Calcium signaling in cardiac ventricular myocytes.

Abstract

Calcium (Ca) is a multifunctional regulator of diverse cellular functions. In cardiac muscle Ca is a direct central mediator of electrical activation, ion channel gating, and excitation-contraction (E-C) coupling that all occur on the millisecond time scale. The key amplification step in E-C coupling is under tight control of very local [Ca]. Ca also directly activates signaling via kinases and phosphatases (e.g., Ca-calmodulin-dependent protein kinase [CaMKII] and calcineurin) that occur over a longer time scale (seconds to minutes), and the co-localization of these Ca-dependent modulators to their targets and to Ca is also critical in distinct signaling pathways. Finally, Ca-dependent signaling is also involved in long-term (minutes to hours/days) alterations in gene expression (or excitation-transcription coupling). These pathways are involved in hypertrophy and heart failure, and they can alter the expression of some of the key Ca regulatory proteins involved in E-C coupling and their regulation by kinases and phosphatases. There may again be physical microenvironments involved in this nuclear transcription, such that they sense a discrete Ca signal that is distinct from that involved in E-C coupling. In this way cells can use Ca signaling in multiple ways that function in spatially and temporally distinct manners.

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86
Ann. N.Y. Acad. Sci. 1047: 86–98 (2005). © 2005 New York Academy of Sciences.
doi: 10.1196/annals.1341.008
Calcium Signaling in Cardiac
Ventricular Myocytes
DONALD M. BERS AND TAO GUO
Loyola University Chicago, Department of Physiology, Maywood, Illinois 60153, USA
A
functions. In cardiac muscle Ca is a direct central mediator of electrical acti-
vation, ion channel gating, and excitation–contraction (E-C) coupling that all
occur on the millisecond time scale. The key amplification step in E-C coupling
is under tight control of very local [Ca]. Ca also directly activates signaling
via kinases and phosphatases (e.g., Ca-calmodulin-dependent protein kinase
[CaMKII] and calcineurin) that occur over a longer time scale (seconds to min-
utes), and the co-localization of these Ca-dependent modulators to their targets
and to Ca is also critical in distinct signaling pathways. Finally, Ca-dependent
signaling is also involved in long-term (minutes to hours/days) alterations in
gene expression (or excitation-transcription coupling). These pathways are in-
volved in hypertrophy and heart failure, and they can alter the expression of
some of the key Ca regulatory proteins involved in E-C coupling and their reg-
ulation by kinases and phosphatases. There may again be physical microenvi-
ronments involved in this nuclear transcription, such that they sense a discrete
Ca signal that is distinct from that involved in E-C coupling. In this way cells
can use Ca signaling in multiple ways that function in spatially and temporally
distinct manners.
BSTRACT
: Calcium (Ca) is a multifunctional regulator of diverse cellular
K
contraction coupling
EYWORDS
: calcium; calmodulin; coupling; dependent protein kinase; excitation–
INTRODUCTION
Calcium (Ca) functions as a ubiquitous intracellular messenger that regulates
many different cellular processes in cardiac myocytes. In cardiac muscle, Ca directly
contributes to electrical and contractile activity and is central in excitation–contrac-
tion (E-C) coupling.
Ca itself exerts important direct regulatory effects on events
during E-C coupling. Ca can also modulate E-C coupling by activation of kinases
and phosphatases, which can modulate gene expression responsible for hypertrophic
signaling and heart failure.
This latter effect is sometimes called excitation–
transcription (E-T) coupling. Changes in [Ca]
on cardiac function. This raises questions about how cardiac myocytes distinguish
different Ca signaling pathways during the large repetitive Ca transients during E-C
coupling. The answer might lie in different localized Ca signaling complexes that
1,2
3
i
produce both acute and chronic effects
Address for correspondence: Prof. Donald Bers, Ph.D., Department of Physiology, Loyola
University Chicago, 2160 S. First Ave, Maywood, IL 60153, USA. Voice: 708-216-1018; fax:
708-216-6308.
dbers@lumc.edu

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87BERS & GUO: CACLCIUM SIGNALING IN MYOCYTES
have strategic locations and time frames of action. This subcellular localization and
temporal coding of Ca signaling can be effectively coupled to specific physiological
or pathological function. Among Ca-dependent proteins, calmodulin (CaM) serves
as a multipurpose intracellular Ca sensor, decoding Ca signals and mediating many
Ca regulated processes.
Here we review the spatiotemporal control of Ca signaling occurring in three dif-
ferent time frames: (1) E-C coupling within milliseconds to seconds, (2) phosphor-
ylation and dephosphorylation over seconds to minutes, and (3) E-T coupling on an
even longer time scale. We will focus on Ca-CaM related modulation related to E-C
and E-T coupling.
4
EXCITATION–CONTRACTION COUPLING
It is well known that Ca plays a pivotal role in cardiac E-C coupling.
membrane depolarization, Ca entry through L-type Ca channels (or dihydropyrodine
receptors [DHPRs]) triggers the Ca-induced Ca release from the sarcoplasmic retic-
ulum (SR) via ryanodine receptors (RyRs) to amplify Ca current (I
rise in global [Ca]
i
activates the myofilaments to produce cardiac contraction. To
allow cardiac muscle to relax, cytosolic [Ca] must decline quickly. This is accom-
plished mainly by transport mediated by the SR Ca ATPase (SERCA), which pumps Ca
back into SR, and the Na/Ca exchanger (NCX), which extrudes Ca out of myocytes.
The apparent global E-C coupling, however, is composed of thousands of syn-
chronized local events. Both DHPRs and RyRs are co-localized in the junctional mi-
crodomain between the SR and sarcolemmal membrane (SL). At each junctional
cleft, a cluster of ~100 individual RyRs and ~25 DHPRs constitute a local SR Ca-
release unit called a junction (or couplon). This local functional unit concept is sub-
stantiated by identification of Ca sparks which originate from a local cluster of RyRs
in the SR, occurring spontaneously in resting myocytes, which can also be spatially
resolved during the E-C coupling upon block of most of the I
events are less overlapping spatially).
Ca influx through I
nel) raises local [Ca]
i
from 0.1 to
>
10
µ
M, and local Ca release from a cluster of
RyRs further increases local cleft [Ca] to
reaches ~1
µ
M (at a later time). A critical aspect of this discrete local signaling is
that as local [Ca]
i
declines between junctions, it is not sufficient to trigger SR Ca re-
lease at neighboring junctions 1–2
µ
m away.
SR Ca release events are synchronized by action potentials and simultaneous activa-
tion of I
Ca
at all junctions to produce a relatively homogenous increase in [Ca]
throughout the cytosol. Therefore, Ca sparks are the elementary units of SR release
both at rest and during the normal Ca transient during E-C coupling.
1,2
Upon
Ca
). The resultant
1
Ca
(even a single chan-
(so the individual
5–7
Ca
>
100
µ
M, whereas global [Ca]
i
only
1
However, physiologically these local
i
1
1,5,8
ACUTE MODULATION OF E-C COUPLING
Ca is not only the central player of ECC, but is also a critical modulator of its own
signaling pathway via Ca-dependent proteins. In this section we focus on acute reg-
ulatory mechanisms in the millisecond-second time frame during E-C coupling. One
of the regulated proteins is the myofilament protein troponin C, which is the main

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88ANNALS NEW YORK ACADEMY OF SCIENCES
Ca target responsible for switching on the contractile machinery in cardiac muscles.
Another ubiquitous Ca sensor is CaM, which deciphers Ca signals and modulates
E-C coupling directly and indirectly through downstream pathways.
The L-type Ca channel is one of the targets modulated by Ca-CaM. I
most of the Ca entry into the cardiac myocyte during the action potential, and it in-
activates rapidly during each pulse. I
Ca
inactivation is highly Ca-dependent and af-
fected by both Ca influx and SR Ca release, such that it provides a negative feedback
to limit the integrated I
Ca
. Since this Ca-dependent inactivation also occurs even
with the presence of cytosolic Ca buffering, it is clear that Ca influx through the
DHPR itself can exert a highly local inactivation on the channels. It is now clear that
Ca-dependent I
Ca
inactivation is mediated by CaM, which is pre-bound during dias-
tole to the carboxy-terminal region (a region between an EF-hand and IQ domain)
of the
α
1 subunit of L-type Ca channel.
is released by RyRs binds to the C-terminal of pre-bound CaM, which then interacts
with IQ domain of the Ca channel to produce inactivation.
duces Ca-dependent I
Ca
facilitation via Ca-calmodulin-dependent protein kinase II
(see following text). Both of these are very local microdomain regulatory phenomena.
RyRs are homotetramers, with each of the 4 subunits having a molecular weight
of ~560 kDa.
RyR2 is the predominant form in heart, and it is both the SR Ca re-
lease channel and a scaffolding protein that strategically anchors numerous regulatory
proteins to the junctional complex (the critical site where ECC occurs).
latory proteins include CaM, Ca-calmodulin dependent protein kinase II (CaMKII),
cAMP-dependent protein kinase (PKA, anchored via mAKAP), phosphatase 1 (PP1
anchored via spinophilin), phosphatase 2A (PP2A anchored via PR130), FK-506
binding protein, sorcin, calsequestrin, junctin, triadin, and homer.
teins are anchored to RyR to form a macromolecular complex, and some of them
may modulate the function of RyR itself highly locally.
CaM binding to RyR2 is Ca-dependent, because higher [Ca] increases CaM:RyR2
stoichiometry and slows down the dissociation of CaM from the RyR (from ~40 s at
low [Ca] to ~9 min at 100
µ
M [Ca]). CaM is reported to decrease RyR2 opening
at all [Ca] (even though not all reports support that) and shift the Ca-dependence of
RyR2 activation to higher [Ca].
This is also the case when Mg-ATP is included
and when redox-state is altered, but the difference is overcome at very high [Ca] and
[ATP].
In our hands, CaM inhibits Ca spark frequency in saponin-permeabilized
mouse ventricular myocytes in a dose-dependent manner and K
lished data). CaM may also affect RyR activity via CaMKII, which we will address
later.
Ca also activates the RyR2 directly from the cytosolic side, but at very high [Ca]
it causes inactivation. Although part of this could be related to CaM, the mM levels
of Ca (or Mg) required suggest that it is not CaM-dependent. Intra-SR [Ca] also
modulates RyR gating (high SR [Ca] activates) and this may be important in the
triggering of “spontaneous” SR Ca release associated with Ca overload and arrhyth-
mias.
This effect may be mediated by intra-SR Ca-binding protein calse-
questrin.
Regulation of RyR gating by luminal SR [Ca] may also be critical in the
termination of SR Ca release, which is otherwise a positive feedback of Ca-induced
Ca release.
That is, as intra-SR [Ca] declines, the RyR shuts off without com-
pletely emptying the SR of Ca.
[Ca]
i
also regulates NCX function by binding to an allosteric regulatory site on
2
4
Ca
carries
4,9
10–16
Upon depolarization, Ca that enters and
10–16
Ca-CaM also pro-
2,8
17
These regu-
17
All of these pro-
2,17
CaM is such an example.
18,19
19,20
19
i
is ~100 nM (unpub-
21–25
26
27

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89BERS & GUO: CACLCIUM SIGNALING IN MYOCYTES
the large intracellular loop of the NCX.
NCX itself) with an apparent K
vation of the protein to participate in Ca transport. As for the Ca channel, this regu-
lation may be part of a Ca feedback system, where the NCX is activated more
strongly to extrude Ca when [Ca]
i
is relatively high. It may also be regulated by local
subsarcolemmal [Ca]
i
rather than global [Ca]
28,29
Ca binding at this site (possibly to the
of ~150 nM [Ca]
i
in myocytes is required for acti-
m
i
.
1
PHOSPHORYLATION OF PROTEINS IN E-C COUPLING
Three Ca-CaM-dependent enzymes that mediate phosphorylation and dephos-
phorylation reactions have significant effects on cardiac function: CaMKII, cal-
cineurin (protein phosphatase 2B), and myosin light chain kinase (MLCK). They
have different Ca sensitivity and localization, and therefore mediate different func-
tions related to E-C coupling. The kinase or phosphatase reactions also incur a tem-
poral delay, such that these mechanisms tend to work on the time frame of seconds
to minutes.
MLCK is associated with actomyosin and phosphorylates regulatory myosin light
chain (MLC2). This Ca-CaM-dependent phosphorylation of MLC2 in cardiac myo-
cytes has two apparent consequences: (1) increase in Ca sensitivity of the myofila-
ment and therefore an improvement in cardiac contractility,
the actin and myosin filaments’ incorporation into the scaffold to form organized sar-
comeres during the cardiac hypertrophy. MLCK is a crucial central mediator of E-C
coupling in smooth muscle, but in cardiac muscle this pathway is thought to be a
minor modulator.
30
and (2) facilitation of
31
CaMKII and ECC
CaMKII is a multifunctional CaMK that may modulate cardiac E-C coupling.
The CaMKII holoenzyme consists of homomultimers (or heteromultimers) of 6–12
kinase subunits (and there are four closely related but distinct genes:
In cardiac myocytes the
δ
isoform is the main isoform (with some CaMKII
Each CaMKII subunit has an amino-terminal catalytic domain, a central regulatory
domain (containing partially overlapping autoinhibitory and CaM-binding regions),
and a C-terminal association domain responsible for oligomerization. Resting
CaMKII is inhibited by the autoinhibitory domain that acts as a pseudosubstrate,
preventing substrates from binding.
Upon activation by increased [Ca]
locks itself into the activated state by auto-phosphorylation on the conserved Thr
in the auto-inhibitory segment of adjacent CaMKII subunits.
tion at Thr
results in an autonomous form of CaMKII (even after the dissociation
of CaM) and increases CaM affinity of the kinase-CaM complex.
fully active with CaM bound regardless of [Ca]
CaM dissociation from this autonomous state.
inant isoform including
δ
B
(contains a nuclear localization signal [NLS]) localized
to nucleus and
δ
C
localized to the cytosol. Different subcellular localizations may
suggest that they have different Ca-CaM sensitivity, specific mode of activation, and
distinct functions. In support of this idea, transient overexpression of wild-type
CaMKII
δ
B
(but not CaMKII
δ
C
) in neonatal rat ventricular myocytes increased ex-
α
,
β
,
γ
, and
γ
).
δ
).
32–34
4,32
i
, CaMKII
286
32–35
Autophosphoryla-
286
35
CaMKII remains
i
level
36,37
35
and 20% to 80% active after
In heart, CaMKII
δ
is the predom-

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90ANNALS NEW YORK ACADEMY OF SCIENCES
pression of atrial natriuretic peptide (a marker of cardiac hypertrophic signaling) and
led to an enhanced transcriptional response to phenylephrine.38 This suggests that
the δB isoform is more related to gene regulation, which will be addressed later. Also,
Bayer et al.39 demonstrated that different CaMKII splice variants have different sensi-
tivity to Ca oscillation. However, the relative roles of CaMKII isoforms and their splice
variants in regulating cardiac function still remain to be extensively studied.34
CaMKII regulates almost all of components related to cardiac ECC. First,
CaMKII phosphorylates L-type Ca channel and results in Ca-dependent ICa facilita-
tion, which is typically observed as an increased ICa amplitude and slower inactiva-
tion over 2 to 5 pulses.40–42 This positive ICa staircase is Ca-dependent and CaMKII-
dependent, because this ICa dependent facilitation could be abolished by CaMKII in-
hibitory peptides and when Ba was the charge carrier. ICa facilitation is also a local
event because it is still seen with 10 mM EGTA in the patch pipette. The molecular
mechanism of facilitation is not clear as to whether the tethered CaM involved in ICa
inactivation is the same one activating CaMKII involved in ICa facilitation. Indeed,
site-specific mutation of isoleucine of the IQ domain that abolishes Ca/CaM depen-
dent inactivation can either increase (Ile to Ala) or abolish (Ile to Glu) Ca-dependent
ICa facilitation.11 ICa inactivation is a rapid negative feedback limiting the Ca entry
at a single heart beat, while ICa facilitation may limit the decrease of Ca channel
availability from beat to beat at higher heart rate (on a longer time scale). Hence, ICa
is tightly controlled by local Ca-CaM-CaMKII signaling.
Phosphorylation of Thr17 on PLB by CaMKII (or Ser16 by PKA) releases
SERCA from the inhibitory influence of PLB and enhances SR Ca uptake.43 It has
also been suggested that CaMKII phosphorylation of PLB may be involved in fre-
quency dependent acceleration of relaxation (FDAR).44,45 Some studies even showed
that direct CaMKII phosphorylation of SERCA stimulates its activity, although
others failed to show the significant stimulatory function of CaMKII on SERCA.4
Finally, CaMKII also co-immunoprecipitates with RyR46 and may alter its activ-
ity.17 The specific sites on RyR2 phosphorylated by CaMKII are controversial.
Witcher et al.47 reported that the unique phosphorylation site was Ser-2809 on RyR2
which regulated channel activity, whereas Rodriguez et al.48 suggested that CaMKII
may phosphorylate at least four sites in addition to Ser-2809. Recently, Wehrens et
al.49 identified a CaMKII site on RyR2 at Ser2815 and a stoichiometry of RyR2
phosphorylation (~1.5) that is lower than that identified in Rodriguez et al.48 Simi-
larly, the real effect of phosphorylation of CaMKII is still unclear. Most studies are
in isolated systems, including single channel recording and SR vesicles, and these
showed CaMKII to either increase47,49,50 or decrease51 RyR opening. It is important
to study RyR behavior in its native cellular environment. In intact voltage clamped
ventricular myocytes endogenous CaMKII increased the amount of SR Ca release
for a given SR Ca content and ICa trigger.52 This effect was Ca- and CaMKII-specific
because smaller conditioning Ca transients (which may not activate CaMKII) pre-
vented KN-93 from having any effect. In addition, in transgenic mice overexpressing
CaMKIIδC, fractional SR Ca release was enhanced and resting spontaneous SR Ca
spark frequency was dramatically increased despite lower SR Ca load and diastolic
[Ca]i.53 Moreover, both acute adenoviral overexpression of CaMKIIδC in adult rab-
bit myocytes and direct application of pre-activated CaMKII to permeabilized
mouse myocytes showed the same activating effect on Ca sparks and fractional Ca
release.54,55 However, Wu et al.56 showed that constitutively active CaMKII inhib-