Cardiovascular Research 38 Ž1998. 559–588
Review
Cardiac protein phosphorylation: functional and pathophysiological
correlates
Stephen T. Rapundalo
)
Department of Biochemistry, Parke-DaÕis Pharmaceutical Research, DiÕision of Warner-Lambert, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Received 19 June 1997; accepted 4 February 1998
Abstract
Protein phosphorylation acts a pivotal mechanism in regulating the contractile state of the heart by modulating particular levels of
autonomic control on cardiac forcerlength relationships. Early studies of changes in cardiac protein phosphorylation focused on key
components of the excitation-coupling process, namely phospholamban of the sarcoplasmic reticulum and myofibrillar troponin I. In more
recent years the emphasis has shifted towards the identification of other phosphoproteins, and more importantly, the delineation of the
mechanistic and signaling pathways regulating the various known phosphoproteins. In addition to cAMP- and Ca2q-calmodulin-dependent
kinase processes, these have included regulation by protein kinase C and the ever-emerging family of growth factor-related kinases such
as the tyrosine-, mitogen- and stress-activated protein kinases. Similarly, the role of protein dephosphorylation by protein phosphatases
has been recognized as integral in modulating normal cardiac cellular function. Recent studies involving a variety of cardiovascular
pathologies have demonstrated that changes in the phosphorylation states of key cardiac regulatory proteins may underlie cardiac
dysfunction in disease states. The emphasis of this comprehensive review will be on discussing the role of cardiac phosphoproteins in
regulating myocardial function and pathophysiology based not only on in vitro data, but more importantly, from ex vivo experiments with
corroborative physiological and biochemical evidence. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Phosphoproteins; Regulatory proteins; Protein kinases; Protein phosphatases; Dephosphorylation; Excitation-contraction coupling
1. Introduction
Phosphorylation and dephosphorylation of proteins is
widely recognized as an important mechanism for regulating cellular function by a variety of physiological stimuli.
It is clear that many hormones, neurotransmitter substances, and other extracellular stimuli mediate their
physiological actions by altering either directly, or indirectly through regulatory proteins, the phosphorylation and
dephosphorylation of many intracellular proteins. In general, phosphorylation of specific residues on target substrates triggers small conformational changes in protein
structure which alter biological properties. Processes as
diverse as membrane transport and permeability,
metabolism, ionic fluxes, contractility, and the transcrip-
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PII S 0 0 0 8 - 6 3 6 3 Ž 9 8 . 0 0 0 6 3 - 7
tion and translation of genes, are all regulated by this
versatile post-translational mechanism w1x.
Protein phosphorylation is intimately involved in the
regulation of myocardial contraction and metabolism w2,3x.
A number of the proteins phosphorylated in the heart have
now been identified, but in many cases the precise mechanisms by which phosphorylation modulates their behavior
is only beginning to be elucidated. Historically, the
cAMP-dependent processes in heart have received the
most attention as modulators of cardiac protein phosphorylation w4–7x. More recently however, investigation has
expanded to include other protein kinases such as Ca2qcalmodulin protein kinase ŽCa2qrCAM-PK. w8,9x, protein
kinase C ŽPKC. w10–12x, cGMP-dependent protein kinase
ŽPKG. w13,14x, tyrosine protein kinases ŽPTKs. w15x, extracellularly regulated kinases ŽERKs. w16x, as well as mito-
Time for primary review 48 days.
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
gen-activated protein kinase ŽMAPKs. w15,17x, and the
related stress-activated or c-jun N-terminal protein kinases
ŽSAPKrJNKs. w18x. Similarly, the process of dephosphorylation by protein phosphatases has taken on new emphasis as investigators have recognized the integral part these
enzymes may play in modulating normal cardiac cellular
function w19–21x, given that the steady-state level of phosphorylation of any protein is therefore a reflection of the
relative activities of protein kinases and phosphatases that
mediate the interconversion process.
The objectives of this review are to present a comprehensive survey of the literature on cardiac phosphoproteins, their role in regulating cardiac function, and their
potential roles in myocardial pathophysiology. In so doing,
some general perspectives on mechanisms of cardiac protein phosphorylation will be presented.
The phosphorylation of cardiac phosphoproteins will be
discussed in this review from an experimental viewpoint.
There are two general experimental approaches used to
study protein phosphorylation of candidate substrates. The
first is in vitro phosphorylation of purified proteins or
mixtures of proteins Že.g. homogenates. by the exogenous
addition of specific kinases, phosphatases, modulating
agents andror cofactors, plus w32 Px-ATP. This type of
reaction defines proteins that are substrates, provides clues
on the specificity of activation, delineates the identity of
phosphorylation sites, and may indicate effects of phosphorylation on the functional properties of the protein. The
second approach is protein phosphorylation of in situ
organs or intact cells preincubated with w32 Px-ATP to label
cellular ATP pools. The tissuercells are then exposed to
various physiological stimuli or pharmacological to elicit
phosphorylation and, it is hoped, clearly measurable biological effects. Information regarding the conditions required for phosphorylation, the extent to which proteins
are phosphorylated in response to stimuli and, sometimes
more clearly than in in vitro studies, the consequences of
phosphorylation, can be gleaned from these ‘ex vivo’
experiments. In vitro or ex vivo, phosphorylated proteins
are often analyzed using one- or two-dimensional Ž2-D.
gel electrophoresis w22x, the increased labeling of proteins
being used to indicate stimuli-mediated phosphorylations.
The investigation of protein phosphorylation is not
straightforward. Problems with the in vitro approach include the use of nonphysiological doses of modulating
agents and cofactors, as well as an altered availability of
substrates due to cell disruption or dilution. Each may lead
to protein phosphorylations not seen ex vivo, and several
examples of this will be described in the context of cardiac
phosphoproteins. Yet another problem may be that the
previous phosphorylation state of a putative protein substrate, as it is extracted from the cell, may influence the
ability of the enzyme itself to utilize the protein as a
substrate. The ex vivo assay suffers from the possible lack
of specificity when using certain modulating agents, and
this may require separate corroborative evidence. Lastly, it
should be noted that phosphorylated proteins cannot be
positively identified as direct substrates, as there may be a
‘cross-talk’ or ‘cascade effect’ between cellular signal
transduction pathways. Technically, the use of one-dimensional gels to analyze protein phosphorylation have been
characterized by poor resolution, particularly when working with protein mixtures, or when trying to identify small,
novel proteins. On the other hand, 2-D gels have been
somewhat problematic in the past because of replication
errors and difficulty in analysis, although recent technological advances have largely ameliorated these issues. It is
important then that the limitations of each type of experimental approach be recognized when interpreting demonstrated results.
Nonetheless, the valuable information that in vivo or ex
vivo experiments can provide is critical, and in conjunction
with corroborative in vitro evidence, a very powerful
means for determining the properties of putative phosphoproteins. For instance, preparations of isolated cardiac
myocytes offer the advantage of performing multiple and
temporal measurements on a uniform cell population. With
perfused hearts sufficient tissue is available to study various proteins and associated biochemical activities. In both
cases useful measurements of mechanical activity are possible, particularly with the advent of sophisticated devices
to quantify contractile parameters in isolated cells. Thus,
the emphasis in this review will be on discussing cardiac
phosphoproteins that have been confirmed through not
only in vitro data, but more importantly, from ex vivo
experiments with corroborative physiological and biochemical evidence.
2. Cardiac phosphoproteins
2.1. Sarcolemma
The plasma membrane or sarcolemma is a semipermeable membrane critically involved in the regulation of the
myocardial contractionrrelaxation process and general cell
homeostasis. Most important are the mechanisms by which
intracellular Ca2q concentrations i.e. wCa2q x i in the cardiac
myocyte are controlled. As a result the sarcolemma contains several key activities for translocating Ca2q into and
out of the cell. These include the voltage-dependent, slow
Ca2q channel for inward Ca2q conductance, NaqrCa2qexchanger and Ca2q-ATPase pump for extruding Ca2q. In
addition, there are a number of other proteins crucial to
myocyte function including membrane-bound protein receptors for neurohormonal factors, enzymes such as adenyl
cyclase and the Naq,Kq-ATPase pump, numerous ion
channel types like the Naq and Kq channels, and a variety
of other proteins which may have regulatory roles associated with the aforementioned major components. A few of
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
the sarcolemmal proteins listed have now been identified
as phosphoproteins and their properties as such are described below.
2.1.1. Phospholemman
Phospholemman ŽPL. is a 72-amino-acid plasma membrane protein of apparent 15 kDa molecular mass as
analyzed by SDS-PAGE w23,24x. It is a highly basic protein and possesses a single membrane-spanning domain
consisting entirely of uncharged residues w24x. The aminoterminus end is oriented extracellularly, whereas the positively charged C-terminus end, which contains protein
kinase phosphorylation sites, projects into the cytoplasm.
PL shows many similarities to phospholamban, the main
regulatory protein of the sarcoplasmic reticulum Žsee section below., including sharing seven out of nine sequence
residues in the critical phosphorylation sites area. In the
transmembrane region, PL exhibits 52% amino acid homology to the g-subunit of Naq,Kq-ATPase w24x.
Previous in vitro studies have shown that PL can be
phosphorylated in cardiac sarcolemma at multiple sites by
the catalytic subunit of the cAMP-dependent protein kinase ŽPKA. w10,25x, as well as being the major plasma
membrane substrate for PKC w10,23,25x. In beating hearts
this protein has been demonstrated to be phosphorylated
upon perfusion with various inotropic agents. Stimulation
of b 1-adrenergic receptors in intact guinea pig myocardium with either isoproterenol w26x or denopamine w27x
resulted in a rapid onset and a 2–3 fold increase in w32 PxPi
incorporation that was correlated to an increase in the
maximal rate of developed tension following drug treatment. Dephosphorylation of PL has been demonstrated in
intact guinea pig hearts, but it occurs very slowly in
contrast to other cardiac phosphoproteins suggesting differential regulation of its activity w28x. Adenosine agonist
treatment of intact guinea pig hearts was also found to
reduce the isoproterenol-stimulated phosphorylation state
of PL w29x.
Activation of a-adrenergic receptors has been associated with an increased phosphorylation state of PL, although some disparities in the literature do exist. Several
groups have found that a-adrenergic stimulation in isolated rat w25,30x and rabbit hearts w11x results in an increased phosphorylation of PL, and in at least the latter
study, was shown to be mediated by PKC activation. The
role of PL as a key plasma membrane substrate for PKC
was also supported by Hartmann and Schrader who showed
that direct treatment with the PKC activator TPA could
stimulate phosphorylation of PL in intact rat cardiac myocytes w30x. Based on this premise, Kranias and colleagues
w10,31x further examined the role of a-adrenergic and
direct PKC activation in effecting PL phosphorylation of
isolated guinea pig hearts, but were unable to observe any
detectable changes in w32 PxPi incorporation. The discrepancy between this study and others noted above is unresolved, though it may simply be due to species differences
561
in signal transduction processes, or perhaps to the complexities of protein kinaserphosphatase interactionŽs. in
the intact cell.
The functional role of PL remains unknown, however it
is possible that alteration of membrane surface charge
secondary to phosphorylation may play a role in its function and may result in effects on activities of various
channels, pumps andror antiporters w24x. Indeed, expression of PL in Xenopus oocytes leads to the occurrence of a
unique chloride current w32x, suggesting that the protein
may be itself be an ion channel. Other investigators have
instead proposed that PL may be a prototypic member of a
new family of membrane proteins capable of regulating
ion channel activity w33x. In this regard, it has also been
suggested that this 15 kDa sarcolemmal protein may modulate increases in the cardiac slow inward Ca2q current
w33x.
As a major phosphoprotein localized in the sarcolemma
and by virtue of its activation by a- and b-adrenergic
processes, PL is most probably involved in positive inotropic effects by as yet undetermined molecular mechanisms. The discrepant findings that this protein may also
mediate negative inotropic actions via PKC-stimulated increases in phosphorylation are puzzling, but may suggest
differential site phosphorylation resulting in opposing
physiological responses.
2.1.2. Na qr Ca 2 q exchanger
Very few reports have addressed the role of protein
phosphorylation in the regulation of the cardiac NaqrCa2q
exchanger ŽNCX1. thereby effecting extrusion of Ca2q
from myocytes w34,35x. When bovine NCX1 was stably
overexpressed in COS or CHO cells no phosphorylation
was detected w34x. In contrast, more recent data has clearly
demonstrated significant basal phosphorylation of canine
NCX1 transfected into both CCL39 cells and rat cardiomyocytes, that was further enhanced by treatment with endothelin-1 ŽET-1., acidic fibroblast growth factor ŽaFGF.,
the phorbol ester, PMA, or the phosphatase inhibitor,
okadaic acid w35x. Additionally it was observed that the
PKC inhibitors, calphostin C and K252a, or EGTA, inhibited phosphorylation. All treatments that increased NCX1
phosphorylation also significantly increased both forward
and reverse modes of NaqrCa2q exchange. It appears
then that the cardiac NCX1 could play an integral role in
some of the reported negative inotropic actions of PKCactivating agents.
2.1.3. Ion channels
Considerable attention has been given to phosphorylation of cardiac ion channels as a means whereby the
activity of ion channels can be regulated, and in most cases
result in the alteration of the myocardial contractile state.
Much of this evidence is based on electrophysiological
studies involving whole-cell patch clamp techniques under
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
phosphorylatable conditions. It has been assumed in many
cases that the regulation is mediated by phosphorylationŽs.
of the channel protein directly or of an associated regulatory protein. Several excellent reviews have summarized in
detail the current level of understanding of ion channel
function, particularly that of slow calcium channels, and
the role of phosphorylation in modulating ion fluxes
w36,37x. Only a brief mention of the pertinent information
is made here.
2.1.3.1. Calcium channels. Cardiac Ca2q channels have
received most of the scrutiny relating to effects of phosphorylation on channel function, and thereby the contractile state of the heart w38x. This is because the Ca2q
channels are the key determinant of intracellular wCa2q x i
levels, which in turn directly effect force of contraction, as
well as acting as a cellular second messenger for other
processes, including Ca2q-dependent protein kinases. A
model by Sperelakis et al. w37x postulated that cAMP
elevation either by receptor-mediated stimulation or indirectly through phosphodiesterase inhibition, activates PKA
which phosphorylates either the slow Ca2q channel or a
contiguous regulatory protein, such that a greater number
of channels are available for voltage activation. Two
mechanisms have been proposed to account for the enhanced channel activation following phosphorylation, the
first being a conformational change in the channel protein
allowing the activation gate to be opened, or secondly, by
directly increasing the effective channel pore diameter. In
this model, phosphorylation markedly increases the probability of channel opening during depolarization. A number
of studies utilizing direct intracellular injections of various
agonist agents, including the catalytic subunit of PKA
w39–41x, and protein inhibitor of PKA w42x, are consistent
with the phosphorylation model of Ca2q channel activation and stimulated I Ca . Furthermore, channel activity can
be reversed upon withdrawal of PKA, suggesting that
regulatory components of the slow Ca2q channels are
either washed away or lose their affinities over time, most
likely through phosphatase action w43x. Thus, any agent
that increases the cellular cAMP level of the myocardial
cell will tend to potentiate I Ca , wCa2q x i , and contraction.
Studies in intact canine myocardium have demonstrated
that isoproterenol and norepinephrine treatment induced
substantial w32 PxPi incorporation into the b-subunit of cardiac L-type calcium channels using back-phosphorylation
techniques, and that this correlated well with positive
inotropic and chronotropic responses and tissue levels of
cAMP w44,45x. Phosphorylation of the Ca2q channel a 1
subunit via PKA-mediated mechanisms has also been
demonstrated, both in liposomes reconstituted with the
Ca2q channel w46x and in CHO cells overexpressing the a 1
subunit protein w47x, which resulted in enhanced Ca2q
efflux.
Other protein kinase systems in addition to PKA also
appear to be involved in the regulation of cardiac slow
Ca2q channels. Direct PKC activation with phorbol esters
and indirect PKC activation by angiotensin II ŽAII. treatment have been shown to stimulate I Ca in chick and rat
hearts w48,49x, but not in guinea pig hearts w50x. Inhibitors
of calmodulin ŽCAM. can inhibit slow Ca2q channel
activity, and this effect can be reversed by subsequent
microinjection of CAM w51x. Thus, it seems that slow
Ca2q channel activation can be achieved by at least three
apparent phosphorylation mechanisms namely, PKA,
Ca2qrCAM-PK, as well as PKC. It remains unclear if all
these phosphorylations occur on the same protein or on
separate proteins, or even if similar phosphorylation sites
are involved.
The myocardial slow Ca2q channels are also regulated
by cGMP, by use of the non-hydrolyzable cGMP analog
and potent PKG stimulator, 8-Br-cGMP, in a manner that
is antagonistic to that of cAMP. This has been demonstrated at both the whole-cell voltage clamp and single
channel level w52,53x. Direct introduction of PKG into
neonatal myocytes is apparently associated with a rapid
inhibition of I Ca w37x. A single protein with approximately
47 kDa mass has been demonstrated to be specifically
phosphorylated by PKG in guinea pig sarcolemmal membranes w54x, suggesting the existence of a putative protein
mediator involved in Ca2q channel regulation by a cGMP
pathway. There has however, been no confirmation of a
cGMP-dependent sarcolemmal phosphoprotein at an in
vivo level.
A brief mention should also be made that protein
phosphatases may also have a direct role in the signal
transduction cascade of Ca2q channel activation. Treatment with the catalytic subunit of protein phosphatases
inhibited PKA-mediated activation of Ca2q channels
w19,55x, whereas exposure to phosphatase inhibitors such
as okadaic acid and microcystin the phosphatase-dependent dephosphorylation w56,57x. Furthermore, in a recent
study Herzig et al. demonstrated that stimulation of protein
phosphatase type 2A activity abolished muscarinic-receptor-mediated inhibition of the b-adrenergic stimulating effects on Ca2q channel activity w58x. This evidence supports
the concept that protein phosphatase stimulation can take
part in the functional antagonism between adrenergic and
cholinergic stimuli in the intact myocardium w20x.
2.1.3.2. Potassium (K q) channels. A second important
class of ion channels involved in the heart is the Kq
channels. While less is generally known about the specific
mechanismŽs. by which these channels are regulated, sufficient electrophysiological evidence now exists to at least
partially implicate phosphorylation as a key modulatory
step. Injection of Xenopus oocytes, co-expressing a cloned
cardiac delayed rectifier Kq channel ŽRAK. and the human b-adrenergic receptor ŽbAR., with isoproterenol
caused a significant increase in the I RA K current w59x,
comparable to that previously seen by adrenergic stimulation of Kq currents in isolated frog myocytes w60x. In
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
separate experiments, similar findings were obtained in
oocytes expressing only the cloned Kq channel and treated
with PKA catalytic subunit w59x. Koumi et al. demonstrated that the inwardly-rectifying Kq channel Ž I Kl . in
human ventricular myocytes could be inhibited by PKAmediated phosphorylation, and that this response was antagonized by either a PKA inhibitor or phosphatase treatment w61x. Since I Kl inhibition led to observed increases in
action potential duration and depolarization of the resting
membrane potential, it is presumed that these were a result
of phosphorylation.
A third type of Kq channel, the ATP-sensitive Kq
ŽK ATP . channel, has recently received increasing attention
because these channels may regulate cardiac function during cellular injury w62x. Little direct evidence exists
demonstrating the role of protein phosphorylation in cardiac K ATP channel activity w63x. Phosphorylation of K ATP
channels is presumed to occur based on the strict requirement for a MgATP-dependent process to maintain these
channels operative w62x. It has been proposed that the K ATP
channel possesses two phosphorylation sites which are
differentially regulated by MgATP, and that their different
phosphorylation states may describe the various activities
displayed by K ATP channels, including resting state Žclosed
channel., spontaneous activity, rundown Ždephosphorylation of the channel., and reactivation Žrephosphorylation of
the channel. w64x. All this may have further implications on
K ATP channel activity with regards to how cellular phosphorylation-dephosphorylation ratios may be effected by
various endogenous neurohormonal and metabolic influences that would be expected in altered inotropic states.
It is unclear presently which specific phosphorylation
sites are involved in the activation or inhibition of K ATP
channel activity. Recent evidence by Kwak et al. showed
that K ATP channel activity in rat cardiac myocytes is
reciprocally modulated by phosphorylation of both Tyr and
SerrThr residues w65x.
The most direct electrophysiological evidence for K ATP
channel phosphorylation to date are the observations that
treatment of ventricular myocytes with various phosphorylating and dephosphorylating agents resulted in modulation
of K ATP channel activity w65–67x. Kwak et al. have shown
that the K ATP channel run-down process was suppressed
by the protein phosphatase inhibitor, okadaic acid, and
accelerated by the protein tyrosine phosphatase inhibitor,
sodium orthovanadate w65x. Following run-down, the
ATP-induced reactivation was enhanced by genistein, a
tyrosine kinase inhibitor. Furthermore, this study provided
the first direct evidence for a regulatory link between K ATP
channel activity and specific phosphorylation sites as activity was reduced by protein phosphatase 2A ŽPP2A. and
increased by tyrosine phosphatase 1B. In a separate study,
PKC phosphorylation resulted in inhibition of ventricular
K ATP channel activity at low cellular ATP levels and
alteration in the stoichiometry of ATP binding to the
channel w66x. However, at physiological ATP levels, PKC
563
upregulated K ATP channel activity and the reversal of this
effect was dependent on the activity of a membrane-associated PP2A w67x. Thus, it appears that the extent of channel
phosphorylation and therefore cardiac K ATP channel activity may be dependent on reciprocal modulation of specific
sites via different signaling mechanisms.
2.1.3.3. Other channels. A brief mention should be made
regarding other cardiac ion channel activities that have
been reported to be regulated by phosphorylation-dephosphorylation mechanisms, including chloride ŽI Cl . currents
w57,68–72x, which are critical for action potential repolarization, as well as connexin43 and connexin45 gap junction channel ŽI J . conductances w73–75x, that are essential
for cell-to-cell communication. For both channel types,
conductances were enhanced following activation of PKAw57,68–71,74,76x and PKC-dependent processes w70,71,74x.
Complete deactivation of channel conductances resulted
from phosphatase action w57,74x, and treatment with phosphatase inhibitors enhanced currents and reversed their
deactivation w57,70,74x. Confirmation that connex43 is a
phosphoprotein was obtained by Laird and coworkers who
showed that the protein is typically present in neonatal rat
cardiac myocytes as a 42 kDa band and constantly incorporates w32 PxPi w77x. A second phosphorylated form, connexin45, was also observed by w32 PxPi labeling at 44 kDa.
Phosphatase treatment of cell lysates eliminated the 42
kDa phosphoprotein band, revealing a non-phosphorylated
40 kDa form. It remains unclear as to whether in intact
myocytes connexin43 is phosphorylated by different kinases or by a single kinase at several sites, although the
primary sequence data indicates consensus sites for PKC
and possibly Ca2qrCAM-PK w78x. Laird et al. postulated
that multiple phosphorylation sites and forms of connexin43
may control various aspects of gap junction
metabolismrfunction, thereby facilitating cell-to-cell communications w77x.
2.1.4. Membrane receptors
Past studies have proposed protein phosphorylation as a
general mechanism in the regulation of receptor function.
The mechanisms involved in receptor phosphorylation are
most likely diverse and still poorly understood w79–81x. In
some cases receptors that signal the formation of second
messengers are themselves regulated by protein kinases
activated by the second messenger w79x. In other instances,
receptors are regulated by soluble receptor-‘specific’ protein kinases or so-called G-protein-coupled receptor kinases ŽGRKs. w82x, as for example, the b-adrenergic receptor kinase or bARK w81,83,84x. The consequences of
receptor phosphorylation include desensitization, i.e. the
diminished responsiveness of receptors to agonists, as well
as receptor internalization and activation w85,86x. The sites
involved in GRK phosphorylation have not been unambiguously identified, though it appears that differential
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
activation can occur subsequent to adrenergic- or PKC-induced phosphorylation by bARK1 on specific serines of
both the b- and a-adrenergic receptors w87–90x. The apparent role of GRKs in myocardial function based on
indirect studies has recently been reviewed w91x.
The muscarinic-cholinergic receptors ŽmAChR. have
been the only receptor family directly studied at the intact
cardiomyocyte or tissue level in terms of their possible
functional regulation by phosphorylation. Hosey and colleagues have demonstrated that the 79 kDa mAChR protein is phosphorylated in an agonist-dependent manner in
intact chick and porcine tissue w92–94x. In both cases,
stimulation of w32 PxPi-labeled tissue with muscarinic agonists led to increased phosphorylation of the mAChRs, and
specifically occurred on Ser and Thr residues w93,94x.
Activation of either PKC or PKA had no effect on receptor
phosphorylation or agonist affinity, nor did the treatment
of CAM antagonists. Furthermore, agonist-dependent
phosphorylation of cardiac mAChRs appeared to correlate
with a decreased agonist affinity and ability to produce a
negative inotropic response. Taken together, the studies by
Hosey and coworkers support the idea that cardiac mAChRs
require agonist occupancy of the receptor and may involve
the participation of a receptor-specific protein kinase. In
addition it appears that phosphorylation of mAChRs in the
heart may be a critical step leading to their desensitization.
In this regard, the mAChRs possess features that are
strikingly similar to that observed for several other members of the G-protein coupled superfamily of receptors,
most notably the b-adrenergic receptors w85x. Studies using
purified mAChRs from chick w95x and porcine heart w96x
have been found to be excellent substrates in vitro for
bARK. Further work is required however, to confirm that
bARK or a related receptor kinase directly phosphorylates
mAChRs in vivo.
2.2. Sarcoplasmic reticulum
Cardiac sarcoplasmic reticulum ŽCSR. consists of a
complex network of anastemosing membrane-limited intracellular tubules which surround the myofilaments as a
network. The CSR, whose main function is the regulation
of cytosolic Ca2q or wCa2q x i , is the most important system
in the cardiac cell that delivers activator Ca2q needed
during contraction for binding to the myofilaments. From
both a structural and a functional standpoint this membrane is divided into two general regions. These are Ža. the
subsarcolemmal cisternae or junctional SR, which refers to
that portion of the CSR that comes into close apposition to
the sarcolemma and transverse tubules, and that contains
the Ca2q release channels through which Ca2q flows to
initiate contraction, and Žb. the much more extensive sarcotubular network or longitudinal Žfree. SR that contains
the Ca2q-ATPase pumprphospholamban protein complexes which regulate active Ca2q transport into the CSR
lumen, and that forms the tubular network around the A
and I bands of myofilaments.
The CSR membrane contains a number of intrinsic
proteins that are key regulators in cardiac excitation-contraction coupling, and specifically modulate wCa2q x i in
determining rates of myofilament contraction and relaxation. These proteins are the Ca2q-ATPase pump ŽSERCA,
predominantly SERCA2 in the myocyte. w97x, phospholamban ŽPLB. w98x, Ca2q release channel ŽCCRC. or
ryanodine receptor w99,100x, and several Ca-binding proteins that include calsequestrin ŽCSQ. w101x, calreticulin,
and the 26 and 170 kDa Ca-binding proteins. Of this
group, the only phosphoproteins identified to date are
PLB, the CCRC and CSQ.
2.2.1. Phospholamban
PLB is the key CSR phosphoprotein involved in the
regulation of the SERCA2 pump, and hence Ca2q transport w102x. PLB is currently viewed as a functional inhibitor of the SERCA2 when it is in an unphosphorylated
state. Once phosphorylated, the inhibition of pump activity
is removed, and the process of active Ca2q transport into
the CSR lumen is allowed to occur. Indeed, recently it has
been demonstrated by Kranias and colleagues that after
ablation of the PLB gene, the Ca2q-uptake rate into CSR
was enhanced in the PLB-deficient hearts compared with
the wild-type mice hearts, with an ensuing elevation in
basal contraction w103x. Despite a considerable amount of
investigation on this protein in recent years, its basic
functional unit and structure in the CSR has not yet been
clearly defined, nor have the molecular mechanisms fully
defined its interactions with the SERCA2 pump w104x.
Nonetheless, recent advances in our understanding of the
SERCA2 protein structure have allowed for the development of a fairly precise mechanistic model of Ca2q transport with phosphorylation playing a key role w105x.
The primary structure of PLB has been deduced from
sequence data obtained from cDNA clones w106x. It is
generally assumed that the quaternary structure of PLB
corresponds to a pentameric structure with an apparent M r
of ; 25 000 based on SDS-PAGE w107x. The oligomeric
structure has been confirmed by the observation that dissociation of PLB into five lower M r species can occur
following boiling in SDS w108x. Each subunit of PLB
contains 52 amino acids, with a calculated M r of 6080
w106x, and is made up of two major structural domains
w106,109x. The highly hydrophobic C-terminal domain most
likely inserts itself into the CSR membrane. The N-terminal sequence of PLB is comprised of an amphiphilic
a-helical cytoplasmic domain which contains the target
sequences for kinase phosphorylation sites. The cytoplasmic domain also possesses the domainŽs. that interactŽs.
with the SERCA2 pump protein. Direct interaction between these two molecules has been demonstrated by
cross-linking studies w110x. Recent data by Cornea et al.
have revealed that the oligomeric states of PLB in an
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
artificial lipid bilayer environment are typically found in a
dynamic equilibrium which is perturbed following in vitro
phosphorylation by PKA catalytic subunit w111x.
A number of studies involving a variety of techniques
have led to a proposed structural model of PLB whereby
the protein exists in its pentameric structure in such a
manner as to form a hydrophobic Ca2q-selective ion channel pore w112–116x. It is unclear at this time how the
putative PLB channel pore is integrated functionally with
the interaction between PLB and the SERCA2 pump.
The concept that phosphorylation of PLB allows for the
removal of the molecule’s inhibition upon SERCA2 activity is supported by several lines of evidence. Proteolysis
studies by Kirchberger et al., demonstrated a correlation
between tryptic digestion of unphosphorylated PLB and an
activation of Ca2q transport by the SERCA2 pump w117x.
Upon phosphorylation, digestion of PLB by proteolytic
enzymes is greatly reduced. Huggins and England suggested that the hydrophobic domain of PLB may in fact
undergo a conformational change w118,119x. This was further supported by Simmerman et al. when discussing their
two-domain model of PLB, where each contains a stable
a-helix, and upon phosphorylation, these helices are believed to rotate relative to each other so as to remove its
inhibition on the SERCA2 w120x. Studies by Wang and
coworkers have implicated residues 7–16 of the N-terminal region of PLB as those essential in the direct regulation
of the SERCA2 w121x. While the exact molecular mechanisms defining the interactions of PLB and SERCA2 are
not currently well understood, it appears certain that the
interaction between them is completely dependent on the
phosphorylation state of PLB w122x. Recent data would
suggest that PLB phosphorylation is associated with enhanced interactions between individual SERCA2 polypeptide chains due to spatial rearrangement and protein-protein interactions w123x.
Phosphorylation of PLB in vitro by PKA w124,125x,
Ca2qrCAM-PK w126,127x, PKC w128,129x, or PKG
w13,130x, has been shown to markedly increase CSR Ca2q
uptake. The substrate site for phosphorylation of PLB by
PKA has been demonstrated to be Ser 16 , whereas Thr 17 is
phosphorylated by the Ca2qrCAM-PK w114x. No definitive phosphorylation sites on PLB have been identified for
PKC, although it did not appear to occur at either Ser 16 or
Thr 17 w128x. In the case of PKG, the Ser 16 residue was
suggested as the target substrate site, since in the presence
of PKA stimulatory effects of both kinases on the SERCA2
activity and Ca2q transport were not additive w130x. The
characteristics of PLB phosphorylation appear to be based
on changes in structural conformation, most likely at the
Ser 16 site w131,132x. Detailed studies have revealed that
PLB phosphorylation of Ser 16 by PKA proceeds via a
random mechanism, while that of Thr 17 by Ca2qrCAM-PK
proceeds via a cooperative mechanism w133x. The latter
process is apparently unaffected by the phosphorylation
status of Ser 16 , and Ca2q accumulation was stimulated in
565
proportion with the stoichiometry of PLB phosphorylation,
regardless of the site of phosphorylation.
Phosphorylation of CSR by endogenous or exogenous
PKA was first shown to be associated with PLB by
Kirchberger et al. w134x. Subsequent studies have confirmed that PKA-dependent phosphorylation of PLB results in the stimulation of the initial rate of Ca2q transport
by lowering the half-maximal concentration of Ca2q
needed for stimulation of SERCA2 activity w125,135,136x.
These findings strongly suggested that the apparent affinity
of the SERCA2 pump for Ca2q was increased during PLB
phosphorylation.
Two possible mechanisms have been proposed to explain the observed stimulation of Ca2q uptake by PKA
dependent phosphorylation, namely an enhanced SERCA2
turnover rate, or an increased efficiency of the SERCA2
for Ca2q Ži.e. an increased coupling ratio. w137,138x. Studies of SERCA2 activity have demonstrated that PKA-dependent phosphorylation of PLB correlated well with stimulation of the SERCA2 while maintaining a stoichiometric
ratio of 2:1 for mole of Ca2q uptakermole ATP hydrolyzed w139x. The enhanced turnover rate of SERCA2 activity by PKA stimulation can be ascribed to changes at two
major reaction steps of the ATP hydrolysis-Ca2q transport
model. Rapid kinetic experiments demonstrated that PKA
phosphorylation of PLB produced a marked increase in the
reaction associated with Ca2q binding to the ATPase
enzyme, and in the rate at which the phosphorylated
intermediate ŽE ; P. was subsequently formed w137,138x.
Stimulation of E ; P formation was found to be associated
with a decrease in the dissociated constant for Ca2q
binding Ži.e. an increase in the affinity of the SERCA2
pump for Ca2q .. The observed alterations in Ca2q affinity
are probably due to an increase in the rate of the slow
conformational change of the SERCA2 enzyme upon Ca2q
binding w140x. Whether the conformational change of the
SERCA2 and its subsequent alteration of intrinsic rate
constants are due to a direct steric interaction of PLB with
the enzyme has not be completely elucidated. Recent
kinetic studies have revealed that PKA-mediated phosphorylation of PLB are correlated to an accelerated rate of
decomposition of the phosphorylated SERCA2 intermediate ŽE 2 P. which contributes to the increase in VmaxŽCa.
w141x. These observations may have some implications for
the possible stimulatory role of PKA-dependent phosphorylation on CSR function in vivo, as will be discussed
later, since it is a slow and rate-limiting reaction step in the
SERCA2 reaction scheme.
PLB phosphorylation by PKA in vitro has also been
shown to stimulate Ca2q efflux from the CSR w142,143x. A
reduction in the amount of Ca2q needed to attain halfmaximal activation of Ca2q efflux was also observed.
These findings suggest that a component of the SERCA2
pump participates in the release of Ca2q from CSR, but no
definitive evidence is available to describe the possible
molecular mechanisms for this process.
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
Phosphorylation of PLB in situ, either in isolated perfused hearts or in isolated cell culture systems, has now
been established in different species using the paradigm of
b-adrenergic stimulation, in conjunction with measurement
of associated functional and biochemical parameters. In
this regard, several groups have made major contributions
over the last two decades to our understanding of PKAmediated stimulation of cardiac regulatory phosphoprotein
activity, including KraniasrSolaro et al. w5,7,28,144–148x,
Watanabe and coworkers w6,8,149,150x, England and colleagues w151–153x, and more recently, Neumann et al.
w29,154x. In most studies, b-adrenergic stimulation was
found to enhance Ca2q transport activities, and observed
PLB phosphorylation appeared to parallel the temporal
change in myocardial relaxation w6,147,155x. Other PKAstimulating agents such as phosphodiesterase inhibitors
w5,156x, adenyl cyclase activators w151,156x, b 2-adrenergic
receptor ligands w157,158x, and Ca2q-sensitizing agents
w159x, have also been studied for their effects on enhancing
PLB phosphorylation. However, there appears to occur a
compartmentalization of cAMP or PKA, or both, in heart
that is differentially coupled Žin some cases not at all. to
PLB phosphorylation andror contraction, based on the use
of PKA-dependent agents w5,348x.
In PLB-ablated transgenic mouse hearts, baseline or
isoproterenol stimulation resulted in similar levels Žalbeit
attenuated in the case of isoproterenol treated animals. in
tissue cAMP levels and the degree of phosphorylation of
other cardiac phosphoproteins when compared to wild-type
hearts w148x. These data that has emerged confirms the
concept of PLB acting as a key regulator of myocardial
relaxation during sympathetic or catecholamine stimulation.
Parasympathetic control through muscarinicrcholinergic-mediated processes have been studied as a corollary to
b-adrenergic stimulatory effects on PLB. Several groups
have provided evidence suggesting that muscarinicr
cholinergic action can reverse b-adrenergic effects of PLB
phosphorylation and function, without much if any alteration to elevated cAMP levels w21,150,156,160–163x. The
effect of adenosine agonists has also been examined on
b-adrenergic-stimulated PLB phosphorylation in intact cardiac myocytes w21,29,162,164–166x. The resulting data
showed that elevated PLB phosphorylation levels were
attenuated following adenosine receptor stimulation, which
supports the idea that adenosine receptor-mediated events
may share similar signal transduction processes to that of
muscarinicrcholinergic pathways in reversing b-adrenergic responses. Similarly, treatment of cardiac myocytes
or isolated hearts with a 1-adrenoceptor stimulation was
found to inhibit b-adrenergic agonist-induced increases in
protein phosphorylation and myocardial contractility w167x.
It was speculated that this modulation may occur via the
PKC-mediated pathway, but no data were provided to
substantiate this.
As in the case with PKA, Ca2qrCAM-PK-dependent
PLB phosphorylation in vitro has been shown to enhance
the initial rates of CSR Ca2q uptake w126,127,137,168x.
This stimulatory effect was most pronounced at low Ž- 1
mM. Ca2q levels and appears to be due to an increase in
the apparent affinity of the SERCA2 for Ca2q w127,168x
Ca2qrCAM-PK-dependent phosphorylation of PLB requires the absolute presence of free Ca2q over a concentration range of 10y7 to 10y5 M w137x, in addition to
exogenous CAM ŽEC 50 s 50 nM. w127x. Kranias and
coworkers have demonstrated that calmodulin can activate
an endogenously bound Ca2qrCAM-PK which phosphorylates PLB, and in turn stimulates the initial rates and
maximal levels of the phosphorylated intermediate, E ; P,
of the SERCA2 w168x. In this regard, the Ca2qrCAM-PK
dependent system is similar to the PKA dependent process
in its kinetic effects on the SERCA2 reaction sequence.
This was confirmed by several groups but it is not clear
whether the cytoplasmic or CSR luminal Ca2q pools are
involved in the Ca2qrCAM-dependent stimulation w169–
171x. Studies by Karczewski et al. have shown recently
that Ca2qrCAM-PK phosphorylated PLB on Thr 17 exclusively, though this was predicated on the use of a synthetic
PLB peptide substrate w172x. The Ser 38 residue on SERCA2
has been identified as the specific site for phosphorylation
by Ca2qrCAM-PK w169x.
Studies in intact hearts indicate that direct alterations in
wCa2q x i levels do not by themselves stimulate PLB phosphorylation, even though Ca2qrCAM levels are raised w8x.
As a result neither CSR Ca2q transport or SERCA2 activities were altered, nor was the observed rate of myocardial
relaxation. However, PLB phosphorylation was attenuated
under conditions where activation of Ca2qrCAM-dependent processes was inhibited w8,9,173,174x. Thus, it is
possible that Ca2qrCAM-dependent mechanisms may be
partially mediating the b-adrenergic cardiac relaxant effect. More recent data has given new insights into the
mechanisms underlying Ca2qrCAM-dependent PLB phosphorylation in the intact heart w175x. Under maximal badrenergic stimulation activation of Ca2qrCAM-PK accounted for approximately 50% PLB phosphorylation exclusively at the Thr 17 site and was closely associated with
increased myocardial relaxation. A recent study by Baltas
et al. demonstrated that CSR Ca2qrCAM-PK is activated
in response to b-adrenergic stimulation, prompting autophosphorylation of its regulatory domain and conversion to
an active Ca2q-independent species w349x. It was suggested
that this could form the basis for potentiation of Ca2q
transients in the heart.
Since CSR function is regulated in vitro by both PKA
and Ca2qrCAM-PK pathways, a clear understanding of
the possible contribution each assumes becomes critical in
determining the interrelationship of these two regulatory
mechanisms. It has been demonstrated that PKA stimulation of Ca2q uptake can occur independently of
Ca2qrCAM-PK dependent phosphorylation w125,176x, with
the converse being true as well w127x. When both protein
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
kinase systems are operating, they appear to have an
additive effect w125x. These findings suggested that at least
in vitro, the presence of dual control systems operating in
an intricate manner to regulate CSR function.
Evidence from intact myocardium studies have suggested that a coordinated dual control system involving
PKA and Ca2qrCAM-PK may not necessarily be the case.
This conclusion is supported by Wegener et al. who
demonstrated sequential PLB phosphorylation, meaning
that Ca2qrCAM-PK phosphorylation occurred only after
PLB had been phosphorylated by PKA-dependent mechanisms. w177x Thus, multi-site and sequential phosphorylation of PLB by two different protein kinases in response to
b-adrenergic stimulation in beating hearts illustrates the
complex and integrated regulation that occurs in the modulation of CSR Ca2q fluxes, and of the myocardial relaxation process.
Mechanistic information on the in vitro stimulatory
effects of PKG on PLB phosphorylation are limited. Based
on kinetic characterization, PLB seems to be an excellent
substrate for PKG with Vm a x values approximately four
times than that seen for PKA w13x. Furthermore, the kinetics of the phosphorylation appear to be cooperative. At the
intact heart or myocyte level the biochemical mechanisms
of how cGMP modulates cardiac contractionrrelaxation is
not completely understood. This is due to disparity in
reported data on effects of PKG on PLB phosphorylation.
In one study, Huggins et al. used cGMP analogs but were
unable to observe enhanced PLB phosphorylation w13x,
whereas Sabine et al. have recently demonstrated that
PKG-dependent agents increased PLB phosphorylation
with an accompanying rise in cellular cGMP levels w14x.
Currently, there is no explanation for the disparity in
results from the two studies.
Studies in vitro have demonstrated that PKC can also
phosphorylate PLB w128,178,179x, in both junctional and
free CSR.w179x This phosphorylation increases the CSR
SERCA2 activity and thus Ca2q uptake w128x. However,
PKC-mediated phosphorylation of PLB appears not to
occur in vivo. Indeed, both Talosi and Kranias w11x, and
Hartmann and Schrader w30x did not observe any PLB
phosphorylation following perfusion of guinea pig hearts
or incubation of isolated rat myocytes with the phorbol
ester, PMA.
In order for the phosphorylation of PLB to play a
physiological role in the regulation of CSR function, and
thereby myocardial contractionrrelaxation, some mechanismŽs. must exist to dephosphorylate the protein and
return it to its role as a functional inhibitor or the SERCA2.
Such a mechanism is fulfilled by protein phosphatases
which hydrolyze the phosphoester bonds formed by protein kinases. As a result, protein phosphatases are generally viewed as being intricately involved in regulating a
variety of signal transduction pathways and cellular proteins w180x.
Studies in the cardiac system have shown that endoge-
567
nous protein phosphatase activity in CSR could dephosphorylate the PKA sites on PLB, and thus cause a decrease
in the degree of stimulation of Ca2q transport activity
w135,181x. Subsequent investigations have confirmed these
initial observations using CSR membrane-bound protein
phosphatase, and were also able to demonstrate that dephosphorylated PLB could be rephosphorylated to full
recovery of Ca2q transport activity w182x. Three distinct
types of protein phosphatases have now been demonstrated
to dephosphorylate PLB to some degree in vitro w183–186x.
Kranias and coworkers have been able to partially purify a
‘PLB-specific’ phosphatase that was capable of dephosphorylating both PKA and Ca2qrCAM-PK activated phosphorylation sites w183,184,186x. A recent in vitro investigation using rat cardiac microsomal preparations has shown
that PP1 is capable of dephosphorylating PLB when the
latter protein is phosphorylated by PKA, but not by
Ca2qrCAM-PK, and that under certain conditions PP2B is
also able to dephosphorylate PKA-activated PLB w187x.
The findings from intact heart studies are in general
agreement with in vitro results on the effects of various
protein kinases, and in particular PKA-dependent phosphorylation, on CSR function. They support the hypothesis
that PLB phosphorylation plays a pivotal role in mediating
sympathetic and other neurohormonal inotropic effects on
the heart. Furthermore, PLB phosphorylation appears to be
only one aspect of a more complex and coordinated regulatory system whereby PKA-mediated effects modulate cardiac function. The full breadth of mechanisms and effects
of this regulatory system are only now beginning to be
understood. As for the roles of Ca2qrCAM-, PKC, or
PKG-dependent protein phosphorylation, their contributions to the overall regulation of myocardial contractile
and relaxation process remain to be fully elucidated, though
some emerging mechanisms are discussed in a later section.
2.2.2. Ca 2 q release channelr ryanodine receptor
In cardiac muscle, Ca2q release from the CSR is mediated by a Ca2q-activated channel called the cardiac Ca2qrelease channel Ž CCRC . or ryanodine receptor
w99,100,188x. The CCRC is regulated by Ca2q influx
through voltage-gated Ca2q channels in the sarcolemma.
This process, termed Ca2q-induced Ca2q release w189x, is
fundamental to cardiac excitation-contraction coupling, the
mechanism that links surface membrane depolarization to
Ca2q activation of the contractile apparatus w190x.
Cloning and sequence analysis of cDNA have suggested
that the CCRC protein is a large polypeptide of approximate 565 kDa mass. The cytoplasmic region of the CCRC
appears to correspond to the ‘foot’ structure Žthe part of
the junctional CSR that bridges the gaps between the CSR
and surface membrane transverse T-tubules. w191x. The
C-terminus region probably forms the CCRC w192x.
The cardiac CCRC appears in vitro to be regulated by
Ca2q w193,194x, Mg 2q w194x, adenine nucleotides w195x,
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
and several protein kinases, including Ca2qrCAM-PK
w196,197x and PKA w197,198x. It has been proposed that
protein kinase-dependent phosphorylation of the CCRC
may be physiologically important for the regulation of the
protein’s activity, and thereby cardiac muscle excitationcontraction coupling. Direct support of in vivo phosphorylation of CCRCs was reported by Yoshida et al., who
observed that w32 PxPi incorporation into CCRCs was enhanced upon isoproterenol treatment of neonatal rat cardiac myocytes w199x. Thus, the PKA-dependent phosphorylation of the CCRC may at least partially account for the
acceleration of the rising phase of transient wCa2q x i in
b-agonist-treated cardiac myocytes.
2.2.3. Calsequestrin
Calsequestrin ŽCSQ. is the major Ca2q-binding protein
of CSR with an apparent mass of 55 kDa based on
SDS-PAGE analysis w101,200x. The protein is mostly localized in the lumen of the junctional SR w201x, where it
has been shown to bind to some protein constituents, for
example the ’foot’ protein and the CCRC w202,203x. Ikemoto et al. have suggested that Ca2q-dependent conformational changes in CSQ affect the junctional SR proteins
and in turn regulate CCRC function w204x. In general, the
primary physiological function of CSQ is thought to be
sequestration of large amounts of Ca2q in the lumen of the
CSR, reducing luminal levels of free Ca2q and facilitating
further Ca2q uptake by the CSR SERCA2 pump, though
details of this process have not been fully elucidated w205x.
Cardiac CSQ has been proposed to be a phosphoprotein
based on the identification of several consensus phosphorylation sites in its primary structure. A unique feature of
the protein is a highly acidic, 31 amino acid C-terminal tail
Žresidues 361–391. which contains three closely spaced
Ser residues that were proposed to act as excellent substrates for casein kinase II w206x. Moreover, cardiac CSQ
was shown to contain endogenous Pi localized to the same
cluster of Ser residues identified in the primary sequence
w207x. In this latter study, Cala and Jones demonstrated that
the same Ser sites of CSQ were rapidly phosphorylated in
vitro by casein kinase II. Similar observations were made
in cultured rat myotubes, validating that CSQ can be
phosphorylated in vivo w208x. There did not appear to be
any marked effect of phosphorylation on CSQ Ca2q-binding capacity or affinity either in vitro or in intact cells.
Thus the functional significance of the phosphorylation
event by casein protein kinase II or similar enzyme upon
cardiac CSQ is presently unknown.
2.2.4. CSR membrane phospholipids
In addition to the phosphorylation of membrane proteins, there have been several in vitro studies suggesting
that phospholipids may also be phosphorylated by both
PKA- and Ca2qrCAM-dependent kinases in cardiac muscle w209,210x. This type of phosphorylation has been implicated in the modulation of wCa2q x i by CSR Ca2q transport
activity, probably via diacylglycerol stimulation of PKC
which is dependent on phospholipid moieties for its activation w128x. There is in vivo evidence that demonstrated an
association between b-adrenergic stimulation of intact
guinea pig hearts with phosphorylation of whole cardiac
and CSR membrane polyphosphoinositides w146x. Specifically, isoproterenol stimulation increased phosphorylation
of phosphatidyl mono- and bi-phosphate, as well as phosphatidic acid. In an associated study from the same laboratory, Edes et al. quantified changes in phosphoinositide
turnover under similar inotropic conditions w144x. Their
findings revealed increases in phosphoinositide cycle intermediates that were not correlated with either increases in
regulatory protein phosphorylation or cAMP levels. A
concomitant decrease in inositol triphosphates was observed which was apparently related to lowered in phosphoinositol-phospholipase C enzymatic activity. The underlying mechanisms of phospholipid phosphorylation in
situ are not presently known, but it suggests that there may
be a complex interrelationship between membrane protein
phosphorylation Že.g. PLB. and phosphoinositide phosphorylation, and respective functional correlates.
2.3. Myofibrillar proteins
The contractile properties of the heart are determined by
the interaction of three major classes of proteins, namely,
contractile proteins Žmyosin and actin., regulatory proteins
Žtropomyosin and troponin complex. and structural proteins ŽC-protein, a-actinin, etc.. w211x. A necessary requirement for cardiac contraction is the ability of myosin
to hydrolyze ATP, liberating the terminal phosphate as a
source of energy for cardiac contraction. The contractile
proteins convert the chemical energy of ATP hydrolysis
into mechanical work through physicochemical changes.
Regulatory proteins bound to actin serve to regulate the
actomyosin cycle such that when wCa2q x i levels are high
Ž100 mM., the regulatory protein complex is inhibited, and
ATP hydrolysis is increased resulting in ‘shortening’ or
contraction. Conversely, when wCa2q x i levels are low Ž0.1
mM., the regulatory protein complex blocks the actinmyosin crossbridge attachment, ATP hydrolysis is reduced, and relaxation ensues. Structural proteins generally
do not participate in the active contractile process, but are
believed to provide some mechanical linkage and stability
properties to the contractile and regulatory proteins.
The three myofibrillar proteins described below represent each of the major classes of proteins mentioned
above. More importantly, they all are proteins in which the
phosphate groups are in rapid equilibrium with wATPxi , and
whose phosphorylation states can change quickly following neurohormonal stimulation.
2.3.1. Troponin I
Troponin I ŽTnI. is the inhibitory subunit of the regulatory troponin complex, along of troponin C ŽTnC. and
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
troponin T ŽTnT.. Tissue-specific isoforms derived from
different gene products have been identified, including the
cardiac muscle type w212x. TnI has a molecular weight of
about 27 000 daltons and serves as the specific inhibitor of
the actomyosin Mg 2q-ATPase.
There are now several lines of evidence indicating that
TnI phosphorylation is a physiologically significant event
in the regulation of myocardial contraction. Early in vitro
studies by several groups demonstrated that TnI was an
excellent substrate for PKA w212–215x, with the N-terminal Ser 20 as a specific phosphorylation site w216x. Several
studies indicated that phophorylation of TnI by PKA decreased the Ca2q sensitivity or activity of actomyosin
Mg 2q-ATPase w145,217,218x, and rate or affinity of Ca2q
binding to TnC w219x. Recent in vitro data from Solaro and
colleagues suggest that phosphorylation of TnI at Ser 23
and Ser 24 residues in the unique NH 2-terminus domain is
both necessary and solely sufficient for the decrease in
myofilament Ca2q-sensitivity associated with PKA-dependent phosphorylation w220x. The latter observations have
been extended by Keane et al. who noted that phosphorylation at these two sites is sequential with Ser 24 being
rapidly phosphorylated followed by a slower phosphorylation of Ser 23 that occurs only after Ser 24 phosphorylation
is almost complete w221x. Furthermore, the Arg 22 residue
appeared to be critical in determining the reaction kinetics
of phosphorylation by PKA resulting in conformational
changes around the paired Ser region. Evidence for conformational changes in N-terminal extension of TnI has recently been demonstrated whereby phosphorylation caused
reductions in the distance between sites located at the Nand C-terminal portion of TnI w222x. In addition, there
appears to be a direct transduction of a PKA-induced
phosphorylation signal from TnI to the regulatory site of
TnC involving a global change in TnI structure w223x.
Together these studies provide a molecular basis for the
change in Ca2q sensitivity of the troponin complex, most
likely through an enhanced off rate for Ca2q exchange
with TnC, following its activation by phosphorylation.
Pioneering work on protein phosphorylation in the intact heart actually began with the demonstration that badrenergic Žcatecholamine. stimulation resulted in TnI
phosphorylation w4,224–226x, specifically at the Ser 20 site
w4,226x. Over the years other groups w7,145,156,227x have
confirmed these early observations and have extended
them to include other PKA-dependent agonists including
forskolin w151,156x, cAMP phosphodiesterase inhibitors
w5,152,154,156x, and Ca2q-sensitizers w159x. An enhanced
sensitivity to b-adrenergic stimulation of TnI as compared
to PLB was observed in isolated hearts, suggesting possible compartmentation of cAMP w5,172x. In all cases where
it was measured, an increase in TnI phosphorylation was
associated with a rise in force of contraction.
The relationship between TnI protein phosphorylation
and the temporal features of associated functional activity
has been well documented. Increases in TnI phosphoryla-
569
tion parallel the rise in force observed with b-stimulation,
but levels of phosphorylation remain high in spite of a
return of force to pre-stimulation levels w28,145x. It may be
then, that TnI phosphorylation is not an absolute requirement for the increase in myofibrillar force, but may serve
to act in concert with other possible mechanisms. Demonstration that the two Ser residues in TnI capable of being
phosphorylated by PKA have differential effects on the
decrease in myofilament Ca2q-sensitivity, may provide an
explanation for the lack of correlation between TnI phosphorylation levels and function during wash-out of badrenergic effects w220x.
Dephosphorylation of TnI is promoted by the addition
of acetylcholine w156,165,225x or adenosine agonists
w29,162,165,228x to hearts or cells stimulated with isoproterenol, in which case force and TnI phosphorylation
fall more or less in parallel. However, in one study no
reduction in TnI phosphorylation was noted following
adenosine agonist treatment w164x. The reason for these
disparate results is unknown. The demonstrated effects of
adenosine and cholinergic agents on b-adrenergic stimulation of TnI phosphorylation have been found to be pertussis toxin sensitive and cAMP-independent, since activated
cellular cAMP levels were unaltered w162x.
Recent experiments by Neumann and coworkers have
focused on the role of phosphatase inhibition on stimulating TnI phosphorylation. Several phosphatase inhibitors
have been characterized, including okadaic acid, calyculin
A, and cantharidin, and all were observed to enhance TnI
phosphorylation and the force of contraction w229–231x.
For the most part it seems that TnI can also act as
superb substrate for PKC, especially in vitro w232–234x.
The major site of phosphorylation for PKC appears to be
Thr 144 , with secondary phosphorylation sites identified at
Ser 43 , Ser 45 and Thr 78 w233x. These latter three residues are
located in the N-terminal region where most of the binding
to TnC occurs w211x. Recent studies have shown that
isozymes of PKC exhibited discrete specificities in phosphorylating distinct sites in TnI w235x. For instance, PKC-d
was uniquely able to phosphorylate Ser 23 and Ser 24 , previously recognized as being PKA phosphorylation sites only,
and thereby reduced myofibrillar Ca2q-sensitivity. In addition, PKC-d, like PKC-a and PKC-´, but not PKC-z,
phosphorylated Ser 43 and Ser 45 and reduced maximal
Mg 2q-ATPase activity.
In intact cardiac myocytes, direct activation of PKC
with phorbol esters, or indirectly with ET-1 and arachidonic acid, thought to be mediated by a PKC-dependent
pathway, served to increase TnI phosphorylation in a
Ca2q-independent manner w12,236,237x. Venema and Kuo
were able to show that the PKC-mediated TnI phosphorylation was associated with the inhibition of myofibrillar
actomyosin Mg 2q-ATPase, similar to that seen with PKA
w12x. However, in beating heart studies Žrat or rabbit.
indirect PKC activation using either ET-1 treatment w238x
or a-adrenergic stimulation w11x, was insufficient to stimu-
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
late TnI phosphorylation, despite being an excellent substrate for PKC in vitro in the latter study. One explanation
for this may be that activation of specific PKC isozymes
are needed to demonstrate in vivo phosphorylation of TnI,
and hence distinct functional effects. Recent studies by
Jideama seem to support this hypothesis since sites on TnI
exclusively phosphorylated by PKC-d were only minimally phosphorylated in a myocyte model w235x.
The phosphorylation of TnI in situ appears to be specific for PKA-dependent and PKC-dependent processes,
and is unaffected by inotropic interventions thought to act
by variations in wCa2q x i . Both Frearson et al., using oubain
and variable amounts of Ca2q w239x, and Ezrailson et al.,
using Ca2q ionophores and paired pulse stimulation w240x,
have showed that TnI is not phosphorylated in beating
heart preparations under these conditions.
2.3.2. Troponin T
Troponin T ŽTnT. is the largest component of the Tn
complex and is named for its ability to bind to tropomyosin
w211x. Early studies by Perry and colleagues found that
TnT existed as a phosphoprotein in cardiac muscle
w213,241x. Like TnI, TnT has also been shown to be
phosphorylated stoichiometrically and at multiple sites by
PKC in vitro w232–234x, leading to reduced Ca2q-stimulated actinomyosin MgATPase activity w234,242x. These
sites are all located at the C-terminus of TnT where
binding to tropomyosin and TnC occurs. More recent
studies have revealed that PKC-z selectively phosphorylated two previously unknown sites in TnT, leading to a
slight increase in Ca2q-sensitivity without affecting the
Mg 2q-ATPase activity w235x. It now appears that PKC-z is
the major isozyme responsible for phosphorylation of TnT
in cardiac myocytes in situ and perhaps in vivo, though
further work is needed utilizing pharmacological and
molecular approaches to decipher the role of specific PKC
isozymes in myofibrillar and cardiac function.
2.3.3. Myosin light chains
The myosin molecule is one of the largest proteins
known, having a mass of ; 500 000 daltons, and is composed of two classes of subunits held together by non-covalent forces, namely two heavy chains Žeach 200 kDa.
and four light chains associated with the head region of the
heavy chain w211x. In cardiac muscle myosin, two types of
light chains are found, one with a M r of about 27 000
daltons and termed LC-I Žessential light chains., and a
second with a M r of 19 000 daltons and referred to as LC2,
regulatory light chain, or phosphorylated-light chain
ŽMLC-2.. Functionally MLC-2 is not essential for enzymatic hydrolysis of ATP by myosin, rather it may act to
modulate the ATPase activity. The actin-activated ATPase
of cardiac myosin changes several-fold with no change in
MLC-2 composition, although removal of MLC-2 caused
an enhanced ATPase, which was restored with the readdition of MLC-2 w243x. Questions remain as to whether or
not phosphorylation of MLC-2 impacts its interaction with
myosin, but readdition of phosphorylated MLC-2 to a
MLC-2 free myosin preparation does not inhibit actinactivated ATPase to the same extent as nonphosphorylated
MLC-2 w243x. It should also be noted that MLC-2 possess
Ca2q-binding regions in its structure homologous to that
seen with other Ca2q-binding proteins like TnC and
calmodulin. Not surprisingly, the kinase that phosphorylates MLC-2 is a specific, Ca2qrCAM-dependent enzyme
termed myosin light chain kinase ŽMLCK., and one that
phosphorylates MLC-2 at a single specific Ser residue.
Much of the understanding for a role of phosphorylation
on MLC-2 function has evolved due to studies on its
phosphorylation properties in intact hearts following inotropic interventions w147,244,245x. Various studies have
found that MLC-2 phosphorylation levels are unaltered
subsequent to treatment with adrenaline w246,247x, or with
increased wCa2q x or wKqx levels w246,248x. However, in
several instances changes in MLC-2 phosphorylation have
been observed under b-adrenergic stimulation w152,248–
250x, but in some of these studies the changes in MLC-2
phosphorylation may be due to experimental artifact Ži.e.
uncontrolled heart rate effects and lower than expected
baseline MLC-2 phosphorylation levels. w248–250x. Despite a general lack of effect on MLC-2 phosphorylation
with short and long term changes in contractility, perfusion
studies do show that the Pi group in MLC-2 is rapidly
being turned over under these conditions w246,251x. This
indicates that the MLCK and putative phosphatases are
both active at the level of MLC-2 in the heart. In fact,
Neumann et al. demonstrated in a recent study that MLC-2
phosphorylation levels were increased following treatment
with okadaic acid, along with other cardiac regulatory
phosphoproteins w229x. Lastly, a mention should be made
of an interesting study whereby arachidonic acid and ET-1
treatment of cardiac myocytes resulted in enhanced PL-C
phosphorylation, via a presumed PKC-dependent process
w237x. Since there have been no other reports of PKC
effects of PL-C phosphorylation or function it is difficult
to interpret these observations.
2.3.4. C-protein
C-protein is one a group of structural proteins found in
striated muscle, including heart, and has a 150 000 dalton
molecular weight by SDS-PAGE w2x. The function of
C-protein is unclear, although both structural and regulatory roles have been suggested. For instance, it has been
reported that C-protein serves to stabilize the thick filament via interactions with the myosin tail regions w252x.
Since C-protein at low ionic strength can inhibit actomyosin Mg 2q-ATPase activity, and activate the Mg 2qATPase at high ionic strength, it has been suggested that
perhaps it may be involved in some sort of regulatory
capacity w253x.
Characterization of cardiac C-protein phosphorylation
in vitro showed that it is an excellent substrate for PKA
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
w254x. Early evidence suggested that C-protein phosphorylation was important in the physiological regulation of
myofilament activity. Jeacocke and England were the first
to provide in vivo evidence for C-protein phosphorylation
mediated by PKA w255x. Since then a number of other
groups have confirmed these results in a variety of
paradigms, including b-adrenergic receptor stimulation
w145,227,256x, and indirect activation of PKA-dependent
processes w151x. Properties of the phosphorylation of Cprotein appear to be similar to that seen with TnI. Recent
reconstitution studies though demonstrated that the decrease in Ca2q-sensitivity associated with PKA phosphorylation is not altered by myofibrils lacking C-protein w220x,
thereby obscuring the role C-protein may play in regulating myofibrillar function and cardiac contractility.
Dephosphorylation of C-protein tracks slowly with TnI
after withdrawal of b-agonist treatment w145x, and could be
stimulated by ACh or adenosine treatment w165,225x. Phosphatase inhibition with calyculin A resulted in a time- and
concentration-dependent increase in C-protein phosphorylation, suggesting that both it and TnI are dephosphorylated by the same protein phosphatase w257x. No evidence
currently exists demonstrating PKC-dependent activation
of the C-protein phosphorylation state. The only study to
examine this found no change in the phosphorylation state
of any cardiac regulatory protein except for PL w11x. Thus,
while C-protein shares many similarities to TnI, little is
known about its function, thereby making it difficult for
any speculation.
2.4. Other cardiac phosphoproteins
2.4.1. Protein phosphatase inhibitor-1
Phosphorylation states of proteins depend on the relative rates of phosphorylation and dephosphorylation w180x.
It is now accepted that protein phosphatases can counteract
the phosphorylation of proteins, and more importantly, are
themselves under strict control by endogenous phosphatase
inhibitor proteins. One such regulatory protein is termed
protein phosphatase inhibitor-1 ŽPPI-1., and is active only
when phosphorylated by PKA-dependent processes. Thus,
these types of phosphatase regulatory proteins are envisioned as participating in a positive feedback system
wherein PKA-induced protein phosphorylation is enhanced
when the PPI-1, itself activated by PKA, can inhibit the
activity of protein phosphatases w180x.
Several reports describe the putative role of PPI-1 in
mediating PKA-dependent phosphorylation processes in
the heart w20,149,258x. Iyer et al. reported that in rat heart
membrane preparations PPI-1 phosphorylation was increased by isoproterenol treatment w258x. Furthermore, PLB
dephosphorylation by exogenous protein phosphatase type
1 was reduced by the enhanced phosphorylation state of
PPI-1. In separate studies, Watanabe and coworkers
demonstrated that isoproterenol and forskolin increased
PPI-1 activity two- and three-fold respectively, effects that
571
could be antagonized by ACh co-administration w20x. Both
PKA-dependent agents also reduced protein phosphatase
type 1 activity intrinsic to the CSR using either w32 PxATP-labeled membrane vesicles or phosphorylase a as
substrates. Co-administration of ACh antagonized these
effects as well, and were reflected as an increase in protein
phosphatase type 1 activity. The mechanisms for the inhibition of PPI-1 activity by muscarinic agents are presently
unclear, but appear to involve a PKA-independent process
since cAMP levels were not effected when hearts were
treated with ACh in the absence or presence of isoproterenol w21x. This same study demonstrated that similar
PPI-1 inhibitory properties were shared by adenosine agonists. In a subsequent study, direct in vivo evidence was
provided for the phosphorylation of the 26 kDa PPI-1 in
response to isoproterenol w149x. Thus, PPI-1 can be modulated in intact heart by autonomic regulatory mechanisms.
2.4.2. Nuclear components
A growing body of evidence now exists that implicates
the phosphorylation of nuclear proteins in the modulation
of gene expression w259x. Many of these nuclear proteins
are known to be transcriptional factors that when subjected
to a variety of stimuli results in post-translational modifications and changes in gene expression. Identification of
these nuclear proteins and characterization of their phosphorylation mechanisms in vivo may lend great significance to their role in normal and diseased cell function.
In cardiac muscle, attention has recently been given to
the characterization of phosphorylation properties of the
cAMP response element binding protein ŽCREB-P.
w260,261x. CREB-P is a key mediator of gene transcription
activation in response to a stimulated cAMP-signaling
pathway w262x. It is a dimeric protein of about 43 000
daltons and contains phosphorylation sites for PKA, PKC,
casein kinase II, and other kinases. These phosphorylation
sites lie within a specific transactivation domain region
which is critical for induction of gene expression. CREB-P
is phosphorylated in vitro by PKA, PKC and Ca2qrCAMPK at the same Se133 site. It is thought that activation of
CREB-P, at least by PKA, causes a conformational change
in the protein which allows the transactivation domain to
interact with its target protein, a TATA-binding factor. In
turn, the TATA-binding factor can effect downstream
changes in gene expression.
CREB-P has been shown to be expressed in the nuclei
of primary embryonic chick heart cell cultures w260x and in
human heart tissue w261x. Following treatment with either
isoproterenol or forskolin Žin the presence of the electrochemical uncoupling agent, 2,3-butanedione monoxine or
BDM, to control drug-induced tension production., Goldspink and Russell found that CREB-P was phosphorylated
for up to 1 hour post-treatment. Also, expression of the
creb gene increased suggesting that both of these events
may be part of the early transcriptional response that takes
place as a result of elevated cAMP levels w260x. This study
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
is the first to support a physiological role of a nuclear
transcription factor, CREB-P, on the stimulation of the
PKA-dependent pathway in cardiac muscle. On the other
hand, Muller et al. showed that chronic in vivo treatment
of rats with isoproterenol led to downregulation of CREB-P
mRNA levels w263x. The discrepant data from these studies
may reflect differences between acute vs. chronic b-adrenergic effects, andror disparities from studying homogenous cultures vs. tissue samples. It seems clear though, that
the role of nuclear transcription factors, particularly those
identifiably involved in specific signal transduction pathways, may play critical roles in the functional regulation of
cardiac proteins w264x.
In an unrelated study, an acid-soluble nuclear protein of
31 kDa mass was found to be phosphorylated in response
to norepinephrine treatment in cultured rat cardiac myocytes w265x. The activation of this nuclear phosphoprotein
could be suppressed by the a 1-adrenergic receptor blocker,
terazosin. No definitive identity of this protein was determined aside from preliminary amino acid analysis. Interestingly, some specificity in effects was observed since
treatment with ET-1, phorbol esters and platelet activating
factor had no phosphorylating effect on the nuclear protein
despite the assumption that their effects were mediated by
PKC.
2.4.3. Phosphorylase kinase
Phosphorylase kinase plays a critical role in cellular
glycogen metabolism as a convergence point for neurohormonal and metabolic signals w266x. Once phosphorylated to
its active form, phosphorylase kinase catalyzes the phosphorylation Ži.e. activation. of phosphorylase b, converting
it to phosphorylase a, which serves as the initiating step
for glycogen breakdown. Breakdown of glycogen by phosphorylase thus can be a main source of energy supporting
muscle contraction, particularly during heightened inotropic states w267x.
Phosphorylase kinase is a complex protein composed of
four each of four subunits, a Žor aX which predominates in
cardiac muscle., b, g, and d w266x. The typical structure is
depicted as Ž a , b, g, d .4 . The g subunit contains the
catalytic site which mediates the Ca2q sensitivity of the
enzyme, and structurally is identical with the Ca2q-binding
proteins calmodulin and troponin C. The other three subunits of phosphorylase kinase are regulatory, with the a
Žor aX . and b subunits in particular able to be phosphorylated by a number of protein kinases, including PKA,
casein kinase I and phosphorylase kinase itself w268x.
Three peptide sites on the aX subunit are key in PKA-dependent phosphorylation of the enzyme, with the site
initially phosphorylated ultimately affecting activity. The
in vitro data have indicated that the phosphorylation of
either subunit cause activation, with phosphorylation of the
b subunit being essential for such activation, and that of
the a subunit amplifying this effect w268x. A complex
interrelationship between the subunits therefore exists in
the regulation of phosphorylase kinase activity by multisite
phosphorylation w269,270x. This may serve a critical role in
determining the enzyme’s response to various physiological stimuli.
Studies have definitively demonstrated that Ca2q is
required for in vitro phosphorylase kinase activity and can
modulate activity in a concentration-dependent manner
w271x. Interestingly, the Ca2q concentrations needed for
activation of phosphorylase kinase seem to be comparable
to those required for Ca2qrCAM-dependent enzymes Žapproximately 1 mM. involved in cardiac muscle contraction,
for instance myosin ATPase. Therefore the proteins which
confer Ca2q sensitivity to both muscle contraction and
cellular glycogen breakdown are similar w267x. Additionally, both enzymes can be regulated by PKA-dependent
phosphorylation mechanisms, thus lending further support
to the concept that phosphorylase kinase can function as an
integral coupling point between energy metabolism and
muscle contraction.
Walsh and coworkers have demonstrated an in vivo role
for protein phosphorylation of phosphorylase kinase activity w272,273x. In the initial report, phosphorylase kinase
was shown to be phosphorylated in perfused rat hearts in
response to catecholamine treatment w272x. Phosphorylation occurred in a time- and concentration-dependent manner in response to norepinephrine, and correlated with
increases in the activation state of the enzyme. Upon
withdrawal of norepinephrine, the enzyme dephosphorylated and activity returned to control levels. In a follow-up
study, changes in phosphorylation of the specific subunits,
aX and b, were measured following treatment with various
inotropic agents, and these were found to correlate with
changes in enzyme activity w273x. Additionally, the role of
altered Ca2q levels was examined in the presence and
absence of inotropic agents, and no differences were observed. This suggested that Ca2q-dependent autophosphorylation of phosphorylase kinase was not a major regulatory determinant of enzyme activity in vivo.
3. Coordinated regulation of cardiac function by phosr dephosphorylation mechanisms
phorylationr
3.1. Functional releÕance of autonomic regulation of protein phosphorylation
The principle physiological modulation of working ventricular myocardium is the control of contractility by the
sympathetic and parasympathetic nerves, and the respective release of the neurotransmitters noradrenaline and
acetylcholine from their nerve terminals. Current opinion
favors the idea that activation of cAMP-dependent processes, can explain the two principal mechanical effects of
catecholamines on cardiac muscle, namely systolic shortening and enhanced contractility. Evidence presented herein
suggests that the PKA system influences cardiac function
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
through specific phosphorylation of regulatory proteins at
a number of levels w274x.
The proposed sequence of events likely begins with the
elevation of cAMP levels by catecholamine stimulation of
the b-adrenergic receptors and activation of adenyl cyclase. In turn, the sarcolemmal slow Ca2q channel protein
itself or a contiguous regulatory type of protein Žperhaps
PL?. associated with the slow Ca2q channel, can become
phosphorylated through a PKA-mediated process w37x.
Phosphorylation of the channel causes a conformational
change allowing the activation gate to be opened and
increases the number of activated slow channels, which
results in an augmented influx of Ca2q. This calcium then
‘triggers’ the release of a greater quantity of Ca2q through
the CCRC Ži.e. calcium-induced calcium release. w189x,
which has also been primed for opening by PKA-dependent phosphorylation w199x. The released free wCa2q x i rises
to sufficient levels for binding to the TnC, intensifying the
TnC-TnI interaction to an extent that TnI-actin interactions
and inhibitory effects of TnI are weakened. This permits
the actinmyosin reaction to occur, allowing crossbridge
cycling to take place. The number of such cross-bridges
cycling establishes the ‘contractility’ of the myocyte, and
is determined by the amount of Ca2q delivered to TnC.
During relaxation, phosphorylation of PLB would stimulate the rate of active Ca2q uptake by the CSR, due to an
increased affinity for Ca2q as well as an increased rate of
some steps in the SERCA2 reactions sequence w137–139x.
It is now apparent that the relative PLB:SERCA2 ratio is a
major determinant of the heart’s overall contractility status
and alterations in this ratio may contribute to myocardial
dysfunction w275x. The increase in Ca2q uptake by the
CSR may explain systolic shortening since the Ca2q would
be removed from TnI at an enhanced rate. The increased
Ca2q uptake would allow for higher levels of Ca2q to be
sequestered in the CSR. As a result more Ca2q could be
made available to the contractile proteins in the subsequent
contraction cycle, thus leading to a positive inotropic
response. This proposed sequence of events appears to be
plausible, since evidence from intact heart studies demonstrates that PLB phosphorylation appears to parallel temporal changes in relaxation times w6,147x. Concomitant to
PLB phosphorylation, PKA-dependent phosphorylation of
TnI exerts an allosteric effect on Ca2q binding to TnC,
decreasing the sensitivity of TnC for Ca2q w218x, and thus
increasing the amount of Ca2q that is required to bind to
TnC in order to produce contraction. In so doing, TnI
phosphorylation facilitates the rate of myocardial relaxation.
Cholinergic control of phosphorylation would be expected to antagonize the b-adrenergic effects of norepinephrine w150x. Evidence now suggests that this can
occur, at least partially, through inhibition of PKA-stimulated PPI-1 phosphorylation w21x and an increase in phosphatase activity w20x. In essence mAChR activation would
attenuate slow Ca2q channel conductance w80x, reduce
573
phosphorylation of other regulatory proteins like PLB and
TnI w156,161–163,165x, and generally reverse the positive
inotropic effects of catecholamines.
In summary, autonomic receptor stimulation exerts a
powerful modulatory effect on the phosphorylation of various cardiac proteins. Involved in this process is the associated regulation by the different kinases and phosphatases.
Much investigation still remains to be done however, to
fully elucidate the coordination that must be involved
among intracellular events to bring about precise control of
cardiac function on a beat-to-beat basis.
3.2. Significance of Ca 2 qr CAM-dependent phosphorylation on function
The physiological significance of Ca2qrCAM-mediated
phosphorylation of putative regulatory proteins is not clear.
Despite several lines of in vitro evidence for regulation of
the CSR SERCA2 pump by Ca2qrCAM-PK mechanisms,
there is no conclusive in vivo evidence to support a role
for this process in the heart w170x. It appears that
Ca2qrCAM-dependent phosphorylation occurs in the intact heart only when cAMP levels are high w177x. This may
suggest that a cooperative interaction with cAMP would
accelerate the removal of activator Ca2q in the intact heart,
and so facilitate Ca2q uptake from the cytosol when Ca2q
entry is increased during sympathetic stimulation.
Frequency-force effects in the heart have long been
thought to be related to the amplitude and time course of
wCa2q x i transient w276x. This is predominantly due to an
increase in the Ca2q influx through the Ca2q channels and
the NaqrCa2q exchanger, in turn augmenting the CSR
Ca2q load so that more Ca2q is available for release and
thereby resulting in greater Ca2q transients, enhanced
relaxation and subsequent contraction. It has been suggested that Ca2qrCAM-dependent phosphorylation of PLB
is involved in regulating the cardiac force-frequency relationship by enhancing CSR Ca2q uptake. Previous work
has not demonstrated any alterations in PLB phosphorylation levels or functional parameters when changes in extracellular Ca2q were used to indirectly alter wCa2q x i , as
would be expected during frequency increases w8x. Recent
direct experiments examining force-frequency effects in rat
ventricular myocytes have also demonstrated no significant
change in phosphorylation levels of the Ser 16 rThr 17 sites
on PLB w277x. The fact that the SERCA2 inhibitor, thapsigargin, but not specific inhibitors of the Ca2qrCAM-PK,
inhibited rate-dependent abbreviation of the Ca2q transient, suggested that this phenomenon is due predominantly to greater Ca2q uptake by the CSR. It appears then,
that Ca2qrCAM-dependent processes do not play a major
role in regulating CSR function.
3.3. Effect of PKC on cardiac contractility
Despite enormous progress towards understanding the
regulation and biochemical properties of PKC in the car-
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
diac system over the last ten years, numerous questions
concerning its function remain to be answered w278x. As
discussed in previous sections, numerous putative PKC
protein substrates have been identified in cardiac tissue,
particularly at the in vitro level. However, little is still
known about whether the phosphorylation of these proteins
occurs in vivo and in a manner that is physiologically
relevant to the homeostatic control of cardiac function.
A key question is whether the various PKC isoforms
expressed in the cardiac myocyte effect specific phosphoproteins, thereby evoking differential physiological responses. Recent evidence by Mochly-Rosen and colleagues
would suggest that this in fact may be a valid hypothesis
w279,280x. In one study, chronic treatment with PMA
selectively down-regulated PKC-b, -d and -´ isozymes,
and was associated with an enhancement of the spontaneous rate of contraction in cardiac myocytes w279x. A
second study demonstrated that a sequence derived from
the V1 fragment of PKC-´, along with a related octapeptide, selectively inhibited the translocation of PKC-´ and
specifically blocked PMA or norepinephrine-mediated regulation of myocyte contraction rate w280x. One can speculate that these actions of specific PKC isozymes on cardiac
myocyte function could be mediated by phosphorylation of
selective phosphoprotein substrates, though this has not
been examined to date.
studies are detailed in sections below within the context of
their respective disease states.
It is important to note that many of the aforementioned
studies dealing with growth-related protein kinases are
based on indirect measurements that nonetheless provide
compelling evidence for the participation of these signalling pathways via phosphorylation in regulating cardiac
function. In only one study to date has there been an
attempt to examine this type of activation at the level of ex
vivo protein phosphorylation using cardiac myocytes w295x.
All the other reported experiments have relied on the
quantification of protein kinase activities using w32 PxPilabeled substrates, or alternatively, antibodies specific to
the phosphorylated forms of the protein kinases. Thus,
while the recent and ever growing efforts to elucidate a
role for growth-related protein kinase-dependent mechanisms in the heart is encouraging, further direct information is required before precise functional roles can be
established. It is interesting to note though that many of
the hypertrophic agents mentioned above have been characterized as positive inotropic agents, with some data
available on their phosphorylation effects of contractile-related proteins Žas described in previous sections above..
4. Pathophysiological alterations in protein phosphorylation
3.4. Role of alternate phosphorylation mechanisms in cardiac function (growth related protein kinases)
4.1. Myocardial ischemia
The field of growth-related protein kinases as it relates
to the heart is still in its infancy w15,281,282x. It is apparent
that the characterization of cardiac growth-related protein
kinase-dependent mechanisms represents the next emerging area of cardiac cellular research. There is now evidence to suggest that protein SerrThr-kinase-dependent
mechanisms transduce stimuli from the cell surface to the
myocyte nucleus, either directly or via cross-talk with
other processes like PKC or the receptor protein tyrosine
kinase system. At the nuclear level a series of molecular
events leading to cellular transformation are then initiated.
Recent evidence suggests that components of the protein SerrThr-kinase- and tyrosine kinase-dependent systems, the best examples being the superfamily of mitogenactivated protein kinases ŽMAPK., and their subfamilies of
stress-activated protein kinasesrc-jun N-terminal
kinasesŽSAPKsrJNKs., extracellular signal-regulated protein kinases ŽERKs., p21-activated protein kinases ŽPAKs.,
cyclin-dependent kinases Žcdks. and Ras GTPases, can be
activated in cardiac tissue or cells by potentially hypertrophic agents Že.g. phenylephrine w283–285x, ET-1
w283,286x, bradykinin w17x, AII w287–289x, growth factors
w286x, and high ATP w290x. as well as stimuli such as
hypoxia w291x, hyperosmotic shock w292x, ischemiarreperfusion w293,294x, and stretch w295x. The latter group of
Ischemia produces depression of myocardial contractile
function, metabolism and alterations in a number of cellular homeostatic processes, particularly that of ionic fluxes
like Ca2q w296–298x. Diminished allosteric effects of ATP
during ischemia would effect all active transport processes
such that sarcolemmal Ca2q fluxes through the slow Ca2q
channels and NaqrCa2q exchanger, CSR SERCA2 activity, Ca2q uptake and release through CCRC, would all be
reduced. This coupled with a desensitization of Ca2q-binding on TnC would impair actin-myosin interactions and
lead to contractile dysfunction. Since regulation of many
of these processes are dependent on protein phosphorylation, it has been assumed that the phosphorylation states of
the key cardiac regulatory proteins are altered as a consequence of ischemia.
Currently, little information is available that deals with
protein phosphorylation during myocardial ischemia. Two
reasons may account for this, and both deal with the ability
to quantify altered levels of w32 PxPi incorporation in labeled
tissuercells. First, dramatic changes in ATP turnover rates
may provide differential states of phosphorylation among
samples, and even during the course of the experimental
period. Secondly, if the ischemic period is severe enough
and ATP levels drop substantially, then the ability to detect
meaningful changes in w32 PxPi will be compromised, partic-
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
ularly if the endogenous pre-treatment wPi x is low to begin
with. Hence, the few studies on protein phosphorylation
and ischemia have relied on either in vitro back-phosphorylation techniques Ži.e. in vitro labeling of mixtures of
proteins or purified proteins derived from intact treated
hearts or cells. or on the measurement of declines in
w32 PxPi in hyper-stimulated Žand thereby hyper-phosphorylated. hearts or cells. In either case, the data derived from
such experiments must be interpreted cautiously as both
sets of conditions may not mimic the true physiological
state, leading to misrepresented findings.
Several in vitro studies have demonstrated reductions in
PLB phosphorylation, and that the magnitude of the reduction reflected the duration of ischemia and presumed severity of the injury w299–301x. Functional recovery following
reperfusion was accompanied by restoration of in vitro
PLB phosphorylation w300x. In the only study where intact
hearts undergoing ischemia were pre-labeled with w32 PxATP, a rapid decrease in the contractile performance was
observed, but with no significant changes in the phosphorylation state of PLB, TnI, or MLC-2 w302x. When hearts
were pre-stimulated with isoproterenol to elevate PLB and
TnI phosphorylation levels ŽMLC-2 levels were unaltered.,
subsequent increasing durations of ischemia resulted in the
phosphorylation responses to be progressively reduced.
Additionally, the attenuation of increased protein phosphorylation was accompanied by a reduction of cAMP accumulation in the ischemic heart. No decreases in ATP
concentration were observed throughout the ischemic periods.
In a more recent study, Li et al. examined mechanical
behavior and TnI phosphorylation in cardiac myocytes 7
days following coronary artery ligation w303x. A reduction
in myofilament isometric tension was observed as was the
TnI protein content, though TnI phosphorylation levels as
measured by w32 PxPi were increased. These data may provide the molecular basis for the decrease in myofilament
Ca2q sensitivity of tension development following not
only MI, but also in heart failure models Žsee section
below..
An apparent feature of myocardial ischemia is the observed drop in intracellular pH leading to a modest state of
acidosis. Since acidosis alters PKA w304x and phosphatase
activity w185x, it seems possible that PLB phosphorylation
could be affected as well. Recent data has demonstrated
that in the absence of b-adrenergic stimulation no acidotic
effect on PLB phosphorylation is observed but that in the
presence of isoproterenol, acidosis increased PLB phosphorylation w305x. In contrast, acidosis increased TnI phosphorylation in the absence and presence of b-adrenergic
agents, and inhibited PP1A. These effects resulted in a
decreased developed force and an accelerated relaxation.
Myocardial injury during reperfusion of the ischemic
myocardium has been partly attributed to the deleterious
effects of oxygen derived free radicals such as H 2 O 2 w306x.
Recent studies whereby w32 PxPi-labelled cardiac myocytes
575
were exposed to H 2 O 2 resulted in a reduction of isoproterenol-stimulated PLB and TnI phosphorylation via decreasing cAMP accumulation. It appears then that blunting of
b-adrenergic-mediated adenyl cyclase stimulation by hydroxyl radicals may account for diminished rates of relaxation in myocardium exposed to oxygen free radicals
w307x.
Taken together, the above data suggest that deficiencies
in the regulation of the CSR pump activity by PLB through
phosphorylation, as well at the level of myofibrillar protein
phosphorylation, particularly that of TnI, may represent
mechanisms underlying ischemia-induced cardiac dysfunction.
Recent studies have implicated PKC as at least one
signaling mechanism involved in the reduced Ca2q responsiveness of myofilaments found in ischemic stunned
myocardium w308x. No changes in TnI phosphorylation
were observed between myofibrils isolated from nonstunned and stunned myocardium. However, maximal
Mg 2q-ATPase activity was stimulated and pCaŽ50. or
Ca2q responsiveness of the myofibrils were enhanced,
thereby increasing contractility profoundly. The lack of
any phosphorylation is indicative of an alternate molecular
basis for the effects of PKC on myofibrillar function.
It is now known that cellular stresses such as
ischemiarreperfusion induce transcriptional changes in the
heart, e.g. increased c-fos and c-jun expression w309,310x.
Recent mechanistic evidence suggests that activation of
such signaling cascades as MAPK, ERK and JNKrSAPK,
and Ras, may be involved in the phosphorylation of the
transactivation domains of various transcription factors,
leading to altered gene expression in response to ischemia
and reperfusion w311,312x. Indeed, in many cases these
kinases are themselves phosphorylated during their activation following cellular stresses. In one study, Mizukami
and Yoshida demonstrated that nuclear MAPK was activated by tyrosine phosphorylation during reperfusion in
perfused rat heart following ischemia w293x. A subsequent
study by the same group revealed that ischemia induced
the translocation of SAPKrJNK1 from the cytosol to the
nucleus in a time-dependent manner but without its activation w294x. Nuclear activity of SAPKrJNK1 was increased
only during reperfusion comparable to that seen with
MAPK in the previous study and as confirmed by phosphorylation of endogenous immediate early genes, c-jun
and c-fos. The activation of the SAPKrJNK1 activation
during reperfusion was most likely due to the significantly
enhanced phosphorylation at Thr 223 of SEK1 ŽSAPKrERK
kinase 1., an upstream nuclear kinase for the SAPKrJNK1
pathway. Given the ability of c-jun and c-fos to activate
the AP-1 complex, it is conceivable that expression of a
number of contractile protein-related genes are induced
during reperfusion, though it is unclear whether this would
be an adaptive or maladaptive response.
Under conditions of hypoxia and hypoxia followed by
reoxygenation, Seko et al. have recently demonstrated a
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S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
rapid activation of various growth related kinases, including that of p65 ŽPAK., p38 MAPK, and SAPKs, in rat
cardiomyocytes using antibodies directed at the phosphorylated forms of these kinases w313x. In turn, these activations caused enhanced phosphorylation of activating transcription factor ŽAFT.-2. In another study, it was determined that reoxygenation, but not hypoxia alone, caused
sustained and significant increases in phosphorylation of
the c-jun transcription factor, apparently through redox
signaling w291x. The activation mimicked treatment with
anisomycin or okadaic acid, and was blocked by the
tyrosine kinase inhibitor, genistein.
4.2. Ischemic preconditioning
Protein phosphorylation has been invoked as a putative
effector mechanism in the infarct size-limiting effect of
ischemic preconditioning, a recently described phenomenon whereby a brief period of cardiac ischemia can
in turn make the myocardium tolerant to a subsequent
sustained and lethal ischemic episode w314,315x. According
to the hypothesis proposed by Downey and colleagues to
describe the mechanisms leading to preconditioning of the
heart, receptor-induced activation of signal transduction
processes such as PKC, may lead to possible protein
phosphorylation of an ‘effector’ proteinŽs. that ultimately
mediates the protective response w316x.
To date, little or no evidence exists to support this idea
of protein phosphorylation involvement in effecting ischemic preconditioning. Brooks et al. demonstrated by
immunoblot analysis the immediate activation in preconditioned rat hearts of the phosphorylated form of the
80KrMARCKS Žmyristoylated alinine-rich C kinase substrate. protein, a major PKC substrate w317x. These preliminary findings suggested that ischemic preconditioning
could potentially upregulate PKC-dependent processes,
thereby resulting in the phosphorylation of specific effector proteins, and in this way, account for the cytoprotective
response. However, this evidence has been neither extended nor confirmed.
The strongest circumstantial support for the role of
phosphorylation in ischemic preconditioning stems from
the work of Armstrong and Ganote, who showed that
treatment with the phosphatase inhibitors okadaic acid,
calyculin A or fostriecin, could mimic preconditioning and
reduce rates of ischemic injury in isolated cardiac myocytes w318–320x. Recent studies have also examined the
possible role of p38 MAPK phosphorylation using Western blot analyses and phosphospecific antibodies in both
rabbit hearts and isolated myocytes undergoing preconditioning protocols w321x. Decreased phosphorylation levels
were noted during ischemia, but were enhanced during
preconditioning, and the latter response was mimicked by
treatment with anisomycin, an activator of p38 MAPK.
While the aforementioned studies are highly suggestive of
a role for phosphorylation in the development of ischemic
tolerance, the direct identification of the putative precondi-
tioning effector phosphoproteinŽs. still remains to be elucidated.
Investigators have implicated a variety of cellular proteins as possible candidate effector phosphoproteins, including the K ATP channel w322,323x, stress proteins
w323,324x, and cytoskeletal proteins w325x, among others.
To date, the only available evidence to support any of
these proteins as potential phosphorylation targets in the
ischemic preconditioned heart has been indirect evidence
based largely on in vitro data involving non-cardiac systems w326x. Nonetheless, recent data by Light et al. provides the first strong suggestion that phosphorylation of
K ATP channels, or some associated protein in the membrane patch, by PKC may act as a link in one or more
receptor-mediated pathways to increase K ATP channel activity and lead to ischemic preconditioning w67x. However,
the fast onset and reversal of the observed PKC effect on
channel activity would argue against the idea that duration
of the protective effect of preconditioning is represented
by the phosphorylated state of the K ATP channel.
4.3. Cardiac hypertrophy
Cardiac hypertrophy has been regarded as a secondary
response of the heart to a sustained increase in overload. It
is believed that various neurohumoral factors, and more
recently, mechanical stretch, can directly regulate the hypertrophic response. Induction of early gene expression
leading to increased protein synthesis appear to be the
molecular mechanisms that ultimately result in larger cell
size w287,327x.
Recent evidence from several groups has suggested that
protein phosphorylation and activation of specific kinase
systems may play a role in the induction of specific early
gene expression observed in hypertrophy. For instance,
studies have shown that mechanical stretching of cultured
rat cardiac myocytes leads to an increase in S6 kinase
activity w295,328x, and ERKs w329x, which is mediated by
the phosphorylation of the 42 kDa MAPK w295x. A model
of stretch-induced protein phosphorylation cascade in cardiomyocytes during the hypertrophic response has been
described by Izumo’s group, and includes the initiating
event Ži.e. stretch. ™ PKC activation™ Raf-1 kinase™
MAPKK™ MAPK™ S6 kinase™ S6 ribosomal phosphorylation™ protein synthesis w287,327,330x.
Other studies using AII as the hypertrophic stimulus
were able to confirm the phosphorylation of various PKC
and growth-related PK signal transduction cascade proteins. Furthermore, in one of these studies, other stimuli
like a 1-adrenergic agonists and ET-1 were found to mimic
the effects of AII in activating tyrosine phosphorylation of
the same protein substrates w330x.
Other cellular stresses such as hyperosmotic shock typically manifested during hypertrophy and other cardiac
pathologies, also appear to differentially activate the
SAPKrJNKs w18,331x, and PAKs w292x. In all cases, phos-
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
phatase inhibition by okadaic acid treatment exacerbated
the stress and further induced the phosphorylation of proteins, particularly ATF2 and c-jun transcription factors
w18x. However, the data is not universal in implicating a
role for the phosphorylation of these kinases and their
products in the generation of hypertrophy. A dissociation
of MAPK activation from receptor-induced hypertrophy in
rat cardiac myocytes was noted when treatment with a
MEPK-specific inhibitor, while blocking kinase activity,
did not suppress atrial natriuretic peptide ŽANF. reporter
gene expression, a recognized marker for hypertrophy
w332x.
In a study investigating contractile proteins, MLC-2
phosphorylation was increased in cultured myocytes isolated from pressure-overloaded rat hearts w333x. A role for
PKC in mediating this response was invoked. However, no
accompanying functional data was presented making interpretations of these results difficult.
4.4. Heart failurer cardiomyopathies
Several important cellular alterations have been found
to occur in heart failure, particularly at the level of contractile regulation. One of the key impairments is to cAMP-related components, including a downregulation of b-receptors, desensitization of adenyl cyclase, and increase in Gi a
proteins, all of which leads to a blunted positive inotropic
response to sympathetic control w334x. Given the integrated
role of cAMP-dependent mechanisms in regulating protein
function, one would expect that changes in protein phosphorylation may underlie contractile dysfunction in failing
hearts.
A recent study by Bohm
¨ et al. examined PLB phosphorylation and PKA activity in both non-failing and failing
human hearts w335x. Concentrations of cAMP were found
to be reduced in failing hearts, but there was no difference
in PKA activity. Both PLB phosphorylation and PLB
levels were similar in non-failing and failing hearts. It was
concluded that impairment of PLB phosphorylation was
not a component of contractile dysfunction during cardiac
failure. However, some caution should be raised when
interpreting these findings, as no steps were apparently
taken by the investigators to control for possible endogenous dephosphorylation of proteins during sample preparation and storage. This concern has been supported by
recent data in animal models whereby careful sample
preparation, and in particular adequate measures to prevent
protein dephosphorylation, resulted in the observation that
PLC phosphorylation is enhanced in cardiac hypertrophy
w336x. In addition, it is presumed that reductions in cAMP
levels might in fact lead to decreases in protein phosphorylation, and therefore the lack of observed protein phosphorylation may suggest that levels in failing hearts were
below endogenous levels found in non-failing hearts.
Somewhat contrary results were obtained by Bartel et
al. who observed a reduced cAMP-generating capacity in
577
isolated trabeculae from failing hearts following treatment
with isoproterenol or the PDE inhibitor, pimobendan w337x.
This was correlated to reduced w32 PxPi-incorporation of
PLB, TnI and C-protein, as measured by back-phosphorylation techniques. It was concluded that these reductions
could be due to b-adrenoreceptor down-regulation and
Gi-protein up-regulation, since treatment with dibutyryl
cAMP was accompanied by accelerated protein phosphorylation suggesting an intact signal transduction system.
More importantly, a direct relationship between the phosphorylation state and contractile activity was established. It
was speculated then that reduced phosphorylation of PLB
may be a key mechanism underlying impaired diastolic
and systolic function in heart failure, thereby accounting
for the failing heart’s diminished capacity to regulate
wCa2q x i levels.
The possibility that other integral phosphoproteins may
be involved in heart failure cannot be excluded. Recent
data by Bodor et al. using a mAb directed to the NH 2terminus phosphorylation site of TnI demonstrated reduced
levels of this protein form in failing human adult myocardium w338x. One can speculate that lower TnI phosphoprotein levels in the failing heart can directly lead to
functional consequences, namely a greater Ca2q sensitivity
of tension development, as previously reported in both
human and animal failure models w339,340x. It remains to
be determined though if the decreased phosphorylation
state of TnI is adaptive, thereby leading to enhanced force
development, or a maladaptive response that could lead to
ventricular diastolic dysfunction.
Studies investigating the role of MLC-2 phosphorylation in heart failure have resulted in ambiguous conclusions w341,342x. On the one hand, experimental failure
induced by myocardial infarction in rats resulted in depressed levels of left ventricular MLC-2 phosphorylation
that is consistent with previously reported decreases in
myofibrillar Mg-ATPase activity w343x. This study also
noted an increase in MLC-2 phosphorylation in the right
ventricle that was concluded to be an adaptive mechanism.
However, no changes in MLC-2 phosphorylation were
observed in patients with either ischemic or dilated cardiomyopathy, though alterations occurred in MLC isoform
expression w342x. In particular, the significant induction of
the atrial light chain, ALC-1, seemed indicative of a
molecular adaptational mechanism to improve cardiac
function.
Since alterations of the b-adrenergic system in heart
failure have been documented w334x, it is possible that
changes in the phosphorylation of nuclear transcription
proteins, such as CREB-P, may form the molecular basis
for functional alterations in a number of cellular proteins.
Muller et al. identified the phosphorylated form of CREB-P
immunologically in both normal and non-failing human
hearts w261x. The study was complicated by the fact that
the non-failing heart donors had received b-adrenergic
agent treatment. Furthermore, these results are discordant
578
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
with rat studies whereby chronic in vivo treatment of
isoproterenol led to downregulation of the CREB-P mRNA
w263x. The differences between acute and chronic b-adrenergic effects on CREB-P are unresolved, but may point to
possible receptor-mediated desensitization effects or the
complex interplay of transcriptional modulating proteins
on CREB-P function under various physiological conditions. Nonetheless, all these findings may have clinical
implications for a possible role of CREB-P and other
transcription factors in heart failure, which deserve further
and more direct investigation.
The role of PKC-mediated phosphorylation in a setting
of experimental cardiomyopathy in chronic diabetes has
recently been investigated w344x. TnI phosphorylation was
increased in cardiomyocytes from diabetic animals and this
correlated with the translocation of PKC-´ from the cytosolic to particulate fraction. These changes were abolished by rendering the animals euglycemic with insulin or
treating with an AngII type-1 receptor antagonist. Evidence from these studies may account at least partially for
the impairment in diastolic relaxation and loss of Ca2q
sensitivity observed in isolated myofibrils from diabetic
animals and humans.
4.5. Hypertension
The only reported study on cardiac phosphoproteins and
regulation of function in hypertension demonstrated that
when the b-adrenergic pathway was activated, PKA-dependent phosphorylation of TnI in myocytes from 26week-old spontaneously hypertensive rats ŽSHR. was
greater than in myocytes from normotensive, nonhypertrophied Wistar-Kyoto ŽWKY. rat hearts w345x. This response
was observed both in the presence or absence of phosphatase inhibition, thereby ruling out the possibility that
TnI phosphorylation was due to decreased phosphatase
activity. The differences were also not due to any effects
on cAMP production or degradation. Interestingly, no
changes in PLB phosphorylation between SHR and WKY
hearts were noted following PKA activation, suggesting
that the response was specific to TnI. A significant rightward shift in the Ca2q-dependence of actomyosin ATPase
activity was associated with the increased TnI phosphorylation in SHR. In other words, at the same free Ca2q
concentration, actomyosin ATPase activity was lower in
SHR vs. WKY rat hearts following PKA-mediated activation, coinciding with previously observed reductions in
force development in SHR hearts w346x. In a follow-up
study examining progression to decompensated hypertrophy in SHR hearts, McConnell et al. demonstrated no
additional increase in TnI phosphorylation at 76 weeks vs.
26 weeks, though differences were noted under b-adrenergic stimulation w350x. A dissociation between TnI phosphorylation and cAMP levels were observed suggesting
compartmentalization of the latter. The evidence would
therefore suggest that a reduced inotropic response to
sympathetic stimulation as mediated by impaired TnI phosphorylation may play a role in hypertensive hypertrophy.
5. Summary and perspectives
The preceding discussion on cardiac phosphorylation
demonstrates the diversity of proteins and functions that
are regulated via phosphorylation, and the mechanistic
complexity by which these functions are controlled. Although numerous phosphoproteins have been identified
and characterized, the list is certainly incomplete, and thus
our understanding of the functional and regulatory integration of signal transduction processes in cardiac contraction
remains somewhat less than clear.
Several common themes emerge however from the
examples cited in this review. First, many of the cardiac
proteins are regulated by phosphorylation with documented effects being stimulatory or inhibitory. Phosphorylation controls different activities of cardiac proteins including ion conductance through channels w37x, active
transport processes in the CSR w145x, and inhibition of
phosphatases w21x, to name a few. Secondly, many cardiac
proteins are phosphorylated at multiple sites by various
protein kinases. This is true in the case of PLB, for
example, which in vitro contains sites for at least three
protein kinases w114x. As a result, different signal transduction processes can converge on a single protein, resulting
in a coordinated regulation of the protein. On the other
hand, differential phosphorylation could yield opposing
effects as may be the case with PL, which can apparently
mediate both positive and inotropic responses w11,26x.
Similarly, in some cases a single residue within the protein
is targeted by more than one protein kinase, as seems to be
the case with CREB-P w262x, indicating that different
signals can share a regulatory mechanism. Lastly, all of the
operative cardiac protein kinases, and presumably the protein phosphatases too, have broad substrate specificities,
suggesting that they may be important for coordinate
control of myocardial contractionrrelaxation events. This
is particularly true of PKA which can control a myriad of
phosphoproteins w2,26,147,149x, thereby underlying sympathetic and parasympathetic autonomic regulation of myocardial function, and determining the inotropic state of
the heart.
At a more defined level, one is able to judge based on
the available data, whether the various cardiac phosphoproteins discussed herein are physiologically relevant or not.
Putative phosphoproteins must satisfy four now wellestablished criteria, put forward by Krebs and Beavo,
before phosphorylation-dephosphorylation properties of a
specific protein can be accepted as physiological control
mechanisms w347x. These criteria include: Ž1. the demonstration in vitro that the substrate can be phosphorylated
stoichiometrically at a significant rate by appropriate pro-
S.T. Rapundalor CardioÕascular Research 38 (1998) 559–588
tein kinaseŽs. and dephosphorylated by a phosphoprotein
phosphatase; Ž2. the demonstration that functional properties of the protein undergo reversible, physiologically
meaningful changes in vitro that correlate with the degree
of phosphorylation; Ž3. the demonstration that the protein
can be phosphorylated and dephosphorylated in vivo or in
an intact system with accompanying functional changes;
and Ž4. that there is a correlation in vivo between the
extent of protein phosphorylation and cellular levels of
effectors of protein kinases andror phosphatases. By these
established standards it is possible to conclude that most of
the major phosphoproteins, e.g. PL, slow Ca2q channel,
mAChR, PLB, TnI, C-protein, and phosphorylase kinase,
described herein satisfy these criteria at least with respect
to their interactions with PKA.
Typically in the cardiovascular field it is the third
criterion Ži.e. in vivo or ex vivo validation. that is the most
essential, but also the most difficult, to meet. The fact that
so many of the cardiac regulatory proteins now identified
can be considered as in vivo regulators, demonstrates the
significant advancements made in the field over the last
decade. Then, only perhaps TnI could safely be considered
as having met all the above criteria for being a physiologically relevant regulatory protein. It is conceivable that
the next few years will bring about the identification of a
host of new phosphoproteins that discriminately control
key processes in normal cardiac function.
Finally, although the past number of years have seen
major advances in our understanding of the control of
cardiac functional and metabolic events by phosphorylation, many more questions remain than have been answered. In several cases, the functional relevance of phosphorylation of known proteins still remains unclear, the
signal transduction processes that modify these proteins
are inadequately characterized, and the signals to which
they respond are poorly understood. New technical advances and greater integration between molecular, biochemical and physiological approaches, however support
the hope that defining the role of phosphorylation, particularly in pathophysiological conditions, may soon be
achievable.
Clearly, much effort has been focused on elucidating
the regulatory mechanisms controlling excitation-contraction coupling. As a result, several areas like nuclear or
cytoskeletal phosphorylation, as well as characteristics of
PKC-SerrThr- and tyrosine kinase-mediated phosphorylation have heretofore been largely ignored or incompletely
studied. Elucidation of their properties will potentially add
much to our understanding of disease processes such as
cardiac ischemia or hypertrophy.
There is considerable scope for future studies in these
fields within the realm of cardiac function in normal and
diseased states, and the ensuing years should provide an
exciting area for research. Undoubtedly this will result in a
better understanding of how the heart functions, and perhaps lead to opportunities for modulating specific mecha-
579
nisms in a therapeutic manner, either at a pharmacological
or molecular level.
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