Am J Physiol Heart Circ Physiol
281: H325–H333, 2001.
Glucocorticoid modulation of protein phosphorylation
and sarcoplasmic reticulum function in rat myocardium
M. K. RAO, A. XU, AND N. NARAYANAN
Department of Physiology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
Received 10 August 2000; accepted in final form 5 March 2001
have suggested an apparent involvement of corticosteroids in the maintenance of
myocardial function. Thus it is well known that adrenalectomized animals, unless supported by maintenance doses of corticosteroids, gradually develop a
form of circulatory decompensation (12). Lefer (19)
observed a marked time-dependent decrease in contractile force development by papillary muscles iso-
lated from adrenalectomized rats. Treatment of adrenalectomized rats in vivo or cardiac muscle in vitro
with the synthetic glucocorticoid dexamethasone prevented the deterioration in contractile performance of
cardiac muscle, and it was suggested that dexamethasone exerted a direct effect on the myocardium, possibly via effects on glycogen metabolism and on electrolyte balance (19). It has also been reported that
dexamethasone treatment significantly enhanced the
development of contractile tension and increased the
velocity of contraction and relaxation in cardiac muscle
from dogs, cats, and rabbits (31). While these observations suggest a likely role for glucocorticoids in the
maintenance of normal contractile function of the
heart, the cellular processes affected by glucocorticoids
and the biochemical mechanisms underlying their action(s) have not been clarified.
By virtue of its ability to control cytosolic Ca2⫹ concentration, the sarcoplasmic reticulum (SR) plays a
central role in contractile force development and the
speed of contraction and relaxation in heart muscle (3).
Conceivably, the ability of glucocorticoids to augment
cardiac contractile function may arise from their ability to influence the Ca2⫹ sequestration and Ca2⫹ release functions of the SR. Consistent with this possibility, we observed previously (23) that cardiac SR
vesicles isolated from adrenalectomized rats exhibit
diminished rates of ATP-energized Ca2⫹ uptake compared with SR vesicles from control rats and that
dexamethasone treatment of adrenalectomized rats results in improved Ca2⫹ uptake activity of SR. The
major Ca2⫹-cycling proteins in the SR include the
Ca2⫹-sequestering ATPase (Ca2⫹-ATPase), the Ca2⫹storage protein calsequestrin (22), the ryanodine receptor/Ca2⫹-release channel (RyR-CRC) (4), and the
Ca2⫹-ATPase-regulatory protein phospholamban (17,
34, 38). In its unphosphorylated state, phospholamban
is thought to diminish the Ca2⫹ sensitivity of Ca2⫹ATPase; phosphorylation of phospholamban by cAMPdependent protein kinase (PKA) or Ca2⫹/calmodulindependent protein kinase II (CaM kinase II) restores
the Ca2⫹ sensitivity (17, 34, 38). Besides phospholamban, calmodulin and CaM kinase are tightly associated
with cardiac SR and have been implicated in the mod-
Address for reprint requests and other correspondence: N. Narayanan, Dept. of Physiology, Medical Sciences Bldg., Univ. of Western
Ontario, London, Ontario, Canada N6A 5C1 (E-mail: njanoor.
[email protected]).
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. Section 1734
solely to indicate this fact.
adrenalectomy; calcium/calmodulin-dependent protein kinase II; calcium adenosinetriphosphatase; calcium transport
A NUMBER OF STUDIES
http://www.ajpheart.org
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H325
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Rao, M. K., A. Xu, and N. Narayanan. Glucocorticoid
modulation of protein phosphorylation and sarcoplasmic reticulum function in rat myocardium. Am J Physiol Heart Circ
Physiol 281: H325–H333, 2001.—To decipher the mechanism(s) underlying glucocorticoid action on cardiac contractile function, this study investigated the effects of adrenalectomy and dexamethasone treatment on the contents of
sarcoplasmic reticulum (SR) Ca2⫹-cycling proteins, their
phosphorylation by endogenous Ca2⫹/calmodulin-dependent
protein kinase II (CaM kinase II), and SR Ca2⫹ sequestration
in the rat myocardium. Cardiac SR vesicles from adrenalectomized rats displayed significantly diminished rates of ATPenergized Ca2⫹ uptake in vitro compared with cardiac SR
vesicles from control rats; in vivo administration of dexamethasone to adrenalectomized rats prevented the decline in
SR function. Western immunoblotting analysis showed that
the relative protein amounts of ryanodine receptor/Ca2⫹release channel, Ca2⫹-ATPase, calsequestrin, and phospholamban were neither diminished significantly by adrenalectomy nor elevated by dexamethasone treatment. However,
the relative amount of SR-associated CaM kinase II protein
was increased 2.5- to 4-fold in dexamethasone-treated rats
compared with control and adrenalectomized rats. Endogenous CaM kinase II activity, as judged from phosphorylation
of ryanodine receptor, Ca2⫹-ATPase, and phospholamban
protein, was also significantly higher (50–80% increase) in
the dexamethasone-treated rats. The stimulatory effect of
CaM kinase II activation on Ca2⫹ uptake activity of SR was
significantly depressed after adrenalectomy and greatly enhanced after dexamethasone treatment. These findings identify the SR as a major target for glucocorticoid actions in the
heart and implicate modification of the SR CaM kinase II
system as a component of the mechanisms by which dexamethasone influences SR Ca2⫹-cycling and myocardial contraction.
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DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
ulation of the Ca2⫹ uptake and release functions of the
SR through direct phosphorylation of Ca2⫹-ATPase
(11, 26–28, 30, 40, 44–47) and RyR-CRC (10, 39, 43).
As part of an attempt to decipher the mechanisms
underlying glucocorticoid modulation of cardiac contractile function, the present study investigated the
effects of adrenalectomy and dexamethasone treatment on the contents of major SR Ca2⫹-cycling proteins, their phosphorylation by SR-associated CaM kinase II, and SR Ca2⫹ sequestration function in the rat
myocardium.
METHODS
nH
⫹ 关Ca2 ⫹ 兴nH兲
v ⫽ V max关Ca2 ⫹ 兴nH/共K0.5
where v is the measured Ca2⫹ uptake rate at a given Ca2⫹
concentration, Vmax is the maximum rate reached, K0.5 is the
Ca2⫹ concentration giving half of Vmax, and nH is the equivalent to the Hill coefficient.
Ca2⫹-ATPase activity of the SR membrane vesicles was
determined as described previously (25) using the assay
conditions specified below. The incubation medium used for
the Ca2⫹-ATPase assay was identical to that described for
Ca2⫹ uptake except that [␥-32P]ATP was used instead of
nonradioactive ATP and nonradioactive CaCl2 was used instead of 45CaCl2. The assays were performed in the absence
and presence of thapsigargin (TG). When present, the concentration of TG in the assay medium was 0.1 ␮M, the
concentration found to produce complete inhibition of Ca2⫹
sequestration by the SR (35). In these experiments, the
TG-inhibitable ATP hydrolysis was defined as the Ca2⫹ATPase activity (designated “TG-sensitive Ca2⫹-ATPase activity” in RESULTS). The ATPase reaction was initiated by the
addition of SR after preincubation of the rest of the assay
components for 3 min at 37°C and was allowed to proceed for
3 min. The longer reaction time used for the measurement of
Ca2⫹-ATPase activity (i.e., 3 min as opposed to 2 min used for
the measurement of Ca2⫹ uptake) permitted better quantitative resolution of Ca2⫹-ATPase activity from the high level
of basal Mg2⫹-ATPase activity associated with rat cardiac SR
vesicles (23).
Immunoblotting of SR Ca2⫹-cycling proteins. Western immunoblotting techniques were used for the detection and
estimation of the relative amounts of SR Ca2⫹-cycling proteins in the rat heart. For immunoassay of RyR-CRC, Ca2⫹ATPase, phospholamban, calsequestrin, and CaM kinase II,
rat heart homogenate (25 ␮g protein/lane) and cardiac SR
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Chemicals. Reagents for electrophoresis were obtained
from Bio-Rad laboratories (Mississauga, ON, Canada),
[␥-32P]ATP was purchased from Amersham (Oakville, ON,
Canada), and 45CaCl2 was obtained from NEN (Mississauga,
ON, Canada). Dexamethasone was obtained from Organon
Teknika (Toronto, ON, Canada). Monoclonal antibodies
against the proteins constituting the RyR, SR Ca2⫹-ATPase,
and calsequestrin were purchased from Affinity BioReagents
(Golden, CO). Antiphospholamban monoclonal antibody was
obtained from Upstate Biotechnology (Lake Placid, NY). Anti-␦-CaM kinase II polyclonal antibody was a generous gift
from H. A. Singer (Albany Medical College, Albany, NY). All
other chemicals were obtained from Sigma (St. Louis, MO).
Animals. Male Wistar rats weighing 250–300 g were purchased from Charles River (St. Constant, PQ, Canada). On
arrival, the rats were housed individually in plastic cages in
the Health Sciences Center animal care facility of this institution at 23°C on a 12:12-h light-dark cycle. The investigations were conducted under guidelines approved by the local
Animal Care Committee in accordance with the standards of
the Canadian Council on Animal Care. The rats were anesthetized with metofane, and bilateral adrenalectomy was
performed as described previously (23). Control animals were
sham operated. The adrenalectomized animals were divided
into two groups: one group received dexamethasone via a
subcutaneously implanted ALZET osmotic mini pump (model
2001, flow rate 1 ␮l/h) that delivered dexamethasone at a
rate of 4 ␮g/h for 7 days, and the second group received no
dexamethasone. The adrenalectomized animals were maintained on normal saline to prevent volume depletion; the
control animals were given tap water. All animals had free
access to food (Purina Chow containing 20% protein). The
animals were killed 7 days after surgery, and the ventricular
myocardium was used for experiments.
Isolation of SR vesicles. SR membrane vesicles were isolated from the ventricular myocardium of control, adrenalectomized, and adrenalectomized/dexamethasone-treated rats
according to the procedure described previously (15). After
isolation, the SR vesicles were suspended in 10 mM Trismaleate (pH 6.8) containing 100 mM KCl, quick-frozen in
liquid N2, and then stored at ⫺80°C. Protein was determined
by the method of Lowry et al. (21) using bovine serum
albumin as standard. The yield of SR membranes from the
hearts of the three groups of rats was similar (⬃1.5 mg
protein/g wet tissue). The relative purity of the cardiac SR
vesicles from the three groups of rats did not differ as judged
from essentially similar protein profiles revealed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE, see RESULTS).
Preparation of muscle homogenates. In addition to the SR,
homogenates from control, adrenalectomized, and adrenalectomized/dexamethasone-treated rat hearts were also used in
some experiments. The homogenates were prepared by ho-
mogenizing the ventricular tissue in 10 volumes (based on
tissue weight) of 10 mM Tris-maleate-100 mM KCl buffer
(pH 6.8) using a polytron PT-10 homogenizer (three 15-s
bursts with 30-s intervals between bursts, setting 6, Brinkman; Westbury, NY). The homogenates were filtered through
four layers of cheese cloth and used for experiments.
Ca2⫹ transport and Ca2⫹ ATPase assays. ATP-dependent,
oxalate-facilitated Ca2⫹ uptake by cardiac SR vesicles was
determined using the Millipore filtration technique as described previously (25). The standard incubation medium for
Ca2⫹ uptake (total volume 250 ␮l) contained (in mM) 50
Tris-maleate (pH 6.8), 5 MgCl2, 5 NaN3, 120 KCl, 0.1 EGTA,
5 potassium oxalate, 5 ATP and 0.1 45CaCl2 (⬃8,000 cpm/
nmol, 8.2 ␮M free Ca2⫹) and cardiac SR vesicles (7.5 ␮g of
protein). In experiments where Ca2⫹ concentration dependence was studied, the EGTA concentration in the assay
medium was held constant at 0.1 mM and the amount of total
45
CaCl2 added was varied to yield the desired free Ca2⫹. The
initial free Ca2⫹ concentration was determined using the
computer program of Fabiato (9). To evaluate the effects of
endogenous CaM kinase II-mediated phosphorylation on
Ca2⫹ uptake, the assays were performed in the absence of
calmodulin and in the presence of 1 ␮M calmodulin in the
incubation medium. Other modifications to the standard assay medium are specified in the figure legends. The Ca2⫹uptake reaction was initiated by the addition of SR to the rest
of the assay components, preincubated for 3 min at 37°C, and
allowed to proceed for 2 min, during which the Ca2⫹ uptake
rates were found to be linear. The data on Ca2⫹ concentration
dependence on Ca2⫹ uptake were analyzed by nonlinear
regression curve fitting using the SigmaPlot scientific graph
program (Jandel Scientific) run on an IBM personal computer. The data were fit to the equation
DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
RESULTS
Effects of adrenalectomy and dexamethasone treatment on the Ca2⫹-sequestration function of cardiac SR.
The ATP-dependent, oxalate-facilitated Ca2⫹ uptake
by SR vesicles is a useful parameter commonly used to
measure the Ca2⫹-pump (Ca2⫹-ATPase) function of SR
in vitro. The results presented in Fig. 1 compare the
rates of ATP-driven Ca2⫹ uptake into cardiac SR vesicles from control, adrenalectomized, and adrenalectomized/dexamethasone-treated rats, measured in the
absence and presence of Ca2⫹-release channel blockers. These assays were performed at a fixed Ca2⫹
concentration (8.2 ␮M) adequate for maximal activation of Ca2⫹ uptake (cf Fig. 2). At concentrations
known to block Ca2⫹ release (5, 48), ruthenium red (25
␮M) and ryanodine (625 ␮M) both stimulated the rates
of Ca2⫹ uptake by SR significantly in control and adrenalectomized/dexamethasone-treated rats. A similar
tendency was also observed in the adrenalectomized
group, but the difference was not statistically significant. The membranes from adrenalectomized rats
showed significantly reduced (⬃40% decrease) rates of
Ca2⫹ uptake compared with the membranes from control animals in both the absence and presence of Ca2⫹release channel blockers. The membranes from adrenalectomized/dexamethasone-treated animals showed
restoration of the higher rates of Ca2⫹ uptake compared with those from adrenalectomized animals in
both the absence and presence of Ca2⫹-release channel
blockers.
In additional experiments, Ca2⫹ uptake by SR was
measured at varying Ca2⫹ concentrations in the presence of ruthenium red (25 ␮M) in the assay medium,
and the results are summarized in Fig. 2. At the wide
range of Ca2⫹ concentrations tested, the rate of Ca2⫹
uptake by cardiac SR vesicles from adrenalectomized
animals was significantly lower than that of the membranes from control animals. A significantly higher
rate of Ca2⫹ uptake by cardiac SR vesicles from adrenalectomized/dexamethasone-treated compared with
Fig. 1. Effect of ryanodine receptor/Ca2⫹-release
channel (RyR-CRC) blockers on ATP-energized
Ca2⫹ uptake rate by cardiac sarcoplasmic reticulum (SR) vesicles from control, adrenalectomized (ADX), and adrenalectomized/dexamethasone-treated (ADX ⫹ DEX) rats. The ATPdependent Ca2⫹ uptake activity was determined
in the absence of RyR-CRC blockers and in the
presence of RyR-CRC blocker ruthenium red
(RR, 25 ␮M) (A), or ryanodine (Ryn, 625 ␮M) (B),
as described in METHODS. The data represent
means ⫾ SE of 6 experiments using separate SR
preparations in each case. *P ⬍ 0.05 (absence vs.
presence of RyR or RR); ⫻P ⬍ 0.05 (control vs.
ADX); #P ⬍ 0.05 (ADX vs. ADX ⫹ DEX).
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vesicles (25 ␮g protein/lane) were first subjected to SDSPAGE in 6% (for RyR-CRC), 10% (for Ca2⫹-ATPase, calsequestrin, and CaM kinase), or 15% (for phospholamban) gels.
The protein samples separated by gel electrophoresis were
then transblotted to nitrocellulose membranes. The membranes were probed with antibodies specific for cardiac RyRCRC [monoclonal (1), dilution 1:2,500], cardiac SR Ca2⫹ATPase [monoclonal (16), dilution 1:2,500], phospholamban
[monoclonal (37), 0.5 ␮g/ml], calsequestrin [monoclonal (18),
dilution 1:1,000], or CaM kinase II (polyclonal, dilution
1:1,000). A peroxidase-linked anti-mouse (for RyR-CRC,
Ca2⫹-ATPase, phospholamban, and calsequestrin) or antirabbit (for CaM kinase II) IgG at a dilution of 1:5,000 was
used as the secondary antibody. Protein bands reactive with
antibodies were visualized using the enhanced chemiluminescence detection system from Amersham. The images of
the protein bands were optimized, captured, and analyzed by
ImageMaster VDS gel documentation system (Pharmacia
Biotech; San Francisco, CA). The Western blotting detection
system was determined to be linear with respect to the
amount of SR/homogenate protein in the range of 10–40 ␮g
using this camera-based densitometry system.
Phosphorylation assay. Phosphorylation of SR proteins by
endogenous CaM kinase II was determined as described
previously (44). The assay medium (total volume 50 ␮l) for
phosphorylation by endogenous CaM kinase II contained 50
mM HEPES (pH 7.4), 10 mM MgCl2, 0.2 mM CaCl2, 0.2 mM
EGTA, 1 ␮M calmodulin, 0.8 mM [␥-32P]ATP (specific activity 200–300 cpm/pmol), and SR (25 ␮g protein). The initial
free Ca2⫹ concentration, determined using the computer program of Fabiato (9), was 5.4 ␮M. The phosphorylation reaction was initiated by the addition of [␥-32P]ATP after preincubation of the rest of the assay components for 3 min at
37°C. Reactions were terminated after 2 min by adding 15 ␮l
of SDS-sample buffer, and the samples were subjected to
SDS-PAGE in 4–18% gradient gels, stained with Coomassie
brilliant blue, dried, and autoradiographed (14). Quantification of phosphorylation was carried out by liquid scintillation
counting after careful excision of the radioactive bands from
the gels (14).
Data analysis. Results are presented as means ⫾ SE.
Statistical significance was evaluated with a single-factor
analysis of variance with the Tukey multiple comparison
test. P ⬍ 0.05 was taken as a level of significance.
H327
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DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
untreated adrenalectomized animals could be observed
at all Ca2⫹ concentrations. The kinetic parameters
derived from the data shown in Fig. 2 are summarized
in Table 1. Adrenalectomy and dexamethasone treatment did not appear to significantly influence the concentrations of Ca2⫹ required for half-maximal velocity
(K0.5) or the Hill coefficient (nH), whereas the Vmax
values were diminished significantly.
Effects of adrenalectomy and dexamethasone treatment on the energy transduction function of Ca2⫹ATPase in cardiac SR. The effect of adrenalectomy and
dexamethasone treatment on the energy transduction
function of the Ca2⫹-ATPase was assessed by measuring TG-sensitive ATP hydrolysis in cardiac SR vesicles.
As shown in Fig. 3, the rate of ATP hydrolysis measured was not affected significantly by adrenalectomy.
Treatment of adrenalectomized animals with dexamethasone, however, led to a significant increase
(⬃70%) in the rate of ATP hydrolysis. The stoichiometry of Ca2⫹ uptake/ATP hydrolysis by cardiac SR vesicles was not improved by treatment of adrenalectoTable 1. Comparison of the kinetic parameters of
Ca2⫹ uptake by cardiac sarcoplasmic reticulum
isolated from control, ADX, and ADX ⫹
DEX-treated rats
Animal
Group
Control
ADX
ADX ⫹ DEX
Vmax
(nmol Ca2⫹ 䡠 mg
protein⫺1 䡠 min⫺1)
71 ⫾ 5
40 ⫾ 4*
67 ⫾ 6
K0.5 for Ca2⫹,
␮M
nH
1.06 ⫾ 0.16
1.34 ⫾ 0.24
1.01 ⫾ 0.12
1.09 ⫾ 0.04
1.29 ⫾ 0.20
1.2 ⫾ 0.10
Values are means ⫾ SE; n ⫽ 6 rats. Values were derived from
data shown in Fig. 2 by using the procedure described in METHODS.
ADX, adenalectomized; DEX, dexamethasone; nH, Hill coefficient.
* P ⬍ 0.05 vs. control or ADX ⫹ DEX.
Fig. 3. Ca2⫹-ATPase activities of cardiac SR from control, ADX, and
ADX ⫹ DEX rats. Thapsigargin (TG)-sensitive Ca2⫹-ATPase activity
was determined as described in METHODS. The results represent
means ⫾ SE of 5 experiments using separate SR preparations in
each case. *P ⬍ 0.05 vs. control or ADX.
mized animals with dexamethasone. The estimated
ratios of Ca2⫹ uptake to TG-sensitive ATP hydrolysis
were as follows: control, 0.59; adrenalectomized, 0.37;
and adrenalectomized/dexamethasone-treated, 0.33.
As discussed elsewhere (23), such low stoichiometry
between Ca2⫹ uptake and ATP hydrolysis has been
reported in several published studies using rat cardiac
SR vesicles; the reasons for the apparently low efficiency of coupling ATP hydrolysis to Ca2⫹ transport in
rat cardiac SR vesicles in vitro remain unclear.
Effects of adrenalectomy and dexamethasone treatment on cardiac SR Ca2⫹-cycling proteins and their
phosphorylation by endogenous CaM kinase II. Major
Ca2⫹-cycling proteins in the SR include the RyR-CRC
responsible for Ca2⫹ release into the cytosol on myocyte excitation to induce muscle contraction (4); Ca2⫹ATPase, which actively sequesters Ca2⫹ back into the
SR lumen to promote muscle relaxation (22); calsequestrin, which serves to bind and store Ca2⫹ within
the SR lumen (22); and phospholamban, which serves
to regulate Ca2⫹-ATPase function (17, 34, 38). In the
present study, we used antibodies specific for each of
these SR Ca2⫹-cycling proteins to perform Western
blotting analysis of their relative amounts in cardiac
SR isolated from control, adrenalectomized, and adrenalectomized/dexamethasone-treated rats. The results
of these experiments are summarized in Fig. 4. No
significant change was evident in the relative amounts
of RyR-CRC, Ca2⫹-ATPase, calsequestrin, and phospholamban in cardiac SR after adrenalectomy or dexamethasone treatment. Similar findings were also obtained in experiments in which Western blot analysis
of these proteins was performed using unfractionated
cardiac muscle homogenates from all three groups (re-
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Fig. 2. Effects of varying the calcium concentration of the incubation
medium on ATP-dependent calcium uptake rate by cardiac SR vesicles from control, ADX, and ADX ⫹ DEX rats. Calcium uptake
assays were performed as described in METHODS. The assay medium
contained 25 ␮M RR, and the free Ca2⫹ concentration was varied as
shown. The data represent means ⫾ SE of 6 experiments using
separate SR preparations in each case.
DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
H329
sults not shown). In additional experiments, Western
blot analysis of cardiac SR membranes from a group of
rats that was not adrenalectomized but received dexamethasone treatment did not show any significant
change in the level of the SR Ca2⫹-cycling proteins
when compared with a corresponding control group
that received no dexamethasone treatment (results not
shown).
CaM kinase II associated with the SR (and present
in the cytosol) is implicated in the regulation of the
Ca2⫹-uptake and -release functions of the cardiac SR
through phosphorylation of phospholamban (17, 34,
38), RyR-CRC (10, 19, 43), and Ca2⫹-ATPase (11, 26–
28, 30, 40, 44–47). We determined endogenous CaM
kinase-catalyzed protein phosphorylation in cardiac
SR vesicles isolated from control, adrenalectomized,
and adrenalectomized/dexamethasone-treated rats.
Figure 5 shows the protein profiles of cardiac SR vesicles isolated from each experimental group and the
corresponding autoradiogram depicting protein phosphorylation. In the presence of Ca2⫹ and calmodulin,
SR-associated CaM kinase II catalyzed the phosphorylation of RyR-CRC, Ca2⫹-ATPase, and phospholamban. No significant change in the phosphorylation of
these Ca2⫹-cycling proteins was evident after adrenalectomy. On the other hand, dexamethasone treatment
of adrenalectomized animals led to a significantly
higher level of phosphorylation of all three CaM kinase
II substrates. In several experiments, phosphorylation
was quantified by determining 32P incorporation into
the protein bands excised from gels (see METHODS). The
results confirmed the significantly higher level (50–
80% increase) of substrate phosphorylation in the dexamethasone-treated group (Fig. 6).
Endogenous CaM kinase II levels in control, adrenalectomized, and dexamethasone-treated rats. We utilized a polyclonal antibody, specific for the ␦-isoform of
CaM kinase II predominantly expressed in the heart
(2, 7, 33), to perform Western blotting analysis of this
enzyme in cardiac SR from control, adrenalectomized,
and adrenalectomized/dexamethasone-treated rats. As
shown in Fig. 7, the relative amount of ␦-CaM kinase II
protein was found to be ⬃2.5- to 4-fold higher in the
adrenalectomized/dexamethasone-treated group compared with control or adrenalectomized groups. No
statistically significant difference was observed in the
level of SR-associated CaM kinase II protein in the
adrenalectomized group compared with the control
group.
Effect of activation of endogenous CaM kinase II on
Ca2⫹ uptake by SR. To compare the effect of endogenous CaM kinase II activation on Ca2⫹ uptake by
cardiac SR from the three groups of rats, ATP-dependent Ca2⫹ uptake by SR was determined in the absence
and presence of calmodulin in the assay medium. Under the experimental conditions employed, the addition
of calmodulin to the Ca2⫹ uptake assay medium promotes phosphorylation of CaM kinase II substrates.
These experiments were performed in the absence of
ruthenium red in the assay medium because this drug
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Fig. 4. Detection and estimation of relative amounts of
Ca2⫹ cycling proteins in cardiac SR isolated from control, ADX, and ADX ⫹ DEX rats by Western blotting.
Identical amounts of SR membranes (25 ␮g protein)
from the three groups of rats were subjected to Western
immunoblotting analysis of Ca2⫹-sequestering ATPase
(Ca2⫹-ATPase), RyR-CRC, calsequestrin, and phospholamban as described in METHODS. Representative immunoblots obtained with 3 separate SR preparations
each from control, ADX, and ADX ⫹ DEX rats are
shown at the bottom. The content of each Ca2⫹ cycling
protein was quantified by computer-assisted analysis of
Western blots using ImageMaster VDS software, and
the results were obtained using 6 separate cardiac SR
preparations from each group of rats are presented as
means ⫾ SE in bar graphs. For each protein, the arbitrary units shown were derived at same software settings form two separate Western blots, each containing
3 different SR preparations from each group of rats.
The values of arbitrary units for individual proteins
varied ⬍10% between the two Western blots used.
Immunoblots for phospholamban show high (H)- and
low (L)-molecular-weight (MW) forms of this protein.
The bar graph for phospholamban includes both highand low-molecular-weight forms.
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DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
has an inhibitory effect on Ca2⫹-ATPase phosphorylation by CaM kinase II (28). As shown in Fig. 8, the
presence of calmodulin (1 ␮M) in the assay medium
resulted in stimulation of Ca2⫹ uptake by cardiac SR
Fig. 6. Endogenous CaM kinase II-mediated phosphorylation of RyR-CRC (A),
Ca2⫹-ATPase (B), and phospholamban (C)
in cardiac SR isolated from control, ADX,
and ADX ⫹ DEX rats. After phosphorylation under standard assay conditions in
the presence of Ca2⫹ and CaM, SR proteins were fractioned by SDS-PAGE, and
32
P incorporation into peptide bands representing RyR-CRC, Ca2⫹-ATPase, and
phospholamban was determined by liquid
scintillation counting as described in
METHODS. The data for phospholamban include 32P incorporation into both LMW
and HMW forms of this protein. Data represent means ⫾ SE of 6 experiments using
separate SR preparations in each case.
*P ⬍ 0.05 vs. control or ADX.
vesicles from control, adrenalectomized, and adrenalectomized/dexamethasone-treated rats. The stimulatory effect of calmodulin was most pronounced in the
dexamethasone-treated group (⬃74% increase) and
was minimal in the adrenalectomized group (⬃25%
increase).
DISCUSSION
In this study, we made the following key observations: 1) The ATP-dependent Ca2⫹ uptake rate (Ca2⫹pump function) of cardiac SR membrane vesicles in
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Fig. 5. Comparison of endogenous Ca2⫹/calmodulin (CaM) kinase
II-mediated protein phosphorylation in cardiac SR isolated from
control, ADX, and ADX ⫹ DEX rats. Phosphorylation reaction was
carried out under standard assay conditions in the presence of Ca2⫹
and calmodulin as described in METHODS. A: Coomassie blue-stained
SDS-PAGE gel depicting protein profile of cardiac SR preparations
from control, ADX, and ADX ⫹ DEX rats. B: autoradiogram of the
same gel depicting protein phosphorylation. Ca2⫹-cycling proteins
undergoing phosphorylation included RyR-CRC, Ca2⫹-ATPase, and
phospholamban (PLN) HMW and LMW forms. No appreciable phosphorylation of these substrates occurred in the absence of Ca2⫹ or
CaM in assay medium (not shown). Results shown are typical of 6
experiments using separate SR preparations each from control,
ADX, and ADX ⫹ DEX rats. Quantitative data on substrate phosphorylation are summarized in Fig. 6.
Fig. 7. Detection and estimation of relative amounts of ␦-CaM kinase II in cardiac SR isolated from control, ADX, and ADX ⫹ DEX
rats by Western blotting. Identical amounts of cardiac SR membranes (25 ␮g protein) from control, ADX, and ADX ⫹ DEX rats were
subjected to Western immunoblotting analysis of CaM kinase II
(␦-isoform) as described in METHODS. Immunoblots obtained with 3
separate SR preparations each from control, ADX, and ADX ⫹ DEX
rats are shown at the bottom. CaM kinase II content was quantified
by computer-assisted analysis of Western blots using ImageMaster
VDS software as described in the legend to Fig. 4, and the results
obtained using 6 cardiac SR preparations from each group of rats are
presented as means ⫾ SE in the bar graph. *P ⬍ 0.05 vs. control or
ADX.
DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
vitro is markedly reduced following adrenalectomy;
treatment of adrenalectomized animals with dexamethasone prevents this decline in SR Ca2⫹ transport
function. 2) The levels of the major Ca2⫹-cycling proteins (Ca2⫹-ATPase, RyR-CRC, calsequestrin, and
phospholamban) in cardiac SR are not altered significantly after adrenalectomy or dexamethasone treatment. 3) Treatment of adrenalectomized animals with
dexamethasone leads to a striking increase in the
amount of ␦-CaM kinase II associated with cardiac SR,
as well as significantly enhanced substrate (Ca2⫹ATPase, RyR-CRC, and phospholamban) phosphorylation by the endogenous CaM kinase II. These findings
clearly identify the SR membrane as a major subcellular target for glucocorticoid actions in the heart, and,
as discussed below, they provide insights into the
mechanisms underlying glucocorticoid modulation of
cardiac contractile function.
Although systematic studies on the rates of contraction and relaxation of cardiac muscle from adrenaldeficient animals seem to be lacking, cardiac muscle
from dexamethasone-treated intact animals has been
shown to display markedly increased contractile tension as well as rates of contraction and relaxation (31).
The present findings suggest strongly that the dexamethasone-mediated increase in the velocity of muscle
relaxation might arise, at least in part, from the ability
of this glucocorticoid to augment the SR Ca2⫹-pump
activity. Because the SR Ca2⫹-pump activity is a major
determinant of SR Ca2⫹ load (and hence, the amount of
Ca2⫹ available for release) (13, 36, 42), the increased
SR Ca2⫹-pump activity may also contribute to the
enhanced velocity of contraction (31) and contractile
tension (19) observed in dexamethasone-treated animals. Conversely, the diminished Ca2⫹-pump activity
of cardiac SR from adrenalectomized animals reported
here correlates well with the depression of myocardial
contractility observed in adrenal deficiency in vivo (41)
and in vitro (19). The Ca2⫹-ATPase content (Fig. 4) and
Ca2⫹-ATPase activity (Fig. 3) of cardiac SR were not
altered significantly by adrenalectomy. Therefore, the
observed decline in SR Ca2⫹-pump function is likely
due to impaired Ca2⫹ translocation rather than energy
transduction. This apparent uncoupling of ATP hydrolysis and Ca2⫹ transport does not appear to be due to
enhanced Ca2⫹ leak from the SR because SR Ca2⫹release channel blockers did not abolish or attenuate
the depression in Ca2⫹-uptake activity of SR from
adrenalectomized animals (Fig. 1). The impaired SR
Ca2⫹-pump function after adrenalectomy and the
improvement after dexamethasone treatment may
involve alterations in the membrane-associated glycogenolytic pathway. Previous studies have demonstrated a marked and selective depletion and restoration of both active and total phosphorylase activities in
the rat heart microsomes after adrenalectomy and
dexamethasone treatment, respectively (24). A strong
association of substantial amounts of phosphorylase,
glycogen, and other enzymes linked to glycogenolysis
with the SR membrane in cardiac muscle has been
documented in earlier studies (8), suggesting that the
glycogenolytic pathway present in the membrane
might serve as a link between excitation-contraction
coupling and intermediary metabolism. It is possible
that the loss of phosphorylase from the SR membrane
in adrenal insufficiency might result in derangement of
the link between excitation-contraction coupling and
intermediary metabolism.
To our knowledge, the present study is the first to
provide evidence of phosphorylation-dependent glucocorticoid modulation of SR function. Our results revealed a significant increase in the CaM kinase IImediated phosphorylation of RyR-CRC, Ca2⫹-ATPase,
and phospholamban after dexamethasone treatment,
although no significant change was observed due to
adrenalectomy per se (Figs. 5 and 6). Because dexamethsone treatment did not significantly alter the levels
of CaM kinase II substrates, this increase in phosphorylation may be attributed to the observed increase in
the amount of ␦-CaM kinase II, which is the predominant CaM kinase II isoform present in cardiac cytosol
and SR (2, 7, 33). The functional consequence of cardiac
RyR-CRC phosphorylation has not been clearly established. Recently, CaM kinase II inhibitors as well as
protein phosphatases have been found to reduce SR
Ca2⫹-release channel activity in intact cardiomyocytes
(6, 20). These findings are consistent with an increased
SR Ca2⫹-release channel activity on RyR-CRC phosphorylation by CaM kinase II. In any case, the observed increase in the RyR-CRC phosphorylation after
dexamethasone treatment can be expected to impact
on the modulation of SR Ca2⫹ release and, therefore,
myofilament activation. Phosphorylation of phospholamban by PKA (at Ser16) and CaM kinase II (at Thr17)
is well known to stimulate Ca2⫹ uptake by SR, apparently by relieving an inhibitory effect exerted by de-
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Fig. 8. Effect of activation of endogenous CaM kinase II by CaM on
Ca2⫹ uptake activity of cardiac SR isolated from control, ADX, and
ADX ⫹ DEX rats. Ca2⫹ uptake reaction was carried out under
standard assay conditions in the absence of CaM (⫺CaM) and in the
presence of 1 ␮M CaM (⫹CaM) as described in METHODS. Ca2⫹
uptake data from experiments using 4 separate cardiac SR preparations from each group of rats are presented as means ⫾ SE. *P ⬍ 0.05
(absence vs. presence of CaM); ⫻P ⬍ 0.05 (control vs. ADX); #P ⬍
0.05 (ADX vs. ADX ⫹ DEX).
H331
H332
DEXAMETHASONE AND SARCOPLASMIC RETICULUM FUNCTION
We thank Bruce Arppe for preparing photographs of illustrations
and Lily Jiang for secretarial assistance.
This work was supported by Grant T-3682 from the Heart and
Stroke Foundation of Ontario.
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