KN-93 did not affect the protein phosphatase activity.

PKC activity. Because the effects of KN-93 on preconditioning observed in this study are at events that are downstream of PKC signaling, it is possible that KN-93 may affect the PKC activity. However, varying concentrations of KN-93 (0.1-5 µM) did not significantly affect the PKC activity in cardiac homogenate obtained from control hearts (Table 3).

                              
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  		Table 3.   Effects of different concentrations of KN-93 on the rat heart homogenate PKC activity


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The SR plays an important role in regulating the intracellular concentration of Ca2+ in cardiomyocytes, whereas the cytosolic Ca2+ overload due to SR dysfunction is considered to be one of the major determinants of the I/R injury (4). Some investigators observed that the development of cytosolic Ca2+ overload due to I/R was delayed in the preconditioned rat heart (23, 26). Furthermore, we (20) reported that the preservation of SR function by IP may contribute to the attenuation of Ca2+ overload in the I/R heart. Because IP has been shown to prevent alterations in the CaMK II-dependent phosphorylation of SR proteins (20), it is possible that SR CaMK II could serve as a major player in protecting the SR function in the ischemic preconditioned heart. In this study we demonstrated that KN-93, an inhibitor of CaMK II, prevented the beneficial effects of IP on cardiac performance as well as SR Ca2+ pump and CaMK II activity. In fact, attenuation of the IP-mediated protection of changes in cardiac function by KN-93 correlated linearly with reduced SR Ca2+ uptake, which in turn correlated well with attenuated CaMK II activity. These results suggest that the SR CaMK II may play a role in mediating the beneficial effects of IP. Because the attenuation of the beneficial effects of IP on SR CaMK II and Ca2+/CaM-dependent Ca2+-uptake activities by KN-93 was almost complete in the reperfusion phase but incomplete in the postischemic phase, it is possible that other mechanisms in addition to CaMK II may participate in the postischemic phase. It should be noted that KN-93 is a synthetic CaMK II inhibitor, and the Ki value of KN-93 for CaMK II is 0.37 µmol/l. Concentrations of KN-93 higher than 30 µmol/l have been reported to inhibit other protein kinases such as protein kinase A, PKC, and myosin light-chain kinase (27). Because the present study employed 1 µmol/l KN-93, it is possible that CaMK II was selectively inhibited without any appreciable effect on other protein kinases under the experimental conditions employed here. In fact, our results indicated that even 5 µmol/l KN-93 did not have any effect on PKC activity in cardiac homogenate preparations, and this excludes the possibility of PKC being an upstream mediator of the KN-93 effects.

In this investigation we show that the enzymatic activity of SR CaMK II in the control hearts was markedly decreased after ischemia and reperfusion, and IP prevented these changes. The Ca2+/CaM-dependent SR Ca2+-uptake activity was also markedly decreased in the control hearts after ischemia and reperfusion but was preserved in the preconditioned hearts. It is noteworthy that Ca2+/CaM-dependent Ca2+-uptake activities were three times higher in the preconditioned ischemic hearts and two times higher in the preconditioned ischemic-reperfused hearts in comparison to the control values. Because the IP-induced preservation of both SR CaMK II and Ca2+/CaM-dependent Ca2+-uptake activities was evident not only at the reperfusion phase but also at the postischemic phase, these results suggest that IP may attenuate the cytosolic Ca2+ overload at an early phase of reperfusion following prolonged ischemia. In fact, the isolated heart studies have consistently demonstrated a transient increase in the intracellular concentration of Ca2+ just after the onset of reperfusion (19). It should also be noted that the Ca2+-uptake activity in the unphosphorylated SR vesicles at the preischemic phase was decreased by IP, whereas the Ca2+/CaM-dependent Ca2+-uptake activity in SR vesicles was unaltered. Such a difference seems to indicate an important role of CaMK II in improving the SR function by IP. The preservation of SR CaMK II activity may therefore contribute to the improved SR Ca2+ uptake and the subsequent recovery of cardiac performance in the preconditioned heart.

A possible mechanism for the preservation of SR CaMK II activity in IP may be the autophosphorylation of SR CaMK II. Brief repetitive Ca2+ signals have been shown to activate CaMK II and stimulate autophosphorylation, which allows this kinase to maintain the activated state beyond the duration of a particular Ca2+ signal (6). Because different investigators have reported (9, 23) that brief ischemia in IP could evoke an increase in the intracellular concentration of Ca2+, it may be possible that the repetitive IP cycles may activate CaMK II and maintain its activity during sustained I/R. In addition, there is a possibility that the phosphatidylinositol signaling pathway, which is involved in preconditioning (3), may activate CaMK II in the heart in a manner similar to that which has been reported to activate CaMK in PC12 cells (14). Nonetheless, it is likely that the observed alterations in SR CaMK II activity are associated with changes at the SR phosphatase level. In fact our results showed a decrease in the SR protein phosphatase activity in I/R hearts; this effect was prevented by IP. The higher activities of SR CaMK II and protein phosphatase in the preconditioned hearts seem to maintain an improved cycle of phosphorylation/dephosphorylation of SR proteins, and this may contribute to efficient intracellular Ca2+ fluxes following I/R.

In summary, we demonstrated that 1) the beneficial effect of IP on the cardiac contractile function in the ischemia-reperfused rat heart correlated well with the SR Ca2+-uptake activity and 2) the SR Ca2+ pump regulated by CaMK II may be one of the pivotal effectors in IP. In fact there was a good relationship between SR Ca2+ uptake and SR CaMK II activities under the experimental conditions employed in this study. These findings suggest the involvement of CaMK II in mediating the beneficial effects of IP on I/R-induced contractile dysfunction and SR Ca2+ transport defects in the isolated rat heart and thereby extend our existing knowledge in the field of IP-induced myocardial protection. Although troponin T, C protein, and Na+-K+-ATPase are also CaMK II substrates in the heart (8, 18, 22), the physiological relevance of these phosphorylations is not clearly established. Nevertheless, this study does not exclude the participation of these non-SR CaMK II substrates in mediating the effects of KN-93. Therefore, the inhibition of the beneficial effects of IP by KN-93 observed in this study may be due to a multifactorial effect.


    ACKNOWLEDGEMENTS

The work reported in this study was supported by a grant from the Medical Research Council of Canada (MRC Group in Experimental Cardiology). N. S. Dhalla holds the MRC/Pharmaceutical Research and Development Chair in Cardiovascular Research supported by the Merck Frosst of Canada.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (E-mail: cvso{at}sbrc.umanitoba.ca).

Received 5 July 1999; accepted in final form 2 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 278(6):H1791-H1798
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