To examine the effects of ischemic preconditioning on ischemia-reperfusion-induced changes in the sarcoplasmic reticulum (SR) function, isolated rat hearts were either perfused with a control medium for 30 min or preconditioned with three episodes of 5-min ischemia and 5-min reperfusion before sustained ischemia for 30 min followed by reperfusion for 30 min was induced. Preconditioning itself depressed cardiac function (left ventricular developed pressure, peak rate of contraction, and peak rate of relaxation) and SR Ca2+-release and -uptake activities as well as protein content and Ca2+/calmodulin-dependent protein kinase (CaMK) phosphorylation of Ca2+-release channels by 25–60%. Global ischemia for 30 min produced marked depressions in SR Ca2+-release and -uptake activities as well as SR Ca2+-pump protein content in control hearts; these changes were significantly attenuated by preconditioning. Compared with the control preparations, preconditioning improved the recovery of cardiac function and SR Ca2+-release and -uptake activities as well as Ca2+-release channel and Ca2+-pump protein contents in the ischemic-reperfused hearts. Unlike the protein kinase A-mediated phosphorylation in SR membranes, the CaMK-mediated phosphorylations at Ca2+-release channels, Ca2+ pump, and phospholamban were depressed in the ischemic hearts; these changes were prevented by preconditioning. These results indicate that ischemic preconditioning may exert beneficial effects on ischemia-reperfusion-induced alterations in SR function by preventing changes in Ca2+-release channel and Ca2+-pump protein contents in the SR membrane.
- calcium/calmodulin-dependent protein kinase phosphorylation
- protein kinase A phosphorylation
- sarcoplasmic reticulum calcium release
- sarcoplasmic reticulum calcium uptake
- sarcoplasmic reticulum phospholamban
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.
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/dt1.
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 cytochromec 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 the45Ca2+-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 μM45CaCl2(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 specific3H-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 (Bmax) and dissociation constant (K d) from the Scatchard plots.
Measurement of Ca2+ uptake.
Ca2+-uptake activity of the SR vesicles was determined by employing45Ca2+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 mM45CaCl2(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 γ-32P-labeled ATP ([γ-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 [γ-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).
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.
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.
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 ⋅ mg−1 ⋅ 15 s−1) and Ca2+-uptake (7.9 ± 0.4 nmol ⋅ mg−1 ⋅ min−1) 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 ⋅ mg−1 ⋅ 15 s−1; Ca2+ uptake: 23.7 ± 2.6 nmol ⋅ mg−1 ⋅ min−1;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).
To gain information regarding the effect of preconditioning on ryanodine-sensitive Ca2+-release channels, Ca2+-induced Ca2+ release from the Ca2+-loaded SR vesicles was determined in the absence and presence of 20 μM ryanodine. The results in Table 1 indicate a marked reduction in ryanodine-sensitive Ca2+ release due to IP during the preischemic phase. Whereas the ryanodine-sensitive Ca2+ release in postischemic and in ischemic-reperfused control hearts was markedly depressed compared with that in the preischemic control hearts, the values in the ischemic and the ischemic reperfused preconditioned hearts were significantly (P < 0.05) higher than those for the preischemic preconditioned hearts. In another set of experiments, specific [3H]ryanodine binding with SR preparations from control and preconditioned hearts was determined, and the results are shown in Table 1. IP was found to decrease the Bmax value for [3H]ryanodine binding in the preischemic phase. The Bmaxvalues for [3H]ryanodine binding in ischemic and ischemic-reperfused control hearts were markedly depressed compared with those in the preischemic control hearts. On the other hand, the Bmax values for [3H]ryanodine binding in the ischemic and the ischemic-reperfused preconditioned hearts were significantly (P < 0.05) higher than those for the corresponding preischemic hearts. It should be mentioned that these changes in [3H]ryanodine binding with SR preparations were not associated with any changes inK d values in all the groups indicated above. These experiments with [3H]ryanodine binding provide evidence that the changes in SR Ca2+ release observed in control and preconditioned hearts are not due to any experimental artifact but rather represent alterations in the SR Ca2+-release channels.
To show whether the observed changes in SR Ca2+ uptake were due to changes in the SR Ca2+-pump mechanism, Ca2+-stimulated ATPase activities in SR preparations from control and preconditioned hearts were determined. The results in Table 2 reveal a marked depression in the SR Ca2+-stimulated ATPase activity in the ischemic and the ischemic-reperfused heart compared with that in the preischemic control heart. Although IP did not change the Ca2+-stimulated ATPase activity in the preischemic phase, the data in Table 2 indicate that the values for Ca2+-stimulated ATPase activities in the ischemic and the ischemic- reperfused preconditioned hearts were significantly (P < 0.05) higher than those in the control hearts. It can be argued that the observed changes in SR Ca2+ uptake and SR Ca2+-stimulated ATPase are due to the isolation and purification of the SR vesicles. Accordingly, oxalate-supported Ca2+-uptake activities in the heart homogenate were studied because they reflect SR function in cardiomyocytes. The results in Table 2 show that IP not only depressed the oxalate-supported Ca2+ uptake in preischemic heart but also prevented the depression in oxalate-supported Ca2+ uptake due to sustained ischemia as well as ischemia-reperfusion. It can be seen from Table 3 that the SR protein yield in the ischemic-reperfused control heart was similar to that in the preconditioned ischemic reperfused heart. Furthermore, the activities of glucose-6-phosphatase and rotenone-insensitive NADPH cytochromec reductase, well-known microsomal enzymes, were purified to an equal extent (∼10-fold) in both control and preconditioned preparations. The activities of ouabain-sensitive Na+-K+-ATPase, a sarcolemmal enzyme, and cytochrome-coxidase, a mitochondrial enzyme, were enriched by 0.6- and 0.5-fold with respect to the heart homogenate activities, respectively (Table3). Similar results were obtained in preischemic and ischemic control and preconditioned hearts (n = 2 in each group). These results show that the SR preparations from control and experimental hearts were equally but minimally contaminated with other subcellular organelles.
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.
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).
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).
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.
The results reported in this study were supported by a grant from the Medical Research Council of Canada (MRC Group in Experimental Cardiology).
Address for reprint requests: N. S. Dhalla, Inst. of Cardiovascular Sciences, St. Boniface General Hospital Research Center, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6.
- Copyright © 1998 the American Physiological Society