From the Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575
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ABSTRACT |
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Phospholamban is a critical regulator of the
sarcoplasmic reticulum Ca2+-ATPase activity and
myocardial contractility. Phosphorylation of phospholamban occurs on
both Ser16 and Thr17 during isoproterenol
stimulation. To determine the physiological significance of dual site
phospholamban phosphorylation, we generated transgenic models
expressing either wild-type or the Ser16 Ala mutant
phospholamban in the cardiac compartment of the phospholamban knockout
mice. Transgenic lines with similar levels of mutant or wild-type
phospholamban were studied in parallel. Langendorff perfusion indicated
that the basal hyperdynamic cardiac function of the knockout mouse was
reversed to the same extent by reinsertion of either wild-type or
mutant phospholamban. However, isoproterenol stimulation was associated
with much lower responses in the contractile parameters of mutant
phospholamban compared with wild-type hearts. These attenuated
responses were due to lack of phosphorylation of mutant phospholamban,
assessed in 32P labeling perfusion experiments. The lack of
phospholamban phosphorylation in vivo was not due to
conversion of Ser16 to Ala, since the mutated phospholamban
form could serve as substrate for the
calcium-calmodulin-dependent protein kinase in
vitro. These findings indicate that phosphorylation of
Ser16 is a prerequisite for Thr17
phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the
-agonist stimulatory responses in the mammalian heart.
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INTRODUCTION |
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Phospholamban (PLB)1 is
a regulator of the affinity of the cardiac sarcoplasmic reticulum (SR)
Ca2+-ATPase for Ca2+. Dephosphorylated PLB is
an inhibitor, and phosphorylation of PLB removes its inhibitory effects
on the SR Ca2+-ATPase. Recently, the critical role of PLB
in the regulation of cardiac contractility has been defined through
gene transfer (1) and knockout (2) technology in the mouse.
Cardiac-specific overexpression of PLB was associated with decreases in
the affinity of the SR Ca2+-ATPase for Ca2+ and
depressed cardiac function, whereas PLB deficiency resulted in
increased Ca2+ affinity of the Ca2+-ATPase and
enhanced myocardial performance. Furthermore, the stimulatory effects
to -adrenergic agonists were more pronounced in the
PLB-overexpressing hearts, whereas these effects were attenuated in the
PLB-knockout hearts compared with wild types (1, 2). These studies
suggested that PLB plays a prominent role in the heart's responses to
-agonists. However, PLB is phosphorylated on both Ser16
and Thr17 during isoproterenol stimulation (3) and the
relative contribution of each site in the altered contractile responses
of the heart is not presently well known. In vitro studies
have shown that Ser16 is phosphorylated by
cAMP-dependent protein kinase, whereas Thr17 is
phosphorylated by Ca2+-calmodulin-dependent
protein kinase (4). Phosphorylation of each site occurs in an
independent manner, although it is not presently clear whether the
stimulatory effects of the two phosphorylations on SR Ca2+
transport are additive (5-9).
In vivo studies have shown that
phosphorylation/dephosphorylation of PLB by
Ca2+-calmodulin-dependent protein kinase has as
a prerequisite the phosphorylation/dephosphorylation by
cAMP-dependent protein kinase (10-15). Furthermore,
elevation of intracellular [Ca2+] to higher levels than
those attained by isoproterenol, which resulted in higher peak tension
than that elicited during -adrenergic stimulation, failed to
phosphorylate PLB (3, 16). However, a recent study, using
phosphorylation site-specific antibodies for PLB, indicated that
cAMP-dependent and
Ca2+-calmodulin-dependent phosphorylation of
PLB can occur in an independent manner and their effects may be
additive in vivo (17). Thus, the functional role of dual
site phosphorylation of PLB is not clear.
The availability of the PLB knockout mouse in conjunction with
site-specific mutagenesis technology have provided us with an excellent
opportunity to further examine the interaction between the
cAMP-dependent and the
Ca2+-calmodulin-dependent pathways of PLB
phosphorylation in the regulation of basal and -agonist stimulated
cardiac contractility in vivo. The aims of the present study
were to: 1) determine whether reintroduction of PLB in the null
background is feasible, and whether it is able to reverse the
hyperdynamic cardiac phenotype of the PLB knockout mouse; and 2)
elucidate the physiological role of Thr17 phosphorylation
in PLB in the absence of Ser16 (Ser16
Ala)
by directing cardiac-specific expression of mutant PLB in the knockout
background.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis--
The site-specific mutation of
Ser16 Ala (TCC
GCC) was
introduced into PLB cDNA by polymerase chain reaction (PCR)
methodology. Briefly, a 0.9-kb SalI fragment containing PLB
cDNA and the SV40 polyadenylation signal sequence (PLB
cDNA-SV40-poly(A)) was released from the
-MHCp-PLB-SV40 fusion
gene (1). This SalI PLB cDNA-SV40-poly(A) fragment was
then subcloned into a pBluescript SKII(
) vector (Stratagene), which
has T3 and T7 primer sites flanking the insert. Polymerase chain
reaction mutagenesis was performed by two consecutive PCR
amplifications using two different sets of primers. For the first PCR
amplification, 100 pg of the subcloned plasmid DNA containing the
0.9-kb SalI fragment was used as template, along with a 5'
end mutant primer (5'-CT ATC AGG AGA GCC GCC ACT
ATT GAA ATG CC-3') corresponding to nucleotides 32-62 of the PLB
coding sequence, and a 3' end T7 primer, to generate a desired mutant
PLB cDNA minor product. Subsequently, an aliquot of the first PCR
product as well as the T3 and T7 primers was used for the second PCR.
The final amplified product was excised and resubcloned into the
SalI site of a second pBluescript SK II(
) vector, which
was then transformed into XL1-Blue-competent cells. The mutated PLB
cDNA-SV40-poly(A) sequence was resubcloned into the SalI
site of the parent PLB overexpression vector pIBI 31 (1).
Generation and Identification of the Transgenic Mice--
The
expression fragment containing the cardiac-specific -myosin heavy
chain (
-MHC) promoter, the mutated PLB cDNA, and the SV40
poly(A) signal sequence was used for pronuclear microinjection of
fertilized eggs derived from the intercrossing of male PLB knockout
(PLB-KO) and female FVB/N mice. The first generation of transgenic mice
was PLB-heterozygous. Mice harboring the mutant PLB transgene were
identified using PCR methodology and Southern blot analysis (1).
Breeding the transgene-positive mice with PLB-KO mice generated
offspring expressing the mutant PLB transgene in the PLB-KO background.
The
-MHCp-PLB-SV40 fusion gene was also used to generate wild-type
PLB (PLB-WT) transgenic mice in a similar manner as the PLB
Ser16
Ala mutant (PLB-MU) transgenic mice.
Western Blot Analysis-- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and quantification of PLB and the SR Ca2+-ATPase were performed as described previously (1, 18). For immunodetection of PLB phosphorylation sites, polyclonal antibodies raised against a PLB peptide (residues 9-19) phosphorylated at Ser16 (1:10,000) or at Thr17 (1:5000) (PhosphoProtein Research) were used.
Langendorff Perfusions-- Hearts from wild-type and transgenic mice were subjected to retrograde aortic perfusion with modified Krebs-Henseleit buffer as described previously (19). After a 30-40-min stabilization period, cumulative concentrations of isoproterenol (0.1 nM to 1 µM) were administered into the buffer flow line at intervals of 7 min.
In Vivo Phosphorylation-- Mouse hearts were perfused in a recirculating system containing 2 mCi of [32P]orthophosphate for 30 min. At the end of this labeling period, isoproterenol (0.1 µM) was administered into the perfusion system and hearts were stimulated for 2 min (19). After stimulation, preparations were freeze-clamped and homogenized in phosphate buffer (50 mM KH2PO4, 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, and 0.3 mM phenylmethylsulfonyl fluoride, pH 7.0) containing 0.5 mM dithiothreitol and 1 µM okadaic acid. Microsomal fractions enriched in SR membranes and myofibrillar proteins were prepared as described previously (20).
In Vitro Phosphorylation--
Cyclic AMP-dependent
protein kinase (PKA) phosphorylation of the cardiac homogenates (60 µg) was carried out at 30 °C in 30 µl of reaction mixture
containing 50 mM K+ phosphate buffer (pH 7.0),
10 mM MgCl2, 5 mM NaF, 0.5 mM EGTA, 0.1 mM ATP, 20 µCi of
[-32P]ATP, and 45 units of the PKA-catalytic subunit.
For endogenous Ca2+-calmodulin-dependent
protein kinase (Ca2+/CAM) phosphorylation of the cardiac
homogenates, 0.5 mM CaCl2, 2 µM
calmodulin, and 1 µM protein kinase inhibitor
peptide-(5-24) amide were added to the above reaction mixture.
Reactions were terminated with 30 µl of SDS sample buffer after 2 min
(PKA) or 5 min (Ca2+/CAM) incubation, which was associated
with optimal phosphate incorporation in PLB. Thirty µg of protein was
subjected to 15% SDS-PAGE and autoradiography.
Statistical Analysis-- Data are expressed as mean ± S.E. Statistical analysis was performed using Student's t test for unpaired observations and analysis of variance followed by Bonferroni's t test for multiple comparisons. Values of p < 0.05 were considered statistically significant.
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RESULTS |
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Reintroduction of Wild-type PLB into the PLB Knockout Mouse
Hearts--
To determine whether the hyperdynamic cardiac function of
the PLB-KO mice can be reversed by reintroduction of the missing PLB
gene, we used the -MHC promoter to direct expression of mouse PLB
cDNA in the cardiac compartment of the PLB knockout mouse. Four
PLB-WT transgenic lines were identified by PCR and Southern blot
analyses of mouse genomic DNA. Northern blot analysis of total RNA
isolated from the hearts of the PLB-WT transgenic mice revealed
expression of the transgene, which migrated at 1.0 kb (1). This 1.0-kb
message was not present in either control or PLB-KO mouse hearts.
However, the control hearts showed the presence of the endogenous PLB
messages migrating at 2.8 and 0.7 kb (data not shown).
Reversal of the PLB Knockout Hyperdynamic Cardiac Function by
Reinsertion of Wild-type PLB--
To determine whether the
reintroduced PLB was capable of reversing the hyperdynamic cardiac
function associated with PLB deficiency, hearts from PLB-WT and PLB-KO
mice were subjected to Langendorff perfusion in parallel with control
hearts. The PLB-KO hearts exhibited significantly enhanced myocardial
performance compared with wild-type controls, as characterized by
significant increases in the maximal rates of cardiac contraction
(+dP/dt) and relaxation
(dP/dt) (Table
I). Reinsertion of PLB in the knockout
background (PLB-WT) was associated with significant depression of the
contractile parameters (Table I). However, since the levels of
reintroduced PLB were 0.7-fold of those present in control hearts,
reversal of the enhanced cardiac contractile parameters was not
complete, or to the levels observed in control hearts (Table I). It is interesting to note that when the relative levels of PLB or PLB/SR Ca2+ pump in the three animal models were plotted against
+dP/dt and
dP/dt, a close
linear correlation was observed (Fig. 1),
consistent with our previous observations in PLB-KO, PLB-heterozygous,
and control hearts (21).
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Generation of Transgenic Mice Expressing Mutant (Ser Ala) PLB
in the Heart--
The reversal of the hyperdynamic contractile
parameters of the PLB-KO hearts by reintroduction of the wild-type PLB
demonstrated the feasibility of reinserting various PLB mutants in the
knockout background and assessing their functional relevance in
vivo. In the present study, mutation of Ser16 to Ala
in PLB was performed by PCR site-directed mutagenesis, and expression
of the mutated PLB was driven by the
-MHC promoter in the PLB-KO
mice, in a manner identical to that for wild-type PLB described above.
Three lines of transgenic mice were generated, which were identified by
PCR and Southern blot analyses. The levels of PLB were 0.7-fold in two
lines and 2.0-fold in one line, compared with control hearts. One of
the lines expressing 0.7-fold PLB, which was similar to the PLB levels
expressed in PLB-WT hearts (Fig. 2), was
selected for breeding and further studies. Analysis of the SR
Ca2+-ATPase levels showed that there was no alteration upon
introduction of mutant PLB in the mouse heart (Fig. 2A).
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In Vivo 32P Incorporation in PLB--
The observation
that the degree of changes in contractile parameters was similar
between PLB-MU and PLB-KO hearts during isoproterenol stimulation,
suggested that mutation of Ser16 to Ala in PLB attenuated
or abolished its regulatory role in the responses to -agonists. This
might be due to lack of phosphorylation of Thr17 in the
mutated PLB form during
-adrenergic stimulation. To determine whether Thr17 was phosphorylated in vivo, PLB-WT
and PLB-MU hearts were perfused in parallel with buffer containing
[32P]orthophosphate. The hearts were stimulated with 0.1 µM isoproterenol as described previously (19). Cardiac
myofibrillar and SR-enriched membrane preparations were isolated and
subjected to SDS-gel electrophoresis and autoradiography (Fig.
4). Examination of the degree of
32P labeling of the proteins in the SR-enriched membrane
fraction indicated that phosphorylation of PLB was pronounced only in
the PLB-WT hearts and incorporation of [32P]phosphate in
this protein was barely detectable in the PLB-MU hearts (Fig.
4A). However, the degree of 32P-incorporation in
troponin I and C-protein in the myofibrillar fraction, isolated from
the same hearts, was similar between PLB-MU and PLB-WT mice (Fig.
4B). Furthermore, PLB-WT and PLB-MU hearts were perfused in
parallel with non-radioactive buffer and stimulated with isoproterenol,
as described above. The SR-enriched membrane preparations were
subjected to SDS-gel electrophoresis and immunoblotting using
antibodies specific to Ser16- or
Thr17-phosphorylated PLB peptides. Both phosphoserine (Fig.
5A) and phosphothreonine (Fig.
5B) were detected in isoproterenol-stimulated PLB-WT hearts.
However, in PLB-MU hearts, there was a very low degree of
phosphothreonine formation (Fig. 5B) but no phosphoserine detection (Fig. 5A), consistent with mutation of this site
to Ala.
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In Vitro Phosphorylation of PLB--
The lack of phosphorylation
of Thr17 in the perfused PLB-MU hearts could be due to
structural alterations in PLB upon mutation of Ser16 to
Ala, rendering the adjacent Thr17 residue inaccessible to
protein kinase. To determine whether this mutation in PLB alters its
ability to become phosphorylated by the
Ca2+-calmodulin-dependent protein kinase,
cardiac homogenates from PLB-WT and PLB-MU mice were incubated with
Ca2+ and calmodulin in the phosphorylation assay buffer and
processed for SDS-polyacrylamide gel electrophoresis and
autoradiography. The degree of 32P incorporation in PLB was
similar between PLB-WT and PLB-MU hearts (Fig.
6A), suggesting that
Thr17 in PLB-MU hearts could be phosphorylated in
vitro. Formation of phosphothreonine in PLB-MU hearts was also
verified by immunodetection, using a polyclonal antibody raised to a
PLB peptide phosphorylated at Thr17 (data not shown). These
data indicate that mutation of Ser16 did not prevent
phosphorylation of the adjacent Thr17 residue in PLB by the
Ca2+-calmodulin-dependent protein kinase.
However, incubation of the same cardiac homogenates with the protein
kinase A catalytic subunit under optimal phosphorylation conditions
indicated that only the PLB in PLB-WT hearts could be phosphorylated,
consistent with the lack of the Ser16 (Ser16
Ala) site in PLB-MU hearts (Fig. 6B).
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DISCUSSION |
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Our results are the first to demonstrate that a cardiac phenotype,
generated by gene targeting, can be reversed by reinsertion of the
missing gene in the null background. In previous studies, we showed
that ablation of PLB resulted in enhanced basal cardiac contractile
parameters assessed at the cellular, organ, and intact animal levels
(2, 18, 22, 23). In this study, we used the -myosin heavy chain
promoter to direct cardiac-specific expression of wild-type PLB in the
knockout background and observed reversal of the hyperdynamic function
of the PLB-deficient hearts. The degree of inhibition of the
contractile parameters was proportional to the expression levels of
PLB, in agreement with our previous studies in PLB-heterozygous and
PLB-homozygous mice (2, 21). The success of PLB transgenesis in the
genetically altered background, accompanied by reversal of the knockout
phenotype, indicated that the PLB-deficient mouse provides an
attractive model system for expression of various PLB mutants in the
heart and elucidation of structure-function relationships in
vivo. With the first mutant we studied, we sought to elucidate the
functional significance of dual site PLB phosphorylation during
-adrenergic stimulation. A site-specific mutation was introduced
into the PLB coding region, converting Ser16 to Ala, and
mutant PLB expression was directed in the knockout background.
Quantitative immunoblotting of cardiac homogenates and SR-enriched
microsomal preparations showed that all the mutant PLB was inserted in
the SR membranes. Transgenic mice, expressing similar levels of
wild-type or mutant PLB in the heart, exhibited similar contractile
parameters under basal conditions, indicating that the mutant PLB was
capable of modulating contractility in a manner similar to that for
wild-type PLB. However, isoproterenol stimulation was associated with
much lower enhancement of the rates of contraction and relaxation in
the PLB-mutant hearts compared with PLB-wild-type hearts, whereas the
heart rate responses were similar between these groups. It is
interesting to note that the maximal increases in contractile
parameters of the PLB-mutant hearts were similar to those of
PLB-knockout hearts under
-adrenergic stimulation. These findings
suggest that mutation of Ser16 in PLB compromised the
contribution of this phosphoprotein in the stimulatory responses of the
heart to isoproterenol. Actually, when
[32P]orthophosphate was included in the perfusate buffer,
there was no 32P labeling of mutant PLB observed, even
under maximal isoproterenol stimulation. However, in the same hearts,
the degree of phosphorylation of troponin I and C-protein in the
myofibrils was similar between hearts expressing mutant or wild-type
PLB. Thus, cardiac phosphoproteins other than PLB were responsible for
mediating the attenuated responses of the PLB-mutant hearts. To exclude
the possibility that the lack of phosphothreonine in vivo
was due to mutation of the adjacent Ser16 to Ala, cardiac
homogenates from PLB-wild-type and PLB-mutant mice were incubated
in vitro under optimal phosphorylation conditions for the
Ca2+-calmodulin-dependent protein kinase. The
degree of mutant PLB phosphorylation was similar to that of wild-type
PLB and phosphothreonine formation was verified using the
phospholamban phosphorylation site-specific antibody, indicating that
the mutant PLB form was capable of being phosphorylated on
Thr17.
The role of PLB phosphorylation by cAMP-dependent and
Ca2+-calmodulin-dependent protein kinases has
been the subject of several studies. Reports by Raeymaekers et
al. (7), Tada et al. (8), and Kranias (6) indicated
that the stimulatory effects of the two protein kinases on sarcoplasmic
reticulum Ca2+ transport can be additive, whereas a study
by Colyer and Wang (9) suggested that maximal stimulation of the
Ca2+ pump occurs by PLB phosphorylation at a single site
and that additional phosphorylation of the other site does not further stimulate pump activity. Furthermore, all the in vitro
studies agree that phosphorylation of PLB by cAMP-dependent
and Ca2+-calmodulin-dependent protein kinases
occurs in an independent manner, whereas in vivo findings
(3, 13, 24) indicate that phosphorylation/dephosphorylation of
Thr17 occurs only subsequent to
phosphorylation/dephosphorylation of Ser16 during
-adrenergic stimulation. Our findings in transgenic animals, demonstrated that: (a) Thr17 in PLB cannot be
phosphorylated in the absence of Ser16 phosphorylation,
even under maximal isoproterenol stimulation of intact, beating hearts;
and (b) phosphorylation of Thr17 in PLB does not
require prior phosphorylation of Ser16 in in
vitro experiments. Thus, phosphorylation of Thr17
occurs independently of Ser16 phosphorylation in
vitro, whereas phosphorylation of the adjacent Ser16
residue appears to be a prerequisite for in vivo
phosphothreonine formation during
-agonist stimulation. This
apparent discrepancy between in vivo and in vitro
findings may be due to differences in the levels of calcium available
to activate the SR Ca2+-calmodulin-dependent
protein kinase. In vitro conditions generally include
optimal calcium concentrations. However, in in vivo studies, phosphorylation of Ser16 may be required to occur first and
enhance the SR Ca2+ uptake rates and, thus, SR
Ca2+ load. This would lead to increased Ca2+
levels released by the SR, activation of the
Ca2+-calmodulin-dependent protein kinase, and
phosphorylation of Thr17 in PLB. The phosphorylation and
activation of the sarcolemmal Ca2+ channels may also
contribute to the increased Ca2+ levels required for
in vivo phosphorylation of Thr17 in PLB (17).
However, the inhibition of the SR-associated protein phosphatase 1 activity, which was suggested to be an important determinant for
Thr17 phosphorylation (17), did not appear to play any role
in our transgenic experiments, even under maximal isoproterenol
stimulation.
The functional significance of Ca2+-calmodulin-dependent phosphorylation of PLB has been previously examined in intact cardiac myocytes (15, 25). Phosphorylation of PLB by Ca2+-calmodulin-dependent protein kinase II was suggested to increase the Vmax of the SR Ca2+-ATPase, whereas phosphorylation by protein kinase A increased the Ca2+ affinity of the pump (15). Furthermore, inhibition of Ca2+-calmodulin-dependent protein kinase II was shown to slow-down sarcoplasmic reticulum Ca2+ uptake and the decline of [Ca2+] even after inhibition of protein kinase A (25), suggesting that the phosphothreonine in PLB may be important in regulation of diastolic Ca2+ and prevention of cytosolic Ca2+ overload especially under pathophysiological conditions (17, 26, 27).
In summary, our findings indicate that the hyperdynamic PLB knockout
phenotype can be reversed by reintroduction of PLB in the null
background, and demonstrate the potential power of this technology in
performing PLB structure-function studies in vivo. Expression of mutant PLB in which Ser16 was replaced by Ala
in the knockout background indicated that the phosphorylation of
Thr17 in PLB requires prior phosphorylation of
Ser16 during -adrenergic stimulation. In the absence of
Ser16 phosphorylation, the degree of the stimulatory
effects by
-agonists was similar to that obtained in PLB knockout
hearts, suggesting that cardiac phosphoproteins other than PLB mediate
these responses. Future studies using transgenic mice harboring the
Thr17
Ala or Ser16-Thr17
Ala-Ala mutations in PLB will further delineate the interrelationship of dual site phosphorylation in PLB and elucidate the functional relevance of each phosphorylation site under physiological and pathophysiological conditions.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Robbins for providing the
murine -myosin heavy chain promoter and J. C. Neumann for
pronuclear microinjection of the transgenic constructs.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL26057, HL22619, and HL52318.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.
To whom correspondence should be addressed: Dept. of Pharmacology
and Cell Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave., P. O. Box 670575, Cincinnati, OH 45267-0575. Tel.:
513-558-2377; Fax: 513-558-2269; E-mail: kraniaeg{at}email.uc.edu.
1 The abbreviations used are: PLB, phospholamban; SR, sarcoplasmic reticulum; PCR, polymerase chain reaction; kb, kilobase(s); MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis; PKA, cAMP-dependent protein kinase.
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REFERENCES |
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