Protein Kinase C epsilon and the Antiadrenergic Action of Adenosine
in Rat Ventricular Myocytes
Koji Miyazaki, Satoshi Komatsu, Mitsuo Ikebe, Richard A. Fenton, James G. Dobson, Jr.
Department of Physiology
University of Massachusetts Medical School, Worcester, MA 01655
Running Head: PKC, and adenosinergic action in rat cardiomyocytes
James G. Dobson, Jr., Ph.D.
Dept. of Physiology, S4-242
Univ. Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655
KEYWORDS: β-adrenergic, cardiomyocyte shortening, transfection, t-tubules, PKC, translocation
Articles in PresS. Am J Physiol Heart Circ Physiol (June 17, 2004). 10.1152/ajpheart.00224.2004
Copyright © 2004 by the American Physiological Society.
Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A1
receptors (A1R). The role of protein kinase C epsilon (PKCε) in the antiadrenergic action of
adenosine was explored with adult rat ventricular myocytes in which PKCε was over-expressed.
Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCε construct
and compared to normal myocytes. The extent of myocyte shortening elicited by electrical
stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCε
was found localized primarily in transverse tubules. The A1R agonist chlorocyclopentyladenosine
(CCPA) at 1 µM rendered an enhanced localization of the PKCε in the t-tubular system. The β-
adrenergic agonist isoproterenol (ISO, 0.4 µM) elicited a 29-36% increase in myocyte shortening in
all three groups. While CCPA significantly reduced the ISO-produced increase in shortening in all
three groups, the reduction caused by CCPA was greatest with PKCε over-expression. The CCPA
reduction of the ISO-elicited shortening was eliminated in the presence of a PKCε inhibitory
peptide. These results suggest that the translocation of PKCε to the t-tubular system plays an
important role in A1R-mediated antiadrenergic actions in the heart.
Adenosine is known to exert numerous effects in the heart. This endogenous nucleoside via
adenosine A1receptors (A1R) causes antiadrenergic (11), antiarrthymogenic (7,18) and
preconditioning (15,19) actions in the myocardium. The antiadrenergic action of adenosine involves
reductions in β-adrenergic catecholamine-induced increases in adenylyl cyclase activity (30,44,45),
cyclic AMP (cAMP) formation (10,51), protein kinase A activation (11), myocardial protein
phosphorylation (17), intracellular Ca2+ transient magnitude (18), and cardiac atrial (12,42) and
ventricular (11,46,51) contractility. Both the A1R-mediated antiadrenergic actions (40) and
preconditioning (29,49) appear to involve protein kinase C (PKC) activity. There are at least 12
isoforms of PKC known to exist in tissues (35) and their activation fosters translocation to anchor
proteins in various subcellular locations (37). The PKCε along with PKCα and PKCδ are the
dominant isoforms present in adult rat cardiomyocytes (9,47). Because A1R stimulation has been
reported to activate PKCδ in freshly isolated rat ventricular myocytes (22), the possibility that this
adenosine receptor activates PKCε was explored.
The present study was undertaken to ascertain if PKCε plays a role in the antiadrenergic
effects caused by A1R stimulation. This possibility was investigated by over-expressing PKCε in
isolated primary cultured rat ventricular myocytes and determining whether the enzyme could be
activated/translocated by A1R stimulation in a manner consistent with its involvement in the
antiadrenergic action of adenosine. The findings indicate that the A1R-elicited activation of PKCε is
associated with potentiation of the antiadrenergic actions of adenosine in ventricular myocytes
transfected with PKCε.
Isolation of ventricular myocytes. Myocytes were isolated from rat hearts using collagenase and
hyaluronidase according to procedures previously reported (44). The harvested rectangular
myocytes displayed no spontaneous contractions, excluded trypan blue, and were capable of
contracting upon electrical stimulation. These myocytes were transfected with εPKC as described
below. Male Sprague Dawley rats (Harlan, Indianapolis, IN or Charles River, Wilmington, MA) 3-4
months of age and weighing 250-325 grams were used to obtain ventricular myocytes for these
studies. The rats were maintained and to obtain used in accordance with recommendations in the
Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal
Resources, National Research Council (DHEW Publication NIH #85-23, Rev. 1996) and the
guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts
Construction of the vector. Rabbit PKCε cDNA was cloned into the BamH1site of the pEGFP-N1
mammalian expression vector (Clontec). The GFP moiety was placed at the C-terminal end of the
PKCε molecule to avoid the potential disruption of the regulatory properties of PKCε and the
binding to the cellular elements that is thought to involve the N-terminal portion of the molecule. To
avoid the effect of the C-terminal GFP moiety on the proper folding of the PKCε molecule, a
flexible linker sequence consisting of a 15 amino acid residue (LQSTVPRARDPPVAT) was
inserted between the two moieties.
Transfection. Isolated ventricular myocyte transfection was performed using GeneSHUTTLE-20
(Quantum Biotechnologies, Montreal, Canada) according to the instructions of the supplier. Briefly,
1 µg of pEGFPN1-PKCε DNA was diluted with 100 µl of minimal essential medium (MEM) and
transferred to the diluted liposome solution containing 5 µl of GeneSHUTTLE-20 in 100 µl of
MEM. After incubation for 30 min, the DNA/liposome complex solutions were added to the
ventricular myocytes in a 35 mm dish. Cells were used in experiments after overnight incubation at
37oC with a 5% CO2atmosphere.
Cos7 cells were transfected with GFP-PKCε expression vector or pEGFPN1 by
electroporation as described previously by us (36). In brief, the cells were treated with ice cold 5%
trichloroacetic acid (TCA) followed by sonication. The samples were then dissolved in a 5% sodium
dodecyl sulfate (SDS), 0.5 M NaHCO3buffer and subjected to SDS PAGE followed by Western
Extraction, immunoprecipitation and assay of PKC. PKCε-GFP was extracted from transfected
Cos7 cells in buffer containing 0.5 M KCl, 10% Glycerol, 0.025% Triton X100, 1 mM ATP, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 0.2 mg/ml Nα-p-tosyl-L-lysine chloromethyl ketone
(TLCK), 1 mM Nα-p-tosyl-L-arginine methyl ester (TAME), 1 mM dithiothreitol (DTT), 10 µg/ml
leupeptin and 30 mM Tris-HCl (pH 7.5). Anti-GFP antibodies conjugated with Protein A beads
were added to perform immunoprecipitation. The immunoprecipitated samples were assayed for
PKC activity in a buffer containing 0.4 mg/ml histone III, 25 mM piperazinediethanesulfonic acid
(PIPES), 1 mM ethylene glycol-bis-aminoethylether tetraacetic acid (EGTA), 0.05 M KCl, 1 mM
MgCl2, 0.1 mg/ml phosphatidyl serine and 0.05 mM [γ-32P]ATP pH 7.0 at 25 oC in the presence and
absence of 50 ng/ml phorbol 12-myristate 13-acetate (PMA). The kinase activity is reported as
µmol of 32P incorporated/min/mg protein.
Immunocytochemistry and imaging. Transfected and non-transfected ventricular myocytes were
cultured for 4 hrs on poly-L-ornithine coated glass coverslips to allow attachment. After attachment,
the myocytes were exposed to an adenosine A1receptor agonist chlorocyclopentyladenosine (CCPA)
and/or the adenosine A1receptor antagonist, dipropylcyclopentylxanthine (DPCPX) as described in
the appropriate figure legend. Myocytes were fixed in 4% formaldehyde for 10 min and
permeabilized with 0.1% Triton X-100 in suffusion solution (SS). SS contained (in mM): 136.4
NaCl, 4.7 KCl, 1.0 CaCl2, 10 hydroxyethylpiperazine-ethanesulfonic acid (HEPES), 1.0 NaHCO3,
1.2 MgSO4, 1.2 KH2PO4, 10 glucose, 0.6 ascorbate and 1.0 pyruvate. After blocking with 3%
bovine serum albumin in SS at room temperature for 1 hr, the preparations were incubated with
rabbit antibodies against PKCε, PKCδ (Calbiochem, San Diego, CA) and GFP (Medical &
Biological Laboratories, Watertown, MA) at 4oC for 12-16 hrs. These antibodies were used to
assess the presence of endogenous PKCε and PKCδ and the level of PKCε transfection in the
myocytes. After washing with SS 3 times, they were incubated with fluorescence-labeled
indodicarbocyanine (Cy5) or fluorescein isothiocyanate (FITC) conjugated anti-rabbit secondary
antibodies (Jackson ImmunoResearch, West Grove, PA or Molecular Probes, Eugene, OR,
respectively) at 37oC for 1 hr. Excess secondary antibody was removed and the samples were
mounted in 3% 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma) / 90% glycerol in SS. The
immunostained samples were viewed using the Leica DM IRBE inverted microscope equipped with
TCS SP2 confocal system, a 65 mwatt argon laser, two helium/neon lasers (1.2 and 10 mwatt) and
differential interference contrast (DIC) accessories (Leica Microsystems Inc., Heidelberg, Germany).
Images were acquired and analyzed with LCS software and Adobe Photoshop 6.0 software (Adobe
Systems Inc., San Jose, CA).
PAGE and Immunoblotting. Ventricular myocytes were dissolved and subjected to PAGE and
immunoblotting as previously described (14). Primary rabbit PKCε and PKCδ antibodies were
used and visualized with a secondary anti-rabbit antibody conjugated to horse radish peroxidase.
PKC? inhibitor peptide. Ventricular myocytes were rendered permeable to PKCε inhibitory
peptide (Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr, EMD Biosciences, La Jolla, CA) by exposure to a
permeabilization buffer (PB) consisting of 20 mM HEPES (pH 7.4), 10 mM EGTA, 140 mM
KCl, 150 µg/ml peptide, 6 mM ATP, 2 µM β-escin and 0.02 % DMSO for 10 min at room
temperature. Following the permeabilization the myocytes were returned to SS by exposing the
cells to gradually increasing concentrations of Ca2+ stepwise every 2 min from 0, 50, 100, 300,
500 to 1,000 µM in SS containing 6 mM ATP and no initially added Ca2+. The myocytes were
suffused with SS for 20 min and stimulated to contract at 12/min.
Myocyte contractile function for over-expression studies. Transfected or non-transfected
ventricular myocytes contained in 35 mm dishes with 3.0 ml of SS were placed on an inverted
fluorescent microscope (Diaphot, Nickon, Japan) equipped with a SIT camera (VE 1000 SIT,
DAGE MTI). A 35 mm plastic ring equipped with platinum wire electrodes was used to initiate
myocyte contraction every 10 sec. Addition the ring also possessed an inflow and aspiration
outflow for continual suffusion of the myocytes with SS at 0.4 ml/min. The SIT camera and a
JVC CR-6004 video cassette recorder (Technical Video Resources, Fairfield, CT) were used to
record myocyte shortening with each contraction. After the transfected and non-transfected
myocytes were delineated in a fluorescent field a visual field was used to assess shortening of the
myocytes. The extent of myocyte shortening was determined by playback and printing of the
relaxed and fully shortened images of each myocyte using a Sony UP-811 video graphic printer.
The difference in the myocyte length between the two images provided the extent of shortening.
Shortening is expressed in microns.
Protocol for assessing myocyte contractile function. The ventricular myocytes were equilibrated
for 30 – 40 minutes without electrical stimulation and continually suffused with fresh SS (Fig 1).
Then a train of four contractions 10 sec apart was recorded as control shortenings. Suffusion was
continued with isoproterenol (ISO), a β1-adrenergic receptor agonist, at 0.4 µM present in the SS for
10 min and a train of four contractions was recorded for the first sequence. In the second sequence
suffusion was continued with the ISO together with the adenosine A1receptor agonist CCPA present
at a concentration of 2 µM in the SS for 10 min. A train of 4 shortenings was recorded. Continuing
with the third sequence, the ISO, CCPA and the adenosine A1receptor antagonist DPCPX (2 µM)
were included together in the SS for 10 min. A final train of four shortenings was recorded.
Myocyte contractile function in the presence of PKC? inhibitor peptide. Myocyte contractile
shortening was determined using IonOptix Contractility System (Milton, MA) in a continuous
suffusion chamber as previously described (13). Briefly, myocytes were stimulated to contract at
12/min and exposed to 0.4 µM ISO, 2 µM CCPA or a combination of these two agents for 4 min in
either the absence or presence of a PKCε inhibitor peptide. Myocyte cell shortening was recorded
and expressed in microns.
Statistical methods. All data are presented as mean ± one standard error of the mean. Analysis of
variance (ANOVA) was performed with additional testing using the Student-Newman-Keuls test
(Statmost, DatAxiom Software Inc., Los Angeles, CA). A probably (P value) of less than 0.05 was
accepted as indicating a statistically significant difference.
Materials. Buffer salts acids and general laboratory reagents were obtained from Fisher Scientific
(Medford, MA). ISO, ascorbic acid, CCPA, DPCPX, HEPES, DMSO, BSA, poly-L-ornithine, β-
escin (Aescin) and formaldehyde were purchased from RBI/Sigma Chemical (St Louis, MO). The
anti PKCε and PKCδ rabbit polyclonal and goat anti-rabbit antibodies were from Santa Cruz (Santa
Cruz, CA). The anti-GFP rabbit polyclonal antibodies were obtained from MBL (Ina, Japan).
Titon-X-100 was from RPI (Mt Prospect, Il) and MEM was from Gibco/BRI (Rockville, MD). ISO
(10 mM) was prepared in 0.1% Na2S2O5fresh daily and upon final dilution is a solution of 1 mM
ascorbic acid. CCPA and DPCPX (10 mM) were prepared in DMSO fresh daily.
Over-expression of GFP-tagged PKC? in cardiomyocytes.
To observe the effects of PKCε on the antiadrenergic action of adenosine A1receptor
stimulation, GFP-tagged PKCε was over-expressed in isolated ventricular myocytes using the
mammalian expression vector pEGFPN1/PKCε. The GFP-tagging makes it possible to identify the
transfected cells during monitoring the contraction. To assure that GFP signals observed in cells
represent GFP-PKCε but not the degradation products, the transfected cells were subjected to
Western blot analysis using anti-GFP antibodies (Fig. 2). We recognized the expressed GFP-tagged
PKC protein as a single band at the expected molecular weight of GFP-PKCε chimera and there
were no degradation products having the GFP moiety. The results indicate that the GFP signals in
cells solely represent GFP-tagged PKCε.
To examine whether the C-terminal GFP hampers PKCε function, PKCε-GFP was extracted
from transfected Cos7 cells, immunoprecipitated and the precipitate assayed for PKC activity as
described in the Methods. The kinase activities of PKCε-GFP in the presence and absence of PMA
were 3.2 µmol/min/mg and 0.25 µmol/min/mg, respectively. The activity was significantly
regulated by PMA suggesting that the PKCε-GFP chimera maintains the authentic PMA dependent
Detection of endogenous PKCε as revealed using a PKCε antibody indicates that upon
exposure of the non-transfected myocyte to CCPA the enzyme translocates to t-tubular-like
structures in the cell (Fig 3 panels a and b). Preincubation of non-transfected myocytes with the
adenosine A1receptor antagonist DPCPX prevents the CCPA-induced translocation of the enzyme
(Fig 3 panel c). This is not unusual because PKCε has been reported to translocate to membrane
caveolae upon activation in rat cardiomyocytes (48). In contrast distribution of endogenous PKCδ
as revealed using a PKCδ antibody indicates that CCPA does not cause this isoform to translocate
(Fig 3 panels g and h). The present results suggest that adenosine A1receptor stimulation using
CCPA elicits translocation of the PKCε to t-tubular-like structures of the ventricular myocytes.
Immunoblotting of endogenous PKCε and PKCδ revealed that both isoforms were present in the
myocytes. While there appeared to be more PKCδ compared to PKCε, the former isoform did not
translocate to t-tubular-like structures with CCPA stimulation of the myocytes.
In transfected myocytes GFP-tagged PKCε also moved to t-tubular-like structures with
CCPA (Fig 4 panels a and b) and it’s translocation is hampered by DPCPX (Fig 4 panel C).
However, the total fluorescent signal in the transfected myocytes was significantly higher than in the
non-transfected cells. These results demonstrate that the CCPA-induced translocation of PKCε
determined by anti-PKCε antibody staining is not a result of non-specific signals and that GFP-
tagging did not interfere with the CCPA-induced translocation of PKCε.
The expressed GFP-tagged PKCε was estimated to be approximately 5-fold (5.5 ± 0.2; n=3
experiments) greater in the transfected myocytes compared to the endogenous PKCε in non-
transfected myocytes. This comparison was obtained by determining the mean value of the total
fluorescent intensities of anti-PKCε antibody signals for non-transfected and transfected myocytes
(n=10 cells). It is reasonable to assume that the overexpression of PKCε should enhance the
activation of relevant downstream pathways as compared to responses in the absence of over-
To more clearly delinate the structure to which PKCε translocated, we compared the
localization of the GFP signal and the bright field image. The localization of GFP was coincident
with the I-band of the myocyte (Fig 5). Since t-tubules are located at the center of the I-band in
myocytes, the results suggest that A1R stimulation, using CCPA, elicits translocation of the PKCε to
t-tubular-like structures of the ventricular myocytes.
Cardiomyocyte contractile activity.
Overexpression of PKCε in ventricular myocytes increases the adenosine A1receptor-
mediated reduction of the β1-adrenergic elicited contractile response. The average resting myocyte
length was 109.5 ± 3.1 microns. The myocytes shortened by approximately 10% upon electrical
stimulation. With ISO administration at 0.4 µM in the suffusion buffer the extent of shortening
increased to approximately 30%. Overexpression of PKCε by transfection in myocytes had no
significant effect on the basal extent of cell shortening (Fig 5). A 10 min exposure to ISO caused a
28.9 ± 3.4 and 36.4 ± 3.7% increase in the extent of myocyte shortening above the basal levels of
normal and transfected myocytes, respectively. The ISO-induced increase in the shortening was not
significantly different between normal and transfected myocytes. In the presence of 2 µM CCPA, an
A1R agonist, the ISO produced increase in shortening was reduced to only an 12.9 ± 0.7% increase
above the basal level of shortening in normal myocytes. However, in transfected myocytes the
CCPA totally inhibited the ISO-elicited increase in the extent of myocyte shortening.
To verify that the CCPA inhibition of the ISO-elicited contractile response was due to
activation of the A1R, the A1R antagonist DPCPX was used in both normal and transfected
myocytes. DPCPX restored the ISO-induced increase in myocyte shortening in both normal and
transfected myocytes to levels not significantly different from the increase in shortening resulting
from the administration of ISO alone. These data suggest that the CCPA-induced reduction of the
ISO-induced increase in shortening is due to A1R stimulation.
Further experiments were conducted transfecting myocytes with PKCε-free (null vector).
The presence of the expression vector devoid of PKCε in the transfected myocytes did not influence
the antiadrenergic action of CCPA. ISO increased shortening by 30.6 ± 1.9% in normal and 32.8 ±
2.5% in null-containing myocytes (Fig 6). The increase in shortening caused by ISO in the presence
of CCPA was 13.1 ± 2.3 and 14.7 ± 2.8% for normal and null vector-containing myocytes,
respectively. These reductions in the ISO responses were not significantly different. DPCPX
restored the ISO produced increase in shortening in the presence of CCPA. These results indicate
that presence of the null expression vector did not influence either the antiadrenergic or ISO-induced
Comparison of the antiadrenergic effects observed in normal, PKCε transfected and null
vector-containing myocytes indicates that myocytes in which PKCε is overexpressed display a
greater antiadrenergic response (Fig 7). The antiadrenergic effect was approximately 2-fold greater
in the PKCε overexposed myocytes suggesting that PKCε plays an important role in the
antiadrenergic action of A1R stimulation.
The reduction of ISO elicited contractile response caused by CCPA was prevented by using a
PKCε inhibitory peptide. Ventricular myocytes rendered permeable to the peptide, as outlined in the
Methods, displayed normal contractile function. Upon permeabilization myocytes that were exposed
to the peptide shortened similarly to those that were not exposed to peptide (Fig 8). ISO at 0.4 µM
increased shortening by 94.7 ± 19.6 and 141.2 ± 15.8% in myocytes exposed and not exposed to the
inhibitory peptide, respectively. While the peptide tended to increase the ISO contractile response it
was not significant. The inhibitory peptide was without effect on myocyte shortening in the
presence of 2 µM CCPA alone. CCPA in the presence of ISO caused only a 46.2 ± 8.9% increase in
shortening when myocytes were not exposed to the peptide. However, in myocytes exposed to the
peptide, CCPA plus ISO increased shortening by 117.5 ± 14.3%. Thus, the PKCε inhibitory peptide
prevented the antiadrenergic action of CCPA in ISO stimulated myocytes.
The present study indicates that adenosine A1receptor stimulation results in a
translocation of endogenous and over-expressed PKCε to t-tubular-like structures of ventricular
myocytes. Furthermore, compared to non-transfected ventricular myocytes and myocytes
transfected with PKCε-free or null vector, over-expression of PKCε in myocytes potentiates the
antiadrenergic action of adenosine A1receptor stimulation in isoproterenol-stimulated
contracting myocytes. Over-expression of PKCε in ventricular myocytes has no effect on the
basal and isoproterenol elicited contractions, but exerts an effect upon stimulation with the
adenosine A1receptor agonist CCPA. While PKCδ was present in the ventricular myocytes,
CCPA did not cause translocation of this isoform of PKC. In addition a PKCε inhibitory peptide
prevented the antiadrenergic effect caused by CCPA in non-transfected myocytes without having
a significant influence on basal or isoproterenol elicited contractions. The results also appear to
indicate that ventricular myocyte PKCε requires adenosine A1receptor stimulation to manifest
an effect. Overall these findings suggest that PKCε may play an important role in mediating the
adenosinergic effects of adenosine A1receptor stimulation in cardiac muscle.
Over the past several years, the importance of PKC in signaling pathways modulating
heart function has become apparent. Studies have shown that PKC activation influences cardiac
contractility (6,31,55), gene expression (37), the development of hypertrophy (1,52) and the
manifestation of preconditioning of the ischemic myocardium (2,54). The PKC isoform PKCε
has been consistently detected in adult ventricular myocytes (23,24,41) and is observed
endogenously in the present study using an antibody to this isoform of the enzyme. Generally,
activation of PKC by extracellular agonists elicits the translocation from the cytosol to the
membrane fraction of cells. In this study PKCε appears to translocate to t-tubular-like structures
with adenosine A1receptor stimulation. This finding is in agreement with previous reports
revealing that PKCε translocates to striated structures thought to be the t-tubules of the
cardiomyocyte as evidenced by the co-localization of the PKC-ε-GFP construct with α-actinin, a
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structural protein marker for the Z-line of the sarcomere (41). However, the importance of this
translocation is not fully understood.
In the myocardium, t-tubule membranes penetrate the cardiomyocytes near the Z-lines
(20). L-type Ca2+ channels, inducible nitric oxide synthase (NOS2; 5), RACK2, the anchor
protein for PKCε, and A-kinase anchor protein (AKAP; 26) have been found to be localized in
the t-tubules. Such a position adjacent to sarcoplasmic reticular ryanodine receptors and
myofibrils creates the potential for an intricate modulation of excitation-contraction coupling via
the regulation of intracellular Ca2+ release and myofilament function (53). With specific
reference to protein kinase A (PKA), the close proximity of AKAP to the L-type Ca2+ channel
ensures that the activation of PKA via extracellular β−adrenergic receptor agonists results in the
phosphorylation and activation of the channel (21). The ensuing increase in Ca2+ current (Ica)
that elicits an increase in cardiomyocyte contractility would be terminated by the
dephosphorylating activity of protein phosphatase 2A also associated with the Ca2+ channel (8).
Giprotein-coupled receptors such as those stimulated by adenosine may potentially
reduce β-adrenergic stimulated (phosphorylated) Ca2+ channel activity and the resulting
augmentation of contractility via several mechanisms. A1R activation has been reported to
reduce the high affinity binding of agonist to myocardial β1receptors (43) as well as reduce
adrenergic stimulation of adenylyl cyclase (30) and cAMP formation (10,51). Each action alone
would result in a reduced adrenergic-stimulated PKA activity (11) and phosphorylation state of
many proteins associated with enhanced contractile function including the L-type Ca2+ channel,
(17). Second, adenosine has been found to manifest antiadrenergic actions via
carboxymethylation of PP2a (34). The resulting increase in enzyme activity would potentially
result in the dephosphorylation of myocardial proteins manifesting the increase in contractility