Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada
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ABSTRACT |
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Although the sarcoplasmic reticulum (SR) is known to regulate the intracellular concentration of Ca2+ and the SR function has been shown to become abnormal during ischemia-reperfusion in the heart, the mechanisms for this defect are not fully understood. Because phosphorylation of SR proteins plays a crucial role in the regulation of SR function, we investigated the status of endogenous Ca2+/calmodulin-dependent protein kinase (CaMK) and exogenous cAMP-dependent protein kinase (PKA) phosphorylation of the SR proteins in control, ischemic (I), and ischemia-reperfused (I/R) hearts treated or not treated with superoxide dismutase (SOD) plus catalase (CAT). SR and cytosolic fractions were isolated from control, I, and I/R hearts treated or not treated with SOD plus CAT, and the SR protein phosphorylation by CaMK and PKA, the CaMK- and PKA-stimulated Ca2+ uptake, and the CaMK, PKA, and phosphatase activities were studied. The SR CaMK and CaMK-stimulated Ca2+ uptake activities, as well as CaMK phosphorylation of Ca2+ pump ATPase (SERCA2a) and phospholamban (PLB), were significantly decreased in both I and I/R hearts. The PKA phosphorylation of PLB and PKA-stimulated Ca2+ uptake were reduced significantly in the I/R hearts only. Cytosolic CaMK and PKA activities were unaltered, whereas SR phosphatase activity in the I and I/R hearts was depressed. SOD plus CAT treatment prevented the observed alterations in SR CaMK and phosphatase activities, CaMK and PKA phosphorylations, and CaMK- and PKA-stimulated Ca2+ uptake. These results indicate that depressed CaMK phosphorylation and CaMK-stimulated Ca2+ uptake in I/R hearts may be due to a depression in the SR CaMK activity. Furthermore, prevention of the I/R-induced alterations in SR protein phosphorylation by SOD plus CAT treatment is consistent with the role of oxidative stress during ischemia-reperfusion injury in the heart.
calcium/calmodulin-dependent protein kinase; cAMP-dependent protein kinase; cardiac sarcoplasmic reticulum; oxidative stress
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INTRODUCTION |
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ALTHOUGH REPERFUSION IS essential for the survival of the ischemic myocardium, reperfusion of the ischemic heart, if not carried out within a certain period, has also been shown to induce further damage to the cardiac muscle (13). The deleterious effects of ischemia-reperfusion injury are evident under different clinical conditions including myocardial infarction and ischemic cardiomyopathy as well as during surgical interventions such as coronary angioplasty, aortocoronary bypass surgery, and cardiac transplantation (13). The occurrence of intracellular Ca2+ overload due to abnormal Ca2+ homeostasis in cardiomyocytes has been suggested as one of the mechanisms underlying ischemia-reperfusion injury (16). The generation of oxyradicals due to a sudden supply of oxygen to the ischemic myocardium has also been implicated as a mechanism responsible for ischemia-reperfusion injury (6). In fact, hearts reperfused after a prolonged period of global or regional ischemia, as well as those exposed directly to free radical-generating systems, have revealed similarities with respect to structural and functional alterations (5, 6, 9).
It is now well established that the sarcoplasmic reticulum (SR) plays a central role in the regulation of intracellular Ca2+ and cardiac contraction-relaxation processes. Although SR dysfunction has been shown to occur in ischemia-reperfused (I/R) hearts (7, 9, 23), the mechanisms underlying these alterations are not fully understood. In this regard, we have recently reported that ischemic preconditioning may render protection against the ischemia-reperfusion-induced damage to the myocardium by improving SR function (23). In view of the critical role of phosphorylations by endogenous Ca2+/calmodulin-dependent protein kinase (CaMK) and exogenous cAMP-dependent protein kinase (PKA) in regulating SR function (3, 8, 12, 20), we have now examined changes in CaMK- and PKA-mediated phosphorylation as well as CaMK- and PKA-stimulated Ca2+ uptake activities in SR preparations from control, ischemic (I), and I/R hearts. Because treatment with superoxide dismutase (SOD) plus catalase (CAT) has been shown to significantly recover the depressed myocardial function in I/R hearts (24, 29), we also examined the effects of a SOD plus CAT treatment on the ischemia-reperfusion-induced changes in CaMK- and PKA-mediated phosphorylation as well as on CaMK- and PKA-stimulated SR Ca2+ uptake activities. Our results suggest that the regulation of the SR Ca2+ pump by protein phosphorylation is impaired in the I and I/R hearts. The observation that SOD plus CAT treatment prevented changes in SR protein phosphorylation in the I/R hearts indicates that oxyradicals may be involved in the ischemia-reperfusion-induced alterations in SR function.
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METHODS |
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Perfusion of isolated heart.
Male Sprague-Dawley rats weighing 320-350 g were used for
experiments. The investigation was conducted under guidelines approved by the local Animal Care Committee in accordance with the standards of
the Canadian Council on Animal Care. Rats were anesthetized with a
mixture of ketamine (60 mg/kg body wt) and xylazine (10 mg/kg). Hearts
were rapidly excised, cannulated to the Langendorff apparatus, and
perfused at 37°C with Krebs-Henseleit (K-H) solution, which was
gassed with a mixture of 95% O2
and 5% CO2, pH 7.4, and which
contained (in mM) 120 NaCl, 25 NaHCO3, 11 glucose, 4.7 KCl, 1.2 KH2PO4,
1.2 MgSO4, and 1.25 CaCl2. The hearts were
electrically stimulated at a constant rate of 300 beats/min, and the
rate of perfusion was 10 ml/min. A water-filled latex balloon was
inserted in the left ventricle and connected to a pressure transducer
(model 1050BP; BIOPAC Systems, Goleta, CA) allowing functional
measurements of the left ventricle systolic pressure (LVSP), left
ventricle end diastolic pressure (LVEDP), and left ventricle developed
pressure (LVDP). LVDP was the difference between systolic and diastolic pressures. LVEDP was adjusted at 10 mmHg at the beginning of the experiment. The left ventricle pressures were differentiated to give
estimates of the rate of ventricular pressure development (+dP/dt) and rate of ventricular
pressure decline (dP/dt).
Data were recorded through an analog-to-digital interface (MP 100; BIOPAC Systems), processed, and stored by using Acknowledge 3.03 software for Windows (BIOPAC Systems).
Isolation of the SR preparation. The SR preparation was isolated by the method of Osada et al. (23) with slight modifications. Briefly, the left ventricle tissue was pulverized and homogenized twice for 20 s each at the half-maximal setting of a Polytron homogenizer (Brinkman, Westbury, NY). The homogenization buffer contained (in mM) 10 NaHCO3, 5 NaN3, 15 Tris · HCl, pH 6.8, and protease inhibitors (in µM) 1 leupeptin, 1 pepstatin, and 100 phenylmethylsulfonyl fluoride. The homogenate was then centrifuged for 20 min at 9,500 rpm (JA 20.0; Beckman), and the supernatant obtained was further centrifuged for 45 min at 19,000 rpm (JA 20.0; Beckman). The resulting supernatant was processed for the isolation of the cytosolic fraction. The pellet obtained was suspended in 8 ml of buffer containing 0.6 M KCl and 20 mM Tris · HCl, pH 6.8, and was recentrifuged at the same speed and for the same duration as in the previous step. The resulting pellet obtained was suspended in a mixture of 250 mM sucrose and 10 mM histidine, pH 7.0. For the isolation of the cytosolic fraction, the supernatant obtained after the first spin (19,000 rpm) was centrifuged at 100,000 rpm for 1 h and the resulting supernatant was employed. All steps were performed in the cold room (0-4°C), and the resulting SR and cytosolic suspensions were later used for various assays. The purity of the SR preparation 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 control, I, and I/R hearts were found to contain minimal (3-5%) but equal levels of cross contamination by other subcellular organelles.
Measurement of phosphorylation by endogenous CaMK and exogenous
PKA.
For phosphorylation experiments, the SR was isolated in the presence of
phosphatase inhibitors to prevent any dephosphorylation from occurring
during the isolation procedure. The homogenization buffer contained 10 nM microcystin LR and 1 mM sodium pyrophosphate. SR protein
phosphorylation by CaMK was determined according to the procedure
described by Netticadan et al. (22). The assay medium (total volume 50 µl) for phosphorylation by endogenous CaMK contained (in mM) 50 HEPES
(pH 7.4), 10 MgCl2, 0.1 CaCl2, 0.1 EGTA, 0.002 calmodulin,
0.8 [-32P]ATP (sp
act 200-300 cpm/pmol), and SR (30 µg protein). Phosphatase inhibitors microcystin LR (10 nM) and sodium pyrophosphate (1 mM) were
also added to the reaction mixture to inhibit any endogenous phosphatase activity. The initial concentration of free
Ca2+ as determined by the computer
program of Fabiato (10) was 3.7 µM. The
Ca2+/calmodulin dependence of
phosphorylation was monitored in parallel assays in which
Ca2+ (1 mM EGTA present) and
calmodulin were lacking in the assay medium. The assay medium (50 µl)
for phosphorylation by PKA contained (in mM) 50 HEPES (pH 7.4), 10 MgCl2, and 0.8 [
-32P]ATP (sp act
200-300 cpm/pmol), as well as SR (30 µg protein) and PKA
(catalytic subunit from the bovine heart; 5.6 µg). The PKA dependence
of phosphorylation was monitored in parallel assays without the PKA
catalytic subunit. The phosphorylation reaction was initiated by the
addition of
[
-32P]ATP after
preincubation of the assay medium for 3 min at 37°C. Reactions were
terminated after 2 min by adding 15 µl of 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
by an imaging densitometer with the aid of Molecular Analyst software,
version 1.3 (Bio-Rad, Hercules, CA).
Measurement of CaMK and PKA activities.
The CaMK and PKA activities of the cytosolic and SR preparations were
measured by using Upstate Biotechnology (Lake Placid, NY) assay kits.
The assay kit for CaMK activity is based on the phosphorylation of a
specific substrate peptide (KKALRRQETVDAL) by the transfer of the
-phosphate of
[
-32P]ATP by CaMK
II. Because the exogenous substrate was also phosphorylated by the SR
and cytosolic CaMK, the activity was calculated as the difference
between the values obtained in the presence and absence of the
exogenous substrate. The assay kit for PKA activity measurement is
based on the phosphorylation of a specific substrate (kemptide) by
using the transfer of the
-phosphate of
[
-32P]ATP by PKA.
The phosphorylated substrates in both assays were then separated from
the residual
[
-32P]ATP with P81
phosphocellulose paper and quantified by using a scintillation counter.
Measurement of Ca2+ uptake activity. The Ca2+ uptake activities of phosphorylated and unphosphorylated SR membranes were determined by the procedure of Hawkins et al. (12) with slight modifications. The standard reaction mixture (total volume 250 µl) contained (in mM) 50 Tris-maleate (pH 6.8), 5 NaN3, 5 ATP, 5 MgCl2, 120 KCl, 5 potassium oxalate, 0.1 EGTA, 0.1 45CaCl2 (12,000 cpm/nmol), and 0.025 ruthenium red. The initial free Ca2+ concentration in this medium, determined by the program of Fabiato (10), was 8.2 µM and was near saturation under the assay conditions (17). Ruthenium red was added to inhibit Ca2+ release channel activity under these conditions. The reaction was initiated by the addition of phosphorylated or unphosphorylated SR membranes to the Ca2+ uptake reaction mixture. The reaction was terminated after 1 min by filtering a 200-µl aliquot of the reaction mixture. The filters were washed, dried at 60°C for 1 h, and counted in a beta liquid scintillation counter. Endogenous CaMK and exogenous PKA phosphorylation assay media were the same as those described in Measurement of phosphorylation by endogenous CaMK and exogenous PKA. To measure Ca2+ uptake by the SR membranes in the absence of endogenous CaMK activators, namely, Ca2+ and endogenous calmodulin, 10 µM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) was included in the phosphorylation assay medium to inhibit the endogenous calmodulin, in addition to the 1 mM EGTA that was already present to chelate Ca2+. The exogenous catalytic subunit of PKA was excluded from the phosphorylation assay medium so that Ca2+ uptake by the SR membranes could be measured in the absence of PKA activator.
Measurement of phosphatase activity. The SR protein phosphatase activity was determined with the Ser/Thr phosphatase assay kit from Upstate Biotechnology. This assay was based on the dephosphorylation of the synthetic phosphopeptide KRpTIRR. The reaction was initiated by adding SR (30 µg) to microtiter wells with and without the synthetic substrate (200 µm) in a total assay volume of 25 µl for 30 min. The reaction was terminated by the addition of 100 µl of Malachite Green solution, and the reaction mixture was kept for 15 min for color development. The absorbance was read at 660 nM to determine the inorganic phosphate released. These experiments were performed both in the presence and absence of the exogenous substrate, and the phosphatase activity was calculated by subtracting the values in the absence of the exogenous substrate from those in its presence. The ratio of the contribution to Pi liberation of endogenous substrate to that of exogenous substrate was ~60:40.
Statistical analysis. Results are expressed as mean ± SE and were statistically evaluated by ANOVA test with post hoc testing using Schaffe's procedure. P < 0.05 was considered the threshold for statistical significance between the control and experimental groups.
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RESULTS |
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Cardiac performance.
Cardiac function in the isolated heart was monitored by measuring LVDP,
LVEDP, +dP/dt, and
dP/dt. Hearts subjected to
global ischemia for 30 min failed to generate LVDP,
+dP/dt, and
dP/dt but showed a marked
increase in LVEDP (Table 1).
Reperfusion of the I hearts for 60 min resulted in the recovery of the
contractile function (LVDP, +dP/dt,
and
dP/dt) to 16-25% of
the respective preischemic values; however, LVEDP was
further increased markedly (Table 1). The recovery of contractile
activity in I/R hearts was markedly improved by SOD plus CAT treatment;
this was reflected by the 80-85% recovery of LVDP,
+dP/dt, and
dP/dt compared with the control
heart preparations, as well as a marked reduction of LVEDP compared
with the I/R hearts (Table 1). It was interesting to observe that
nearly complete restoration of changes in LVDP, +dP/dt, and
dP/dt due to I/R occurred after
treatment with SOD plus CAT, whereas the reduction in LVEDP was not as
pronounced; in fact, LVEDP in these treated hearts was not appreciably
different from that observed in the I hearts (Table 1). Although LVEDP is considered to be a major determinant of LVDP, the observations in
this study suggest differences in the underlying mechanisms for these
changes in contractile parameters due to ischemia-reperfusion injury and SOD plus CAT protection.
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Phosphorylation by CaMK.
Endogenous CaMK phosphorylation of SR proteins in control, I, and I/R
hearts, in addition to that in I/R hearts treated with SOD plus CAT,
was studied. Figure
1A
shows the typical SDS-PAGE protein profile of SR preparations in
control, I, I/R, and SOD plus CAT treatment groups. From Fig.
1A it can be seen that a 97-kDa
protein band, unlike most of the other protein bands, was markedly
diminished in I and I/R hearts. Although the identity of this band
remains to be established, the reduction in its intensity was prevented
by treatment of the I/R hearts with SOD plus CAT. Figure
1B is an autoradiogram depicting SR
protein phosphorylation; the analysis of the phosphorylation of the
ryanodine receptor (RyR), the Ca2+
pump ATPase (SERCA2a), and phospholamban (PLB) in the different groups
is given in Fig. 1C. Identification of
RyR, SERCA2a, and PLB as the phosphorylated substrates was carried out
as described previously (12, 22, 23). CaMK phosphorylation of all three SR proteins was significantly decreased in the I and I/R hearts in
comparison to controls. Treatment of the I/R hearts with SOD plus CAT
significantly recovered the phosphorylation of all three SR proteins
when compared with the phosphorylation of I/R heart proteins. Because CaMK phosphorylation has been shown to
regulate the activity of cardiac SR
Ca2+ uptake (12), we examined the
effect of CaMK phosphorylation on
Ca2+ uptake. For the purpose of
comparison, Ca2+ uptake in the
unphosphorylated SR preparations in the presence of 10 µM W-7
and 1 mM EGTA to inhibit endogenous calmodulin and chelate endogenous
Ca2+, respectively, was also studied. Figure
2A shows Ca2+ uptake
activities of SR membranes phosphorylated in the presence and absence
of CaMK activators Ca2+ and
calmodulin. CaMK-stimulated Ca2+
uptake was depressed significantly in the I and I/R hearts compared with that in controls. SOD plus CAT treatment of the I/R hearts significantly improved the CaMK-stimulated
Ca2+ uptake compared with that for
I/R hearts; the levels of stimulation in control, I, I/R, and SOD plus
CAT treatment hearts were 303, 142, 212, and 338%, respectively. The
low Ca2+ uptake values for the
unphosphorylated (without Ca2+ or
calmodulin) SR membranes from control, I, I/R, and SOD plus CAT
treatment hearts indicate that the calmodulin present in all groups was
fully inhibited by the presence of W-7 and that endogenous Ca2+ was chelated by the presence
of EGTA in the assay medium.
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Phosphorylation by PKA.
To test if the observed changes in SR protein phosphorylation were
limited to the CaMK system, the phosphorylation of SR proteins by PKA
in control, I, I/R, and SOD plus CAT treatment I/R hearts was also
studied. Because appreciable PKA activity was not detectable in the SR,
exogenous PKA was used to examine the PKA phosphorylation of SR
proteins. Figure
3A shows
the typical SDS-PAGE protein profile of SR preparations in control, I,
I/R, and SOD plus CAT treatment groups. Figure
3B is an autoradiogram depicting SR
protein phosphorylation, and Fig. 3C
shows the analysis of the phosphorylation of PLB for the different
groups. The identification of PLB as the phosphorylated substrate was
established as previously shown (23). PKA phosphorylation of PLB was
significantly decreased only in the I/R hearts compared with controls.
Treatment of the I/R hearts with SOD plus CAT significantly prevented
changes in the phosphorylation of PLB when compared with results for
the I/R group. Because PKA phosphorylation has been shown to regulate
Ca2+ uptake (20), we investigated
the effect of PKA phosphorylation on SR
Ca2+ uptake. Figure
4A shows
the Ca2+ uptake of SR membranes
phosphorylated in the presence and absence of exogenous PKA. It should
be pointed out that the control SR Ca2+ uptake activity in the
absence of PKA was markedly less than that reported by us earlier (23);
however, the pattern of changes due to ischemia or
ischemia-reperfusion was similar to that observed previously
(23). The reason for these low
Ca2+ uptake values appears to be
the batch of animals and reagents employed in this study. The
PKA-stimulated Ca2+ uptake was
significantly depressed in the I/R hearts compared with controls.
Treatment of hearts with SOD plus CAT significantly improved the
PKA-stimulated Ca2+ uptake
compared with results for the I/R group; the levels of stimulation of
Ca2+ uptake in control, I, I/R,
and SOD plus CAT treatment hearts were 150, 150, 124, and 200%,
respectively. Because both the exogenous PKA phosphorylation and
PKA-stimulated Ca2+ uptake
were significantly depressed in the I/R hearts, PKA activities in the
cytosolic fractions from all groups were estimated. Figure 4B shows that the cytosolic PKA activity was not affected by
ischemia and ischemia-reperfusion.
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SR phosphatase activity.
Because the observed changes in endogenous CaMK phosphorylation in the
I and I/R groups may be associated with dephosphorylation, the
endogenous phosphatase activity in the SR was measured. A significant
decrease in the phosphatase activity in the I and I/R groups compared
with controls was observed (Fig. 5). SOD
plus CAT treatment of the I/R hearts significantly recovered the
phosphatase activity in the SR compared with results for the I/R group.
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DISCUSSION |
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Our results show a depression of cardiac function and decreased SR Ca2+ uptake in hearts subjected to ischemia as well as ischemia-reperfusion. Impaired cardiac performance and depressed SR Ca2+ uptake have been reported to occur in I/R hearts (9, 23). In this study we have shown that the CaMK-mediated phosphorylation of SR proteins is depressed in both the I and I/R groups of rat hearts, whereas the PKA-mediated phosphorylation is decreased in the I/R hearts only. These results are in agreement with earlier reports concerning the effects of ischemia-reperfusion on changes in CaMK-mediated phosphorylation in rat hearts (23) as well as in PKA-mediated phosphorylation in pig hearts (27, 31). It should be noted that CaMK is known to phosphorylate SR proteins such as SERCA2a, RyR, and PLB but that PKA phosphorylates PLB only (12, 22, 23, 34). Whereas the CaMK-mediated phosphorylation of SERCA2a has been shown to result in an increase in ATP hydrolysis and Ca2+ transport (12, 30, 34), the phosphorylation of PLB by CaMK and PKA has been reported to relieve the inhibitory action of PLB on SERCA2a, resulting in an increase in the affinity of SERCA2a for Ca2+ (8, 26). In addition, the phosphorylation of PLB by PKA has also been shown to increase the maximal velocity of Ca2+ uptake (3, 17, 18). Because both PKA- and CaMK-mediated phosphorylations have been demonstrated to occur in vivo (32), it appears that the phosphorylation of SR proteins plays a critical role in regulating the SR function. Accordingly, the observed alterations in SR protein phosphorylations due to ischemia as well as ischemia-reperfusion can be seen to represent important biochemical abnormalities in cardiac dysfunction in ischemic heart disease.
It is pointed out that CaMK is endogenous to the SR membrane and has
been identified as the -isoform (4). The decrease in CaMK-mediated
phosphorylation of SERCA2a and PLB during ischemia for a period
of 30 min and during reperfusion for 1 h is in agreement with our
previous report (23). Such a depression may explain the observed
decrease in the CaMK-stimulated
Ca2+ uptake in the SR vesicles in
the I and I/R hearts. In fact, the decrease in CaMK activity in the SR
membrane due to ischemia may account for the reduced
phosphorylation of the SERCA2a and PLB. Because cytosolic CaMK activity
was not altered during ischemia, our results indicate a
specific impairment of the SR CaMK only. Because phosphorylation and
dephosphorylation are complementary regulatory mechanisms, the
decreased SR phosphorylation activity observed in the I and I/R hearts
may be due to an increase in the endogenous phosphatase activity.
Contrary to our expectations, the endogenous phosphatase activity in
the SR membrane was depressed in the I and I/R hearts. It has been
shown that the endogenous phosphatase is a type 1 phosphatase (28),
which dephosphorylates PLB at both the PKA and the CaMK sites,
resulting in decreased SR Ca2+
uptake (19). Our results indicate that ischemia for 30 min did
not affect the phosphorylation of PLB by exogenous PKA or the
PKA-stimulated Ca2+ uptake
activity. This observation is in agreement with another report (27)
showing that there was no change in the PKA phosphorylation of PLB in
the pig heart when the ischemic period was of 30-min duration; however,
longer periods of ischemia depressed the SR PLB
phosphorylation. In our study PKA phosphorylation of PLB and PKA-stimulated Ca2+ uptake
activities were found to be depressed in the I/R hearts. The reduced
phosphorylation of PLB by PKA observed in the I/R heart is also
consistent with another report indicating a similar decrease after 30 min of myocardial ischemia followed by 2 h of reperfusion (31).
Although PKA phosphorylation of PLB was reduced in the I/R heart, the
cytosolic PKA activity was unaltered because of
ischemia-reperfusion, indicating no impairment of the cytosolic PKA. The depressed phosphorylation by CaMK and PKA in the I/R hearts
may be due to a decrease in the PLB protein content of the SR membrane
(data not shown). Because the endogenous CaMK and endogenous
phosphatase activities were depressed in both the I and I/R hearts, it
may be suggested that the balance of the phosphorylation-dephosphorylation cycle in the I and I/R hearts was not
altered. To the best of our knowledge, this is the first report
demonstrating simultaneous impairment of the SR CaMK and CaMK-stimulated Ca2+ uptake as
well as the SR phosphatase in the I and I/R hearts.
It is now well accepted that SR Ca2+ cycling in the cardiac muscle is dependent on the rate of Ca2+ uptake and release. Although defects in SR Ca2+ release in the I/R hearts have been reported (7, 9), the mechanisms underlying these alterations are not yet clear. The RyR has been shown to undergo phosphorylation by several protein kinases, and maximal incorporation of 32Pi has been achieved with CaMK phosphorylation (15). Besides, a unique phosphorylation site for CaMK (serine 2809 residue) on the cardiac RyR has been identified (33). Although CaMK- and PKA-mediated phosphorylation has been shown to occur under physiological conditions (32), PKA phosphorylation of the SR membranes under the experimental conditions used in this study resulted in weak phosphorylation of the RyR. In the present study we observed that the phosphorylation of RyR by endogenous CaMK was depressed in both the I and I/R hearts. An increase in the Ca2+ release channel activity in intact cardiac myocytes during excitation-contraction coupling resulting from SR CaMK-mediated phosphorylation of RyR has been recently reported (21). In view of the role of endogenous CaMK phosphorylation in Ca2+ release, it may be suggested that depressed RyR phosphorylation in both the I and I/R hearts may account for the impaired Ca2+ release from the SR vesicles.
Although the beneficial effects of antioxidant treatment for the I/R hearts have been documented (29), there is some controversy regarding the effectiveness of SOD plus CAT in preventing contractile dysfunction due to ischemia-reperfusion. There are reports showing beneficial effects in the I/R hearts (11) and in the oxyradical-perfused hearts (14), but no protection for the I/R hearts has also been reported (25). In the latter study, 15 min of ischemia depressed SR Ca2+ uptake, which was normalized completely by reperfusion with or without SOD plus CAT treatment; however, the contractile dysfunction due to ischemia-reperfusion was not prevented. The difference in the results obtained in this and our study with respect to contractile dysfunction could be due to 1) the ischemic models used, i.e., global ischemia vs. regional ischemia, 2) the animals used (rats vs. dogs), 3) the durations of ischemia and reperfusion (30 min of ischemia and 1 h of reperfusion vs. 15 min of ischemia and 15 min of reperfusion, and 4) the duration of SOD plus CAT treatment, 80 vs. 161/2 min. In our study, not only did SOD plus CAT treatment improve the cardiac performance but also the reductions in endogenous CaMK activity, CaMK phosphorylation, and CaMK-stimulated Ca2+ uptake, as well as exogenous PKA phosphorylation and PKA-stimulated Ca2+ uptake in the I/R hearts were attenuated. In addition to preventing changes in the endogenous CaMK activity, SOD plus CAT also attenuated alterations in the endogenous phosphatase activity; these effects would render protection to the SR phosphorylation-dephosphorylation cycle in the I/R hearts. Furthermore, SOD plus CAT treatment was effective in recovering the endogenous CaMK phosphorylation of RyR in the I/R hearts, suggesting an improved SR Ca2+ release. Our results indicating beneficial effects of SOD plus CAT treatment are further supported by a recent report showing the protective effects of membrane phosphorylation on the cardiac SR SERCA2a against chlorinated oxidants in vitro (2). Nonetheless, in view of the well-accepted role of SOD plus CAT in scavenging oxyradicals and active species of oxygen (11, 14, 29), it is likely that oxidative stress may be playing a crucial role in inducing SR dysfunction in the I/R hearts. This is a new observation because there is no report showing the beneficial effects of SOD plus CAT in preventing alterations in SR phosphorylation and dephosphorylation in the I/R hearts.
In conclusion, the SR CaMK, unlike the cytosolic PKA, is affected by ischemia and ischemia-reperfusion. Moreover, alterations in SR protein phosphorylation may result in altered SR Ca2+ uptake and release. Treatment with SOD plus CAT may improve SR Ca2+ uptake and release in the I/R hearts by protecting changes in the SR protein phosphorylation. Because SOD plus CAT treatment is known to scavenge oxyradicals and other species of active oxygen metabolites (6, 24, 29), it is reasonable to presume that the observed beneficial effects of this intervention are due to attenuation of oxidative stress in the I/R hearts. However, it should be noted that the depressions of CaMK- and PKA-mediated phosphorylations, SR CaMK activity, SR Ca2+ uptake activities in the absence or presence of PKA, and SR phosphatase activity in the I/R hearts were not greater than those observed in the I hearts. In view of the fact that the degree of oxidative stress in the I/R hearts is greater than that in the I hearts (6, 13, 24), it can be argued that the changes in the above-mentioned parameters in both I and I/R hearts may not be entirely due to the function of oxyradicals and oxidants. Thus the involvement of other ischemic effects such as a decrease in the intracellular pH, depletion of high-energy phosphate stores, the occurrence of intracellular Ca2+ overload, and proteolysis (9, 13, 14, 16, 29) cannot be ruled out when indicating the role of oxidative stress in cardiac depression and SR dysfunction.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Medical Research Council of Canada (MRC Group in Experimental Cardiology). R. Temsah was a predoctoral fellow of the Univ. of Manitoba, M. Osada was a postdoctoral fellow of the Heart & Stroke Foundation of Canada, and N. S. Dhalla held the MRC/PMAC Chair in Cardiovascular Research supported by Merck Frosst Canada.
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FOOTNOTES |
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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, Inst. of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, Manitoba R2H 2A6, Canada (E-mail: cvso{at}sbrc.umanitoba.ca).
Received 26 October 1998; accepted in final form 9 May 1999.
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