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Integrative Physiology |
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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Key Words: Ca2+/calmodulin-dependent protein kinase cAMP-dependent protein kinase sarcoplasmic reticulum myocardial infarction congestive heart failure
| Introduction |
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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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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
Ser16phosphorylated 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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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
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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
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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
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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 CaMKrelated 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.
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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.
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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.
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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.
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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
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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.
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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.
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| Discussion |
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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
studies21 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
studies16 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 studies37 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 reports45 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 studies47 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 32Pi 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 |
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-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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