Review

Calcium Signaling Series

Donald M. Bers, Guest Editor

Calcium Signaling and Transcriptional

Regulation in Cardiomyocytes
Matthias Dewenter,* Albert von der Lieth,* Hugo A. Katus, Johannes Backs

Abstract: Calcium (Ca2+) is a universal regulator of various cellular functions. In cardiomyocytes, Ca2+ is the central
element of excitation–contraction coupling, but also impacts diverse signaling cascades and influences the regulation
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of gene expression, referred to as excitation–transcription coupling. Disturbances in cellular Ca2+-handling and

alterations in Ca2+-dependent gene expression patterns are pivotal characteristics of failing cardiomyocytes, with
several excitation–transcription coupling pathways shown to be critically involved in structural and functional
remodeling processes. Thus, targeting Ca2+-dependent transcriptional pathways might offer broad therapeutic
potential. In this article, we (1) review cytosolic and nuclear Ca2+ dynamics in cardiomyocytes with respect to
their impact on Ca2+-dependent signaling, (2) give an overview on Ca2+-dependent transcriptional pathways in
cardiomyocytes, and (3) discuss implications of excitation–transcription coupling in the diseased heart.   (Circ Res.
2017;121:1000-1020. DOI: 10.1161/CIRCRESAHA.117.310355.)
Key Words: calcium ■ calcium-calmodulin dependent protein kinase II ■ calcineurin ■ calmodulin
■ excitation transcription coupling

C alcium (Ca2+) is a ubiquitous key signaling element in all

eukaryotic cell types. It regulates diverse essential physi-
ological processes such as cell differentiation, proliferation
and motility, apoptosis, secretion, excitation, contraction, and
neuronal plasticity.1 In the heart, the most obvious crucial role
of Ca2+ is its involvement in electric activity and cardiac con-
tractility, acting as the central player of excitation–contraction
(EC) coupling.2 Besides being directly involved in EC cou-
pling, Ca2+ is an important element in various signaling cas-
cades, regulating the activity of diverse downstream effectors.
Thereby, Ca2+ can exert acute effects but also influence the
regulation of gene expression, called excitation–transcription
(ET) coupling. These ET coupling pathways play a critical
role not only in cardiac homeostasis but also in cardiac disease
development, since diseased cardiomyocytes show vast altera-
tions in Ca2+-handling and Ca2+-dependent transcriptional pat-
terns.3,4 Among these pathways, especially signaling cascades
involving CaMKII (Ca2+/calmodulin-dependent kinase II) and
the Ca2+/calmodulin-dependent serine/threonine phosphatase
calcineurin have been extensively characterized on their role
in cardiac hypertrophy and remodeling processes.5,6 Yet, we
are just beginning to understand how the multiple ET coupling
pathways are differentially activated, how they interrelate, and
how the multiple targets in this complex network relatively
contribute to failure of cardiomyocytes.
This review gives an overview on Ca2+-dependent tran-
scriptional mechanisms in cardiomyocytes and their role in
cardiac disease. First, we discuss cytosolic and nuclear Ca2+
dynamics as the basis for the regulation of Ca2+-dependent
signaling. Then we consider the key players and pathways in-
volved in ET coupling in cardiomyocytes. Finally, we review
implications of ET coupling in the diseased heart and give an
outlook on possible therapeutic perspectives.
Ca2+ Dynamics in Cardiomyocytes
Cytosolic Ca2+
Given the central role of Ca2+ in controlling EC coupling, a
strict regulation of Ca2+-handling is crucial for cardiac func-
tion. Ca2+ transport mechanisms in cardiomyocytes include
both Ca2+ cycling between the cytosol and the extracellular
space and Ca2+ cycling between the cytosol and intracellular

From the Department of Molecular Cardiology and Epigenetics (M.D., A.v.d.L., J.B.) and Department of Cardiology (H.A.K.), Heidelberg University,
Germany; and DZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Germany (M.D., A.v.d.L., H.A.K., J.B.).
*These authors contributed equally to this article.
Correspondence to Johannes Backs, MD, Department Molecular Cardiology and Epigenetics, University Hospital Heidelberg, Im Neuenheimer Feld
669, 69120 Heidelberg, Germany. E-mail Johannes.backs@med.uni-heidelberg.de
© 2017 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.117.310355

1000
 Dewenter et al   Calcium Signaling and Transcription   1001
Nonstandard Abbreviations and Acronyms
ANGII angiotensin 2
AP-1 activator protein 1
AR adrenergic receptor
ATF1 activating transcription factor 1
CaMKII Ca2+/calmodulin-dependent kinase II
CAMTA calmodulin-binding transcription activator
CnA calcineurin subunit A
CnB Ca2+-binding regulatory subunit B
CREB cAMP response element binding protein
EC excitation–contraction
ET excitation–transcription
ET-1 endothelin-1
HDAC histone deacetylase
HP1 heterochromatin protein 1
IGF-1 insulin-like growth factor 1
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IP3 inositol 1,4,5-trisphosphate
IRF8 interferon regulatory factor 8
LTCC L-type Ca2+ channel
MCIP1 myocyte-enriched calcineurin-interacting protein 1
MEF2 myocyte enhancer factor 2
NCX Na+/Ca2+ exchanger
NFAT nuclear factor of activated T cells
NFκB nuclear factor κB
NPC nuclear pore complex
PKA protein kinase A
PKC protein kinase C
PLC phospholipase C
PLN phospholamban
RyR ryanodine receptor
SERCA SR Ca2+ ATPase
SOCE store-operated Ca2+ entry
SR sarcoplasmic reticulum
SRF serum response factor
STIM1 stromal interaction molecule 1
TRP transient receptor potential
TXNIP thioredoxin-interacting protein
Ca2+ stores, in particular the sarcoplasmic reticulum (SR). A
critical cellular microdomain for cytosolic Ca2+ cycling is the
dyadic junction, where invaginations of the plasma membrane
(T-tubules) and the SR are in close proximity.7 On depolariza-
tion of the cell membrane, LTCC (L-type Ca2+ channels; also
known as dihydropyridine receptors) in the plasma membrane
are activated, allowing Ca2+ to enter the cytosol. This Ca2+ en-
try raises local Ca2+ concentration from 0.1 to >10 μmol/L
in the junctional cleft, which triggers the activation of Ca2+
release channels (RyR [ryanodine receptors]) in the SR mem-
brane, a process often referred to as Ca2+-induced Ca2+ release.
The SR Ca2+ release raises local cleft Ca2+ to >100 µmol/L and
global cytosolic Ca2+ concentration from 0.1 µmol/L during
diastole to 1 µmol/L during systole.8 This increase in cytosolic
Ca2+ causes contraction via Ca2+-binding–induced conforma-
tional changes in the troponin–tropomyosin complex, which
allows the myofilaments actin and myosin to slide past one
another.9 For relaxation to occur, Ca2+ must be removed from
the cytosol. The 2 main mechanisms are Ca2+ transport back
into the SR by SERCA (SR Ca2+ ATPase) and Ca2+ extrusion
from the cell via NCX (Na+/Ca2+ exchanger) in the plasma
membrane. A small amount of Ca2+ is also removed by Ca2+
ATPases in the plasma membrane and by mitochondrial Ca2+
uniporters.10
These Ca2+ cycling processes are finely tuned in response
to extra- or intracellular stimuli by various regulators that
affect the activity state of Ca2+-handling proteins. Central
modulators of cardiomyocyte Ca2+-handling proteins are PKA
(protein kinase A) and CaMKII. PKA is activated in response
to sympathetic stimulation of β-AR (β-adrenergic receptors)
and phosphorylates LTCC, RyR, and the SERCA inhibitor
PLN (phospholamban), thereby increasing Ca2+ influx, SR
Ca2+ release, and SR Ca2+ reuptake. CaMKII shares common
targets with PKA and is also activated in response to sym-
pathetic activation; however, CaMKII-dependent mechanisms
have been suggested to exert more long-term effects on Ca2+
cycling.4 These kinase-mediated effects on EC coupling pro-
teins are counterbalanced by protein phosphatases, especially
protein phosphatase 1 and 2A.11 In addition, the activity state
of Ca2+-handling proteins and also of their upstream regula-
tory kinases can be modulated by multiple other posttrans-
lational modifications, such as oxidation, nitrosylation, and
glycosylation.12–15
Besides the above-mentioned elements of Ca2+-handling,
other Ca2+ cycling proteins have been characterized in car-
diomyocytes that are suggested to play a less pivotal role in
the classical concept of EC coupling but are implicated in
Ca2+-dependent signaling mechanisms; in particular, IP3 (ino-
sitol 1,4,5-trisphosphate) receptors, TRP (transient receptor
potential) channels, and SOCE (store-operated Ca2+ entry)
controlled by STIM1 (stromal interaction molecule 1). IP3
receptors are Ca2+ channels activated by IP3, which is gener-
ated through PLC (phospholipase C)–dependent hydrolysis of
phosphatidylinositol-4,5-bisphosphate after Gq protein–cou-
pled receptor stimulation.16 Typical activators of IP3 signaling
in cardiomyocytes are ET-1 (endothelin-1), catecholamines,
and ANGII (angiotensin 2), which presumably modulate spe-
cialized pools of Ca2+ via this mechanism.17 Localized prefer-
ably in the perinuclear region and the nuclear envelope, IP3
receptors are suggested to play a major role in local nuclear
Ca2+ cycling. Further details in this regard will be discussed
below. TRP channels are ubiquitously expressed nonselective
cation channels with variable Ca2+ permeability. In cardiomy-
ocytes, TRP channels, especially TRPCs, have been suggested
to act as fine-tuners of Ca2+ cycling and were demonstrated
to control Ca2+-dependent signaling in response to neurohu-
moral stimulation.18–20 Besides the activation via Gq protein–
coupled receptor stimulation (receptor-operated Ca2+ entry),
TRPCs also participate in SOCE.21 SOCE involving STIM1
and Ca2+ channel Orai1 has been characterized as a key ele-
ment of Ca2+-dependent signaling in nonexcitable cells.22 In
cardiomyocytes, STIM1-dependent Ca2+ entry was observed
to coexist with the global Ca2+ transients. Increasing evidence
indicates that STIM1 is important for Ca2+ response signal-
ing within microdomains, inducing changes in cardiac tran-
scriptional profiles.23 Apart from association with Orai1 and
TRPC channels, recent data suggest that STIM1 interacts with
 1002  Circulation Research  September 29, 2017
several other Ca2+ cycling proteins, including LTCC, SERCA,
plasma membrane Ca2+ ATPases, and RyR.24–27
This highly complex system of cardiomyocyte Ca2+ cy-
cling on the one hand allows global continuous intracellular
Ca2+ oscillations and on the other hand controls local pools
of Ca2+ within spatial microdomains, and the regulation of
nuclear Ca2+ adds an additional layer of complexity.
Nuclear Ca2+
The nucleus is a subcellular compartment surrounded by 2
phospholipid bilayers, referred to as the nuclear envelope.
This nuclear envelope separates cytoplasm from nucleoplasm
and is structurally and functionally subdivided. The outer nu-
clear membrane faces the cytosol and merges with the mem-
brane of the SR. Therefore, both the outer nuclear membrane
and the SR membrane share the same characteristics on their
composition, and the perinuclear space, which is in between
the outer nuclear membrane and the inner nuclear membrane,
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resembles the SR in ion and protein content.28,29 In contrast
to the outer nuclear membrane, the inner nuclear membrane,
which is directed to the nucleoplasm, shows a distinct com-
position unique to the nucleus. The nuclear envelope forms
nuclear invaginations that reach deep into the nucleus, creat-
ing complex structures that in their entirety are termed the nu-
clear reticulum.28,30 In close proximity to the nuclear envelope,
T-tubules reaching deep into the cytosol and mitochondria can
be found, constituting microdomains for Ca2+ cycling and sig-
nal responsiveness.31
The nuclear membranes are poorly permeable to ions and
water-soluble molecules. Transport and diffusion is facilitat-
ed by nuclear pore complexes (NPCs), which are distributed
throughout the nuclear envelope.32,33 Proteins and RNAs ≤39
nm in diameter are actively shuttled in and out of the nucleus
by a transport system associated with the NPC.34,35 Ions and
molecules smaller than 9 nm and 40 kDa can diffuse through
the NPC.34 Thus, the NPCs function as both barriers and selec-
tive filters for the substances trafficking between nucleoplasm
and cytoplasm.
In cardiomyocytes, every cytosolic Ca2+ transient is ac-
companied by a nuclear Ca2+ transient.36 However, nuclear
Ca2+ transients, compared with cytosolic transients, seem
to have a slower and delayed upstroke, a lower peak, and a
prolonged return back to baseline.37,38 These findings led
to the hypothesis that cytosolic Ca2+ passively diffuses into
the nucleus during systole, thereby causing the nuclear Ca2+
transients. Interestingly, to date none or only inconsiderable
numbers of primary active pumps for Ca2+ reuptake have been
found on the inner nuclear membrane.33,39 Therefore, the idea
is that Ca2+ mostly diffuses out of the nucleus via NPCs and is
then taken up by SERCA on the outer nuclear membrane and
the junctional SR, by nearby mitochondria or is extruded by
NCX on T-tubules in proximity to the nuclear envelope.31,40,41
This would also explain the observation that the nuclear base-
line [Ca2+] increases with higher beating frequencies. Because
the decay of nucleoplasmic Ca2+ transients is slower compared
with cytoplasmic Ca2+ transients, Ca2+ noticeably builds up
within the nucleus when diastole is shortened.37
Nuclear Ca2+ not only follows whole-cell Ca2+ oscilla-
tion but also small perinuclear Ca2+ release events. Lipp et al
showed that even Ca2+ puffs in the perinuclear area within 3
µm of the nuclear envelope can lead to an increase in nuclear
Ca2+ while leaving the rest of the cytosol unaffected.42 This
clearly states the importance of the perinuclear SR when
considering nuclear Ca2+ regulation and already implies that
nuclear Ca2+ is influenced by many aspects beyond whole-cell
Ca2+ oscillation.
However, passive diffusion is only 1 aspect of nuclear
Ca2+ cycling. Ion diffusion can also be subject to regulation
at the level of NPCs. Ambient Ca2+ and ATP are essential for
the maintenance of NPC diffusion capacity; thus, decreasing
Ca2+ or ATP in the area surrounding the NPC slows down its
conductance, but not to the extent of a full diffusion block-
ade.43 Furthermore, the distribution and position of NPCs on
the nuclear envelope, as well as their distance to nuclear Ca2+
channels on the outer and inner nuclear membrane, seem to
have an important effect on local Ca2+ diffusion.44 The amount
or density of NPCs in the nuclear envelope can vary between
10 and 20 pores/µm2 in vertebrates, and, thus, total Ca2+ diffu-
sion capacity varies accordingly.44,45
Functional and structural evidence further indicates that
nuclear Ca2+ can be actively controlled. IP3 receptor type 2,
the predominant subtype in cardiomyocytes, is concentrated
in the perinuclear area and can be found on the junctional
SR, the outer and the inner nuclear membrane.46 These nu-
clear IP3 receptors seem to account for the widely described
observation of a diastolic nucleoplasmic-to-cytoplasmic
[Ca2+] gradient.37 Experiments on isolated nuclei, permea-
bilized, and intact cardiac myocytes consistently show that
the activation of IP3 receptors preferably increases nuclear
Ca2+.46,47 IP3, generated after stimulation of Gq protein–
coupled receptors in the plasma membrane, acts not only
on plasmalemmal IP3 receptors but can also diffuse to the
nucleus and activate IP3 receptors in the outer and inner nu-
clear membrane.48,49 In addition, there is increasing evidence
that IP3 can also be directly generated at the inner nuclear
membrane and, therefore, selectively act on nuclear IP3 re-
ceptors to increase nuclear Ca2+, especially in response to
ET-1, ANGII, and α-adrenergic stimuli.47,50–52 Notably, ET-1
receptors, ANGII receptors, and α-AR have been identified
not only on the plasma membrane but also on the nuclear
envelope.50–53
Several more receptors were proposed to play a role
in nuclear Ca2+ regulation. For example, it has been dem-
onstrated that activation of the IGF-1 (insulin-like growth
factor 1) receptor, a receptor tyrosine kinase localized deep
within the T-tubules in proximity to the nucleus, leads
to PLC-dependent production of perinuclear IP3.54 This
consequently causes a selective increase in nuclear Ca2+.
Moreover, the existence of β1- and β3-AR on the nuclear
membrane was demonstrated,55 suggesting that β-adrenergic
signaling might have an influence on nuclear [Ca2+] within
microdomains via localized perinuclear PKA activation
and subsequent phosphorylation of PKA target structures.56
Although there is substantial evidence for the influence of
nuclear β-ARs on transcriptional processes, the exact mech-
anisms involved remain unknown.57 Another suggested reg-
ulator of nuclear Ca2+-handling is the RyR. It is primarily
located on the SR and only to a lesser extent on the outer
 Dewenter et al   Calcium Signaling and Transcription   1003

nuclear membrane.58 Intranuclear RyR localization remains Ca2+-Dependent Regulation of Gene

controversial, yet it was described for neonatal cardiac myo-
cytes.58 Perinuclear RyRs, however, were reported to play a
pivotal role in generation of Ca2+ waves originating from the
perinuclear region.59
In conclusion, nuclear Ca2+ is basically subject to cyto-
solic Ca2+ oscillations. The mere passive character of cytosolic
to nuclear Ca2+ diffusion is, however, enriched by different
modifying mechanisms incorporating influences and specific
pathways of various origins. Moreover, nuclear, perinuclear,
and cytosolic structures, such as nuclear invaginations, NPCs,
perinuclear reticulum, T-tubules, and the distribution of, for
example, receptors, channels, and pumps, create Ca2+ micro-
domains that offer a vast array of possibilities for spatially and
temporally restricted Ca2+ control.
Thus, cytosolic and nuclear Ca2+ cycling are part of a
highly sophisticated system which controls Ca2+-dependent
signaling by modifications of global Ca2+ oscillations as well
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Expression in Cardiomyocytes
Several Ca2+-dependent pathways are described that modu-
late gene expression by signal transduction to transcriptional
regulators. In cardiomyocytes, well-characterized signaling
patterns in this regard involve calmodulin, CaMKII, and cal-
cineurin. Figure 1 illustrates the major findings summarized
in this section.
Calmodulin
Calmodulin is a highly conserved Ca2+ sensor protein in eu-
karyotic cells with no innate enzymatic function. It is a small
16 kDa protein that consists of a C-terminal and an N-terminal
lobe with 2 Ca2+-binding EF-hands each. Different Ca2+ af-
finities of the N-terminal and C-terminal EF-hands and intra-
molecular structures cooperatively facilitate a differentiated
response to a broad range in Ca2+ concentrations. When Ca2+
binds to the lobes, conformational changes occur and thereby

as Ca2+ regulation within microdomains. In the following, we protein interaction sites are released.60 To date, >300 pro-
focus on pathways that link cardiomyocyte Ca2+ cycling with teins have been shown to bind to calmodulin. Various bind-
transcriptional regulation. ing motifs and mechanisms of interaction have been identified

Figure 1. Schematic diagram of the Ca2+-dependent pathways mediating transcriptional regulation in cardiomyocytes. For details,
see main text. CaM indicates calmodulin; CAMKII, Ca2+/calmodulin-dependent kinase II; CAMTA, calmodulin-binding transcriptional
activator; CaN, calcineurin; cPKC, conventional protein kinase C; cPKCct, conventional protein kinase C c terminal; CREB, cAMP
response element binding protein; DAG, diacylglycerol; H3S10, histone 3 serine 10; HDAC4, histone deacetylase 4; HDAC5, histone
deacetylase 5; HSF1, heat shock factor 1; IκB, inhibitor of κB; IKK, inhibitor of κB kinase; INM, inner nuclear membrane; IP3, inositol
1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; LTCC, L-type Ca2+ channel; MEF2, myocyte enhancer factor 2; NFAT,
nuclear factor of activated T cells; NFκB, nuclear factor κB; NPC, nuclear pore complex; ONM, outer nuclear membrane; RyR, ryanodine
receptor; SRF, serum response factor; STIM1, stromal interaction molecule 1; and TRPC, classical transient receptor potential cannel.
 1004  Circulation Research  September 29, 2017
resulting in local regulation of target activity at very different
Ca2+ concentrations.61,62
Besides its regulatory function in activating Ca2+-sensitive
enzymes such as CaMKII and calcineurin, calmodulin exerts
effects on gene expression via CAMTA (calmodulin-binding
transcriptional activator).63 In humans, 2 different CAMTA
genes, CAMTA1 and CAMTA2, have been characterized.
Their expression seems to be most prominent in heart and
brain tissue.64 In the heart, CAMTA2 raised interest because of
its noteworthy ability to induce the expression of atrial natri-
uretic peptide—a surrogate parameter for cardiac hypertrophy
and marker of heart failure–related diseases. Mechanistically,
it was shown that CAMTA2 binds to the homeobox pro-
tein NKX2-5, which is also implicated in hypertrophic re-
sponse, and coactivates NKX2-5–dependent transcription.
Furthermore, the Olson laboratory demonstrated that HDAC5
(histone deacetylase 5) represses CAMTA2 through a direct
interaction. With PKD (protein kinase D)–dependent HDAC5
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phosphorylation and consequent nuclear export, CAMTA2 is
released and executes its coactivating function.63,65 Therefore,
CAMTA is not only directly regulated by Ca2+ via binding of
Ca2+/calmodulin but also indirectly via its interaction with
HDAC5. The relevance of CAMTA2 in human was only re-
cently confirmed by showing that certain genetic polymor-
phisms in the coding region of CAMTA2 alter the risk of
developing cardiac hypertrophy.66
CaMKII
CaMKII is a serine/threonine kinase with the isoforms CaMKIIδ
and CaMKIIγ being expressed in the heart. The splice vari-
ant CaMKIIδC primarily localizes within the cytosol, whereas
CaMKIIδB has a nuclear localization sequence. CaMKII be-
comes activated upon Ca2+/calmodulin binding with a Kd ≈50
nmol/L.67 An important regulatory mechanism of CaMKII activ-
ity dynamics is its autophosphorylation, which is sensitive to the
frequency and duration of Ca2+ spikes.68 Autophosphorylation at
Thr 286/287 enhances the affinity of CaMKII toward calmodu-
lin and prevents autoinhibition of CaMKII, thereby maintaining
enzyme activity independent of Ca2+/calmodulin binding.69,70
Analogous to autophosphorylation, several other posttransla-
tional modifications, such as oxidation, glycosylation, and ni-
trosylation, were described to further increase CaMKII activity
after initial opening by Ca2+/calmodulin binding.15,71,72 CaMKII
exerts its molecular effects by binding and phosphorylation of
target proteins. Regarding the regulation of transcriptional pro-
cesses, CaMKII has been shown to phosphorylate transcription
factors, epigenetic regulators, and histones.
To date, several transcription factors that are activated in
a CaMKII-dependent manner have been identified. CREB
(cAMP response element binding protein) is a ubiquitously
expressed nuclear transcription factor involved in cardiac in-
tegrity, inflammation processes, and metabolic signaling.73–75
CREB activity is regulated via various signaling pathways, and
especially PKA-mediated phosphorylation of Ser-133 has been
shown to be critical for its activation. CaMKII either increases
activation of CREB by phosphorylation of Ser-133 or decreas-
es CREB activity by phosphorylation of Ser-142, suggesting
a modulatory role beside cAMP/PKA-dependent CREB regu-
lation.76 Notably, CaMKII has recently been demonstrated
to mediate CREB activation in response to neurohumoral
stimulation by ET-1 and phenylephrine.77 In addition, ATF1
(activating transcription factor 1), that shares huge homology
with CREB and that is also modulated by cAMP/PKA, can be
phosphorylated by CaMKII, remarkably only at its activation
site Ser-63 (corresponding to Ser-133 of CREB).78 However,
up to now the specific functional consequences of CaMKII-
dependent modulation of these cAMP-regulated transcription
factors in cardiac myocytes remain elusive.79
The functional role of other CaMKII-dependent tran-
scription factor activation events has been more specifically
characterized, for example, on apoptosis and inflammation.
Interestingly, whereas the CaMKIIδ splice variant CaMKIIδC
has been suggested to mediate stress-induced proapoptotic
effects via classical mitochondrial pathways,80,81 the splice
variant CaMKIIδB seems to play an antiapoptotic role.
Mechanistically, CaMKIIδB has been suggested to activate
the transcription factor heat shock factor 1 by phosphorylation
at Ser-230, inducing increased transcription of cell-protective
factor inducible heat shock protein 70.82 This putatively pro-
tective role of the CaMKIIδB splice variant is supported by
the observation that it is required for binding of transcription
factor GATA4 to the promotor region of antiapoptotic protein
Bcl-2 and consequently is crucial for reducing doxorubicin-
mediated cardiotoxicity.83
Emerging evidence points to an important role of CaMKII
in inflammation. For instance, CaMKIIδ has been described
to mediate activation of the transcription factor NFκB (nu-
clear factor κ B).84,85 The underlying mechanism most likely
involves a more indirect pathway via inhibitor of κB kinase
activation and subsequent phosphorylation and degradation
of Inhibitor of κB, leading to nuclear translocation of NFκB.
This pathway has been investigated, for example, in the set-
ting of ischemia/reperfusion injury, suggesting a detrimental
proinflammatory role of CaMKIIδ.86 In the same setting of
ischemia/reperfusion injury, we found that CaMKII induces
transcription and secretion of the chemokines CCL2 and
CCL3 from cardiomyocytes, thereby triggering an intrinsic
chemoattractant cardiomyocyte signaling cascade that is as-
sociated with scar formation and cardiac fibrosis.87
In addition, CaMKII was demonstrated to interact with
SRF (serum response factor), a transcription factor implicated
in cardiac integrity as genetic models have revealed.88,89 It has
been shown that CaMKII phosphorylates SRF at Ser103 and
Thr160 in skeletal muscle90; however, the functional role of
these events in cardiomyocytes is not clear yet. Furthermore,
myocardin, a transcriptional coactivator of SRF that has been
causatively associated with cardiac hypertrophy,91 is activated
via Ca2+-dependent signaling involving CaMKII.92
CaMKII modulates ion currents not only by direct interac-
tion and phosphorylation of ion channels but also by transcrip-
tional regulation. Studies reported that CaMKII is critical for
β-AR–stimulated upregulation of NCX1 by transcription fac-
tor AP-1 (activator protein 1) activation.93 Moreover, CaMKII
has been demonstrated to repress the expression of LTCC by
activating downstream regulatory element binding transcrip-
tion factor DREAM, thus constituting a negative feedback
mechanism on Ca2+ influx.94
 Dewenter et al   Calcium Signaling and Transcription   1005
CaMKII may also affect transcriptional patterns on a more
global scale by regulating transcription factor MeCP2 (meth-
yl CpG binding protein 2). MeCP2, which was implicated
in regulating the genome-wide chromatin state, was shown
to be phosphorylated by CaMKII in neurons.95,96 Notably,
MeCP2 is also expressed in the heart, where its relevance in
human and experimental heart failure was recently demon-
strated.97 Looking at these studies, it is worth considering that
CaMKII signaling to MeCP2 may depict another important
Ca2+-dependent aspect of transcriptional regulation in cardiac
myocytes.
Besides the phosphorylation of transcription factors,
CaMKII is also involved in chromatin modification. On the
one hand, CaMKIIδB can directly phosphorylate histones,
in particular histone 3 at Ser10 (H3S10). This phosphoryla-
tion event has been associated with induction of hypertrophic
growth, based on increased chromatin accessibility for prohy-
pertrophic gene regulation.98,99 It will be interesting to see in
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the future whether CaMKII-mediated histone phosphorylation
at H3S10 or at other potential histone phosphorylation sites
directly results in the regulation of specific gene programs.
Chromatin immunoprecipitation of CaMKII-phosphorylated
histones followed by massive genomic sequencing might
enable one to study this fascinating possibility. On the other
hand, CaMKII has been identified to interact with chroma-
tin modifying enzymes, in particular HDAC4. HDAC4 is a
class IIa HDAC that itself has only a weak deacetylase activ-
ity and exerts transcriptional regulation by binding of tran-
scription factors or recruitment of other chromatin modifying
enzymes.100 One of the critical transcription factors regulated
by HDAC4 is MEF2 (myocyte enhancer factor 2). MEF2 ac-
tivation has been implicated in cardiac remodeling, fetal gene
reprogramming, and inflammation,101,102 and HDAC4 acts
as a repressor in this scenario. CaMKII has been identified
as a specific kinase for HDAC4, since HDAC4 has a unique
docking site for CaMKII that is absent in other HDACs.
Nuclear CaMKIIδB-dependent phosphorylation of HDAC4
(at Ser-467 and Ser-632) promotes chaperone 14-3-3 bind-
ing and subsequent nuclear export of HDAC4, whereas cy-
tosolic CaMKIIδC-dependent phosphorylation of HDAC4
blocks nuclear import (Figure 2).103–105 As a consequence,
HDAC4-dependent repression of MEF2 is diminished, re-
sulting in enhanced MEF2-dependent transcriptional activ-
ity.103 Aside from MEF2, HDAC4 also acts as a repressor
of the above-mentioned SRF, indicating a more complex
functional consequence of CaMKII-mediated HDAC4 shut-
tling.106,107 Via HDAC4 binding, CaMKII is also capable of
regulating HDAC5. Although HDAC5 alone is not respon-
sive to CaMKII, hetero-oligomerization of HDAC5 with
HDAC4 enables CaMKII to phosphorylate HDAC5, result-
ing in nuclear export and subsequent activation of MEF2.108
CaMKII-regulated nuclear export of HDAC4 seems to affect
transcriptional patterns even further. For example, it has been
demonstrated that methylation of H3K9 at the promoter site
of atrial natriuretic peptide depends on HDAC4 shuttling.
Mechanistically, HP1 (heterochromatin protein 1) and histone
methyltransferase SUV39H1 associate with class IIa HDACs
to build a corepressor complex.109 HDAC4, when shuttled out
of the nucleus, releases the corepressor complex and, there-
fore, prevents further methylation. Subsequently, the H3K9
site becomes demethylated, which relieves chromatin conden-
sation and facilitates gene expression.110
Calcineurin
Calcineurin (protein phosphatase 2B) is a serine/threonine
phosphatase that is structurally composed of 2 subunits: CnA
(calmodulin-binding catalytic subunit A) and CnB (Ca2+-
binding regulatory subunit B).111 Like CaMKII, calcineurin is
activated by binding of Ca2+/calmodulin, notably with a much
higher affinity (Kd<<1 nmol/L).67 The classical calcineurin-
dependent transcriptional regulation pathway involves NFAT
(nuclear factor of activated T cells). On cytosolic dephosphor-
ylation by calcineurin, NFAT is imported into the nucleus,
where it acts in conjunction with transcription factor GATA4.
This calcineurin-dependent pathway has been elegantly dem-
onstrated to play a crucial role in cardiac development and in
the adult cardiac hypertrophic response.112,113 Calcineurin has
also been observed to regulate NFAT directly in the nucleus.
Mechanistically, nuclear calcineurin was shown to suppress
exportin protein Crm1 binding to NFAT, with nuclear NFAT
consequently accumulating.114,115
Downstream effectors of calcineurin signaling may also
include MEF2, as this is implied by findings from skeletal
Figure 2. Proposed interplay between
Ca2+/calmodulin-dependent kinase II
and calcineurin and their downstream
signaling pathways in cardiomyocytes.
CaMKII indicates Ca2+/calmodulin
dependent kinase II; CaN, calcineurin;
HDAC4, histone deacetylase 4; MEF2,
myocyte enhancer factor 2; Mrj,
mammalian relative of DnaJ; and NFAT,
nuclear factor of activated T cells.
 1006  Circulation Research  September 29, 2017
muscle cells, neurons, and lymphocytes.116–118 As an under-
lying mechanism, it was proposed that calcineurin is able to
dephosphorylate MEF2, thereby blocking its sumoylation and
consequently promoting MEF2-dependent transcription.119 In
addition, it was shown that calcineurin possesses the ability
to enhance MEF2 transcriptional activity via a calcineurin/
mAKAP/MEF2 complex formation.120 Another possible path-
way involves prominent calcineurin downstream target NFAT,
since Blaeser et al described activated calcineurin to induce
the formation of a MEF2/NFATc2 transcriptional complex.118
Irrespective of the exact mechanism, the functional improve-
ment of MEF2 antagonization in a calcineurin-induced heart
failure model argues for a relevant calcineurin-dependent
MEF2 effect also in cardiomyocytes.121 Yet, one has to con-
sider that MEF2 activation in this model of calcineurin over-
expression may also be a secondary phenomenon during the
development of heart failure, which is not based on a direct
signaling of calcineurin to MEF2. Another aspect of inter-
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relation between the HDAC/MEF2 axis and NFAT was em-
phasized by Dai et al,122 showing that NFATc3 is negatively
regulated by class II HDACs through the DnaJ-Related Factor
Mrj. Notably, calcineurin activity can be negatively regulated
by direct CaMKII-dependent phosphorylation, causing dimin-
ished NFAT translocation.123 This phenomenon has been dem-
onstrated by us to account for hypertrophic cardiac growth
in CaMKIIδ/CaMKIIγ double knockout (KO) mice.124 It has
moreover been suggested that calcineurin-dependent dephos-
phorylation of CaMKII at its autophosphorylation site vice
versa constitutes another negative crosstalk pathway between
these 2 enzymes.125 This pathway may also involve a more
indirect mechanism via calcineurin-dependent dephosphory-
lation of protein phosphatase 1 inhibitor-1.126 Thereby, protein
phosphatase 1 activity is increased, which would consequently
reduce autophosphorylation of CaMKII.127 Figure 2 illustrates
the potential interplay between CaMKII and calcineurin and
their downstream signaling pathways.
In addition to CaMKII, calcineurin via NFAT was also
reported to have an impact on the transcriptional coactivator
myocardin.128 Li et al129 could further show that NFAT binds
to the promotor region of myocardin, induces its expression,
and synergistically with myocardin enhances expression of
the LTCC α subunit.
Beyond the regulation of NFAT, calcineurin was revealed
to interact with the transcription factor nuclear factor Y and
affect gene expression of TXNIP (thioredoxin-interacting pro-
tein). Nuclear factor Y binds to the CCAAT element in promo-
tor regions, and depending on the particular DNA sequence
either is an activator or repressor of gene expression.130 It
has been proposed that calcineurin dephosphorylates nuclear
factor Y, therefore, decreases its affinity to the DNA and at-
tenuates its transcriptional repression of one of its targets—
TXNIP.131 TXNIP, an endogenous inhibitor of antioxidant
thioredoxin, is considered to be a relevant player in control-
ling energy metabolism and reactive oxygen species in cardiac
pathologies.132,133
Calpain
The nonlysosomal cysteine protease calpain gets only acti-
vated at relatively high ambient Ca2+ concentrations of about
1 µmol/L.134 Otherwise, it is regulated by its endogenous in-
hibitor calpastatin. Although some physiological functions
have been demonstrated, calpain is mainly described to cleave
proteins in pathological settings. In regard to cardiomyocytes,
especially ischemia reperfusion settings and heart failure con-
ditions depict situations of Ca2+ overload where calpain was
shown to be more active.135
Calpain was described to cleave PKCα, the most abundant
PKC isoform in the heart, in its V3 hinge region, thereby sepa-
rating the C-terminal catalytic from the N-terminal regulatory
domain.136–138 Accordingly, without this regulatory domain
permanently inhibiting the catalytic function, the C-terminal
domain is constitutively active. Furthermore, the C-terminal
fragment of PKCα gains the ability to phosphorylate addition-
al targets and to translocate to the nucleus. Here, the catalytic
fragment leads to HDAC5 phosphorylation, which results in
nuclear export of HDAC5, and consequent activation of MEF2-
dependent genes.139 Aside from PKC, 2 other prominent cleav-
age targets of calpain have been described in regard to their
influence on transcriptional regulation. For the phosphatase
calcineurin, calpain was reported to cleave the autoinhibitory
domain, which results in translocation of calcineurin to the
nucleus, causing increased and prolonged NFAT-dependent
gene transcription.140 In addition, the calpain-mediated calci-
neurin cleavage product was described to be independent of
Ca2+/calmodulin binding and, therefore, constitutively active
in its function.141 Moreover, calpain cleavage of LTCC was
proposed. Its distal C-terminal fragment was shown to local-
ize to the nucleus and alter transcriptional activity.142 Via this
mechanism, the LTCC, for example, autoregulates its own ex-
pression in cardiomyocytes.143
Direct Effects of Ca2+
An interesting finding in terms of direct interaction between
Ca2+ and chromatin is the observation that certain GC se-
quences in the DNA have the ability to bind Ca2+, which leads
to local changes in the DNA structure.144 The physiological
relevance of this finding is still unclear, yet one has to consider
that Ca2+, locally enhanced in nuclear microdomains, might
bind to a very specific region of the DNA and recruit Ca2+-
dependent enzymes to spatially modify chromatin structure
and potentially change transcription.
ET Coupling in the Diseased Heart
Ca2+ Cycling in Heart Failure
Defects in cellular Ca2+-handling and alterations in Ca2+-
dependent signaling pathways are molecular hallmarks of
heart failure. The underlying mechanisms of cardiac dysfunc-
tion may vary depending on the different causes and stages of
the disease, yet several consistent findings on the dysregula-
tion of Ca2+ cycling have been characterized. On the one hand,
structural changes in Ca2+-handling microdomains regularly
occur such as decreases in T-tubule and nuclear invagination
density and rearrangements of the nuclear transport machin-
ery.33,145–148 On the other hand, several Ca2+-handling proteins
show altered expression patterns and activity states. In par-
ticular, defective SR Ca2+-handling is considered an important
aspect of heart failure pathophysiology. On a molecular level,
 Dewenter et al   Calcium Signaling and Transcription   1007
impaired SERCA function, based on reduced protein expres-
sion and increased SERCA inhibition by PLN, and increased
open probability of RyR, based on hyperphosphorylation by
PKA and CaMKII, seem to be causative.149 Therefore, SR
Ca2+ load is decreased because of diminished SR filling and
increased diastolic RyR Ca2+ leak. As a consequence of defec-
tive SR Ca2+ cycling and SR Ca2+ depletion, Ca2+ transients
in failing cardiomyocytes show distinct alterations: the am-
plitude decreases, the time to peak prolongs, the rate of Ca2+
removal is reduced, and the resting [Ca2+] is elevated.150 NCX
is increased in expression and activity in various heart failure
conditions151 and may influence these alterations in different
ways. Shifting the balance of cytosolic Ca2+ removal toward
enhanced Ca2+ extrusion via NCX might further impair SR
Ca2+ load. In contrast, NCX can switch into reversed mode at
high intracellular [Na+] as observed in heart failure, causing
Ca2+ influx which may contribute to diastolic Ca2+ overload.150
Besides these global Ca2+ disturbances, distinct local changes
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in Ca2+ have been observed in heart failure. Ljubojevic et al
demonstrated that nuclear Ca2+ transients display pathological
patterns earlier in disease development than the cytosolic Ca2+
transients,33 suggesting an important role for Ca2+-dependent
maladaptive gene program activation. Consistent with this ob-
servation, changes in nucleocytoplasmic trafficking processes
were observed to precede cardiac functional deterioration in
experimental heart failure.148 Moreover, increasing evidence
indicates that also other Ca2+ channels, less implicated in EC
coupling, significantly contribute to disturbances in Ca2+-
dependent pathways, as IP3 receptors and several TRPC
channels have been demonstrated to be upregulated in experi-
mental and human heart failure.152–155
Changes in Ca2+-Dependent Transcriptional
Regulation in Heart Failure
Ca2+-dependent transcriptional pathways involving key regu-
lators such as CaMKII and calcineurin are overactive in the
diseased heart and suggested to drive structural changes and
functional deterioration by inducing remodeling processes.
Table 1 gives an overview on in vivo studies on proteins in-
volved in these pathways with respect to their impact on cardi-
ac hypertrophy, cardiac function, cardiac fibrosis, and cardiac
inflammation.
CaMKII activity is consistently increased in human and in
experimental heart failure,156,202 and activation of CaMKII has
been observed in response to multiple neurohumoral signals
that are enhanced in heart failure, including ANGII, ET-1, α-
AR, and β-AR agonists.71,80,203–209 This neurohumoral activation
of CaMKII has been linked to the induction of hypertrophic
and fetal gene programs.204,210–212 In addition, CaMKII was
shown to act as a critical downstream mediator of noncanoni-
cal Wnt signaling via dishevelled, which represents a widely
unrecognized but central pathway in myocardial remodeling
processes.166 A causative role of CaMKII in heart failure devel-
opment has been derived from cardiomyocyte-specific genetic
mouse models.87,124,162,163,165 Cardiac-specific overexpression of
cytosolic CaMKIIδC and nuclear CaMKIIδB induces dilated
cardiomyopathy and cardiac hypertrophy, respectively.156,161
Given the multiple targets of CaMKII in cardiomyocytes,
specifically estimating the relevance of effects based on Ca2+
cycling protein modification versus effects on transcriptional
regulation components is challenging. However, deciphering
the role of nuclear versus cytosolic CaMKII might deliver in-
sights into distinct mechanisms. By comparative approach, it
was shown that overexpression of both splice variants induces
activation of MEF2, mechanistically via phosphorylation of
HDAC4, whereas SR Ca2+ cycling is only affected by cyto-
solic CaMKIIδC.105 Accordingly, CaMKIIδ KO mice are pro-
tected from cardiac remodeling upon pressure overload.162,163
In our studies on CaMKIIδ KO mice, we found a marked
reduction in CaMKII-dependent HDAC4 phosphorylation
and attenuated induction of the fetal gene program, while we
could not see any genotype-related disturbances in intracellu-
lar Ca2+ transients under either unstressed conditions or in the
presence of transverse aortic constriction.163 These results in-
dicate that effects on Ca2+-handling are not absolutely required
for the protective CaMKIIδ KO–dependent phenotype. In
contrast, Ling et al demonstrated in a different CaMKIIδ KO
mouse model that the increased SR Ca2+ leak after transverse
aortic constriction is completely abolished via prevention of
RyR2-S2814 hyperphosphorylation, suggesting that restored
SR Ca2+ might underlie the beneficial effects of CaMKIIδ
deletion.162 However, attempts to rescue the cardiomyopathic
phenotype in CaMKIIδC TG mice by restoring SR function
to our knowledge failed to date. SR-targeted CaMKII inhibi-
tion indeed significantly reduced the SR Ca2+ leak but accel-
erated the development of cardiac remodeling in the TG.158
In addition, restoring SR load in CaMKIIδC TG by genetic
KO of PLN showed no rescue effect but resulted in exacerba-
tion of cardiac dysfunction and mortality in the TG.157 These
findings strongly indicate that even cytosolic CaMKIIδC ex-
erts maladaptive effects in heart failure via SR-independent
mechanisms.
The critical involvement of the class IIa HDAC/MEF2 axis
in the induction of cardiac hypertrophy and remodeling has
also been demonstrated in genetic mouse models. HDAC9 KO
mice are sensitized to hypertrophic signals including calcineu-
rin overexpression, show an increased activation of the fetal
gene program, and display a hypersensitive MEF2 activity.171
Similar findings have been reported for HDAC5 KO mice.170
Regarding MEF2, characterization of MEF2A and MEF2C
TG revealed a dosage-dependent cardiomyopathic phenotype
and reduction in ventricular performance,172 as well as an in-
creased susceptibility to calcineurin overexpression-induced
hypertrophy.121 In addition, MEF2D overexpression induces
pathological cardiac remodeling and drives the fetal gene
program, whereas MEF2D KO are protected against remod-
eling and exhibit attenuated fetal gene activation in response
to pressure overload and chronic β-adrenergic stimulation.101
Calcineurin is overactive not only in human heart failure
but also in compensated cardiac hypertrophy.213,214 Notably,
IRF8 (interferon regulatory factor 8), which acts as an in-
hibitor of calcineurin downstream target NFATc1, has been
described to be downregulated in dilated and hypertrophic
cardiomyopathy.215 In vitro data consistently indicate that
pharmacological and genetic calcineurin inhibition at-
tenuates cardiomyocyte hypertrophy induced by neurohu-
moral stimulation.113,204,216 This antihypertrophic effect was
 1008  Circulation Research  September 29, 2017

Table 1.  Overview on In Vivo Studies Investigating Ca2+-Dependent Signaling Proteins Involved in Transcriptional Pathways
With Respect to Their Impact on Cardiac Hypertrophy, Cardiac Function, Cardiac Fibrosis, and Cardiac Inflammation
Protein/Mutant Genetic Pharmacological/ Cardiac
Intervention Cardiac Growth Cardiac Function Cardiac Fibrosis
Name Model Peptide-Based Inhibition Inflammation
CaMKIIδC ↑(156,157)
none ↓↓(156–160) ↑(156)
TG ↑↑(159,160)
MI ↓↓(86) ↑(86)
CaMKIIδB TG none ↑( )161
↓( )161

CaMKIIδ TAC ↓( 162,163

) ↑(162) ↓(162,163)
KO MI ↑(85) ↓(85)
ISO →( ) 164
↑( )164
↓( )
164

CaMKIIδ/ TAC →( 124,165

) ↑( 124,165
) ↓(124,165)
CaMKIIγ
MI →(87) ↑(87) ↓↓(87) ↓(87)
DKO
ISO →(124) ↓↓(124)
DVL TG ↓( )166
↑( )166
↓(166)
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CaMKII MI ↑(167) ↓(168)

AC3-I TG CnA TG →(169) ↑(169)
ISO ↓(167) ↑(167)
KN93 MI ↑(167)
HDAC5 none ↑(170)
KO TAC ↑(170)
ISO →(170)
HDAC5/
CnA (constitutively KO/TG none ↑↑↑(170)*
active CnA)
HDAC9 none ↑(171)
KO TAC ↑(171)
ISO →(170)
HDAC9/
CnA (constitutively KO/TG none ↑↑↑(171)*
active CnA)
HDAC5/
DKO none ↑(170)
HDAC9
MEF2A ↑↑(172)
none ↓↓(172)
TG ↑(121)
TAC ↑(172) ↓↓(172)
MEF2C TG none ↑(172) ↓↓(172)
MEF2D TG none ↓(101) ↑↑(101)
MEF2D TAC ↓(101) ↑(101) ↓↓(101)
KO
ISO ↓( )101
↑( )101

CnA (constitutively →(121,175)

active form) ↑↑(113,121,152,173–177)
TG none ↓(173,178,179) ↑(113,178)
↑(178–180) ↓↓(174,177)
CnAß TAC ↓( )181

KO ISO ↓(181)
ANG ↓(181)
dnCnA TG TAC ↓(182) ↓(182)

(Continued )
 Dewenter et al   Calcium Signaling and Transcription   1009

Table 1. Continued
Protein/Mutant Genetic Pharmacological/ Cardiac
Intervention Cardiac Growth Cardiac Function Cardiac Fibrosis
Name Model Peptide-Based Inhibition Inflammation
CaN →(183–186) ↑(188)
TAC →(187)
↓(187–190) →(189)
→(191) →(192,193)
MI →(191,192)
CsA ↓(192–194) ↓(191,194)
↓(113,177,195)
CnA TG ↑↑(177)
↓↓(113,177,195)
ANGII ↓(196)
→(184,185)
TAC ↓↓(197)
FK506 ↓(197)
CnA TG ↓↓(195)
TAC ↓(198)
ΔCain TG
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ISO ↓(198)
TAC ↓(198)
ΔAKAP TG
ISO ↓(198)
TAC ↓(199) →(199) →(199)
MI ↓( )
200
↑( )
200
↓↓(200)
MCIP1 TG
CnA TG ↓(174) ↑↑(174)
ISO ↓(174)
Zaki-4β TG TAC ↓(201) →(201)
NFATc3 Δ317 TG none ↑( )
113
↑(113)
NFATc3 TAC ↓(173)
KO CnA TG ↓(173) →(173)
ANGII ↓( )
173

NFATc4 TG none ↑(177)

NFATc4 KO CnA TG →(173)
CAMTA2 TG none ↑↑(65)
CAMTA2 TAC ↓(65)
KO ISO ↓(65)
ANGII ↓(65) ↓(65)
Calpain1 KO MI ↑( )
137

ANGII indicates chronic angiotensin II infusion; AKAP, A-kinase anchoring protein; CaMKII, Ca /calmodulin-dependent kinase II; CAMTA, calmodulin-binding
2+

transcriptional activator; CaN, calcineurin; CnA, calcineurin subunit A; CsA, cyclosporin A; DKO, double knockout; dnCnA, dominant negative calcineurin subunit
A; DVL, dishevelled; HDAC, histone deacetylase; ISO, chronic isoprenaline infusion; KO, knockout; MCIP, myocyte-enriched calcineurin-interacting protein; MEF2,
myocyte enhancer factor 2; MI, myocardial infarction; NFAT, nuclear factor of activated T cells; PE+ANGII, chronic application of phenylephrine and angiotensin
II; TAC, transverse aortic constriction; TG, transgenic overexpression; ↑, <2-fold increase compared with respective control; ↑↑, >2-fold increase; ↑↑↑, >3-fold
increase (*Compared with the wild-type control group); →, no significant alterations; ↓, <50% decrease; and ↓↓, >50% decrease.

confirmed in various in vivo models.181,187,195,198 Further evi-
dence for a causative role of calcineurin and its downstream
effector NFAT in heart failure was obtained from cardiac-
specific genetic mouse models. Calcineurin and NFAT3 TG
develop cardiac hypertrophy and heart failure, with NFAT
and GATA4 synergistically activating the fetal gene pro-
gram.113 Of note, calcineurin TG mice were shown to ex-
hibit cardiac remodeling despite increased SR function.217
The critical involvement of NFAT in cardiac hypertrophy
was additionally investigated by studying NFATc3 KO mice,
revealing that NFATc3 deletion protects against calcineurin
overexpression-induced, pressure overload–induced, and
ANGII-induced cardiac hypertrophy.173
There are, however, some inconsistent findings about the
role of the calcineurin/NFAT pathway in physiological ver-
sus pathological hypertrophy because several studies also
suggest calcineurin-dependent pathways to be involved in
nonmaladaptive growth. Interestingly, calcineurin upregula-
tion is more pronounced in compensated cardiac hypertrophy
compared with heart failure.214 Eto et al reported calcineurin
 1010  Circulation Research  September 29, 2017
upregulation in adaptive cardiac hypertrophy upon exercise
training in rats.218 In line with this finding, calcineurin inhi-
bition by MCIP1 (myocyte-enriched calcineurin-interacting
protein 1) overexpression in mice has been shown to attenuate
exercise-induced cardiac hypertrophy.174 Moreover, calcineu-
rin activity was suggested to be a requirement for adaptive hy-
pertrophy during pregnancy.219 Another observation that might
challenge the view of calcineurin as an overall detrimental ET
coupling player is related to its role in dilated cardiomyopa-
thy. KO mice lacking the stress responsive isoform of calci-
neurin exhibit enhanced cardiomyocyte apoptosis and cardiac
dysfunction in a genetic mouse model of muscle LIM protein
deficiency. Conversely, expression of activated calcineurin
improves function and adverse remodeling in this model, sug-
gesting a rather protective role in this scenario.175
Also findings in the context of the interrelation between
CaMKII and calcineurin signaling indicate calcineurin ac-
tivation in nonpathological cardiac growth. In line with the
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above-mentioned detrimental role of overactivated CaMKII,
CaMKIIδ/CaMKIIγ double KO mice are protected against
cardiac dysfunction and interstitial fibrosis upon pressure
overload. However, these mice do develop cardiac hypertro-
phy, but remarkably without signs of pathological remodel-
ing, establishing a dissociation of cardiac hypertrophy on the
one hand and pathological remodeling on the other hand. In
the same study, these mice also show an increased activity
of endogenous calcineurin and exaggerated cardiac hyper-
trophy upon exercise, again without signs of heart failure.
Mechanistically, calcineurin activation was shown to account
for cardiac growth in this model because of completely dimin-
ished CaMKII-dependent phosphorylation at the inhibitory
calcineurin phosphosite Ser-411 in the CaMKIIδ/CaMKIIγ
double KO mice. Notably, this striking effect was only ob-
served in the CaMKIIδ/CaMKIIγ double KO model, which
lacks any measurable CaMKII activity in the heart, but not
in the CaMKII single KO models, because CaMKIIδ and
CaMKIIγ are able to compensate for each other.124
In contrast to the indicated crucial involvement of the cal-
cineurin/NFAT pathway activation in adaptive cardiac growth,
Hainsey et al reported no effect of calcineurin inhibitor cyclo-
sporin A on exercise-induced cardiac hypertrophy in wild-type
mice.220 Other data even suggest that cardiac antiremodeling
effects of exercise training involve deactivation of the calci-
neurin/NFAT pathway.221 Consistent with the latter, Wilkins et
al also observed a decrease in NFAT activity upon exercise.222
An aspect that should be taken into consideration when
trying to explain the discrepancies on the role of calcineurin
in nonmaladaptive growth versus decompensated heart failure
is the applied genetic model. The studies investigating calci-
neurin TG mice used a constitutively active form of calcineu-
rin for the transgenic overexpression. These mice show severe
cardiac remodeling accompanied by contractile dysfunction
and increased collagen deposition. However, the fact that the
overexpressed calcineurin is constitutively active might ac-
count for some divergences in the results compared with stud-
ies investigating the modulation of endogenous calcineurin.
Taking all of the studies on genetic and pharmacological cal-
cineurin and NFAT inhibition into account (Table 1), the rele-
vance of the calcineurin/NFAT axis for cardiac hypertrophy is
striking, but not so the evidence for its role in pathological re-
modeling and contractile dysfunction. Also the time course of
calcineurin activation during the compensated versus the de-
compensated phase of heart failure supports this perspective.
For instance, our laboratory showed that calcineurin activation
precedes CaMKII activation upon the induction of pathologi-
cal pressure overload and is, in contrast to CaMKII, down-
regulated during the transition to heart failure.124 These data
may indicate that increased calcineurin/NFAT activity plays
an important role in compensated and early cardiac hypertro-
phy rather than in long-term cardiac remodeling processes.
The observation that calcineurin is upregulated in exercise-
induced hypertrophy and required for adaptive cardiac growth
supports from our point of view this conclusion.
Taken together, it will be important in the future to detect
dynamic changes of different Ca2+-dependent signaling mol-
ecules in the course of heart failure development but also to
increase our understanding of the distinct Ca2+ sources that
account for the differential regulation in adaptive versus mal-
adaptive signaling cascades.
Increasing evidence indicates that IP3 receptors, TRPC
channels, and SOCE dysregulation significantly contribute to
detrimental enhancement of the above-mentioned transcrip-
tional pathways in heart failure. Table 2 gives an overview
on in vivo studies investigating these Ca2+-handling proteins
with respect to their impact on cardiac hypertrophy, cardiac
function, cardiac fibrosis, and the suggested involvement in
transcriptional regulation.
IP3R2 TG mice develop cardiac hypertrophy in re-
sponse to chronic isoproterenol infusion, Gαq overexpres-
sion, and exercise stimulation, which is blocked via genetic
calcineurinAβ deletion.17 Moreover, local nuclear envelope
Ca2+ release via IP3 receptors in response to ET-1 has been
shown to affect the HDAC5/MEF2 axis independent of the
global Ca2+ transients.231
Also Ca2+ entry via TRPC channels has been linked to
calcineurin activation. TRPC3 TG develop hypertrophy and
contractile dysfunction in response to pressure overload,
which is abrogated in a calcineurinAβ KO background.225 In
addition, overexpression of TRPC6 was demonstrated to in-
duce cardiomyopathy and accelerate cardiac remodeling, ac-
companied by an increase in NFAT-dependent expression of
the β-myosin heavy chain.152 Of note, Seo et al demonstrated
that only combined deletion of TRPC3 and TRPC6 reduces
the hypertrophic response to pressure overload, whereas indi-
vidual gene deletion is not protective.226 Moreover, studies on
dominant-negative TRPC3, TRPC6, and TRPC4 revealed that
inhibition of TRPCs reduces the activity of the calcineurin/
NFAT pathway and attenuates hypertrophic response in vitro
and in vivo.228 Interestingly, protection against neurohumor-
al-induced cardiac hypertrophy in TRPC1/C4 KO mice was
shown to involve a reduction not only in calcineurin activity
but also in MEF2-dependent gene activation.227
Recent experimental data also suggest SOCE to play an
important role in activating transcriptional pathways involved
in cardiomyocyte hypertrophy. STIM1-dependent Ca2+ signal-
ing was revealed to activate the calcineurin/NFAT pathway
and CaMKII signaling in vitro.232,233 Hulot et al emphasized
the in vivo relevance of STIM1 in cardiac hypertrophy,
 Dewenter et al   Calcium Signaling and Transcription   1011

Table 2.  Overview on In Vivo Studies Investigating Ca2+-Handling Proteins With Respect to Their Impact on
Cardiac Hypertrophy, Cardiac Function, Cardiac Fibrosis, and the Suggested Involvement in Transcriptional
Pathways
Protein/Mutant
Name Genetic Model Intervention Cardiac Growth Cardiac Function Cardiac Fibrosis Suggested Pathway
IP3R2 none ↑(17) →(17)
TG TAC ↑(17) Calcineurin-NFAT17
ISO ↑(17)
IP3R2 KO TAC →(223) →(223)
TRPC1 Calcineurin-
KO TAC ↓(224) ↑(224) ↓(224)
NFATc3224
TRPC3 none ↑(225) ↓(225)
TG TAC ↑(225) ↓(225) Calcineurin-NFAT225
PE+ANGII ↑( )
225

TRPC3 KO TAC →(226) →(226)

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TRPC6 none ↑(152)

TG Calcineurin-NFAT152
TAC ↑(152) ↓(152)
TRPC6 KO TAC →(226) →(226)
TRPC3/ →(227)
TRPC6 TAC ↑(226)
↓(226)
DKO Calcineurin-NFAT226
ISO →(227)
ANG →(227)
TRPC1/ TAC ↓(227) ↑(227) ↓(227) Calcineurin-NFAT227
TRPC4
DKO ISO ↓(227)
CaMKII-MEF2227
ANG ↓(227)
dnTRPC3 TAC ↓(228) ↑(228) ↓(228)
TG Calcineurin-NFAT228
PE+ANGII ↓(228)
dnTRPC4 TG TAC ↓(228) Calcineurin-NFAT228
dnTRPC6 TAC ↓(228) ↑(228) ↓(228)
TG Calcineurin-NFAT228
PE+ANGII ↓(228)
STIM1 none ↑↑(229) ↓(229) Calcineurin-NFAT229
TAC ↑( )
229
↓( )
229

TG
ISO ↑( )
229
CaMKII-MEF2229
PE+ANGII ↑(229) ↑↑(229)
STIM1 none →(23,230) ↓(23,230) ↑(230) FAK-Akt23
KO
TAC ↓( )
23
→( ) 23
ERK1/223
ANGII indicates chronic angiotensin II infusion; CaMKII, Ca2+/calmodulin dependent kinase II; DKO, double knockout; dnTRPC, dominant
negative transient receptor potential C; IP3R, inositol-1,4,5-trisphosphate (IP3) receptor; ISO, chronic isoprenaline infusion; KO, knockout; MEF2,
myocyte enhancer factor 2; NFAT, nuclear factor of activated T cells; PE+ANGII, chronic application of phenylephrine and angiotensin II; STIM1,
Stromal interaction molecule-1; TAC, transverse aortic constriction; TG, transgenic overexpression; TRPC, transient receptor potential C; ↑, <2-
fold increase compared with respective control; ↑↑, >2-fold increase; →, no significant alterations; ↓, <50% decrease; and ↓↓, >50% decrease.

demonstrating that shRNA-mediated STIM1 gene silencing
protects against cardiac growth in response to pressure over-
load, associated with a reduction in nuclear NFATc3 trans-
location.234 Conversely, it was shown that overexpression of
STIM1 results in cardiac hypertrophy and contractile dysfunc-
tion.229 The cardiac-specific STIM1 KO mice from Parks et al
exhibited similar results with regard to attenuation of hyper-
trophic response, but additionally presented impaired baseline
contractility and rearrangements of cytoskeletal structures
under unstressed conditions.23 The important role of STIM1 in
cardiac integrity was further illustrated by Collins et al, dem-
onstrating that cardiac-restricted deletion of STIM1 leads to
increased ER stress, mitochondrial disorganization, and pro-
gressive cardiac remodeling in mice.230 This shapes an overall
complex picture of STIM1-dependent cellular effects with
broad implications that are up to now still poorly understood.
 1012  Circulation Research  September 29, 2017
In conclusion, Ca2+-dependent transcriptional regula-
tors are evidently involved in heart failure pathophysiology.
The majority is overactive in the state of heart disease and in
experimental in vivo models, and numerous studies empha-
sized that Ca2+-dependent ET effectors and pathways are able
to dramatically change the cardiac phenotype. Nevertheless,
whereas some few downstream targets are well characterized,
most of them remain elusive just as the interaction of different
Ca2+-dependent pathways and the question whether 1 path-
way is solely maladaptive or does also possess some adaptive
functions.
Therapeutic Implications
Current pharmacological treatment options in heart failure (be-
ta-blockers, angiotensin-converting enzyme inhibitors, angio-
tensin receptor blockers, and aldosterone inhibitors) are in line
with the paradigm of neurohumoral overactivation. Indeed,
angiotensin, endothelin, and catecholamines are reportedly
Downloaded from http://circres.ahajournals.org/ by guest on October 8, 2017
upregulated in heart failure,235–238 and exposure to excessive
amounts has detrimental effects on the cellular level.239–241
Conversely, neurohumoral blockade effectively enhances heart
function, improves clinical outcome, and reduces hospitaliza-
tion.242,243 Despite this, the overall mortality of heart failure is
still high. In addition, tolerability of the optimal doses of stan-
dard medications is often limited because of contraindications,
interactions, and adverse effects. This underlines the necessity
to find novel and relevant therapeutic targets.
Different neurohumoral stimuli were indeed shown to af-
fect cellular Ca2+, and neurohumoral inhibition consequently
improves Ca2+-handling, possibly thereby contributing to
clinical amelioration of contractility.244,245 The fact that Ca2+ is
regulated by a multitude of pathways, and the finding that Ca2+
itself affects various cellular functions such as excitation, con-
traction, metabolism, and transcription, turns it into a nexus
of signaling cascades in cardiomyocytes. On the background
of altered Ca2+-handling in heart failure, it was thus proposed
that restoring Ca2+ cycling back to the physiological state is a
promising therapeutic goal. However, defining a potent target
turned out to be rather challenging.
As mentioned above, increased SR Ca2+ leak is 1 char-
acteristic finding in failing hearts; thus, restabilization of the
RyR is thought to be a rationale concept.246,247 Still, the func-
tional role and the relative impact of the PKA- and CaMKII-
dependent phosphorylation of RyR remain controversial.
Studies on PKA-phospho-resistant RyR mutant mice could
not consistently demonstrate the relevance of this phospho-
site for SR Ca2+ leak or cardiac function in experimental heart
failure models.248–250 In contrast, the association of CaMKII-
dependent RyR phosphorylation with Ca2+ sparks is well es-
tablished.251,252 Findings on RyR mutants that prevent or mimic
CaMKII-dependent phosphorylation offered insights into the
functional relevance of this phosphorylation site at S2814.
The phospho-resistant S2814A mutation was shown to inhibit
Ca2+ leakage from the SR, improve SR Ca2+ load, and to be
protective in the setting of transverse aortic constriction.253 In
contrast, however, this RyR mutation had no protective im-
pact on cardiac dysfunction upon myocardial infarction.253
Conversely, the S2814D mutation depicts a constitutively ac-
tivated form of the CaMKII-dependent RyR phosphosite.254
Mice with this mutation exhibit pathological SR Ca2+ release
events and reduced SR Ca2+ load. Interestingly, this seems to
primarily result in an increase in arrhythmia susceptibility
but causes only a slight decrease in contractile function with
aging. Thus, it seems evident that CaMKII-dependent RyR
phosphorylation at the 2814 site causes pathological altera-
tions in SR Ca2+-handling and arrhythmias. However, a clear
picture with respect to the pathogenesis of all aspects of heart
failure including contractile dysfunction cannot yet be drawn.
Given the complexity of RyR regulation in a macromolecular
complex with multiple posttranslational modifications, target-
ing proteins and interaction partners, future research will need
to shape an integrated view on RyR regulation, causes of RyR
dysfunction, and their relevance in the pathogenesis of heart
failure.
Because decreased Ca2+ uptake into the SR is a character-
istic observation in heart failure, another promising approach
may be enhancement of SR Ca2+ reuptake to rescue patho-
logically altered Ca2+ cycling. In this regard, gene therapy ap-
proaches may offer yet unexplored possibilities. Especially
SERCA, SUMO-1, and S100A1 vectors proved to have bene-
ficial effects on heart failure in animal models.255–257 However,
the outcome of the latest clinical trial for adeno-associated
virus 1–delivered SERCA gene therapy (CUPID 2) could not
yet successfully establish this approach in the clinical set-
ting.258 Although these results do not argue against SERCA as
a promising target, ongoing difficulties with the gene therapy
approach regarding vector design, delivery methods, and is-
sues with humoral immunity need to be solved to use gene
therapy for the re-expression of downregulated Ca2+-handling
proteins such as SERCA or S100A1.259
Moreover, the major Ca2+-dependent signaling enzymes,
CaMKII and calcineurin, are focus of attempts to pharmaco-
logically treat heart failure. In case of CaMKII, it has been
demonstrated that induced genetic KO slows down the pro-
gression of heart failure development in response to pressure
overload, herewith providing further evidence for its thera-
peutic potential.165 In addition, recent findings suggest that
CaMKII blockade would complement the effect of the current
standard therapy.159 However, given the fundamental role of
CaMKII in various physiological cellular functions involving,
for example, synaptic plasticity,260 fertility,261 and immuno-
logic memory,262 pharmacologically inhibiting global CaMKII
might presumably be accompanied by serious adverse ef-
fects. Thus, for a successful application, the issues of selec-
tive organ targeting and CaMKII isoform specificity have to
be addressed as well as the issue of target-specific inhibition,
which requires a deeper understanding of the critical CaMKII
downstream mechanisms in heart failure. Inhibitor develop-
ment programs with focus on myocardial CaMKII have been
launched by several companies,263 but the way into clinical
application is not clear to date.
Cardiac calcineurin is predominantly described to be in-
volved in pathological settings. However, this picture is con-
troversial and has been challenged repeatedly, as discussed
above. Referring to Table 1, the evidence for calcineurin be-
ing involved in cardiac hypertrophy is indeed overwhelming,
but especially with regard to contractile function, the evidence
 Dewenter et al   Calcium Signaling and Transcription   1013
for the benefit of calcineurin inhibition is not consistent. In
part, this could be related to different models of experimental
heart failure, inhibitor strategies, doses, treatment time points,
and treatment periods used in the various studies. These in-
consistencies might also explain why, despite the accessibility
of established pharmaceutical compounds, translation of the
findings around calcineurin inhibition into the clinics was not
successful yet. A recent phase III trial with single application
of CsA in patients after myocardial infarction somewhat de-
picts this translational dilemma and shows no improvement
in clinical outcomes or ventricular remodeling compared with
the control group.264
Even though the recent clinical trials following the prem-
ise to rescue cellular Ca2+ cycling did not yet meet the ex-
pectations, and also pharmacological inhibition of the major
Ca2+-dependent CaMKII and calcineurin pathways could
not be translated into clinical application to date, these Ca2+-
regulated enzymes were convincingly shown to be important
Downloaded from http://circres.ahajournals.org/ by guest on October 8, 2017
players in cardiac pathologies and, therefore, their cascades
present promising therapeutic targets. Concurrently, these
findings illustrate the major problems we face when dealing
with the paradigm of globally disturbed Ca2+ cycling and Ca2+-
dependent signaling pathways, that is, deciphering (1) how
Ca2+-dependent pathways influence different pathological
processes at varied time points, (2) how specifically and se-
lectively these Ca2+-dependent pathways mediate maladaptive
features, and (3) how Ca2+-dependent pathways interrelate.
To circumvent the complexity of CaMKII or calcineu-
rin signaling and their time-dependent and isoform-specific
peculiarities, another therapeutic approach would be to sys-
tematically search for downstream effectors, elucidate their
pathophysiological relevance, and focus on the targets solely
mediating maladaptive features. Pharmacological interven-
tions further downstream bear the potential to offer several
advantages like a higher specificity in the mechanism of ac-
tion and, therefore, a lower possibility of on-target adverse
effects. Given the various disease causes of heart failure and
the complex interplay between disturbed signaling pathways,
deciphering nodal points of maladaptive Ca2+-dependent sig-
naling might finally lay the foundation for effective novel
therapeutic interventions.
Acknowledgments
We thank Dr Christiane Vettel for her valuable suggestions.
Sources of Funding
H.A. Katus and J. Backs were supported by grants from the DZHK
(Deutsches Zentrum für Herz-Kreislauf-Forschung—German Centre
for Cardiovascular Research) and by the BMBF (German Ministry of
Education and Research).
Disclosures
None.
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 Calcium Signaling and Transcriptional Regulation in Cardiomyocytes
Matthias Dewenter, Albert von der Lieth, Hugo A. Katus and Johannes Backs
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Circ Res. 2017;121:1000-1020

doi: 10.1161/CIRCRESAHA.117.310355
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