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Calcium Signaling in Cardiac Myocytes

Claire J. Fearnley1,3, H. Llewelyn Roderick1,2,3, and Martin D. Bootman1,3

1Laboratory of Signalling and Fate, The Babraham Institute, Babraham, Cambridge CB22 3AT, United Kingdom 2Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom Correspondence: [email protected]; [email protected]

Calcium (Ca2þ) is a critical regulator of cardiac myocyte function. Principally,Ca2þ is the link between the electrical signals that pervade the and contraction of the myocytes to propel blood. In addition, Ca2þ controls numerous other myocyte activities, including gene transcription. Cardiac Ca2þ signaling essentially relies on a few critical molecular players—ryanodine receptors, voltage-operated Ca2þ channels, and Ca2þ pumps/transport- ers. These moieties are responsible for generating Ca2þ signals upon cellular , recovery of Ca2þ signals following cellular contraction, and setting basal conditions. Whereas these are the central players underlying cardiac Ca2þ fluxes, networks of signaling mechanisms and accessory impart complex regulation on cardiac Ca2þ signals. Subtle changes in components of the cardiac Ca2þ signaling machinery, albeit through mutation, disease, or chronic alteration of hemodynamic demand, can have profound con- sequences for the function and phenotype of myocytes. Here, we discuss mechanisms under- lying Ca2þ signaling in ventricular and atrial myocytes. In particular, we describe the roles and regulation of key participants involved in Ca2þ signal generation and reversal.

OVERVIEW OF THE contraction of these chambers, forcing blood into the ventricles. On reaching the atrioven- he mammalian heart is a complex organ tricular (AV) node, the depolarization pauses Tconsisting of four chambers—the left and for a short time period (0.1 s in humans) right atria and the left and right ventricles. to ensure completion of atrial . Impor- Through a highly coordinated series of events, tantly, the AVnode acts as an electrical insulator the muscular heart pumps blood through the between the atria and ventricles. The AV node pulmonary and systemic vasculature (Fukuta prevents the transfer of aberrant contraction and Little 2008). During , all four cham- patterns to the ventricles, such as the spontane- bers are relaxed. Systole is initiated by prop- ous electrical activity occurring during atrial agation of a depolarizing fibrillation. The lower portion of the AV node from the sino-atrial node located in the apex is designated the , which then of the right , through the right and then splits into the left and right branches, allow- the left atrium. This depolarization induces ing activation of the left and right ventricles,

3All three authors contributed equally to this article. Editors: Martin Bootman, Michael J. Berridge, James W. Putney, and H. Llewelyn Roderick Additional Perspectives on Calcium Signaling available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a004242 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004242

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C.J. Fearnley et al.

respectively. These branches give rise to thin et al. 2001; Berridge 2003). When Vm reaches filaments called Purkinje fibers, composed of a critical threshold (240 to 250 mV), plasma noncontractile cells that distribute the action membrane L-type Ca2þ channels are opened 2þ potential to ventricular myocytes and enable (ICa,L), allowing a large influx of Ca into the the heart to contract in a coordinated fash- and increasing the . Transduction of the depolarization signal to þ10 mV. It is this depolarization signal through the His-Purkinje system causes ventric- that is transmitted from the SA node through ular systole. The contraction wave, traveling up the cardiac conduction system, culminating in from the ventricular base, expels blood into cardiac myocyte contraction. Within the SA the pulmonary then on to the lungs, or node cells, ICa,L activates an outward potassium through the aorta into the arterial system. Ret- current (IK) which hyperpolarizes the mem- rograde flow of blood is prevented by valves brane and curtails the action potential. The between the atria and ventricles. hyperpolarization leads to activation of If, As indicated above, the SA node situated T-type Ca2þ channels and Ca2þ sparks, to begin at the apex of the right atrium is responsible the next conduction cycle. for initiation of the cardiac action potential. At rest, the membrane potential starts around EXCITATION-CONTRACTION COUPLING –70mV (V ) and slowly depolarizes until an m (EC-COUPLING) action potential is triggered. Ca2þ signals may play a key role in action potential generation, EC-coupling is the process pairing myocyte although there is considerable debate regard- depolarization with mechanical contraction. ing the major mechanisms controlling the rate Ca2þ is the critical intermediary (Bers 2008). of SA node depolarization (see Lakatta and Indeed, since Ringer’s experiments more than DiFrancesco 2009). One primary component a century ago, Ca2þ has been known to be an of SA node depolarization is known as If (f essential mediator of this process (Ringer stands for funny) (Brown et al. 1979), an ion 1883). As the action potential sweeps over the current mediated by hyperpolarizing-activated heart, the plasma membrane () of cyclic nucleotide-gated (HCN) channels (Di- each myocyte becomes depolarized (290 mV Francesco 1993). Because this current is trig- to þ20 mV) thereby causing concerted open- gered by hyperpolarization, it is activated at ing of L-type VOCCs (“long-lasting current;” 2þ the start of diastole and slowly declines Cav1.2). Ca flows via the VOCCs into a throughout the pacemaker period. HCN chan- restricted space between the sarcolemma and nels are relatively nonselective, and they there- the underlying (SR) fore generate an inward current depolarizing known as the “junctional zone” or “dyadic 2þ Vm toward the threshold for firing an action cleft.” The accumulation of Ca during potential. Intracellular Ca2þ cycling has also an action potential increases the Ca2þ con- been proposed to act as a primary regulator of centration within this microdomain from SA node depolarization. Imaging SA node cells 100 nM to 10 mM. This elementary Ca2þ reveals spontaneous elementary Ca2þ signals influx signal, derived from the activation of known as Ca2þ sparks arising from the SR and VOCCs is known as a “Ca2þ sparklet” (Fig. 1) preceding action potential generation (Huser (Wang et al. 2001). The distribution of Ca2þ et al. 2000). Ca2þ sparks reflect the concerted sparklet magnitudes suggests that one or sev- opening of a cluster of RyRs. The Ca2þ sparks eral VOCCs can give rise to such signals within activate sodium/calcium exchange (NCX), myocytes (Cheng and Wang 2002). which promotes membrane depolarization Ca2þ sparklets themselves are not adequate because three Naþ ions enter for each Ca2þ to cause substantial contraction. However, ion that leaves. T-type Ca2þ channels (“tran- they are sufficient to induce opening of RyRs sient current;” Cav3) may also provide a source (type 2 RyRs) on the closely apposed SR, via a of Ca2þ for triggering Ca2þ sparks (Bogdanov process known as “Ca2þ-induced Ca2þ release”

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Calcium Signaling in Cardiac Myocytes

Ai L-type VOCC ii RyR distribution distribution

Bi ii L-type VOCC iii RyR Diastole activation activation Action potential arrives- sarcolemmal depolarization Ca2+ sparklet Ca2+ spark T-tubule SR Dyadic cleft Ci ii Control myocyte Detubulated 12 12 myocyte Edge of myocyte Center of myocyte 8 8

4 4 emission (f/f0) emission (f/f0)

Fluo4 fluorescence 0 Fluo4 fluorescence 0 0123 0123 Time (s) Time (s)

Figure 1. Excitation contraction coupling in ventricular myocytes. Panel A illustrates the distribution of L-type VOCCs (Ai) and type 2 RyRs (Aii) in a section of a ventricular myocyte. The distributions of these proteins are essentially overlapping at the level of the light microscope. Panel B is a cartoon sequence of events leading to the generation of a Ca2þ signal within a ventricular myocyte. A small section of a ventricular myocyte is depicted with two T-tubule projections (T-tubule spacing 1.8 mm). During the diastolic phase (Bi), the L-type VOCCs (red channels on the T-tubule membranes) and RyRs (blue channels on SR membrane) are silent. Arrival of the action potential causes depolarization of the sarcolemma and activation of the L-type VOCCs thereby generating “Ca2þ sparklets” (Bii). The Ca2þ sparklets trigger activation of the RyRs thereby producing “Ca2þ sparks” (Biii). Panel Ci depicts the consistent, global Ca2þ responses observed in an electrically paced ventricular myocyte. The black and gray traces indicate the Ca2þ concentration (measured with fluo4) at the center and edge of the myo- cyte. The profile of the Ca2þ signal was essentially the same in both locations. Panel Cii illustrates what happens in a ventricular myocyte following detubulation (using formamide treatment). Detubulation decreases the amplitude of systolic Ca2þ transients, and provokes spatial heterogeneity of the resultant Ca2þ signals. The black and gray traces indicate the Ca2þ concentration at the center and edge of the myocyte. Whereas the Ca2þ responses in the edge of the myocyte were reasonably consistent, the signals in the center of the cell showed beat-to-beat variation in amplitude. Such Ca2þ signal alternans are a potential cause of cardiac .

(CICR). Seminal studies in the 1970s showed following activation of L-type Ca2þ channels that RyR activity is dependent on cytosolic falls within the range of that required for chan- Ca2þ levels, with low concentrations (1– nel activation, thus facilitating CICR. 10 mM) being activatory and high concentra- The activation of a cluster of RyRs, and tions (.10 mM) inhibiting the channel consequent mobilization of Ca2þ from the SR, (Fabiato and Fabiato 1972; Fabiato 1983). The produces an elementary Ca2þ release signal concentration of Ca2þ at the dyadic cleft know as a “Ca2þ spark” (Fig. 1) (Cheng and

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C.J. Fearnley et al.

Lederer 2008). As with Ca2þ sparklets, Ca2þ is Ca2þ sparklets do not trigger Ca2þ release spark magnitudes can vary, indicating that from the SR. Rather, L-type VOCCs (Cav1.1) different numbers of RyRs participate in their on the sarcolemma have a direct physical inter- generation. Release of Ca2þ through the action with RyRs (type 1 RyRs) on underlying RyRs increases the concentration of Ca2þ to SR forming the triadic junction (Rios and .100 mM in the dyadic cleft. It is estimated Brum 1987; Block et al. 1988). Depolarization that 25 L-type Ca2þ channels and 100 RyRs of the sarcolemma induces a conformational are closely associated within the dyadic cleft to change in the VOCCs, which allosterically acti- form a “couplon” (Bers and Guo 2005). Ca2þ vates the RyRs. ions diffuse out of the cleft to engage the con- tractile machinery, thereby promoting cell REGULATION OF L-TYPE VOCCs shortening to provide the force for pumping blood. During a single action potential, thou- The channels and homeostatic processes under- sands of Ca2þ spark sites are simultaneously lying Ca2þ release and Ca2þ clearance are activated by their corresponding Ca2þ sparklet subject to regulation by multiple signaling triggers (Cheng and Lederer 2008). Diffusion pathways. These signaling pathways can rapidly of Ca2þ ions, and their subsequent spatial and alter the amplitude and/or spatial properties temporal summation, produces an average of myocyte Ca2þ signaling to acutely modulate global Ca2þ increase of 500 nM to 1 mM. . For example, L-type VOCCs C (TnC), the Ca2þ-binding compo- are subject to regulation by cAMP-dependent nent of the contractile filaments, is sensitive ( kinase A; PKA) downstream to Ca2þ concentrations over that range thereby from b-adrenergic stimulation, e.g., allowing coupling of the AP-mediated Ca2þ during the fight or flight response. PKA phos- transient and contraction. At the end of an phorylation increases channel activity, thereby action potential Ca2þ transients are rapidly ter- contributing to the increased cardiac contrac- minated, and the cells return to resting diastolic tion (positive inotropic response) evoked by levels in preparation for the next depolarization. b-adrenergic stimuli. PKA-mediated phos- In addition to L-type VOCCs, contractile phorylation increases opening of the VOCC cardiac myocytes express a T-type current by a twofold mechanism—it increases both (Cav3 family), which is so named because of the number of channels in an activatable its transient nature. As described in the section state, and their activation probability (Catterall on pacemaking above, this current is activated 2000). PKA phosphorylates Ser-1928 in the at a more negative membrane potential than amino terminal of the a1 subunit (Perets et al. the L-type (Nowycky et al. 1985). Although 1996), in addition to residues on the b2 subunit ICa,T plays a role in depolarizing SA node (Curtis and Catterall 1985; Gerhardstein et al. cells, under normal physiological situations its 1999). Rapid dephosphorylation of the channel role is negligible within ventricular and atrial is provided by the Ser/Thr phosphatases 1 and cardiac myocytes as most Ca2þ enters through 2A (PP1 and PP2A, respectively) (Kamp and the L-type channels. However, T-type VOCC Hell 2000). expression is up-regulated during cardiac L-type VOCCs are also phosphorylated by hypertrophy, and they may provide a critical C (PKC) in response to activa- 2þ Ca signal to drive hypertrophic remodeling. tion of Gq-coupled receptors, e.g., a1-adrener- For example, Cav3.2 knockout mice did not dis- gic, endothelin, and II receptors. play cardiac hypertrophy in response to pressure The targets for PKC phosphorylation are pro- overload or angiotensin II (Chiang et al. 2009). posed to be two Thr residues in the amino ter- EC-coupling is also medi- minal of the a1 subunit (Shistik et al. 1998; ated by elevations in intracellular Ca2þ, and McHugh et al. 2000). The effects of PKC phos- has some similarities to the cardiac scheme phorylation are less clear than for PKA phos- described above. The key difference in this tissue phorylation. It is proposed that the effect may

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Calcium Signaling in Cardiac Myocytes

be reliant on the particular PKC isoform Ca2þ entry once a Ca2þ sparklet is forming. activated, the expression of which varies in a However, once global Ca2þ is elevated, Ca2þ complex developmental, species-dependent, also binds to the amino terminal lobe of and disease-regulated manner in the heart. CaM leading to CDI and termination of Ca2þ Additional regulation of the L-type Ca2þ chan- entry. nel is provided by cGMP-dependent protein Additional modification of ICa,L is provided kinase G (PKG), which has been shown to by Ca2þ/CaM-dependent kinase II (CaMKII), have an inhibitory effect, thus opposing the which potentiates the influx of Ca2þ (Anderson effects of PKA (Hartzell and Fischmeister et al. 1994; Xiao et al. 1994; Yuan and Bers 1994; 1986; Abi-Gerges et al. 2001). Wu et al. 2001b). CaMKII interacts with and Inactivation of the L-type Ca2þ channel is phosphorylates the carboxyl terminal of the mediated by both membrane repolarization a1 subunit (Hudmon et al. 2005). CaMKII and by Ca2þ itself (Ca2þ-dependent inacti- remains tightly bound to the channel even in vation, CDI). The latter acts as a negative feed- the absence of Ca2þ, although it is only active back loop, and is thought to be the more when it has Ca-CaM bound. Importantly, important of the two mechanisms (Lee et al. Ca-CaM can remain bound to CaMKII, and 1985). CDI is controlled by the kinase active, even after global Ca2þ has (CaM), which is constitutively bound to the declined, thereby allowing CaMKII to act as a channel (Peterson et al. 1999; Qin et al. 1999). detector of Ca2þ spike frequency (Hudmon The CaM binding site is a canonical “IQ” CaM- et al. 2005). binding motif within the carboxyl terminus of the a subunit that binds the Ca2þ-free form 1 REGULATION OF RyRs AND CICR of CaM (apo-CaM) (Rhoads and Friedberg 1997; Zuhlke and Reuter 1998). Ca2þ entering There are three mammalian RyR isoforms through L-type channels during EC-coupling (RyR1–3). RyR2 is predominant in cardiac binds to apo-CaM to form Ca-CaM, which myocytes, with significantly lesser amounts of inactivates the channel (Tang et al. 2003). Inter- RyR3. The RyR channel is a large homotetra- estingly, Ca-CaM can also enhance Ca2þ entry meric assembly of 2 megadaltons (each through L-type Ca2þ channels by Ca2þ- subunit has a molecular mass of 560 kDa) dependent facilitation (CDF). CDF is also (Lanner et al. 2010). Structural studies have dependent on the IQ motif in the cytoplasmic revealed four-fold symmetry, with a four-leaf tail of the a1C subunit (Zuhlke et al. 1999). clover or mushroom morphology formed by This capacity for dual regulation is caused the transmembrane domain and bulky cyto- by the presence of both high and low affinity plasmic domain (Anderson et al. 1989; Sery- Ca2þ binding sites (EF-hands) within CaM, sheva 2004). Binding of Ca2þ is proposed to in the carboxyl and amino terminal lobes, cause conformational changes that evoke chan- respectively. It is believed that CDI depends nel gating in a mechanism similar to that of on Ca2þ bound to the amino terminal EF- a camera iris, with twisting of the transmem- hands, whereas CDF depends on the carboxyl brane regions opening the ion pore (Serysheva terminal EF hands (DeMaria et al. 2001). Sub- et al. 1999). sequently, it was revealed that the two lobes RyRs are bound by a multitude of accessory of CaM can detect Ca2þ arising from distinct proteins, comprising a macromolecular signal- sources—the high affinity carboxyl terminal ing complex. These interactions determine the site sensing local Ca2þ arising within the nano- efficiency and specificity of signaling to and domain of the channel mouth, whereas the low from RyRs, and between other signal trans- affinity amino terminal lobe detects global duction cascades. Moreover, scaffolding of Ca2þ signals (Tadross et al. 2008). It therefore proteins to RyRs recruits and concentrates appears that Ca2þ binding to apo-CaM at the important regulatory proteins in the junctional carboxyl terminus of L-type VOCCs potentiates zone where they are ideally located to modulate

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C.J. Fearnley et al.

and/or be regulated by EC-coupling. These node-evoked AP) (Viatchenko-Karpinski et al. interactions occur on both the lumenal and 2004). Another mutation, arginine to glu- cytosolic face of the RyR (Lanner et al. 2010). tamic acid at residue 33 (CSQR33Q), decreases In the lumen of the SR, RyRs interact with the interaction between the RyR and CSQ, (CSQ), the major Ca2þ binding/ resulting in abnormal regulation of the RyR by storage protein of muscle. CSQ is a low- lumenal Ca2þ and increased Ca2þ release (Ter- affinity Ca2þ storage protein that maintains entyev et al. 2006). A substantial proportion of the SR lumenal free Ca2þ concentration be- CPVT patients (50%) express mutated RyRs tween 100–500 mM (Yano and Zarain- that have altered association with accessory Herzberg 1994; Berridge 2002). The critical proteins. role of CSQ in Ca2þ storage was shown by the CaM is an important regulator of the RyR in increase or decrease in SR Ca2þ load observed cardiac myocytes, both in its Ca2þ bound and in experiments in which CSQ expression was Ca2þ free form (Ca-CaM and apo-CaM, respec- enhanced or suppressed, respectively (Terentyev tively). The binding site for CaM on the RyR et al. 2003). In addition to acting as the major was mapped to the carboxyl terminal of the Ca2þ storage protein in , CSQ receptor by site-directed mutagenesis (Porter also regulates RyR channel activity (Prins Moore et al. 1999a; Porter Moore et al. and Michalak 2011). CSQ reversibly changes 1999b), in agreement with cryo-EM studies between monomeric to oligomeric forms in (Wagenknecht et al. 1997). Binding of CaM response to changes in luminal Ca2þ concentra- decreases Ca2þ efflux through these channels tion. It has been suggested that CSQ oligomers (Meissner and Henderson 1987). This is are present when the SR is replete, and that these thought to be facilitated by a reduction in the mainly serve to buffer Ca2þ. However, when opening probability and in the Ca2þ-dependent RyRs open and SR luminal Ca2þ declines, activation of the channel (Balshaw et al. 2001). Ca2þ unbinds from calsequestrin and the oligo- Further Ca2þ-dependent modification of meric protein dissociates. The calsequestrin RyRs is provided by CaMKII-dependent phos- monomers bind to RyRs (via a protein inter- phorylation. Sequence analysis revealed six mediate called triadin) and inhibit channel consensus phosphorylation sites in the RyR activity. This is an important component of (Zucchi and Ronca-Testoni 1997). Both Ser- the mechanisms that terminate Ca2þ release 2809 (Witcher et al. 1991) and Ser-2815 during each heartbeat (Gyorke et al. 2009). (Wehrens et al. 2004) have been shown to be The key role of CSQ in regulating Ca2þ crucial for CaMKII-dependent phosphoryla- storage and RyR function is highlighted by a tion. CaMKII phosphorylation increases RyR pathological condition known as catecholami- activity (Witcher et al. 1991; Wehrens et al. nergic polymorphic ventricular tachycardia 2004), although some studies have contested (CPVT) that is observed in patients with CSQ this idea (Lokuta et al. 1995; Wu et al. 2001a). mutations. CPVT is a life-threatening form of Phosphorylation by CaMKII is emerging as cardiac dysrhythmia typically brought about the dominant mode of regulation of RyR activ- by emotional or physical stress. Recessive CSQ ity during adrenergic stimulation and in the mutations are found in 3% of CPVT patients greater contractility associated with increased (Katz et al. 2009). The effects of some CPVT- frequency of myocyte contraction (Wu et al. inducing CSQ mutations on calcium fluxes in 2009; Grimm and Brown 2010). cardiac myocytes are known. For example, Ca2þ release through RyRs is regulated by mutation of the aspartate to histidine at residue interaction with FK binding proteins (FKBPs), 307 in CSQ (CSQD307H) causes decreased SR named because of their binding of the immu- Ca2þ storage and release, and increases the nosuppressant drug FK506. Cardiac myocytes frequency of delayed afterdepolarizations express two isoforms of the 12 kDa FKBP, (DADs; spontaneous electrical depolarization namely FKBP12 and FKBP12.6 (also known of cardiac myocyte independent of the SA as calstabin1 and calstabin2, respectively). The

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Calcium Signaling in Cardiac Myocytes

cardiac RyR2 binds FKBP12.6 with a higher A cAMP phosphodiesterase (PDE) has also affinity (Timerman et al. 1996; Jeyakumar been identified within the RyR macromolecular et al. 2001). FKBP12.6 binding stabilizes the complex (specifically, the PDE4D3 isoform). coordinated gating of RyR subunits within a The presence of this provides a mecha- tetramer, thereby enabling channels to transi- nism to tightly regulate cAMP, and thus PKA tion between the fully closed and the fully activity, in the vicinity of the receptor. The levels open state, while also shifting the Ca2þ depend- of this PDE isoform have been reported to be ence of channel opening to a higher Ca2þ reduced in failing (Lehnart et al. 2005). concentration (Bers 2004). CPVT-inducing Type 2 RyRs are associated with phospha- mutations within the type 2 RyR channel tases to mediate rapid receptor dephosphoryla- decrease the association between the receptor tion and return it to basal levels of activity. and FKBP12.6, although only under conditions For example, calcineurin is suggested to be an of b-adrenergic stimulation. FKBP12.6 dissoci- accessory protein of cardiac RyRs (Bandyopad- ation may lead to increased Ca2þ release from hyay et al. 2000), and is proposed to decrease the SR (Wehrens et al. 2003). Ca2þ channel activity. In addition, the phos- RyRs can also be phosphorylated by PKA, phatases PP1 and PP2A are associated with which is tethered by an A kinase anchoring cardiac RyRs (Marx et al. 2001). PP1 associates protein (mAKAP) (Marx et al. 2000). PKA- with RyRs via an interaction with spinophilin, dependent phosphorylation of the receptor whereas PP2A binds to a targeting protein was reported to increase its responsiveness to PR130, which then anchors it to RyRs (Marx Ca2þ (Valdivia et al. 1995), although the precise et al. 2001). consequences of PKA-dependent phosphoryla- tion have been controversial. Awidely discussed CARDIAC MYOCYTE CONTRACTION model proposed that PKA phosphorylation of RyRs causes dissociation of FKBP12.6, thereby Contraction of cardiac myocytes is facilitated leading to increased probability of channel by myofilaments organized into sarcomeres, opening (Marx et al. 2000; Wehrens et al. situated along the long axis of the cell. The sar- 2003). This model of RyR regulation would comere consists of -containing thick fil- not only be relevant physiologically, coupling aments surrounded by a hexagonal array of thin b-adrenergic stimulation with enhanced Ca2þ filaments, which are made up of polymers release, but would also be important in disease and troponin/a-tropomyosin (Tn/Tm) regula- conditions in which elevated PKA phosphoryla- tory units (Parmacek and Solaro 2004). Addi- tion had been reported to decrease FKBP12.6- tionally, each thin filament is separated at the RyR associations and result in increased Z-line by an actin binding protein, a-actinin. spontaneous diastolic RyR activity and DADs. Every seventh actin monomer comprising However, the dependence of FKBP12.6 associa- the thin filament is bound to a Tn/Tm complex. tion with the RyR on PKA phosphorylation Tn is composed of three subunits: TnC, Tropo- has been much disputed (Xiao et al. 2007). nin I (TnI), and Troponin T (TnT) (Greaser and Indeed, a recent report provides substantial Gergely 1971). TnC has a similar structure to evidence that the association of FKBP12.6 inter- CaM, and contains Ca2þ binding EF-hands action with the RyR is insensitive to the degree of (Parmacek and Solaro 2004). TnI is an inhibi- PKA phosphorylation (Guo et al. 2010). tory subunit, and TnT constitutively interacts PKA may also regulate RyR activity by with Tm. Binding of Ca2þ to TnC causes a con- phosphorylation of Sorcin, another RyR-inter- formational change in associated TnI. This en- acting protein. Sorcin is a ubiquitously ex- ables Tn/Tm to slide into the groove between pressed 22 kDa Ca2þ binding protein that actin monomers, allowing the myosin thick inhibits Ca2þ release via the RyR. This inhibi- filament to bind actin, thus forming a cross- tory effect is lost following its phosphorylation bridge. By repetitive, transient actin-myosin by PKA (Lokuta et al. 1997). interactions and utilizing energy from ATP

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hydrolysis, the two filaments slide relative to dysfunction, which also leads to heart failure each other, thus shortening the cell. Coordi- and sudden cardiac death. RCM is linked to nated shortening of the entire myocyte popula- diastolic dysfunction, although there is little or tion by the spreading AP leads to cardiac no effect on systolic function or ventricular contraction. As cytosolic Ca2þ levels decline, wall thickness. Mutations leading to all three Ca2þ is released from TnC leading to cross- of these myopathies have been identified in the bridge detachment, and the thick and thin contractile proteins of the sarcomere, although filaments slide past each other back to their interestingly none have yet been found that are original positions. Cross-bridges cannot form linked to ARVD/C (Morimoto 2008). Consid- between the filaments as they travel past in ering TnT, for example, 27 mutations have that direction. been identified that have been linked to HCM, Troponin proteins are regulated by phos- which act by increasing the Ca2þ sensitivity of phorylation, which alters their activity and cardiac . Two TnTmutations thus affects cardiac myocyte contraction. PKA have been linked to DCM, the functional conse- phosphorylation downstream from b-adrener- quence being decreased Ca2þ sensitivity and gic stimulation is of particular importance, as an increased affinity of TnT for tropomyosin. this would facilitate altered cardiac contraction Thirty-three mutations in TnI have been found during, for example, physical exertion. Much that are linked with HCM, DCM, and RCM, work has been completed in elucidating the mainly causing increased Ca2þ sensitivity and functional outcome of this modification, and impaired interaction of TnT with TnI. Regard- the consensus of opinion is that PKA phosphor- ing TnC, a HCM-causing mutation has been ylation leads to increased cardiac contraction identified that abolishes its interaction with (for a review see Metzger and Westfall 2004). TnI, thus losing the altered Ca2þ sensitivity PKC phosphorylates regions on TnT and TnI. imparted by PKA-dependent phosphorylation. Early studies reported divergent results regard- Thirteen tropomyosin mutations have been ing the effects of PKC phosphorylation, al- identified, leading to HCM and DCM in the though more recent studies point toward an manner described previously. inhibition of contractile function following PKC phosphorylation (Takeishi et al. 1998; Ca2þ EFFLUX Sumandea et al. 2004). A large number of mutations have been EC-coupling events are short-lived—atrial and identified in Ca2þ-dependent contractile pro- ventricular myocytes reach peak contraction teins that are linked with specific cardiomyopa- within a few tens of milliseconds of action thies (reviewed in Morimoto 2008). Clinically, potential initiation at the SA node. After cyto- cardiomyopathies can be divided into four solic Ca2þ has activated the contractile units, main groups: hypertrophic cardiomyopathy it is rapidly extruded from the cytosol in prep- (HCM), dilated cardiomyopathy (DCM), aration for the following action potential restrictive cardiomyopathy (RCM), and ar- (Shannon and Bers 2004). The main efflux rhythmogenic right ventricular dysplasia/cardi- mechanisms in cardiac myocytes are the plasma omyopathy (ARVD/C). HCM is characterized membrane NCX and the sarco/endoplasmic by thickening of the ventricular walls with reticulum Ca2þ ATPase (SERCA) pump on accompanying decreases in ventricular cham- the SR. The plasma membrane Ca2þ ATPase ber volume. In this condition, systolic function (PMCA) and the mitochondrial uniporter is preserved at the expense of diastolic function, may also play a more minor role. The relative which is responsible for symptoms of heart contribution of these mechanisms varies in a failure and death in these patients. Thickened species-dependent manner, for example in ventricular walls are also observed in DCM, rabbit ventricles 70% of cytosolic Ca2þ is in addition to an increase in chamber removed by SERCA2a, 28% by NCX, and the volume. The clinical outcomes include systolic remaining 2% by the mitochondrial uniporter.

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Calcium Signaling in Cardiac Myocytes

A similar pattern is apparent in ventricles from low for NCX activity. This is facilitated by the dog, cat, guinea pig, and human. Alternately, action of the sodium potassium ATPase, which in mouse and rat ventricles, SERCA2a removes extrudes Naþ entering the cell during the action 92% of the Ca2þ, leaving only 7% for potential. Inhibition of this pump by cardiac the NCX (Bassani et al. 1994; Bers 2001). The glycosides such as and ouabain results difference in the relative contributions of in an accumulation of intracellular Naþ, leading the Ca2þ clearance pathways is reflected by to a suppression of NCX activity and thereby the observed kinetics of NCX activity between attenuating Ca2þ efflux. As a consequence, different species (Sham et al. 1995). NCX cur- Ca2þ transient amplitude and myocyte contrac- rent density is lowest in myocytes from rat, tion are increased. Because of these positive and highest in those from guinea pig. The inotropic effects, digoxin has long been used adaptability of this system was shown in a as therapy for failing heart. However, this ma- recent report showing minimal cardiac dysfunc- nipulation can also lead to arrhythmia and tion in a cardiac-specific SERCA2 KO mouse myocyte death. (Andersson et al. 2009). Ca2þ fluxes were main- NCX has been shown to be phosphorylated tained by increased activity of the L-type Ca2þ by PKC and PKA suggesting that its activity channel and NCX, demonstrating that these may be regulated by these posttranslational proteins can compensate for a major reduction modifications (Iwamoto et al. 1996; Ruknudin in SERCA levels (,5% SERCA2 protein re- et al. 2000). NCX interacts with a number of mained in myocardial tissue 4 weeks after accessory proteins. In particular, it interacts gene excision). with the transmembrane protein phospholem- In its forward mode, NCX uses the electro- man, which exerts an inhibitory effect (Zhang chemical gradient across the sarcolemma to et al. 2003; Ahlers et al. 2005; Cheung et al. translocate three Naþ ions into the cytosol 2007). Phosphorylation of phospholemman is and expel one Ca2þ. In this situation, NCX is reported to occur on residues within the cyto- a depolarizing current. If Naþ levels are high, plasmic carboxyl terminal of the protein, specif- the exchanger may change to its reverse mode, ically Ser-68, and acts to inhibit NCX (Song and bring Ca2þ into the cell. The exchanger et al. 2005a; Zhang et al. 2006; Cheung et al. was discovered around 40 years ago in the squid 2007). This appears to be mediated by PKC giant axon (Baker et al. 1969) and in the mam- and not PKA (Zhang et al. 2006). NCX has malian heart (Reuter and Seitz 1968). Following been identified within a macromolecular cloning of the canine NCX (Nicoll et al. 1990), complex containing PKA and its anchoring three isoforms were identified (NCX1-3). Of protein mAKAP, together with PKC and the these, NCX1 (110 kDa) is predominant in car- phosphatases PP1 and PP2A (Schulze et al. diac myocytes. It is responsible for exporting 2003), providing a robust mechanism for the an amount of Ca2þ approximately equivalent phosphorylation and regulation of NCX and/ to the flux entering via L-type Ca2þ channels or phospholemman. (Bridge et al. 1990). The adaptability of cardiac Ca2þ reuptake into the SR is mediated by myocytes is again shown in the NCX KOmouse, the SERCA pump, which uses energy from in which EC-coupling is maintained by a com- ATP hydrolysis. Molecular cloning has identi- pensatory reduction in Ca2þ influx (Pott et al. fied three SERCA isoforms (SERCA1–3), all 2007). of which undergo alternative splicing. The alter- Cardiac NCX is regulated by the concentra- nately spliced isoforms of SERCA2 (SERCA2a tion of cytoplasmic ions, specifically activation and SERCA2b) possess distinct carboxyl termi- by Ca2þ (Hilgemann 1990) and inhibition by nal residues. SERCA isoforms differ in their Naþ (Hilgemann et al. 1992). Maintenance of relative affinity for Ca2þ and their transport the ionic gradient between the extracellular rates (Lytton et al. 1992). SERCA2a, the main space and cytosol is critical for NCX activity. cardiac isoform, possesses a K1/2 of 0.4 mM; In particular, intracellular Naþ must remain however, in vivo, this is 0.9 mM because of

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its association with its regulatory transmem- myocytes, the depolarization signal culminates brane phosphoprotein, (PLB). in activation of the sarcomeric contractile units Cardiac SERCA pumps are usually situated on by elevating intracellular Ca2þ. The spatial a region of the SR separate from the junctional properties of the Ca2þ increase depending on zone where the RyRs are located. During recov- the structure of the different myocytes within ery of a Ca2þ transient, Ca2þ is pumped into the heart. For example, adult ventricular the SR at the location of the SERCA . myocytes possess numerous invaginations of The Ca2þ ions return to the dyadic SR lumen the sarcolemma, which form a regular array by tunneling through the SR network. of inwardly directed membranous structures SERCA regulation in the heart is primarily known as transverse tubules (T-tubule) (Fig. 1) governed by PLB. Inactive, phosphorylated (Song et al. 2005b). T-tubules are narrow PLB exists as a pentamer. It depolymerizes (200 nm diameter) and occur at regular inter- when not phosphorylated and can then interact vals of 2 mm (Brette and Orchard 2003). with SERCA (Kimura et al. 1997). Unphos- Additional branches project from the main phorylated PLB monomers decrease the affinity T-tubules to give a complex network of sarco- of SERCA for Ca2þ (Tada et al. 1974). Sub- lemmal intrusions (Ayettey and Navaratnam sequent phosphorylation of PLB prevents its 1978). T-tubules are a feature of mammalian interaction with SERCA, increasing the ap- ventricular myocytes, and are absent in the ven- parent activity of the pump and the rate of tricles of birds (Bossen et al. 1978), reptiles, and Ca2þ accumulation within the SR. PLB can be amphibians (Bossen and Sommer 1984). phosphorylated on three sites: Ser-16 by PKA, The presence of T-tubules facilitates homo- Thr-17 by CaMKII, and Ser-10 by PKC (Movse- genous Ca2þ transients during EC-coupling in sian et al. 1984; Simmerman et al. 1986), ventricular myocytes. T-tubules serve to create facilitating regulation by a variety of signaling dyadic junctions deep within the volume of a pathways. CaMKII is present in a multiprotein ventricular myocyte. In this way, Ca2þ sparks complex with SERCA2a, and can directly phos- can be triggered simultaneously throughout a phorylate the Ca2þ pump (Toyofuku et al. 1994; cell. The alternative to T-tubules is observed in Narayanan and Xu 1997) leading to enhanced neonatal myocytes and atrial myocytes, where activity (Xu and Narayanan 1999; Xu et al. dyadic junctions occur solely at the periphery 1999). Modulation of PLB-mediated SERCA of the cells (Fig. 2). T-tubules also act as impor- inhibition is a major mechanism for acute tant scaffolding regions for many of the proteins enhancement of cardiac function following essential for Ca2þ signaling (Chase and Orchard b-adrenergic receptor activation. As a result of 2011). For example, the sarcolemmal L-type reduced PLB interaction with the pump, the Ca2þ channels and NCX are abundant on rates of clearance of the Ca2þ transient and T-tubule membranes (Orchard and Brette relaxation is increased (positive lusitropic 2008). It has been estimated that .75% of ICa response), and SR store loading is enhanced. flows into a myocyte through the T-tubules Because of greater Ca2þ within the store, the because of the high concentration of L-type magnitude of Ca2þ fluxes is elevated thereby Ca2þ channels in this region. As mentioned in producing enhanced contraction. the previous section, Ca2þ-dependent inactiva- tion of ICa is one of the main mechanisms to curtail Ca2þ influx. This feedback mechanism SUBCELLULAR ORGANIZATION OF appears to be more potent at T-tubules, mean- CARDIAC MYOCYTES ing ICa at T-tubules is large but inactivates As described previously, the action potential rapidly. In contrast, inactivation of ICa at the generated at the SA node sweeps rapidly peripheral sarcolemma is slower overall, result- through the heart, coordinating cardiac con- ing in more Ca2þ entering the cell across the traction by activating atrial and then ventricu- outer region of a cell than at T-tubule sites. lar myocytes in synchrony. Within individual This prolonged Ca2þ entry occurs during the

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Calcium Signaling in Cardiac Myocytes

iA L-type VOCC ii RyR distribution distribution

iB ii L-type VOCC iii RyR Diastole activation Ca2+ activation sparklet Dyadic cleft Ca2+ spark Action potential arrives- sarcolemmal depolarization

Z-tubule iC Control myocyte 12

Edge of myocyte 8 Center of myocyte

4 emission (f/f0)

Fluo4 fluorescence 0 0123 Time (s)

Figure 2. Excitation contraction coupling in atrial myocytes. Panel A illustrates the distribution of L-type VOCCs (Ai) and type 2 RyRs (Aii) in a section of an atrial myocyte. The pattern of L-type VOCC expression is clearly different from that in ventricular cells. The distribution of RyRs is similar to that in ventricular cells, except that there is an evident population of peripheral RyRs around the edge of the myocyte. Solely these peripheral RyRs align with the L-type VOCCs to produce functional dyads. Panel B is a cartoon sequence of events leading to the generation of a Ca2þ signal within an atrial myocyte. A small section of an atrial myocyte is depicted. There are no T-tubules, but instead two prominent SR tubules with a spacing of 1.8 mm. Such SR tubules have previ- ously denoted as “Z-tubules,” as they occupy the Z-line (just like T-tubules). During the diastolic phase (Bi), the L-type VOCCs (red channels on the T-tubule membranes) and RyRs (blue channels on SR membrane) are silent. Arrival of the action potential causes depolarization of the sarcolemma and activation of the L-type VOCCs thereby generating “Ca2þ sparklets” at the periphery of the cell (Bii). The Ca2þ sparklets trigger activation of nearby RyRs thereby producing “Ca2þ sparks” (Biii). Panel Ci depicts the gradient of Ca2þ typically observed during electrical pacing of atrial myocytes. The Ca2þ signal at the edge of the cell (black trace) is larger and more rapidly rising than the central response (gray trace). The extent to which the Ca2þ signal occurs in the cen- ter of the cell (and thereby causes contraction) is dependent on the inotropic status of the cell. Application of a b-adrenergic agonist can make atrial Ca2þ signals become homogenous.

2þ latter stages of ICa, and it has been proposed to balance between ICa,Ca release from the SR promote SR Ca2þ loading in preparation for the and Ca2þ efflux via NCX. Acute changes in following next cycle of EC-coupling. any one of these fluxes will exert compensatory Cardiac myocytes display an important changes in the others that bring systolic Ca2þ homeostatic principle known as “autoregula- transients back to normal levels. For example, tion” (Eisner et al. 1998). This is essentially a under conditions of increased SR, Ca2þ release

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following RyR phosphorylation Ca2þ influx the centripetal Ca2þ waves. The atrial myocytes 2þ through ICa is reduced and Ca efflux via in some mammalian species display a relatively NCX is increased, thus maintaining the steady- high degree of T-tubule membrane. Why some state systolic Ca2þ transient. Taking into con- atrial cells should rely on T-tubules when others sideration that Ca2þ-dependent inactivation do not is unclear. However, it has been shown following SR Ca2þ release occurs predom- that the presence of T-tubules within individual inantly at the T-tubules, and NCX is also con- atrial myocytes is correlated with cell diameter, centrated in this region, it is evident that suggesting that tubulation acts to coordinate occurs mainly at the T-tubules. Ca2þ signaling in larger cells (Smyrnias et al. Because of the principle of autoregulation, pro- 2010). longed changes in inotropic status require sus- Neonatal rat ventricular myocytes are simi- tained changes in more than one component lar to atrial cells in that they lack a fully formed 2þ of the ICa/Ca release/NCX triumvirate (Eis- T-tubule network. The T-tubules appear pro- ner et al. 2009). gressively through development (Sedarat et al. The T-tubular network of atrial myocytes is 2000). Just as with atrial myocytes, the main generally not as well developed as that observed region of VOCC-RyR interaction within a neo- in ventricular myocytes, especially in the atrial natal myocyte is at the periphery of the cell. myocytes of small . The sarcolemmal Therefore, Ca2þ signals within a neonatal myo- L-type Ca2þ channels of atrial myocytes provide cyte closely resemble those in an adult atrial a triggering Ca2þ signal for a small population cell, and change to become homogenous Ca2þ of “junctional” RyRs situated below the sarco- signals when T-tubules arise. lemma at the periphery of the cells (Bootman Ventricular myocytes from hearts that are et al. 2006). The consequence of this arrange- progressing toward failure show decreased 2þ ment is that the initial Ca influx (ICa) acti- organization of their T-tubules, and loss of vates Ca2þ sparks solely from the peripheral T-tubules (Fig. 3) (He et al. 2001). As a result, SR (Figs. 1 and 2). In the absence of a positive there is reduced coupling efficiency between inotropic agonist, the Ca2þ signal remains con- the L-type VOCCs on the sarcolemma and fined to the cellular periphery and contraction RyRs on the underlying SR. The loss of T-tubule is minimal (Bootman et al. 2011). membranes means that dyadic junctions are In addition to the junctional RyRs, atrial lost and RyRs become “orphaned.” This leads myocytes express a major population of non- to abnormal EC-coupling characterized by an junctional RyRs that form a 3-dimensional increased propensity for arrhythmia and lattice of Ca2þ release sites within the cells decreased magnitude of the Ca2þ response (the distribution of RyRs is actually very similar and contraction (Louch et al. 2004). EC-cou- between ventricular and atrial myocytes—the pling in detubulated ventricular myocytes re- location of L-type VOCCs is different) (Figs. 1 sembles that observed in atrial myocytes, and 2) (Chen-Izu et al. 2006; Schulson et al. whereby Ca2þ signals are initiated at the plasma 2011). Because these nonjunctional RyRs are membrane and propagate through the myocyte not located within a dyadic junction, they are by the relatively slow process of CICR (Smyrnias not activated by ICa (Mackenzie et al. 2001). et al. 2010). However, they can be activated by CICR if the peripheral Ca2þ signal is sufficient to act as a ADAPTATION TO DISEASE trigger for a Ca2þ wave. The depth that such centripetal Ca2þ waves propagate within an In response to pressure or , atrial myocyte determines the extent of contrac- damage, or genetic factors, the heart mounts tion—the deeper a Ca2þ wave spreads the more an adaptive hypertrophic response. Because contractile filaments will be engaged (Macken- cardiac myocytes are terminally differentiated, zie et al. 2004). The extent of atrial myocyte con- enlargement of the heart results mainly from traction is regulated by controlling the spread of growth of existing myocytes and not as a result

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Calcium Signaling in Cardiac Myocytes

of proliferation. For many hypertrophic stimuli, of SERCA dysfunction to the decrease in the remodeling of the heart is initially beneficial cardiac function during failure has led to by acting to increase cardiac output. However, the development of viral-mediated SERCA under conditions in which stress persists, for overexpression for gene therapy (Lyon et al. example, because of hypertension, the remod- 2011). eled heart undergoes a process known as Failing hearts are also characterized by more “decompensation.” Specifically, the thickness arrhythmic Ca2þ signals. This is perhaps of the wall diminishes, and the ability surprising given that SR store loading—a key of the heart to supply the cardiovascular determinant of RyR activity—is decreased. requirements of the organism is lost. Such RyRs isolated from failing hearts and incorpo- pathological cardiac hypertrophy has a poor rated into lipid bilayers are more spontaneously prognosis and leads to cardiac failure and active than RyR isolated from control hearts, sudden death (Levy et al. 1990). perhaps explaining this conundrum (Kubalova A widely supported hypothesis is that et al. 2005). Indeed, myocytes from failing changes in Ca2þ cycling mediate the hypertro- hearts showed decreased store loading, in- phic response (Molkentin 2006). Although creased spontaneous RyR opening (observed initial modifications of Ca2þ handling are as Ca2þ sparks) and decreased SR Ca2þ reup- beneficial, as the heart progresses to failure take. Another mechanism for increased they may contribute to pathology (Fig. 3) arrhythmic Ca2þ signaling during heart failure (Roderick et al. 2007). A general feature of cal- is the expression of inositol 1,4,5-trisphosphate 2þ cium fluxes during adaptive hypertrophy is an (InsP3) gated Ca release channels (InsP3Rs). 2þ increase in the amplitudes of the Ca transient, InsP3Rs are typically .50-fold less abundant and also of the Ca2þ sparks that underlie than RyR in healthy myocytes (Kockskamper them. Contributing to this is an increase in SR et al. 2008). However, their expression increases store loading mediated by increased SERCA significantly during hypertrophy and heart pump activity. SERCA activity is enhanced failure. In particular, their expression within through several mechanisms: increased SERCA dyadic junctions increases (Fig. 3) (Harzheim expression, decreased PLB expression, and in- et al. 2009, 2010). The up-regulation of InsP3Rs creased PLB phosphorylation. A decrease in provides a positive-feedback loop that can NCX current and an increase in RyR activity promote further hypertrophic remodeling have also been detected during this stage of (Nakayama et al. 2010). Although InsP3Rs are hypertrophy. not capable of mounting significant Ca2þ As the heart progresses to failure, further signals by themselves, their location next to changes in Ca2þ regulation and flux are ob- RyRs within dyadic junctions means that they served. Contributing to the decreased contrac- can trigger CICR and thereby augment EC- tility of the failing heart is a general decrease coupling. The expression of InsP3Rs may be in the amplitude of each action potential- an initially beneficial adaptive aspect of evoked Ca2þ transient. Underlying this reduced myocyte remodeling in that they can help to Ca2þ signal are a number of factors, foremost increase systolic Ca2þ transients and evoke of which is a decrease in SERCA activity greater contraction. However, concomitant (Fig. 3) (Hoshijima et al. 2006). SERCA activity with this potentially beneficial aspect of en- may be modified through a reduction in its hanced InsP3R expression is an undesirable expression, increased PLB expression or de- increase in the propensity of cells to show spon- creased PLB phosphorylation. As a result of taneous Ca2þ release events that may cause this decrease in SERCA function, diastolic arrhythmia. This is because of the fact that 2þ Ca is elevated (a common feature of the fail- InsP3Rs are not solely tuned to the activation ing heart) while myocyte relaxation is pro- of ICa, but can open independently whenever longed and SR store content is diminished cytosolic InsP3 levels are sufficient (Harzheim (Roderick et al. 2007). The key contribution et al. 2009).

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Myocyte Λ GPCR Δ PMCA NCX function Sarcolemma IP Gq Gs 3 30% < 70% cAMP 2+ [Ca ]i ~ 100 nM

T-Tubule PKA Mito PLB Ca2+ CSQ2 CSQ2

2+ DHPR μ [Ca ]SR ~ 500 M Healthy muscle Healthy

CICR

IP3 Gq Gs < 30% cAMP > 70%

PKA

P 2+ Mito PLB Ca CSQ2 CSQ2 Compensated muscle

IP 3 Gq Gs > 30%

< 70% 2+ [Ca ]i ~ 200 nM

Mito PLB Ca2+ CSQ2 CSQ2 Decompensated muscle

2+ [Ca ]0 ~ 2 mM

L-type VOCC Type 2 InsP3R SERCA CSQ Calsequestrin

Ryanodine receptor PLB Phospholamban Ca2+ Δ Membrane depolarization

[Ca2+] Ca2+ store PMCA NCX

Wider dyadic cleft Orphaned RyR Gq-coupled GPCR

Figure 3. Excitation contraction coupling in healthy and decompensated hypertrophic cardiac muscle. The figure depicts the architecture and molecular composition of a cardiac dyad in a normal, healthy myocyte (upper panel), a compensated hypertrophic situation (middle panel), and a decompensated failing myocyte (lower panel). A key depicting the symbols used to represent the major players in EC-coupling is provided at the bottom of the figure. Depolarization of the plasma membrane results in Ca2þ influx through L-type voltage gated channels in the T-Tubule, which stimulates Ca2þ release via RyRs located on the juxtaposed SR. Following diffusion out of the dyadic cleft, Ca2þ encounters the contractile filaments causing myocyte contraction. Myocyte relaxation is then brought about by Ca2þ recycling back into the SR by the SERCA pump or extrusion across the plasma membrane via NCX. (See facing page for legend.)

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The importance of cellular architecture to are elevated during hypertrophy, may also con- EC-coupling is highlighted by its contribution tribute to overcoming the decreased efficiency to Ca2þ dysregulation during cardiac failure. of coupling. In myocytes from failing heart, the synchro- nicity of the initiation of the Ca2þ transient is reduced and the amplitude of the transient CONCLUSION diminished (Gomez et al. 1997). Work from a The raison d’etre of cardiac myocytes is con- number of laboratories using live and fixed trolled contraction in response to repetitive cell staining approaches, have now established electrical depolarization signals—a function that T-tubules are lost or atrophied as hypertro- that is fundamentally controlled by Ca2þ. phy develops, whereas the distribution of RyRs Although there are a limited number of key on the SR is unaffected (Gomez et al. 2001; players—ICa, NCX, SERCA, RyRs, TnC— Song et al. 2005b). As a consequence of this involved in generating and reversing Ca2þ sig- membrane remodeling, RyRs become progres- nals, they are subject to numerous levels of sively orphaned. Deficiencies in EC-coupling regulation and an array of interactions with because of T-tubule remodeling have also been other proteins. Furthermore, the cellular loca- observed during the earlier stages of hypertro- tion of the Ca2þ signaling systems is critical in phy, albeit with less dramatic consequences determining the spatial properties of the Ca2þ (Xu et al. 2007). During this stage, the width signals during EC-coupling. Cardiac Ca2þ sig- of the dyadic cleft is marginally increased, alter- nals can be acutely altered to provide rapid ing the kinetic properties of coupling between changes in cardiac output. The heart responds membrane depolarization and Ca2þ release to long-term increased hemodynamic demand from the SR. Inotropic stimuli such as adrena- by remodeling. This remodeling encompasses line, which serve to increase Ca2þ influx and both structural changes and altered gene expres- sensitivity of RyRs to release, overcome this sion. Depending on the stimulus, this can be an deficiency in EC-coupling thus indicating that adaptive, reversible remodeling that promotes the Ca2þ signaling machinery is still intact. Sim- Ca2þ signaling and cardiac function. Alterna- ilarly, dyadic InsP3Rs, which as indicated earlier tively, a heart can become committed to an

Figure 3. (Continued) Neurohormonal activation of Gaq-coupled receptors leads to the formation of InsP3, 2þ 2þ which stimulates Ca release via InsP3Rs located in the dyad. This InsP3-stimulated Ca release sensitizes neighboring RyRs, causing enhanced Ca2þ fluxes and increasing the frequency of arrhythmic events. Activation of b-adrenergic receptors increases intracellular cAMP,which activates PKA leading to phosphorylation of PLB. On phosphorylation, PLB dissociates from SERCA thereby enhancing Ca2þ transport activity. Compensated/ adaptive hypertrophy is associated with enhanced EC-coupling. Significantly contributing to this phenotype is an increase in SR store loading. This is brought about by an up-regulation of SERCA activity mediated by either an increase in SERCA or decrease in PLB expression. Alternatively, as a result of PKA-dependent phos- phorylation, PLB interaction with SERCA and suppression of Ca2þ transport may also be decreased. Increased SERCA activity also serves to increase the rate of relaxation thereby allowing more rapid cycles of myocyte con- traction. The width of the dyadic cleft may also marginally increase at this stage of hypertrophic remodeling. 2þ However, IP3Rs are up-regulated in the dyad during hypertrophy supplementing the Ca signal arising via RyRs to possibly further support EC-coupling. During decompensated hypertrophy, myocyte architecture and protein expression are remodeled. Specifically, the width of the dyadic cleft is increased making it harder for Ca2þ arising via VOCCs to activate Ca2þ release from RyRs. T-Tubules also atrophy resulting in orphaned RyRs. The SERCA-PLB ratio is also modified to favor decreased SERCA activity. Notably, InsP3R expression in the dyad is increased. As a result, more of the RyRs that are located in this region are close enough to InsP3Rs to 2þ be affected by Ca arising from them. Overactivation of these InsP3Rs, for example, by the elevated levels of circulating ET-1 present during heart failure, promotes thereby contributing to the pathology asso- ciated with heart failure.

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Calcium Signaling in Cardiac Myocytes

Claire J. Fearnley, H. Llewelyn Roderick and Martin D. Bootman

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a004242 originally published online August 29, 2011

Subject Collection Calcium Signaling

The Endoplasmic Reticulum−Plasma Membrane Primary Active Ca2+ Transport Systems in Health Junction: A Hub for Agonist Regulation of Ca 2+ and Disease Entry Jialin Chen, Aljona Sitsel, Veronick Benoy, et al. Hwei Ling Ong and Indu Suresh Ambudkar Calcium-Handling Defects and Neurodegenerative Signaling through Ca2+ Microdomains from Disease Store-Operated CRAC Channels Sean Schrank, Nikki Barrington and Grace E. Pradeep Barak and Anant B. Parekh Stutzmann Lysosomal Ca2+ Homeostasis and Signaling in Structural Insights into the Regulation of Ca2+ Health and Disease /Calmodulin-Dependent Protein Kinase II (CaMKII) Emyr Lloyd-Evans and Helen Waller-Evans Moitrayee Bhattacharyya, Deepti Karandur and John Kuriyan Ca2+ Signaling in Exocrine Cells Store-Operated Calcium Channels: From Function Malini Ahuja, Woo Young Chung, Wei-Yin Lin, et al. to Structure and Back Again Richard S. Lewis Functional Consequences of Calcium-Dependent Bcl-2-Protein Family as Modulators of IP3 Synapse-to-Nucleus Communication: Focus on Receptors and Other Organellar Ca 2+ Channels Transcription-Dependent Metabolic Plasticity Hristina Ivanova, Tim Vervliet, Giovanni Monaco, et Anna M. Hagenston, Hilmar Bading and Carlos al. Bas-Orth Identifying New Substrates and Functions for an Calcium Signaling in Cardiomyocyte Function Old Enzyme: Calcineurin Guillaume Gilbert, Kateryna Demydenko, Eef Dries, Jagoree Roy and Martha S. Cyert et al. Fundamentals of Cellular Calcium Signaling: A Cytosolic Ca2+ Buffers Are Inherently Ca2+ Signal Primer Modulators Martin D. Bootman and Geert Bultynck Beat Schwaller

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Role of Two-Pore Channels in Embryonic Organellar Calcium Handling in the Cellular Development and Cellular Differentiation Reticular Network Sarah E. Webb, Jeffrey J. Kelu and Andrew L. Wen-An Wang, Luis B. Agellon and Marek Michalak Miller

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