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(Circulation Research. 2000;86:596.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Sarcoplasmic Reticulum Ca²⁺/Calmodulin-Dependent Protein Kinase Is Altered in Heart Failure

Thomas Netticadan, Rana M. Temsah, Kenichi Kawabata, Naranjan S. Dhalla

From the Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.

Correspondence to Naranjan S. Dhalla, Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, 351 Taché Ave, Winnipeg, Manitoba R2H 2A6, Canada. E-mail cvso{at}sbrc.umanitoba.ca

	Abstract

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Abstract—Although Ca²⁺/calmodulin-dependent protein kinase-II (CaMK) is known to phosphorylate different Ca²⁺ cycling proteins in the cardiac sarcoplasmic reticulum (SR) and regulate its function, the status of CaMK in heart failure has not been investigated previously. In this study, we examined the hypothesis that changes in the CaMK-mediated phosphorylation of the SR Ca²⁺ cycling proteins are associated with heart failure. For this purpose, heart failure in rats was induced by occluding the coronary artery for 8 weeks, and animals with >30% infarct of the left ventricle wall plus septum mass were used. Noninfarcted left ventricle was used for biochemical assessment; sham-operated animals served as control. A significant depression in SR Ca²⁺ uptake and release activities was associated with a decrease in SR CaMK phosphorylation of the SR proteins, ryanodine receptor (RyR), Ca²⁺ pump ATPase (SR/endoplasmic reticulum Ca²⁺ ATPase [SERCA2a]), and phospholamban (PLB) in the failing heart. The SR protein contents for RyR, SERCA2a, and PLB were decreased in the failing hearts. Although the SR Ca²⁺/calmodulin-dependent CaMK activity, CaMK content, and CaMK autophosphorylation were depressed, the SR phosphatase activity was enhanced in the failing heart. On the other hand, the cAMP-dependent protein kinase–mediated phosphorylation of RyR and PLB was not affected in the failing heart. On the basis of these results, we conclude that alterations in SR CaMK-mediated phosphorylation may be partly responsible for impaired SR function in heart failure.

Key Words: Ca²⁺/calmodulin-dependent protein kinase • cAMP-dependent protein kinase • sarcoplasmic reticulum • myocardial infarction • congestive heart failure

	Introduction

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It is now well known that changes in intracellular Ca²⁺ handling by cardiomyocytes are intimately associated with defects in cardiac performance in the failing heart.^{1 2} Because the sarcoplasmic reticulum (SR) plays a pivotal role in regulating the intracellular concentration of Ca²⁺, abnormalities in cardiac contractile activity are generally explained on the basis of changes in SR function.^{2 3 4} Although previous studies have reported that SR Ca²⁺ uptake and release activities are depressed in the failing heart,^{5 6 7 8} the mechanisms underlying these alterations in heart failure are far from clear. Although a decrease in the SR Ca²⁺ cycling protein levels has been implicated as a factor underlying changes in the contractile function in the failing heart,^{9 10} no alterations in the SR Ca²⁺ pump ATPase (SR/endoplasmic reticulum Ca²⁺ ATPase [SERCA2a]) or phospholamban (PLB) proteins were observed in heart failure by some investigators.¹¹ Because Ca²⁺/calmodulin-dependent protein kinase-II (CaMK) and cAMP-dependent protein kinase A (PKA) are involved in the regulation of SR function via phosphorylation of different SR Ca²⁺ cycling proteins,¹² it is probable that a defect in these regulatory mechanisms may be of significance in the development of cardiac SR dysfunction and subsequent heart failure.

There is considerable evidence to suggest that protein phosphorylation and dephosphorylation play a critical role in regulating various cellular processes under physiological and pathophysiological conditions.^{12 13 14 15} We have recently reported that a defect in SR protein phosphorylation by endogenous SR CaMK may be partly responsible for SR dysfunction in ischemia-reperfused hearts.^{16 17} We have also reported that SERCA2a activity in the presence of Ca²⁺/calmodulin was depressed in congestive heart failure due to myocardial infarction.¹⁸ In view of these considerations, we sought to examine the status of CaMK phosphorylation of SR Ca²⁺ cycling proteins such as the Ca²⁺ release channel or ryanodine receptor (RyR), SERCA2a, and PLB in control and failing rat hearts. Our results indicate that endogenous CaMK phosphorylation of SR Ca²⁺ cycling proteins is depressed in heart failure due to myocardial infarction. It is suggested that this alteration may in part be responsible for abnormalities in SR function and subsequent attenuated cardiac performance in the failing heart.

	Materials and Methods

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The experimental protocol in the present investigation was approved by the Animal Care Committee of the University of Manitoba and conforms with Canadian Council on Animal Care Concerning the Care and Use of Experimental Animals (vol 1, 2nd ed, 1993).

Experimental Model
Myocardial infarction was induced by occlusion of the left anterior descending coronary artery in male Sprague-Dawley rats (175 to 200 g) according to the procedure described earlier.^{5 19} Sham-operated animals were treated in the same way, except that the coronary artery was not ligated. After 8 weeks of inducing myocardial infarction, randomly chosen animals were used for studying general characteristics and hemodynamic performance under in vivo conditions.^{5 19} Animals showing scar weight <25% of the left ventricle including septum were excluded.

Isolation of SR Vesicles
The SR preparation was isolated by the method of Netticadan et al.¹⁷ The purity of the membrane preparation was determined by measuring the activities of marker enzymes according to methods described earlier.⁵ The protein concentration was measured by the procedure of Lowry et al.²⁰

Measurement of Ca²⁺ Pump and Release Activities
Ca²⁺ uptake activities of the SR vesicles was determined by the procedure of Hawkins et al.²¹ Ca²⁺-stimulated ATPase activity was measured according to the method described elsewhere.¹⁸ The concentration of free Ca²⁺ in the assay medium for Ca²⁺ pump activities, as determined by the program of Fabiato,²² was 8.2 µmol/L. Ca²⁺ release activities of the SR vesicles was determined by the procedure of Temsah et al.²³

Measurement of Phosphorylation by Endogenous and Exogenous CaMK and Exogenous PKA
SR protein phosphorylation by endogenous and exogenous CaMK, as well as exogenous PKA, was determined by the procedure described by Netticadan et al.^{17 24}

Western Blot Analysis
The protein content of RyR, SERCA2a, and PLB was determined according to the procedure described by Temsah et al.²³ The SR -CaMK and ₃-CaMK (a subclass of the -CaMK subunit containing a second variable domain expressed predominantly in the heart) contents were determined by the procedure of Xu et al.²⁵

Analysis of CaMK Autophosphorylation
SR membranes were phosphorylated by endogenous CaMK, resolved on 4% to 18% SDS-polyacrylamide gradient gels, subjected to Western blot analysis, and probed for -CaMK.

Determination of PLB Phosphorylation at Thr17 and Ser16 Residues
SR membranes phosphorylated by endogenous CaMK and exogenous PKA were separated on 15% gels and subjected to Western blot analysis. Membranes were probed with antibodies specific to Thr17– and Ser16–phosphorylated PLB.

Measurement of CaMK and Phosphatase Activities
The CaMK and phosphatase activities of SR and cytosolic fractions were measured by using Upstate Biotechnology assay kits.¹⁷

Statistical Analysis
Results are expressed as mean±SE and statistically evaluated by the ANOVA test followed by the Student t test. P<0.05 was considered as the threshold for statistical significance between the control and experimental groups.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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The data given in Table 1 reveal an increase in heart weight, heart/body weight ratio, and right ventricle weight, indicating the presence of cardiac hypertrophy in the 8-week infarcted animals. The experimental animals showed the presence of ascites in the abdomen; 32% of the left ventricle wall plus septum mass was scar tissue. Lung congestion in the infarcted animals was evident from the increased wet weight/dry weight ratio of the lung. Hemodynamic assessment revealed an increase in the left ventricular end diastolic pressure without any change in the left ventricular systolic pressure or heart rate, whereas both the rate of pressure development and the rate of pressure fall were depressed in the infarcted animals (Table 1). These general characteristics and hemodynamic changes in the experimental animals are consistent with the presence of congestive heart failure in rats with a large infarct of the left ventricle 8 weeks after occlusion of the coronary artery.^{5 19}

View this table: [in this window] [in a new window]   Table 1. General Characteristics and Hemodynamic Parameters of Sham Control and Infarcted Rats

The yield of SR proteins from the viable (noninfarcted) left ventricle was not different from the control values (Table 2). The marker enzyme activities revealed minimal but equal cross-contamination with other subcellular organelles in both control and failing heart SR preparations (Table 2). Both the Ca²⁺ uptake and Ca²⁺ release activities in the SR preparations from the failing hearts were depressed (Table 2). Likewise, the SR Ca²⁺ pump ATPase activity in the failing heart was decreased (Table 2). The Ca²⁺ pump ATPase activities in both control and failing heart SR preparations were inhibited by 95% to 98% by thapsigargin (20 µmol/L) and cyclopiazonic acid (50 µmol/L), the well-known inhibitors of the SR Ca²⁺ pump. In another set of experiments, hearts from 4 sham control and 4 infarcted rats were used to determine SR Ca²⁺ uptake, Ca²⁺ release, and Ca²⁺ pump ATPase activity on assessing these animals hemodynamically. Changes in hemodynamic parameters and SR Ca²⁺ transport activities in animals with heart failure were similar to those reported in Tables 1 and 2.

View this table: [in this window] [in a new window]   Table 2. SR Proteins, Marker Enzymes, Ca2+-Uptake, Ca2+-Release, and Ca2+-Pump ATPase in Sham Control and Infarcted Rats

Because the SR Ca²⁺ uptake and release activities are regulated by CaMK and PKA phosphorylations of the Ca²⁺ cycling proteins,¹² the endogenous CaMK phosphorylation of SR proteins was studied in the sham control and failing hearts. Figure 1A shows the SR protein profile, Figure 1B depicts the corresponding autoradiogram of SR protein phosphorylation, and Figure 1C shows the analysis of phosphorylation of the RyR, SERCA2a, and PLB proteins. CaMK-mediated phosphorylation of all 3 proteins was significantly decreased (25% to 50%) in the failing heart SR membranes in comparison with controls. The identity of phosphorylated proteins of the SR preparations was established as reported earlier.^{16 17 21 24 25 26}

View larger version (34K): [in this window] [in a new window]   Figure 1. Endogenous SR CaMK-mediated phosphorylation of RyR, SERCA2a, high molecular weight (H) PLB, and low molecular weight (L) PLB in sham control (S) and failing (MI) hearts. A, SR protein profile. B, Corresponding autoradiogram showing phosphorylated substrates. C, Analysis of CaMK phosphorylation of RyR, SERCA2a, and PLB. PLB phosphorylation was the sum of PLB (H) and PLB (L) phosphorylations. Results are mean±SE of 4 separate experiments in each group. *P<0.05 vs control.

In view of the decrease in endogenous CaMK phosphorylation in the failing heart, endogenous SR CaMK and cytosolic CaMK activities were determined. Figure 2A shows that the endogenous Ca²⁺/calmodulin-dependent CaMK activity was significantly depressed, whereas the Ca²⁺/calmodulin-independent CaMK activity was not altered in the failing heart SR preparations. Figure 2B shows that the Ca²⁺/calmodulin-dependent cytosolic CaMK activity was not significantly changed in the failing hearts. To ensure that changes in SR endogenous CaMK phosphorylation and CaMK activity in the SR preparations are related to heart failure, 4 sham control and 4 infarcted rats were assessed hemodynamically and their hearts used for the analysis of SR CaMK–related activities. The results of this experiment were similar to those reported in Figures 1 and 2. Because the endogenous Ca²⁺/calmodulin-dependent CaMK activity was depressed in the failing hearts, the -CaMK and ₃-CaMK isoform contents of the SR were assessed in the sham control and failing hearts. Figures 3A and 3B show that both the -CaMK and the ₃-CaMK isoform contents were significantly depressed in failing heart SR preparations in comparison with controls. Figures 3C and 3D show that the concentrations of the SR proteins loaded for Western blot analysis of both the -CaMK (25 µg) and ₃-CaMK (20 µg) isoform contents were in the linear range.

View larger version (19K): [in this window] [in a new window]   Figure 2. A, Endogenous SR Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent CaMK activity in sham control (S) and failing (MI) hearts. B, Cytosolic Ca2+/calmodulin-dependent CaMK activity. Results are mean±SE of 4 separate experiments in each group. *P<0.05 vs control.

View larger version (35K): [in this window] [in a new window]   Figure 3. A and B, Autoradiogram depicting SR -CaMK and SR 3-CaMK protein bands and analysis of their contents in sham control (S) and failing (MI) hearts. Results are mean±SE of 4 separate experiments in each group. *P<0.05 vs control. C and D, Protein dependency of immunochemical detection of SR -CaMK and SR 3-CaMK.

Figure 4A shows ³²P incorporation into the 55-kDa fused-doublet band; this represents the Ca²⁺/calmodulin-dependent and Ca²⁺/calmodulin-independent autophosphorylated -CaMK band in the control and failing hearts. The identity of the -CaMK band is in agreement with other studies.^{25 27} A significant reduction in the Ca²⁺/calmodulin-independent as well as Ca²⁺/calmodulin-dependent autophosphorylation of CaMK was observed in the failing heart SR membranes in comparison with control (Figure 4B). This decrease seems to be nonspecific, because all of the other protein bands also exhibited a similar reduction.

View larger version (26K): [in this window] [in a new window]   Figure 4. Autophosphorylation of endogenous SR CaMK in sham control (S) and failing (MI) hearts. A, Autoradiogram depicting inducible SR CaMK autophosphorylation. B, Analysis of Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent CaMK autophosphorylation. Results are mean±SE of 4 separate experiments in each group. CM indicates calmodulin. *P<0.05 vs control.

To examine whether the kinase substrates remained capable of phosphorylation by CaMK after the isolation procedure, a set of control experiments with exogenous CaMK was carried out. The autoradiogram in Figure 5A shows that RyR, SERCA2a, and PLB in both groups were capable of undergoing phosphorylation. For comparative purposes, the endogenous CaMK phosphorylation of these proteins has been done in both groups, and the results observed are similar to those observed in Figure 1. It must, however, be noted that because of the different exposure times and the marked differences in intensities of the phosphorylated protein bands by the endogenous and exogenous CaMK, only the SERCA2a and PLB phosphorylated bands (by endogenous CaMK) are visible in Figure 5, whereas the phosphorylated RyR band is not visible in both groups. Figure 5B shows that the exogenous CaMK phosphorylation of RyR, SERCA2a, and PLB was reduced in the failing heart SR preparation by 10% to 15% only. In view of the 25% to 50% decrease in the endogenous CaMK phosphorylation of SR proteins (Figure 1), it appears that the endogenous CaMK in the failing heart SR is defective.

View larger version (45K): [in this window] [in a new window]   Figure 5. Exogenous SR CaMK-mediated phosphorylation of RyR, SERCA2a, high molecular weight (H) PLB, and low molecular weight (L) PLB in sham control (S) and failing (MI) hearts. A, Autoradiogram showing endogenous and exogenous phosphorylated substrates. B, Analysis of exogenous CaMK phosphorylation of RyR, SERCA2a, and PLB. PLB phosphorylation was the sum of PLB (H) and PLB (L) phosphorylations. Results are mean±SE of 3 separate experiments in each group. *P<0.05 vs control.

Because the decrease in the endogenous CaMK phosphorylation observed in the failing heart may also be due to a reduction in the protein content of the phosphorylated substrates, the protein contents of RyR, SERCA2a, and PLB were examined in the sham controls and failing hearts. Figure 6 shows that the levels of all 3 SR proteins were depressed in the failing heart SR preparations in comparison with controls. In view of the alterations observed in the failing heart SR protein content, it was necessary to establish the changes in phosphorylation per unit amount of substrate protein. Table 3 shows that RyR, SERCA2a, and PLB phosphorylation by CaMK as well as CaMK autophosphorylation were depressed because of a decrease in the protein content.

View larger version (20K): [in this window] [in a new window]   Figure 6. A through C, Autoradiograms depicting SR RyR, SERCA2a, and PLB protein bands and analysis of their contents in sham control (S) and failing (MI) hearts. Results are mean±SE of 3 separate experiments in each group. *P<0.05 vs control.

View this table: [in this window] [in a new window]   Table 3. Relative Ratio of CaMK Phosphorylation of SR Proteins to Their Respective Protein Contents

To establish the specificity of CaMK-mediated changes in SR phosphorylation, PKA phosphorylation of SR proteins was examined in the control and failing hearts. Figure 7A shows the SR protein profile, Figure 7B depicts the corresponding autoradiogram of SR protein phosphorylation, and Figure 7C shows the analysis of phosphorylation in both control and experimental groups. PKA phosphorylation of RyR and PLB in the failing heart SR preparation was not significantly different from that observed in the control. The identity of the phosphorylated proteins was established as reported earlier.^{16 17}

View larger version (32K): [in this window] [in a new window]   Figure 7. PKA-mediated phosphorylation of RyR as well as high molecular weight (H) PLB and low molecular weight (L) PLB in sham control (S) and failing (MI) hearts. A, SR protein profile. B, Corresponding autoradiogram showing phosphorylated substrates. C, Analysis of RyR and PLB phosphorylation. PLB phosphorylation was the sum of PLB (H) and PLB (L) phosphorylations. Results are mean±SE of 4 separate experiments in each group. *P<0.05 vs control.

To further confirm the decrease in the endogenous CaMK phosphorylation of PLB in the failing hearts, the sham control and failing SR membranes were probed with an antibody that specifically recognizes the CaMK-phosphorylated Thr17 residue in PLB. Figure 8A shows a significant reduction in the phosphothreonine content of PLB in the failing heart SR preparations in comparison with controls. These results are consistent with the decrease observed in PLB phosphorylation by the endogenous CaMK in the failing hearts shown in Figure 1. To establish that the exogenous PKA phosphorylation of PLB is not altered in the failing hearts, the sham control and failing SR membranes were probed with an antibody that specifically recognizes the PKA-phosphorylated Ser16 residue in PLB. Figure 8B shows no significant decrease in the phosphoserine content of the PLB in the failing heart SR preparations in comparison with controls. These results in Figure 8 confirm the unaltered status of PLB phosphorylation by exogenous PKA in the failing hearts.

View larger version (26K): [in this window] [in a new window]   Figure 8. A and B, Autoradiogram and analysis depicting Thr17-phosphorylated PLB and Ser16-phosphorylated PLB in sham control (S) and failing (MI) hearts. Results are mean±SE of 3 separate experiments in each group. *P<0.05 vs control.

To assess whether alterations occurring in the endogenous CaMK phosphorylation and protein content in the failing heart are associated with dephosphorylation, the endogenous SR and cytosolic phosphatase activities were determined in the control and failing hearts. A significant increase in the SR and cytosolic phosphatase activities were observed in the failing hearts in comparison with the control (Table 4). The contributions of the individual phosphatases in the total SR phosphatase activities were examined in the SR isolated from the sham control and failing hearts. A dose-dependent inhibition of the SR phosphatase activities in sham and failing hearts was observed with increasing concentrations of okadaic acid (Table 4). Approximately 20% to 30% of the SR phosphatase activity was inhibited by 100 µmol/L EGTA in sham and failing hearts. However, there were no significant differences between the sham and failing hearts.

View this table: [in this window] [in a new window]   Table 4. Phosphatase Activities in Sham Control and Infarcted Rats

	Discussion

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It is now well established that SERCA2a is inhibited by the unphosphorylated form of PLB in the SR membrane. When PLB undergoes phosphorylation by PKA at Ser16 or by CaMK at Thr17, the inhibitory effect of PLB on SERCA2a is relieved, and this results in an increase in the affinity of SERCA2a for Ca²⁺ as well as the V_max for Ca²⁺ transport.^{28 29 30 31} In addition to the phosphorylation of PLB, direct phosphorylation of SERCA2a by the endogenous CaMK has been reported to result in a 2-fold increase in V_max for ATP hydrolysis and Ca²⁺ transport.^{21 26 32} It should be mentioned that although SERCA2a phosphorylation appears to be an attractive mechanism for the regulation of SR Ca²⁺ pump, it has been a subject of controversy. On one hand, using Western blot as well as immunoprecipitation techniques, Xu et al²⁶ demonstrated SERCA2a as a substrate for the endogenous CaMK phosphorylation in SR vesicles isolated from the rabbit heart. This finding was further corroborated when Toyofuku et al³² used site-directed mutagenesis approaches to identify Ser38 as the CaMK phosphorylation site on SERCA2a. Moreover, serine phosphorylation of SERCA2a has been recently demonstrated in the intact beating heart.³³ On the other hand, Reddy et al³⁴ have reported that neither the purified reconstituted cardiac Ca²⁺-ATPase nor the Ca²⁺-ATPase in canine cardiac SR vesicles was phosphorylated by CaMK. Because the identity of a 100- to 105-kDa protein that was lightly phosphorylated in the longitudinal SR and appreciably phosphorylated in the junctional SR by the endogenous CaMK was not checked by Reddy et al,³⁴ it is difficult to draw any meaningful conclusion on the basis of their results. Nonetheless, the data in the present study support the observations of other investigators.^{26 32}

The functional consequence of SERCA2a phosphorylation has also been challenged by Odermatt et al,³⁵ who raised a technical concern regarding the use of EGTA in the absence of Ca²⁺ in previous studies^{21 32} to demonstrate the endogenous CaMK-mediated stimulation of Ca²⁺ uptake in the phosphorylated SR membranes. This concern was primarily due to the instability of SERCA2a in the absence of Ca²⁺ and presence of EGTA. In this regard, Xu and Narayanan³⁶ have demonstrated a 2-fold endogenous CaMK-mediated stimulation of Ca²⁺uptake when the control medium contained 5.4 µmol/L free Ca²⁺. Moreover, in this latter study,³⁶ the CaMK-stimulated Ca²⁺ uptake was observed to be due to the selective phosphorylation of SERCA2a by the endogenous CaMK without any contribution of CaMK phosphorylation of PLB. Under our experimental conditions, we have demonstrated in this study as well as in previous studies^{16 17} that SERCA2a undergoes phosphorylation by the endogenous CaMK in rat cardiac SR vesicles. Furthermore, we have observed a significant stimulation of SR Ca²⁺ uptake¹⁷ as well as Ca²⁺-ATPase activities¹⁸ in the presence of Ca²⁺ and calmodulin. In the present study, we observed a decrease in CaMK phosphorylation of SERCA2a and PLB in the failing heart. This decrease in the SR preparation may be due to a specific depression in the Ca²⁺/calmodulin-dependent endogenous CaMK activity, because the cytosolic Ca²⁺/calmodulin-dependent CaMK activity was found to be unaltered in the failing hearts. In contrast, Kirchhefer et al³⁷ have reported an increase in the CaMK activity in the ventricular homogenates isolated from failing human hearts due to dilated cardiomyopathy but no change in the CaMK activity in failing human hearts due to ischemic heart disease. Although the SR Ca²⁺/calmodulin-dependent CaMK activity was depressed in the failing hearts, the Ca²⁺/calmodulin-independent CaMK activity was unaltered. It should be pointed out that the Ca²⁺/calmodulin-independent activity was only one tenth the Ca²⁺/calmodulin-dependent activity, and its role in heart function is not clear at present. CaMK has been shown to undergo autophosphorylation at Thr236/Thr237 in the presence of Ca²⁺ and calmodulin.³⁸ Because autophosphorylation has been shown to be essential for the complete activation of the enzyme,³⁹ the depression in the Ca²⁺/calmodulin-dependent CaMK autophosphorylation may account for the reduced endogenous Ca²⁺/calmodulin-dependent CaMK activity observed in the failing heart. Because the CaMK that is endogenous to the SR has been recently identified as the -isoform,²⁷ the observed decrease of CaMK autophosphorylation and activity in the failing hearts may be attributed to a reduction in the -CaMK content as well as the ₃-CaMK content, ₃-CaMK being a subclass of the -CaMK subunit containing a second variable domain expressed predominantly in the heart. In contrast, Hoch et al⁴⁰ have recently demonstrated enhanced transcript levels as well as expression of ₃-CaMK in the total cardiac tissue homogenates isolated from the failing human hearts as a result of dilated cardiomyopathy. Although the apparent differences observed in CaMK activities as well as the CaMK contents in our study and the above-mentioned studies^{37 40} could be attributed to the difference in species as well as stage and type of heart failure, the contribution of changes in non-SR CaMK associated with particulate fractions cannot be ruled out at this time.

Because phosphorylation and dephosphorylation are complementary regulatory mechanisms, the decreased endogenous CaMK activity observed in the failing heart may be associated with an enhanced phosphatase activity. As expected, the endogenous SR and cytosolic phosphatase activities were increased in the failing hearts. Thus, the increased dephosphorylation due to enhanced phosphatase activity may be another factor responsible for the reduced CaMK phosphorylation of the SR proteins. Our results are supported by a recent study⁴¹ that reported an increase in the protein phosphatase-1 (PP1) activity in ventricular membrane vesicles isolated from the failing human heart. It has been shown that the endogenous phosphatase dephosphorylates PLB at the CaMK sites, resulting in decreased Ca²⁺uptake.⁴² It has been reported that submicromolar concentrations of okadaic acid inhibit PP1 activity, whereas this agent in nanomolar concentrations inhibits type 2A (PP2A) activity,⁴³ and 100 µmol/L EGTA inhibits type 2B phosphatase (PP2B) activity.⁴⁴ Our results indicate that the major contribution of the SR phosphatase activity is from PP1, whereas PP2A and PP2B contribute to 20% and to 20% to 30% of the total phosphatase activity, respectively, observed in both the sham and the failing hearts. However, it should be mentioned that it is difficult to estimate the exact relative amounts of the different types of phosphatases in our membrane preparations.

In view of the observations that the endogenous CaMK activity was depressed and the endogenous phosphatase activity was increased, it is evident that the delicate balance in the SR phosphorylation-dephosphorylation cycle may be disrupted in the failing heart. Our results also indicate that the phosphorylation of PLB by exogenous PKA was unaltered in the failing heart; this observation is in agreement with previous reports^{45 46} showing no change in PKA phosphorylation of PLB⁴⁵ or PKA-phosphorylated PLB-mediated Ca²⁺uptake⁴⁶ in the failing human heart. However, recently, Schmidt et al⁴⁷ showed a depression in PKA phosphorylation of PLB, whereas Schwinger et al⁴⁸ reported a decrease in Ser16 PLB phosphorylation in failing human hearts. The apparent differences observed in PKA phosphorylation in these studies^{47 48} and ours could be attributed to differences in species and type of heart failure. In view of the unaltered status of PKA phosphorylation of PLB in the failing hearts, it is suggested that alterations in SR Ca²⁺ uptake may occur partly but specifically because of some defects in the CaMK-mediated phosphorylation.

Although SR Ca²⁺ release is subjected to regulation by phosphorylation of RyR by different protein kinases, maximal incorporation of ³²P_i in this protein has been achieved with CaMK phosphorylation.⁴⁹ In the present study, we observed that the phosphorylation of RyR by endogenous CaMK but not the exogenous PKA was depressed in the failing heart. Decreased CaMK phosphorylation of RyR may be due to the depressed endogenous CaMK activity and increased endogenous phosphatase activity observed in the failing heart. Although Hain et al⁵⁰ earlier reported the inactivation of the Ca²⁺ release channel by the endogenous CaMK phosphorylation in SR vesicles incorporated into planar lipid membranes, Li et al⁵¹ recently showed that phosphorylation of RyR by the endogenous CaMK increases Ca²⁺ release channel activity in the intact cardiac myocytes during excitation-contraction coupling. Moreover, a unique phosphorylation site, Ser2809, for CaMK has been identified on the cardiac RyR, and the CaMK phosphorylation has been shown to activate the Ca²⁺ release channel in cardiac junctional SR vesicles or partially purified RyR fused into planar bilayers.⁵² In view of the role of endogenous CaMK phosphorylation in Ca²⁺ release, it is likely that the observed depression in RyR phosphorylation in the failing heart may result in the impairment of SR Ca²⁺ release. Because PKA phosphorylation of RyR was unaltered in the failing hearts, it is suggested that the alterations in SR Ca²⁺ release may occur partly but specifically as a result of some defects in the CaMK-mediated phosphorylation.

The alterations in endogenous CaMK phosphorylation of the RyR, SERCA2a, and PLB could also be attributed to a reduction in their respective protein contents observed in the failing heart. These decreases are consistent with our previous report.⁵³ However, it should be noted that the exogenous CaMK phosphorylation of these substrates in the failing hearts was minimally affected (10% decrease in SERCA2a and PLB phosphorylation and 15% decrease in RyR phosphorylation). This is in contrast to a relatively marked decrease in the endogenous CaMK phosphorylation of these substrates (25% to 30% decrease in SERCA2a and PLB, 50% decrease in RyR phosphorylations). These data confirm that in addition to alterations in the CaMK substrates due to heart failure, the endogenous CaMK is significantly impaired. Nevertheless, we cannot rule out the possibility of an incomplete recovery of the endogenous CaMK from the failing hearts due to the isolation procedure.

It is pointed out that our study is the first to demonstrate alterations in the endogenous CaMK and CaMK phosphorylation of SR Ca²⁺ release and Ca²⁺ pump proteins in heart failure. It appears that depression in the CaMK activity, reductions in the CaMK substrates, and increased phosphatase activity may represent major defects of the regulatory mechanisms governing the SR Ca²⁺ uptake and Ca²⁺ release processes in the failing heart. Because the observed changes in SR Ca²⁺ transport and CaMK activities in the failing hearts are similar to those reported under acute conditions of ischemia-reperfusion,^{16 17} it is possible that such alterations are due to oxidative stress.¹⁷ However, a detailed time-course study regarding the relationship between changes in SR function and the degree of oxidative stress is required to draw any meaningful conclusion in this regard.

	Acknowledgments

 
The work reported in this study was supported by a grant from the Medical Research Council of Canada (MRC Group in Experimental Cardiology). R.M.T. is a predoctoral fellow of the Heart and Stroke Foundation of Canada, and N.S.D. holds the MRC/Pharmaceutical Research and Development Chair in Cardiovascular Research supported by Merck Frosst Canada. We thank Dr H.A. Singer, Weis Center for Research (Danville, Pa), for providing us with the -CaMK antibody. We are also grateful to Dr P. Karczewski, Max Delbruck Center for Molecular Medicine (Berlin, Germany), for providing us with the ₃-CaMK antibody.

Received November 10, 1999; accepted December 1, 1999.

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