Just Accepted by Scandinavian Cardiovascular Journal

Cardiomyocyte Ca2+ dynamics: Clinical perspectives Jan Magnus Aronsen, MD, William E. Louch, PhD, Ivar Sjaastad, MD PhD Doi: 10.3109/14017431.2015.1136079 Abstract In the heart, Ca2+ signals regulate a variety of biological functions ranging from contractility to gene expression, cellular hypertrophy and death. In this review, we summarize the role of local Ca2+ homeostasis in these processes in healthy cardiac muscle cells, and highlight how mismanaged Ca2+ handling contributes to the pathophysiology of conditions such as cardiac arrhythmia, ischemic heart disease, cardiac hypertrophy and heart failure. Aiming to provide an introduction to the field with a clinical perspective, we also indicate how current and future therapies may modulate cardiomyocyte Ca2+ handling for the treatment of patients.

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Cardiomyocyte Ca2+ dynamics: Clinical perspectives

Jan Magnus Aronsen1 MD, William E. Louch2 PhD, and Ivar Sjaastad2 MD PhD.

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Bjørknes College, Oslo, Norway

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Institute for Experimental Medical Research, Oslo University Hospital Ullevål and University of Oslo,

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Oslo, Norway.

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Running title: Cardiomyocyte Ca2+ dynamics

Address reprint request to Dr Aronsen, Institute for Experimental Medical Research, Oslo University

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Hospital Ullevål, Oslo, Norway, Kirkeveien 166, 0407 Oslo, Norway. Telephone: +47 23016800. Fax:

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+47 23016799. E-mail: [email protected].

Abstract In the heart, Ca2+ signals regulate a variety of biological functions ranging from contractility to gene expression, cellular hypertrophy and death. In this review, we summarize the role of local Ca2+ homeostasis in these processes in healthy cardiac muscle cells, and highlight how mismanaged Ca2+ handling contributes to the pathophysiology of conditions such as cardiac arrhythmia, ischemic heart disease, cardiac hypertrophy and heart failure. Aiming to provide an introduction to the field with a clinical perspective, we also indicate how current and future therapies may modulate cardiomyocyte

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Ca2+ handling for the treatment of patients.

1. Cardiomyocyte Ca2+ dynamics: local control Ca2+ serves as a central intracellular messenger in the heart. In cardiac muscle cells, called cardiomyocytes, Ca2+ signals regulate contraction but also a host of other cellular processes including gene regulation, cellular growth and death. How could a single ion precisely control such diverse cellular processes? Our understanding of this issue is incomplete, but there is an emerging appreciation that the magnitude and temporal signature of Ca2+ signals is critical, as is the cellular localization of these signals. Cardiomyocytes contain subcellular compartments, or microdomains, where Ca2+ levels are regulated

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distinctly from bulk intracellular Ca2+ concentration (Ca2+i). The effects of Ca2+ within each

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compartment are thus decided by local Ca2+ and the Ca2+ responsive proteins that are present in the compartment. By this model, Ca2+ transporters (Ca2+-permeant ion channels and Ca2+ pumps)

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control cardiac function by determining the balance between Ca2+ influx and efflux in each compartment. There are four key Ca2+ microdomains which are known to regulate cardiomyocyte

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function (Figure 1):

Sarcoplasmic reticulum: The sarcoplasmic reticulum (SR) is an intracellular store for Ca2+. As will be discussed in Section 2, coordinated release of Ca2+ through the ryanodine receptors (RyRs) in

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the SR membrane triggers cardiomyocyte contraction as Ca2+ binds to the myofilaments. The SR Ca2+ ATPase 2 (SERCA2) recycles released Ca2+ back into the SR (Figure 1), which helps maintain

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low cytosolic resting free Ca2+ (≈100 nM) and high SR Ca2+ content (≈1 mM). Thus, there is a large Ca2+ gradient from the SR to the cytosol, which is essential for enabling rapid release, but which also results in a constant leak of Ca2+ from the SR via RyRs. 

Dyadic cleft: RyR Ca2+ release occurs at junctions between the sarcolemmal and SR membranes. These physical junctions are called dyads, and they occur both at the cell surface and at invaginations of the membrane called t-tubules (Figure 1). The dyadic microdomain serves to

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Figure 1: Ca microdomains in cardiomyocytes. The diverse actions of Ca in cardiomyocytes are made 2+

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possible by the presence of Ca microdomains, where Ca levels are discretely controlled. 2+

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1) Sarcoplasmic reticulum (SR): The SR serves as a key Ca store, with Ca levels set by the activity of release 2+

channels called ryanodine receptors (RyRs) and reuptake via the SR Ca ATPase 2 (SERCA2). 2+

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2) Dyadic cleft: Narrow dyadic junctions between t-tubules and SR restrict Ca diffusion, and enable Ca influx 2+

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via L-type Ca channels (LTCCs) to trigger RyR Ca release. Contraction is elicited as Ca binds to the 2+

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myofilaments. Ca efflux from the cell is enabled by the Na -Ca exchanger (NCX). 2+

3) Mitochondria: Ca -dependent regulation of metabolism is controlled by mitochondrial uptake via the 2+

mitochondrial Ca uniporter (MCU) and extrusion by mitochondrial NCX. The mitochondrial permeability 2+

transition pore (MPTP) opens in response to elevated [Ca ]i. 2+

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4) Nucleus: Ca -dependent gene transcription is regulated by local nuclear Ca levels set by release of Ca from inositol triphosphate receptors (IP3Rs) in the nuclear envelope.

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regulate cardiac contractility and signaling. Measuring only 12-15 nm across, the tight dimensions of the dyadic cleft restrict the diffusion of Ca2+ to and from this space. Indeed, with RyR Ca2+ leak, resting Ca2+ in the dyadic cleft is significantly higher than the bulk cytosol [1]. Dyadic Ca2+ levels are additionally controlled by influx of Ca2+ via L-type Ca2+ channels (LTCCs) in the t-tubules, and removal of Ca2+ by the nearby Na+-Ca2+ exchanger (NCX). 

Mitochondria: The Ca2+ in the mitochondria critically regulates cardiac metabolism. Ca2+ is primarily taken up into mitochondria by the mitochondrial Ca2+ uniporter (MCU) and removed by a specialized NCX located in the mitochondrial membrane (Figure 1). Most data indicate that mitochondrial Ca2+ uptake and removal is relatively minor in healthy cells on a beat-to-beat basis

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[2]. However, under pathological conditions the mitochondria overload with Ca2+, which can

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trigger opening of the mitochondrial permeability transition pore and induce cell death [3]. Nucleus: The Ca2+ in the nucleus regulates gene transcription in cardiomyocytes. Importantly,

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Ca2+ release from the nuclear envelope is suggested to regulate gene expression independent of bulk Ca2+i, enabling high-fidelity of local control [4] (Figure 1). Several transcription factors and

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chromatin structure are regulated by Ca2+-dependent kinases.

This review will summarize current understanding regarding local control of cardiomyocyte Ca2+

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homeostasis, aiming to serve as an introduction to the field with a clinical perspective. We will first examine Ca2+ handling in healthy cardiomyocytes, and then focus on the role of mismanaged Ca2+ in

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cardiac arrhythmia, ischemic heart disease, cardiac hypertrophy and heart failure. Finally, we will examine how future therapies may therapeutically modulate cardiomyocyte Ca2+ handling for the benefit of patients.

2. Cardiac ECR coupling Cardiac excitation-contraction-relaxation coupling (ECR coupling) is the physiological basis for the heartbeat. ECR coupling employs Ca2+ to link the action potential to mechanical activation of cardiomyocytes, and the coordinated contraction of the cardiomyocytes in the atria and ventricles results in the ejection of blood from these chambers.

2.1 Phases in ECR coupling ECR coupling can be divided into distinct stages: 

Excitation: Opening of voltage-gated Na+ channels initiates the action potential in

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cardiomyocytes, which rapidly depolarizes the cell membrane from its resting membrane

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potential. LTCCs are opened as a result of the membrane depolarization, which allows Ca2+ influx from the extracellular space into the cytosol (Figure 1). Ca2+ entry continues until the LTCCs close,

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which occurs mainly in a negative feedback manner due to the resulting high Ca2+ in the dyadic cleft.

Ca2+ induced Ca2+ release: Ca2+ entering through LTCCs diffuses across the dyadic cleft, and binds

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to clusters of RyRs triggering them to open and release additional Ca2+. This process is termed Ca2+ induced Ca2+ release (CICR). The Ca2+ released from the RyRs rapidly increases the Ca2+ in

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the dyadic cleft, which within tens of milliseconds diffuses into the rest of the cytosol and leads to an increase in the average Ca2+i. This temporary increase in bulk Ca2+ level is referred to as

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the Ca2+ transient.

Myofilament movement: An increase in [Ca2+ near the myofilaments results in Ca2+ binding to troponin-C, which activates myofilament sliding and contraction of the cell. The “Ca2+ sensitivity” of the myofilaments describes the relationship between the Ca2+i and the resulting force generation, and inotropic effects can in principle be increased either by increasing the Ca2+i or by increasing the Ca2+ sensitivity of the myofilaments.

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Ca2+ removal: Following Ca2+ influx and release, Ca2+i must be returned to its resting level to allow cardiomyocyte relaxation. As the Ca2+ transient predominantly results from SR Ca2+ release, recycling of Ca2+ into the SR by SERCA2 is the principal determinant of cytosolic Ca2+ removal. By setting the SR Ca2+ content available for Ca2+ release in the next beat, SERCA2 activity determines not only relaxation, but also contractility. Its function is regulated by binding of the small inhibitory protein phospholamban (PLB in Figure 1) as discussed in later sections. Ca2+ is also extruded from the cell by NCX, which moves one Ca2+ ion over the cell membrane in exchange for 3 Na+. NCX activity is therefore determined by the membrane potential, as well as by the transmembrane gradients for both Na+ and Ca2+. Thus, NCX activity couples cellular [Na+]i to

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control of cardiac contractility. Of note, under conditions of elevated [Na+]i and/or depolarized

2.2 Regulation of ECR coupling

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membrane potentials, NCX function can reverse, promoting Ca2+ influx and Na+ extrusion.

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Cardiac contractility and relaxation must be continuously adjusted to meet the requirements of the body for blood supply to the metabolizing tissues. A variety of control mechanisms exist, which we will discuss with accompanying clinical perspectives.

2.2.1 The Ca2+ balance and negative feedback loops

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To achieve steady state contractions, Ca2+ extrusion over the sarcolemma must equal the Ca2+ influx

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during the action potential. Also, the SR Ca2+ reuptake must equal the Ca2+ released from the SR during CICR. Two negative feedback mechanisms lead to maintenance of stable steady-state contractions on a beat-to-beat basis [5]: 

Ca2+-dependent inactivation of LTCCs: As LTCCs and RyRs are co-localized in the dyadic cleft, the release of Ca2+ from RyRs during CICR rapidly increases the local Ca2+ close to the LTCCs, triggering their inactivation. This response is graded by the amount of Ca2+ released from the SR,

thus setting up a localized negative feedback loop whereby a large Ca2+ release into the cytosol restricts further Ca2+ influx through the LTCCs. 

SERCA-NCX balance: The balance between the SERCA- and NCX-mediated Ca2+ removal from the cytosol determines the amount of Ca2+ that is recycled into the SR or extruded, respectively. High SERCA2 activity and low NCX activity will increase SR Ca2+ load and thus increase the Ca2+ transient magnitude in the next heartbeat. As NCX activity is regulated by the transmembrane gradient for Ca2+, high Ca2+i promotes NCX-mediated Ca2+ extrusion which tends to reduce SR Ca2+ load. This mechanism can be viewed as a second negative feedback loop controlling steady

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state ECR coupling [5].

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2.2.2 Neurohumoral control of ECR coupling

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Cardiac contractility is under constant neurohumoral regulation as a response to the body demands for blood supply, which for example could be altered by physical activity. The following

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neurohumoral pathways are important modulators of cardiac contractility with clinical roles: The sympathetic nervous system: Sympathetic stimulation leads to chronotropic, inotropic, and lusitropic activation of the heart. Inotropic effects are mediated by the stimulation of α-and βadrenergic receptors. While α-adrenergic receptor activation increases contractility by

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modulating myofilament Ca2+ sensitivity [6], β-adrenergic receptor activation augments

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contraction by increasing Ca2+ transient magnitude. SERCA activation additionally speeds relaxation [1]. 

Nitrogen oxide: Nitrogen oxide (NO) is a central intracellular and intercellular signaling molecule with clinically important roles due to the widespread use of nitrates in ischemic heart disease. NO reduces myocardial O2 consumption in part by reducing Ca2+ transient magnitude. The intracellular response to NO is not completely understood, but seems to involve direct

nitrosylation of Ca2+ transporters (ie. by direct coupling of nitrosyl groups to the ion transporter), as well as indirect effects, mediated by increased levels of the intracellular messenger cGMP [7]. 

Natriuretic peptides (NPs): Natriuretic peptides (ANP, BNP and CNP) are usually secreted in response to high cardiac workload. Of these, BNP has gained considerable prominence as a biomarker for congestive heart failure. Traditionally, NP signaling has been believed to increase concentration of cGMP as two of the three NP-receptors (NPR-A, NPR-B, but not NPR-C) are directly coupled to guanylate cyclase. BNP-mediated effects on cardiac contractility appear to follow this scheme, as an observed lowering of Ca2+i has been linked to the inhibitory actions of cGMP on the L-type Ca2+ channel [8, 9]. A more complex picture has emerged for CNP, which

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shown to amplify β-adrenoceptor-mediated inotropy [11].

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appears to increase Ca2+i while conversely reducing contractility [10]. CNP has, in addition, been

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Activity in the neurohumoral pathways are coupled to activity of the specific Ca2+ transporters by specific intracellular second messengers. Two second messenger systems with notable clinical

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implications are:

cAMP-PKA-signaling: Activation of certain G protein-coupled receptors leads to increased cytosolic concentration of the second messenger cAMP, which activates protein kinase A (PKA).

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PKA-dependent phosphorylation of LTCCs and phospholamban (SERCA2’s endogenous inhibitor) increases Ca2+ transient magnitude [1]. cGMP-PKG-signaling: NO and NPs both elevate the concentration of the second messenger

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cGMP, which activates protein kinase G (PKG) leading to phosphorylation of various target proteins.

While “classical” signaling models are based on uniform cAMP and PKA activation inside the cells, newer data convincingly show that cAMP signaling is differentially regulated in microdomains [12]. Akinase anchoring proteins (AKAPs) are scaffolding molecules that by definition couple PKA to its substrate. An important feature of these macromolecules is that they contain not only PKA, but also

other signaling molecules such as phosphodiesterases (PDEs). PDEs are enzymes that exlusively degrade cAMP and cGMP, which thus limits local cAMP concentrations and modulate cAMP-PKA effects on the target protein. With PKA and signaling molecules located in the same macromolecules as the Ca2+ transporter, local cAMP concentration is thought to control local Ca2+ and thus ECR coupling. Examples of macromolecules which control local Ca2+ include: 

L-type Ca2+ channels: LTCCs are organized into two known macromolecules, where AKAP15/18 and AKAP79/150 orchestrate two different pools of LTCC proteins in the cytosol. AKAP79/150 is reported to be necessary for allowing sympathetic stimulation to increase contractility, by

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binding to a subpopulation of LTCCs [13]. PKA-dependent phosphorylation of LTCC increases Ca2+

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currents, while PKG-dependent phosphorylation of a different site than PKA decreases LTCC current [8].

Ryanodine Receptors: RyRs and the muscle-AKAP (mAKAP) are parts of a macromolecular

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complex. RyRs are phosphorylated by both PKA and Ca2+/calmodulin-dependent kinase II

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(CaMKII). Increased CaMKII activity has been reported to increase RyR open probability, with consequences for arrhythmogenesis (discussed in Section 3). A notable recent finding is that high glucose concentration in cardiomyocytes, as observed in diabetic patients, is able to activate CaMKII by O-GlcNAcylation and induce ventricular arrhythmias by activation of RyRs [14]. SERCA2: Inhibition of SERCA2 by phospholamban is relieved when phospholamban is

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phosphorylated by PKA. Phospholamban is also coupled to the anchoring protein AKAP18δ [15],

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which appears to constitute a localized signaling domain including specific phosphodiesterases (PDE3A and PDE4D) [16, 17] and phosphatases [18]. 

NCX: NCX is part of a macromolecular complex where Ankyrin-B brings together NCX, the Na+/K+ ATPase (NKA) and IP3-receptors. Mutations in Ankyrin-B can induce long QT syndrome (LQTS4), also known as the Ankyrin-B syndrome, characterized by life threatening ventricular arrhythmias [19].

2.2.3 Na+ balance in cardiomyocytes NCX activity is regulated by the intra- and extracellular concentrations of Na+ and Ca2+, which allow the cytosolic concentration of Na+ ([Na+]i) to fine-tune ECR coupling. Increased [Na+]i favors reversemode NCX activity, ie. Ca2+ influx, which loads the SR with Ca2+, leading to larger Ca2+ transients and positive inotropy. This role may be further augmented since Ca2+ entry via NCX can serve as a weak trigger for SR Ca2+ release during the early phase of action potential [20, 21]. Of note, [Na+]i also regulates activity of the mitochondrial NCX (Figure 1). At high [Na+]i, Ca2+ efflux from the mitochondria may be favored, which decreases metabolism [22]. With the knowledge that [Na+]i thus regulates both metabolism and contractility, it is important to understand how [Na+]i is

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regulated by cellular Na+ influx and extrusion:

Na+ influx: Due to the transmembrane Na+ gradient with low [Na+]i compared to the outside of

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the cell, Na+ constantly leaks into cardiomyocytes as a result of opening of voltage gated Na+ channels and via forward-mode activity of the sarcolemmal NCX. Na+ extrusion: The NKA(Figure 1), which uses ATP to pump 3 Na+ out of the cell in exchange for 2

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K+, is the main Na+ extrusion mechanism in cardiomyocytes. At steady-state, NKA activity balances the Na+ influx into the cell during a normal ECR-cycle. NKA is the target for cardiac glycosides such as digitalis, with inhibition of its function leading to increased [Na+]i, increased

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[Ca2+]i via sarcolemmal NCX, and thereby positive inotropy. The NKA exists in several isoforms, and emerging data indicate that the NKA α2 isoform in t-tubules is particularly important for

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regulation contractility and Ca2+-dependent arrhythmias [23, 24]. 2.2.4 Long term modulation of ECR coupling Long-term regulation of cardiac ECR coupling is achieved by controlling protein levels of Ca2+ transporters and their regulatory proteins. Control points include modulation of gene transcription, gene translation and protein degradation, which have potential implications for future clinical applications:

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Cardiac gene transcription: An emerging appreciation of cardiomyocyte gene regulation has pointed to a key role for histone acetylation and regulatory roles of non-coding RNAs on cardiac disease responses [25], and methodological advances are likely to provide new insight into transcriptional regulation and orchestration of specific gene programs in cardiomyocytes.

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Cardiac gene translation: Micro-RNAs (miRNAs) are short non-coding mRNAs which bind to and promote degradation as well as block the translation of specific mRNA molecules. Thus, high miRNA levels will reduce mRNA levels and protein synthesis. miRNAs are increasingly understood to be powerful regulators of cardiac function. For example, miRNA-25 counteracts SERCA2 expression, and blocking miRNA-25 by gene therapy upregulates SERCA2 function, improves

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cardiac function and survival in mice with heart failure [26]. miRNA antagonism may therefore

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provide an opportunity to selectively restore dysfunctional Ca2+ fluxes in disease, as discussed in section 3.

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Cardiac protein degradation: Cardiomyocytes orchestrate protein degradation predominately via proteolysis and lysosomal/autophagy systems. Proteins are marked for proteolytic digestion by

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tagging with ubiquitin. In contrast, SUMOylation (coupling of the SUMO-protein to lysine residues) may exert opposite effects by inhibiting protein degradation. Such actions of SUMOylation have been reported for SERCA2a, indicating that reduced SUMOylation can lead to

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reduced SERCA2 levels in congestive heart failure [27].

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3. Role of Ca2+ fluxes in cardiac disease 3.1 Ca2+ and cardiac arrhythmias Cardiac arrhythmias can be divided into those associated with abnormally slowed or accelerated heart rates, respectively termed bradyarrhythmias and tachyarrhythmias. Tachyarrhythmias such as atrial fibrillation and ventricular tachycardia are elicited by an initial triggering event, combined with a reentrant circuit. Cardiac Ca2+ fluxes are considered to be a primary trigger of tachyarrhythmias by inducing delayed or early afterdepolarizations (DADs, EADs) between two regular action potentials. 3.1.1 Delayed afterdepolarizations

Spontaneous SR Ca2+ release: RyRs open stochastically at a given frequency, which is low in

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two regular action potentials. DADs are generated in three phases [28]:

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DADs are phasic episodes of depolarization from the resting membrane potential that occur between

resting conditions. However, increased RyR opening frequency between regular contractions can

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occur as a result of high SR Ca2+ content, or RyR phosphorylation which lowers the threshold for RyR opening. RyR mutations can also increase spontaneous channel opening resulting in specific inherited arrhythmias (catecholaminergic polymorphic ventricular tachycardia type 1, CPVT1). 

Ca2+ waves: Ca2+ spontaneously released into the cytosol might be rapidly pumped back into the

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SR by SERCA2, or may diffuse along the SR membrane where it reaches other RyRs, triggering their opening. Thus, Ca2+ release can propagate between RyRs, moving across the cell as a “Ca2+

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wave” in a feed-forward cycle (Figure 2). Membrane depolarization: Ca2+ released during a wave is extruded from the cell by the NCX. As each Ca2+ ion is removed in exchange for an influx of 3 Na+, an inward current is induced which leads to depolarization of the cell membrane (Figure 2A). If this depolarization is sufficiently large, a spontaneous action potential is initiated which can trigger a tachyarrhythmia.

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Figure 2: Cellular mechanisms for delayed and early afterdepolarizations (DADs and EADs). A) DADs are 2+

depolarizing events that occur from resting membrane potential, when spontaneous RyR Ca release triggers a 2+

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Ca wave. This is illustrated in a model “line-scan image” in which Ca levels are recorded across the cell over time. In this example, the wave is initiated at one edge of the cell and travels across its width. Depolarization 2+

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results from extrusion of the released Ca by NCX, since 1 Ca is removed in exchange for 3 Na . B) EADs occur 2+

during the action potential, and result from depolarization due to re-opening of L-type Ca channels (LTCCs) or 2+

Ca waves. When DADs or EADs are sufficiently large, an extra action potential is triggered.

3.1.2 Early afterdepolarizations Early afterdepolarizations (EADs) are abnormal depolarizing events that occur during the downstroke

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of the action potential. These occur most commonly when action potential duration is prolonged. In

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humans, EADs are believed to be due to one or two of the following Ca2+-dependent mechanisms (Figure 2B, reviewed in [29]):

L-type Ca2+ channels: Re-opening of L-type Ca2+ channels after closure leads to a second bout of

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Ca2+ influx during the action potential, and a phasic depolarization. NCX: Spontaneous SR Ca2+ release during the action potential can trigger EADs by a mechanism

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similar to DADs; ie. a Ca2+ wave that triggers Ca2+ extrusion by NCX, resulting in depolarization.

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3.1.3 Ca2+ and reentry

Ca2+ signaling is reported to promote reentrant circuits in atrial fibrillation by the following two

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mechanisms:

Electrical remodeling of atrial cardiomyocytes: Rapid stimulation of cardiomyocytes in atrial fibrillation typically activates the Ca2+-dependent calcineurin-NFAT pathway (as discussed in detail in section 3.3), which leads to lower expression of LTCCs and increased expression of K+ channels. These changes induce an abbreviation of the action potential in atrial cardiomyocytes,

shortening the refractory period for which they can be re-activated by a returning electrical stimulus. 

Tissue remodeling of atrial myocardium: Proliferation and differentiation of atrial fibroblasts are central in the tissue remodeling that occurs during atrial fibrillation, and these processes are reported to be dependent on Ca2+ entry through TRP3-channels in atrial cells [30].

3.2 Ca2+ and ischemic heart disease Coronary heart disease is a leading cause of cardiac disease and death. Coronary artery occlusion

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results in ischemic damage of cardiomyocytes, and coronary intervention with reperfusion leads to

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additional reperfusion injury of the myocardium. Ischemic and reperfusion injury is both linked to Ca2+ overload in cardiomyocytes, which might induce cell death (infarction) and arrhythmias as

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discussed in section 3.2.2. However, despite being targeted in several clinical trials, a protective role of inhibiting Ca2+ loading for prevention of reperfusion injury has yet to be demonstrated in patients

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[31].

3.2.1 Roles of Ca2+ in myocardial ischemic damage Cellular Ca2+ overload during prolonged myocardial ischemia leads to necrosis and death of

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cardiomyocytes. It is now well established that Ca2+ accumulation is precipitated by increased [Na+]i due to [31](Figure 3):

Reduced NKA activity: Ischemia gradually depletes cellular ATP levels as the lack of O2 abolishes

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oxidative phosphorylation. Since NKA requires high ATP concentration to maintain low [Na+]i, ATP depletion during ischemia leads to reduced NKA activity and Na+ accumulation. 

Anaerobic glycolysis: The lack of O2 leads to anaerobic metabolism, with production of lactic acid and H+ accumulation. A resulting activation of the Na+/H+-exchanger results in cellular Na+ gain as protons are extruded.

Accumulation of Na+ in the cytosol drives NCX to extrude Na+ in exchange for influx of Ca2+. The resulting increase in Ca2+i promotes cell injury due to the activation of Ca2+-dependent enzymes. For example, Ca2+-dependent proteases have been shown to degrade cytoskeletal components and to trigger apoptosis [32]. Ca2+ overload also damages mitochondria, allowing high energy electrons to leak out of the electron transport chain. The resulting formation of oxygen-derived free radicals can cause widespread damage in the cell. Furthermore, opening of the MPTP during conditions of elevated [Ca2+]i abolishes the mitochondrial membrane potential and thus the ability of mitochondria to produce ATP. Very low ATP synthesis subsequently leads to cellular swelling and induces cellular

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necrosis [3].

3.2.2 Roles of Ca2+ in reperfusion injury

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Perhaps somewhat counterintuitively, reperfusion of ischemic myocardium leads to further cellular damage. However, this fact raises the potential that treatments administered at reperfusion may

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attenuate myocardial injury [33]. Interestingly, most of the hallmarks of reperfusion injury can be mimicked by reintroduction of Ca2+ to the perfusate of myocardium perfused with Ca2+-free solutions, suggesting that Ca2+ is a key pathological mediator in reperfusion [34]. Ca2+i is elevated by ischemia, and is further increased upon reperfusion [35]. It is believed that Ca2+ entry via NCX is a key

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contributor to this additional Ca2+ gain (Figure 3); rapid recover of intracellular pH quickly increases NCX activity during reperfusion, while delayed recovery from Na+ overload promotes reverse-mode

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transport [36]. Restored SR Ca2+ release is also believed to contribute to reperfusion injury, since SERCA2 inhibitors have been shown to be protective [37]. Increased Ca2+i during reperfusion can have three detrimental cellular outcomes [38]: 

Lethal reperfusion injury: Reperfusion, particularly following a prolonged ischemic period, can promote significant cardiomyocyte death. Indeed, current models suggest that the majority of the final myocardial infarction is due to reperfusion injury and not ischemia per se [38]. As during

the ischemic period, the mechanisms of Ca2+-dependent cellular injury during reperfusion include damage caused by Ca2+ -dependent proteases, oxygen-derived free radicals, and opening of the MPTP. Preclinical studies with therapeutic agents aiming at lowering Ca2+ levels in reperfused myocardium have reduced infarct size by up to 50%, but clinical trials employing similar interventions have so far been unsuccessful [38]. 

Myocardial stunning: Myocardial stunning denotes “mechanical dysfunction that persists after reperfusion despite the absence of irreversible damage and despite restoration of normal or near-normal coronary flow” [39]. This depression of contractility can last for hours to weeks in animal models depending on the length of the ischemic period. While the issue remains debated,

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the majority of studies have observed that Ca2+ transient magnitude is normal in late stages of

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reperfusion [40, 41]. Rather, it is believed that the cause of reduced contraction magnitude in stunned myocardium is decreased responsiveness of the myofilaments to Ca2+. Myofilament de-

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sensitization appears to be triggered by Ca2+- and free radical-induced damage to these proteins, particularly during the early stages of reperfusion [39].

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Arrhythmias: Reperfusion-induced arrhythmias, such as ventricular fibrillation, are frequent and can be traced, at least in part, to alterations in cardiomyocyte Ca2+ handling. Ca2+ overload during early reperfusion combined with recovery of SR Ca2+ re-uptake results in spontaneous SR Ca2+

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release and DADs [40, 41]. Elevation of [Ca2+]i is also known to promote re-entrant arrhythmia by delaying inter-cellular conduction, and thus decreasing conduction velocity [29].

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Figure 3: Mechanisms for ischemia- and reperfusion-induced injury. Ischemia and early reperfusion promote significant cell damage, and often cell death. The mechanism underlying this injury involves sequential +

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increases in [H ]i, [Na ]i, and [Ca ] i. Decreased O2 availability during ischemia results in a shift to anaerobic +

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metabolism, and lactate production by glycolysis. Removal of produced H by the Na -H exchanger increases +

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[Na ]i. Na loading is further precipitated by decreased activity of the NKA due to reduced ATP levels. With +

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increased [Na ]i, reverse-mode NCX function promotes Ca overload, with numerous deleterious 2+

consequences including activation of Ca -dependent proteases, formation of oxygen-derived free radicals, and opening of the mitochondrial permeability transition pore (MPTP). During early reperfusion, rapid recovery +

2+

from acidosis, but not from Na overload, causes additional Ca entry by NCX, and recovery of SERCA activity 2+

may enable SR function to contribute to Ca -dependent injury.

3.3 Ca2+ and cardiac hypertrophy Cardiac hypertrophy refers to a thickening of the myocardium, and is usually a response to increased workload. Hypertrophy can be either pathological or physiological depending on the initiating stimulus; physiological hypertrophy is induced by exercise training, while pathological hypertrophy most commonly occurs following hypertension or aortic valve stenosis. Despite its ability to normalize cardiac wall stress, pathological hypertrophy is a marker of poor clinical outcome. Hypertrophy is initiated by neurohumoral or mechanical stimuli, which activate intracellular signaling pathways triggering cellular growth. Two Ca2+-dependent signaling pathways are believed to be central in inducing myocardial hypertrophy, as illustrated in Figure 4 [42, 43]: The calcineurin-NFAT-pathway: The transcription factor Nuclear Factor of Activated T-cells (NFAT)

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is usually present in the cytosol in an inactive state. Ca2+ activates the Ca2+-dependent phosphatase calcineurin, which by dephosphorylation allows NFAT translocation into the nucleus,

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signaling transcription of pro-hypertrophic genes. This process can be initiated by activation of the α-adrenoceptor, receptors for angiotensin II or endothelin [43], as well as mechanosensors

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such as the stretch-activated transmembrane protein Syndecan-4 [44]. Local Ca2+ pools are believed to trigger calcineurin activation, since the calcineurin-NFAT pathway can be activated even during normal global Ca2+ transients [45]. Proposed trigger sources of Ca2+ include elevated

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dyadic [Ca2+]i resulting from LTCC and RyR opening, and local Ca2+ entry via non-dyadic transient receptor potential channels (TRPCs) and LTCCs (Figure 4, Reviewed in [4]). Recently, a localized

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pool of cAMP regulated by PDE2 has been demonstrated to counteract NFAT translocation into the nucleus and thus prevent cardiac hypertrophy [46], highlighting PDE2 inhibitors as a potential antihypertrophic agents. 

The CaMKII-HDAC pathway: Activation of nuclear CaMKII can trigger hypertrophy by stimulating histone deacetylase (HDAC) class II. HDAC activation, in turn, induces de-condensation of chromatin and dis-inhibits the transcription factor Myocyte Enhancer Factor-2 (MEF2), allowing

DNA to be transcribed [47]. While CaMKII activity is Ca2+-dependent, it is important to note that

2+

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local Ca2+ levels near nuclear CaMKII appear to be insulated from global, cytosolic changes in

Figure 4: Ca -dependent signaling pathways leading to cardiac hypertrophy. Transcription of hypertrophic 2+

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genes is regulated by the transcription factors NFAT and MEF2 via two pathways. 1) Local Ca signals activate calcineurin, which de-phosphorylates NFAT, allowing it to translocate to the nucleus. A variety of local Ca 2+

2+

2+

signals have been proposed to trigger this response, including dyadic cleft Ca (resulting from Ca flux via 2+

LTCCs and RyRs) and non-dyadic Ca influx in caveolae via transient receptor potential channels (TRPCs) and 2+

LTCCs. 2) MEF2 activity, on the other hand, is controlled by nuclear Ca . Stimulation of G-protein coupled 2+

receptors by endothelin, angiotension II, or adrenalin leads to the production of IP 3 and release of Ca from IP3 receptors (IP3R) in the nuclear envelope. A resulting activation of CaMKII phosphorylates HDAC, dis-inhibiting MEF2 and enabling transcription. IP3R activation additional leads to hypertrophic gene transcription by reducing levels of miR-133a.

[Ca2+]i. Rather, activation of CaMKII, and thus HDAC and hypertrophy signaling, seem to be triggered by Ca2+ released from IP3 type-2 receptors on the nuclear membrane [48]. Furthermore, IP3 receptor-mediated Ca2+ release decreases the level of antihypertrophic miR-133a, which leads to a further increase in IP3 receptor 2 levels (IP3R2) and perinuclear Ca2+ release. IP3R2-mediated decrease of mir-133a thus initiates a positive feedback circuit that drives the cardiac hypertrophic response to pathological stress [49].

3.4

Ca2+ and chronic heart failure

Dysfunctional cardiomyocyte Ca2+ handling is considered a hallmark of heart failure, which has

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consequences both for the ECR cycle, arrhythmogenesis and cell signaling.

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3.4.1 Dysfunctional ECR coupling

Reduced power of the heartbeat is a hallmark of the failing myocardium, and is characterized by both

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reduced maximal force generation and slowing of force generation. Both alterations can be traced, at least in part, to reduced and slowed cardiomyocyte Ca2+ transients and contractions [1, 50, 51]. The

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precise mechanistic details of these alterations continue to be unraveled, but include (Figure 5): Reduced SR Ca2+ content: Decreased Ca2+ transient magnitude in failing cells partly results from reduced SR stores, as a consequence of decreased SERCA2 expression and/or activity, and

Dyadic disruption: T-tubules are lost and/or disorganized in failing cells [52]. This results in the formation of orphaned RyRs, which do not have paired LTCCs (Figure 5). Such dyadic disruption

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increased RyR Ca2+ leak [1]

decreases the “gain” of Ca2+-induced Ca2+ release; that is the ability of Ca2+ influx via LTCCs to trigger Ca2+ release from RyRs [53]. Thus, the magnitude of the Ca2+ transient is reduced. However, Ca2+ release is also de-synchronized, as Ca2+ released at intact dyads must diffuse to

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Figure 5: Mechanisms for dysfunctional Ca signaling and contractile deficit in failing cardiomyocytes. In 2+

heart failure, decreased SERCA activity, greater SR Ca leak via RyRs, and increased NCX expression have 2+

2+

all been reported to reduce the SR Ca store available for release. However, Ca release is also impaired 2+

2+

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as a result of t-tubule disruption, which reduces the ability of Ca influx via LTCCs to trigger SR Ca release 2+

and de-synchronizes the Ca transient. Poor cardiomyocyte relaxation in failing cardiomyocytes may result 2+

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from reduced Ca removal by SERCA, but also from impairment of NCX activity due to elevated [Na ]i. Na +

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accumulation due to reduced Na -K ATPase expression additionally promotes NCX-dependent Ca

+

2+

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removal from mitochondria which impairs ATP production. Interestingly, Na accumulation during heart 2+

failure has been linked to extrusion of Ca from the mitochondria via the mitochondrial NCX. The resulting 2+

loss of Ca -dependent ATP production is proposed to starve the failing cell of energy required for the generation of contractile force.

trigger orphaned RyRs [54, 55]. De-synchronized Ca2+ release results in a slower Ca2+ transient and contraction [52]. Reduced gain of CICR in failing cells may additionally result from alterations in action potential configuration [56], dispersion of RyR clusters [57], and loss of LTCCs [58].

Heart failure is associated not only with impaired ventricular contractility, but frequently also with impaired relaxation. Indeed, recent data have indicated that roughly half of patients exhibit heart failure with preserved ejection fraction (HFpEF), associated primarily with impaired relaxation and filling of the ventricle [59]. Reduced and slowed myocardial relaxation has been attributed to [60]: Decreased cardiomyocyte Ca2+ removal: Decreased SERCA activity is widely reported to slow Ca2+

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removal following release in failing cells. However, Ca2+ removal by NCX may also be impaired

Stiffening of myocardial tissue: Depending on the precise failing phenotype, impaired myocardial

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due to accumulation of intracellular Na+ following down-regulation of the NKA [23, 61].

relaxation may result from stiffening of titin, a giant elastic protein component of the

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myofilaments [62], or stiffening of the extracellular matrix due to collagen accumulation [63]. Recently, syndecan-4 has been shown to be a regulator of the fibrotic response in hypertrophic hearts [64, 65].

Ca2+-dependent arrhythmogenesis in heart failure

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3.4.1

Congestive heart failure increases the risk for ventricular tachycardia and fibrillation, with DADs and

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EADs thought to be a key underlying mechanism. DADs result from increased RyR Ca2+ sensitivity during heart failure, which increases the occurrence of spontaneous SR Ca2+ release and Ca2+ waves [66]. Reduced SERCA2 activity might also promote DAD generation; although SERCA loss in and of itself is reported to decrease the occurrence of Ca2+ waves [67], a simultaneous increase in adrenergic stimulation during heart failure may counteract these effects by decreasing the threshold for RyR Ca2+ release [68]. When spontaneous Ca2+ release occurs during the action potential it can

result in EADs, as described in Section 3.1.2. Indeed, with reduced repolarization reserve during heart failure, cardiomyocytes also has less ability to counteract these depolarizing events [69]. Action potential prolongation during heart failure additionally promotes EAD generation by facilitating reopening of L-type Ca2+ channels [29].

3.4.2 Dysfunctional Ca2+ signaling in heart failure While abnormalities in Ca2+ homeostasis are believed to play a central role in contractile and electrical dysfunction during heart failure, Ca2+ also importantly serves as a pathogenetic signaling

Cardiac remodeling: Various Ca2+-dependent signaling pathways are central in triggering

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molecule in this condition:

cardiomyocyte remodeling in failing hearts, as discussed in section 3.3. Of note, the hypertrophy-

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inducing calcineurin-NFAT signaling pathway may be activated in failing cardiomyocytes due to elevated dyadic cleft [Ca2+] (Figure 4). This local elevation in [Ca2+ ]i is thought to result from RyR

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leak, impaired SR Ca2+ re-uptake, and/or impaired NCX-mediated Ca2+ extrusion (Figure 5)[1]. With augmented endothelin, angiotensin II and -adrenergic signaling in heart failure, the CaMKII/HDAC prohypertrophic pathway is also activated (Figure 4). 

Attenuated metabolism: Attenuated metabolism in cardiomyocytes is linked to depletion of

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mitochondrial Ca2+, as mitochondrial Ca2+ ions promote metabolism. Cardiomyocytes in heart failure typically have increased Na+ levels due to reduced NKA levels [23, 60], which in turn is

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suggested to increase extrusion of Ca2+ in exchange for Na+ into mitochondria through mitochondrial NCX [22](Figure 5). 

Progressive cardiomyocytedeath: Emerging data indicate that a decrease in the number of cardiomyocytes in the viable myocardium is an important pathogenic factor in congestive heart failure. Such cell loss is caused by an imbalance between pathways contributing to cell survival versus cell death [70]. Both apoptotic and necrotic pathways are stimulated in failing

cardiomyocytes, and these pathways are critically regulated by Ca2+. Apoptosis during heart failure involves activation of Ca2+ -dependent caspases, and can be attenuated by blocking the activity of these enzymes [70]. As discussed above in regards to ischemic myocardium, necrosis

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may be triggered in heart failure as elevated [Ca2+]i causes opening of the MTPT [3].

4. Ca2+ and therapy of heart disease The majority of pharmacological agents presently used to treat chronic heart disease, alter cardiomyocyte Ca2+ fluxes either directly or indirectly. Future therapies are expected to more specifically target Ca2+ -handling proteins based on their emerging role is disease pathogenesis [71].

4.1 Commonly used drugs which modulate Ca2+ fluxes 

Cardiac glycosides: Cardiac glycosides, such as digitalis, inhibit the NKA and have been used for centuries in the treatment of heart failure. NKA inhibition increases [Na+]I, which results in

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elevation of Ca2+i through secondary effects on the NCX. The resulting increase Ca2+i improves

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cardiac contractility, but also increases the risk for DADs and arrhythmias [66].

Other inotropic drugs: Common inotropic agents, such as noradrenaline and dobutamine, also

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induce inotropy by increasing Ca2+i in cardiomyocytes. In addition, Ca2+ sensitizers promote inotropy by augmenting the force generated by the myofilaments for a given rise in Ca2+i. Betablockers: Betablockers exert beneficial effects on the heart by a variety of mechanisms. By

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inhibiting the ability of β-adrenergic signaling to increase Ca2+ transients, these agents tend to lower Ca2+i, which reduces metabolic demand and inhibits arrhythmogenesis. However, these

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drugs can also produce side effects, such as fatigue related to reduction of cardiac output. Betablockers can also inhibit Ca2+-dependent signaling pathways which control gene regulation,

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and these effects are thought to promote cell survival in heart failure [59]. NO donors/nitrates: NO donors and nitrates exert antianginal effects by a combined action on vessels and cardiac contractility. In cardiomyocytes, NO reduces Ca2+i by direct nitrosylation of Ca2+ transporters and by signaling increased cGMP concentration [7]. 

Calcium channel blockers: Calcium channel blockers reduce the influx of Ca2+ via LTCCs, and thus decrease the magnitude of the Ca2+ transient and contraction. These agents provide an

antianginal and antihypertensive treatment option, and may also protect against Ca2+-dependent arrhythmia [72]. 

Neprilysin inhibitor: The clinical roles of the natriuretic peptides are highlighted by recent reports that inhibitors of neprilysin, an enzyme that degrades natriuretic peptides, improve clinical outcome in heart failure patients [73]. The exact mechanism by which neprilysin inhibitors counteract heart failure is not known.

4.2 Future therapy

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Although they have not yet reached clinical use, a number of new therapies aimed at curbing Ca2+

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dysregulation during disease are currently being investigated. Several targets are being explored: SERCA2: Since reduced SERCA2 activity has been tightly coupled to contractile dysfunction in

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chronic heart disease, increasing SERCA activity is an important therapeutic goal. Early clinical trial data suggested improved clinical outcome in congestive heart failure patients treated with a

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single administration of SERCA2 gene via adeno-associated virus (AAV), with a minimum of side effects [74]. However, yet unpublished data from a larger follow-up study have not supported such beneficial effects of AAV1-based SERCA2a gene transfer as initially was suggested. Another

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strategy to counteract low SERCA2 activity in failing hearts is by SERCA2 activating agents, but presently no such compounds exist. A possible approach to develop such agents is to create

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disruptor molecules of protein-protein interactions coupling inhibitory molecules from the SERCA2 macromolecular complex [75]. This strategy could upregulate SERCA2 activity by releasing inhibitory molecules such as PDEs, which in theory could work as future, specific SERCA2 activators. 

CaMKII: As CaMKII activation has been shown to contribute to a variety of cardiac pathologies, there is considerable current interest in investigating CaMKII inhibitors. Recent work has shown that secretoneurin, a member of the granin family of proteins, improves Ca2+ homeostasis in

cardiomyocytes by inhibiting CaMKII activity, and in addition provides prognostic information as a biomarker for heart failure and arrhythmia [76]. 

NCX: By inhibiting Ca2+ extrusion, various NCX inhibitors have been shown to increase [Ca2+]i and thus inotropy, which may be therapeutic in heart failure patients with systolic dysfunction. NCX blockade may also inhibit arrhythmia generation by reducing EADs and DADs associated with Ca2+ extrusion [69]. In the context of ischemia and reperfusion, NCX blockade may be beneficial by inhibiting pathogenic Ca2+ entry via reverse-mode activity.

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RyRs: Since increased RyR Ca2+ leak impairs Ca2+ release and promotes afterdepolarizations, RyR inhibitors may be beneficial in conditions such as heart failure. Indeed, many of the benefits of

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CaMKII inhibition may result from reduced Ca2+ sensitivity of the RyR. RyR blockade may also be

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beneficial during early reperfusion, when SR Ca2+ release is believed to contribute to cellular injury.

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T-tubule structure: Disruption of T-tubules during heart failure and associated disturbances of Ca2+ homeostasis may be prevented by stabilizing the structure of the dyad. Recent data have

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indicated that dyadic disruption is signaled by elevated ventricular workload, and includes loss of junctophilin, a key dyadic anchor. Strategies which decrease workload and stretch-dependent

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signaling or increase junctophilin expression may therefore be therapeutic.

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5. Conclusion Ca2+ is a central second messenger which regulates cardiac function at multiple levels. Ca2+ is an important participant in ECR coupling, and in long term regulation of cardiac function by modulation of gene expression and cell survival. Altered Ca2+ fluxes play a primary role in a variety of cardiac diseases including tachyarrhythmias, ischemic heart disease and heart failure, and may serve as key targets for future therapies.

6. Conflict of interest

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Two of the authors (JMA, IS) have filed patent applications for specific agents modulating SERCA2

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activity.

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