Cardiac sodium transport and excitation–contraction coupling

Journal of Molecular and Cellular Cardiology 61 (2013) 11–19

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Review article

Cardiac sodium transport and excitation–contraction coupling J.M. Aronsen a,b,c, F. Swift a,b, O.M. Sejersted a,b,⁎ a b c

Institute for Experimental Medical Research, Oslo University Hospital Ullevål and University of Oslo, Oslo, Norway KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Oslo, Norway Bjørknes College, Oslo, Norway

a r t i c l e

i n f o

Article history: Received 17 April 2013 Received in revised form 17 May 2013 Accepted 5 June 2013 Available online 14 June 2013 Keywords: EC-coupling Sodium Microdomains Calcium

a b s t r a c t The excitation–contraction coupling (EC-coupling) links membrane depolarization with contraction in cardiomyocytes. Ca2+ induced opening of ryanodine receptors (RyRs) leads to Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) into the dyadic cleft between the t-tubules and SR. Ca2+ is removed from the cytosol by the SR Ca2+ ATPase (SERCA2) and the Na,Ca-exchanger (NCX). The NCX connects cardiac Ca2+ and Na+-transport, leading to Na+-dependent regulation of EC-coupling by several mechanisms of which some still lack firm experimental evidence. Firstly, NCX might contribute to CICR during an action potential (AP) as Na+-accumulation at the intracellular site together with depolarization will trigger reverse mode exchange bringing Ca2+ into the dyadic cleft. The controversial issue is the nature of the compartment in which Na+ accumulates. It seems not to be the bulk cytosol, but is it part of a widespread subsarcolemmal space, a localized microdomain (“fuzzy space”), or as we propose, a more localized “spot” to which only a few membrane proteins have shared access (nanodomains)? Also, there seems to be spots where the Na,K-pump (NKA) will cause local Na+ depletion. Secondly, Na+ determines the rate of cytosolic Ca2+ removal and SR Ca2+ load by regulating the SERCA2/NCX-balance during the decay of the Ca2+ transient. The aim of this review is to describe available data and current concepts of Na+-mediated regulation of cardiac EC-coupling, with special focus on subcellular microdomains and the potential roles of Na+ transport proteins in regulating CICR and Ca2+ extrusion in cardiomyocytes. We propose that voltage gated Na+ channels, NCX and the NKA α2-isoform all regulate cardiac EC-coupling through control of the “Na+ concentration in specific subcellular nanodomains in cardiomyocytes. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes.” © 2013 Elsevier Ltd. All rights reserved.

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Introduction — “The digitalis paradigm” . . . . . . . . . . Overview of EC-coupling and Na+-fluxes in cardiomyocytes 2.1. Cardiac EC-coupling . . . . . . . . . . . . . . . . 2.2. Na+ fluxes in cardiomyocytes . . . . . . . . . . . Cellular organization of cardiac EC-coupling . . . . . . . . 3.1. T-tubules as main regulators of cardiac EC-coupling . 3.2. Voltage gated Na+ channels . . . . . . . . . . . . 3.3. Na,Ca-exchanger . . . . . . . . . . . . . . . . . 3.4. Na,K-pump . . . . . . . . . . . . . . . . . . . . 3.5. Anchoring proteins regulate EC-coupling . . . . . . Ionic microdomains in cardiomyocytes . . . . . . . . . . 4.1. A Ca2 + microdomain controls cardiac EC-coupling . 4.2. Potential Na+ microdomains in the cardiomyocyte . 4.3. Biophysical basis for subcellular ion gradients . . .

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⁎ Corresponding author at: Institute for Experimental Medical Research, Oslo University Hospital Ullevål and University of Oslo, Kirkeveien 166, 0407 Oslo, Norway. Tel.: +47 93401058. E-mail address: [email protected] (O.M. Sejersted). 0022-2828/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.06.003

J.M. Aronsen et al. / Journal of Molecular and Cellular Cardiology 61 (2013) 11–19

Sodium and regulation of CICR . . . . . . . . . . . . . . . . . . . 5.1. CICR and Na+; fuzzy space and sodium hotspots . . . . . . . 5.2. Voltage gated Na+-channels and cardiac EC-coupling . . . . . 5.3. Reverse mode NCX as mediator of CICR . . . . . . . . . . . . 5.4. A possible role for NKA α2-isoforms in regulating CICR? . . . . 5.5. Future perspectives . . . . . . . . . . . . . . . . . . . . . 6. Sodium and regulation of Ca2 + extrusion . . . . . . . . . . . . . . 6.1. NCX–SERCA2-balance as regulator of Ca2 + homeostasis . . . . 6.2. NKA–NCX-interplay controls Ca2 + extrusion in cardiomyocytes 6.3. NKA α2 isoforms regulate Ca2 + extrusion in cardiomyocytes . . 7. Conclusion and future perspectives . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction — “The digitalis paradigm” The clinical effects of digitalis were described over 200 years ago [1], but it was not until the early 1950s that it was found that this drug inhibits transport of Na+ and K+ [2]. Some years later the Na,K-pump (NKA) was discovered, and eventually it became clear that this membrane protein was a high affinity receptor for digitalis [3,4]. However, the effect of digitalis on cardiac contractility remained a mystery until the link between intracellular Na+ and Ca2+ was described [5–7]. Eventually, this led to the cloning of the Na,Ca-exchanger (NCX) [8], and finally it was shown that digitalis has no effect on cardiac contractility in NCX-deficient mice [9]. Together, the membrane proteins NKA and NCX form an electroneutral, ATP-driven system, which pumps one Ca2+ ion out of the cell in exchange for two K+ ions at the same time as three Na+ ions are cycled across the membrane. The link to Na+ transport introduces a voltage sensitivity, which is most pronounced for NCX. Also, the two transporters are functionally coupled through intracellular Na+, and the concentration of Na+ at the intracellular sites is an important determinant of transport rate of both proteins. Inhibiting NKA with digitalis will thus cause the heart to lose K+, intracellular Na+ concentration to increase and SR Ca2+ load to increase [10,11]. This simple scheme explaining the effect of digitalis on cardiac contractility with two tightly linked key players – NKA and NCX – could be coined the digitalis paradigm implying that the concentrations of Na+ and Ca2+ are linked in the bulk cytosol. Later, it has become apparent that the picture is more complicated, especially since digitalis can increase contractility without a change in global cytosolic Na+ concentration [12,13]. We can probably no longer regard intracellular Na+ as one single pool. The purpose of this review is to describe the regulatory role of Na+ on the excitation–contraction coupling (EC-coupling) in cardiomyocytes in light of the current knowledge about Na+ and Ca2+ transporters and their localization. We will first describe Na+ fluxes and the proteins that carry Na+. Then we will discuss the concept of ionic microdomains, the existence of which so far is mainly based on indirect evidence. Finally follows a perusal of the evidence that Na+ regulates the EC-coupling and the extrusion of Ca2+ from the cell. 2. Overview of EC-coupling and Na+-fluxes in cardiomyocytes

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mechanism called Ca2+ induced Ca2+ release (CICR) [14]. Contraction of the cardiomyocytes is initiated by binding of Ca2+ to Troponin C of the myofilaments, inducing a conformational change which allows cross-bridges to form between myosin and actin. Since the peak of the intracellular Ca2+ concentration is at a steep part of the force–pCa curve, a larger Ca2+ transient will activate more cross-bridges and therefore induce a stronger contraction and vice versa. In order for the muscle to relax, Ca2+ must be removed from the cytosol. Ca2+ reuptake into the SR is mediated by SERCA2 and Ca2+ is also extruded out of the cell mainly via NCX. 2.2. Na+ fluxes in cardiomyocytes Intracellular Na+-concentration in cardiomyocytes is a primary determinant of cardiac contractility, and even minor changes in intracellular global Na+-concentration have a large impact on contractility [15,16]. Cardiomyocytes have a large electrochemical transmembrane Na+-gradient, which is the basis for secondary active transport of a variety of molecules including Ca2+ and H+, connecting Na+-fluxes in cardiomyocytes to EC-coupling and pH-regulation. The intracellular Na+-level in cardiomyocytes is 4–16 mM, and is determined by the balance between Na+ influx (“leak” rate) and efflux (“pump” rate) [17], as illustrated in Fig. 1. As the Na,K-pump (NKA) is the only Na+ efflux mechanism using ATP to pump Na+ against its electrochemical gradient, the properties and transport kinetics of NKA will be one determinant of the intracellular Na+ concentration and thereby regulate

Na+ influx

Vmax

Na+ influx

5.

Na+ transport rate (NKA)

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Na+ affinity

2.1. Cardiac EC-coupling The EC-coupling in cardiomyocytes links electrical depolarization to contraction. Membrane depolarization is initiated by opening of voltage gated Na+ channels, leading to Na+-influx (INa) which in a feed-forward manner further depolarizes the membrane. Voltage gated L-type Ca2+ channels (LTCCs) subsequently open to allow Ca2+ influx from the cell exterior. Ca2+ ions entering the cytosol bind to and open ryanodine receptors (RyRs) in the membrane of the SR, leading to rapid release of Ca2+ into the cytosol. Thus, a small Ca2+ influx via LTCCs initiates a large Ca2+ release from the SR through a feed-forward amplification

a

b

[Na+]i

Fig. 1. Intracellular levels of Na+ are determined by the balance between Na+ influx and efflux. Na+ influx through voltage gated Na+-channels, NCX and other transport proteins is balanced by Na+ efflux through the NKA. NKA-mediated efflux of Na+ is dependent on the Na+ concentration, the Na+ affinity of NKA and the maximal transport rate (Vmax) for NKA. By this scheme, the Na+-concentration could be altered by either altered NKA kinetics (at a given Na+-influx) as suggested by a), or alterations in Na+-influx at a given NKA transport rate as suggested by b).

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cardiac EC-coupling. As shown in Fig. 1, the NKA transport rate can be affected in two ways, either by altering maximal transport capacity (Vmax) or the Na+-affinity for the pump. The number of active NKA pumps in the cell is a major determinant of Vmax, while for example phosphorylation of the regulatory protein phospholemman mediates alterations in Na+-affinity [18]. In contrast to cellular Na+-efflux, several transport proteins mediate + Na -influx, and to draw the relationship between intracellular Na+ concentration and influx rate as a straight line is clearly an oversimplification, but serves the purpose of showing the principle of the “pump-leak” concept. Voltage gated Na+-channels, NCX, Na+,H+-exchanger (NHE), Na+, HCO−-cotransporter (NBC) and Na+,K+,2Cl−-cotransporter (NKCC) all mediate a significant Na+ influx in resting cardiomyocytes [17]. In the beating cardiomyocyte, most of the Na+-entering the cardiomyocyte enters the cell through voltage gated Na+-channels and NCX [17]. Voltage gated Na+-channels have a short open time, meaning that most channels close a few milliseconds after initiation of the action potential. However, a small subgroup of the Na+ channels remains active at a longer timeframe and reopens during the last phase of the AP, contributing to a small, sustained Na+ influx named the late sodium current (INa,Late). Although this current is small compared to the regular INa [19], it has been shown to be able to induce significantly Na+ influx throughout the AP [20]. Several kinases including PKA, PKC and to Ca/Calmodulin-dependent kinase II (CaMKII) have been shown to modulate voltage gated Na+-channels,

a

and CaMKII-dependent phosphorylation has been shown to upregulate INa,Late [21]. The relative contribution of the various Na+ transport proteins varies in different situations. For example intracellular acidosis and ischemia represent important pathological states that alter the balance between the different Na+ influx modes. In both resting and beating cells, the overall intracellular Na+-concentration is determined by the intersection of leak and pump rates as illustrated in Fig. 1. Despa and coworkers [22] have investigated the relationship between Na+ influx and Na+ efflux. In their study, rat cardiomyocytes had larger Na+ efflux, but still higher cytosolic Na+-levels than rabbit cardiomyocytes, indicating that the Na+ influx in rat cardiomyocytes was very high. The mitochondrial membrane also contains NCX and NHE allowing for Na+ and Ca2+ transport between the cytosol and mitochondrial compartment, that at least temporarily can affect the cytosolic Na+ concentration [23,24]. 3. Cellular organization of cardiac EC-coupling 3.1. T-tubules as main regulators of cardiac EC-coupling The cell membrane in ventricular cardiomyocytes is characterized by its many invaginations forming transverse (T-) tubules, constituting a network mainly at the z-lines of the cells (Fig. 2). One important

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surface membrane cytosol

T-tubule membrane

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longitudinal section T-tubule NKA

jSR

actin

jSR

network SR

NCX

RyR (feet)

myosin

RyR

LTCC z-line I-band

z-line A-band

Na+-channel

I-band

NCX

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NKA

high Ca2+

low Ca2+

Fig. 2. Structural basis for EC-coupling in ventricular cardiomyocytes. a) T-tubules are invaginations of the cell membrane, and these invaginations allow close interaction between the sarcoplasmatic reticulum (SR) and the sarcolemma. The SR is divided into junctional SR (jSR) close to the t-tubules and network SR between two t-tubules. The myofilaments with actin and myosin are regularly organized in sarcomeres between two Z-lines, surrounded by the SR. b) The dyadic clefts are close connections between the junctional SR and the t-tubule membrane. L-type Ca2+-channels (LTCCs) in the t-tubular membrane are closely apposed to groups of ryanodine receptors (RyRs), creating the structural basis for Ca2+ sparks during membrane depolarization. A subset of such LTCC–RyR-couplons has also been shown to colocalize with NCX, supporting that NCX can promote CICR in cardiomyocytes. However, the localization of voltage gated Na+ channels, NCX and Na,K-pump (NKA) is not known in detail, and the positions of these membrane proteins relative to the dyadic cleft are of crucial importance for cardiac EC-coupling (see for instance Lines et al. [89]). Experimental data indicates functional roles for both voltage gated Na+-channels and NKA α2-isoforms in close proximity to the LTCC–RyR-couplon. c) The concentration gradient of Ca2+ in the cytosol is a major regulator of cardiac EC-coupling. CICR rapidly increases the local Ca2+ concentration in the dyad (bright color), leading to Ca2+ diffusion out of the dyadic cleft to increase the global Ca2+ levels in the cell (dark color), which triggers contraction.

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function of the t-tubules is to provide proximity between the LTCCs in the cell membrane and RyRs in the SR membrane [25,26]. The junctional part of SR is coupled to the t-tubular membrane at regular intervals, where the narrow (~10 nm [26]) dyadic clefts between the SR and t-tubular membranes provide the structural basis for CICR. At the dyad, clusters of ~100 RyRs at the SR membrane are connected to LTCCs in the t-tubular membrane in subunits known as couplons [27]. The Ca2+ release from one RyR cluster is known as a Ca2+ spark, and as membrane depolarization rapidly spreads through the t-tubules during an AP, a large amount of Ca2+-sparks from different RyR clusters together constitutes the Ca2+-transient in the cell, initiating contraction [28,29]. In the following sections we will discuss the placement of Na+ transport proteins which tend to be clustered in the t-tubules and how they influence CICR. Atrial cardiomyocytes which mostly lack t-tubules also have less NCX and NKA expression than ventricular cardiomyocytes [30]. 3.2. Voltage gated Na+ channels Voltage gated Na+-channels exist in several isoforms in adult cardiomyocytes, including the neuronal types (Nav1.1, Nav1.2, Nav1.3 and Nav1.6), the skeletal muscle isoform Nav1.4 and the cardiacspecific isoform Nav1.5 [31–34]. The cardiac isoform is less TTXsensitive than the other isoforms, and was shown to contribute to 92% of the total INa in cardiomyocytes [32]. Importantly, the different Na+ channel isoforms seem to differ in their subcellular distribution and the cardiac isoform has been shown to localize to t-tubules and intercalated discs, in contrast to neuronal types mostly localized to t-tubules (for review, see Ref. [33]). 3.3. Na,Ca-exchanger The NCX exists in three isoforms (NCX1, NCX2, NXC3), but NCX1 is the only isoform present in the heart [35]. NCX1 appears more concentrated in t-tubules than in the surface sarcolemma [36–45], and an important question in regulation of cardiac EC-coupling is the localization of NCX relative to LTCCs, RyRs and the dyadic cleft. Several reports have reported a small proportion of the t-tubular NCX-molecules to be colocalized with RyRs [41,42,44,46,47], indicating a possible role for NCX in modulating CICR. Further, Schulson and coworkers [46] used triple-labeling of rat atrial cells, and showed that NCX was clustered to some of the LTCC–RyR-couplons, but not all. Most of the NCXmolecules in the t-tubules appear to be extradyadic, possibly involved in Ca2+-extrusion. However, Thomas et al. [42] using an immunogold technique showed that several NCX molecules were found as close to RyRs as the LTCCs. Importantly, NCX-molecules have been colocalized with NKA in the t-tubules [48–50], especially with the NKA α2-isoform [48,49], indicating a special role for this NKA-isoform in controlling NCX-activity [13,51,52]. 3.4. Na,K-pump The NKA is composed of an α and β-subunit [53], and α1 and α2 isoforms constitute the majority of NKAs in cardiomyocytes [54]. The α-subunits of NKA contain both the Na+- and K+-binding sites, the ATP-catalytic site and the ouabain binding site [53]. As the different NKA α-isoforms show different affinities for extracellular K+ [55], glycosides [56] and possibly intracellular Na+ [57], the different NKA isoforms could have different roles in regulating cellular function. The subcellular distribution of NKA α-isoforms has been extensively examined, and evidence shows that the α1-isoform is homogenously distributed in the sarcolemma, while the α2-isoforms appear to be more concentrated in the t-tubules [13,52,58]. As will be discussed later, the localization of NKA α2-isoforms in t-tubules fits well with a large amount of evidence of the NKA α2 isoform as a major regulator of EC-coupling [13,42,52,59–61].

3.5. Anchoring proteins regulate EC-coupling There is now clear evidence that many proteins form macromolecular complexes consisting of multiple proteins highly organized by specific protein–protein-interactions and held in place by anchoring proteins. Ca2+ handling proteins have been shown to be tethered by various A-kinase anchor proteins (AKAPs), which in that way orchestrates macromolecular complexes that closely regulate the function of for instance LTCCs, RyRs or SERCA2 [62]. Similarly, Na+-transport proteins have been shown to be anchored to the cell membrane by Ankyrin-proteins. Ankyrin-G is necessary for membrane targeting and proper function of Nav1.5. [63], while Ankyrin-B is shown to scaffold NCX, NKA and IP3-receptors [50,64], extending previous findings showing that NCX can colocalize with both α1 and α2 NKA-isoforms [48,49]. By bringing NCX and NKA close together, we speculate that ankyrin-B could create an ATP driven 1Ca2+:2K+ pump in which the pumping activity of both proteins might be coordinated by the Na+ concentration in a common microdomain. Also, local Na+-concentration at the cytosolic site of this protein cluster rather than the global ion concentrations could regulate NCX-activity. 4. Ionic microdomains in cardiomyocytes 4.1. A Ca2+ microdomain controls cardiac EC-coupling An important feature of cardiac Ca2+ homeostasis is that tight control of the local dyadic Ca2+ concentration secures SR Ca2+ release to only occur during the AP. Further, rapid and large efflux of Ca2+ out of the dyad is necessary to synchronously activate the actin–myosin complex in cardiomyocytes. These apparently contrasting requirements led to the concept of “local control” put forward by Stern in 1992 [65], where the dyad can function as a subcellular microdomain that during a brief period may have a different ion concentration than the bulk cytosol. Influx of Ca2+ during the AP rapidly increases the local Ca2+-concentration, but apparently in a focused way so that the various couplons induce synchronized Ca2+ release that results in an increase in global Ca2+-concentration. Given the low volume of the dyadic space, the number of Ca2+ ions that is required to trigger Ca2+ release is quantitatively low [66]. 4.2. Potential Na+ microdomains in the cardiomyocyte Less established, but nevertheless extensively investigated, is the possible existence of subcellular microdomains of Na + in cardiomyocytes. Several microdomains with different roles during the heartbeat have been proposed in the literature (Fig. 3). Experimental evidence indicates that Na+ flux through voltage gated Na+ channels, NCX or NKA has the ability to alter cardiac contractility without detectable accompanying changes in cytosolic Na+-concentration. This has been taken to show the existence of Na+ microdomains. Three main types of Na+ microdomains involved in cardiac ECcoupling have been suggested in the literature (see Fig. 3): 1) A microdomain involving TTX-sensitive Na-channels, NCX and RyRs, originally referred to as a “fuzzy space”, proposed by Lederer and coworkers in 1990 [27]. This was thought to be a microdomain where TTXsensitive Na+-current increased Na+-concentration at the cytosolic site of NCX, causing sufficient Ca2+-influx to lead to CICR [27,67]. 2) The subsarcolemmal space. Several electrophysiological approaches, including comparison of NKA- and NCX-mediated currents with global Na+-concentrations, have been applied to study the possible Na+-gradient between the bulk cytosol and the subsarcolemmal space. In several cardiomyocyte models these two compartments have been introduced [68]. Overall, these experiments suggest that the subsarcolemmal Na+-concentration can be several-fold higher than in the bulk cytosol [68]. This has also been reported in studies using

J.M. Aronsen et al. / Journal of Molecular and Cellular Cardiology 61 (2013) 11–19

15

ion channel

hotspot

coldspot

fuzzy space

crosstalk

microdomain

subsarcolemmal space bulk cytosol

NCX NKA Na channel

dyad

Ca channel microdomains RyR SERCA Fig. 3. Suggested Na+ gradients in cardiomyocytes. The subsarcolemmal space appears to have higher Na+ concentration than the bulk cytosol. In addition, Na+ “hotspots” and “coldspots” could be created as a result of localized Na+ influx and efflux by Na+ channels or transporters. An interesting possibility is that Na+ transport proteins could share a restricted micro or nanodomain that provides crosstalk for example between ankyrin-B anchored NKA and NCX molecules through the local Na+ concentration. In addition the subsarcolemmal space and the originally proposed fuzzy space involving a microdomain with NCX, voltage gated Na+-channels and RyRs are illustrated. The fuzzy space could overlap with the dyadic cleft.

electron probe X-ray microanalysis [69,70]. 3) NKA-mediated Na+ depletion, where the activation of NKA seems to establish a gradient between an undefined volume surrounding the cytosolic site of NKA and the bulk cytosol. Despite extensive experimental work, many controversies exist regarding these hypothetical microdomains, including whether they can function as regulators of EC-coupling in intact cardiomyocytes. Interestingly, Silverman and coworkers [69] reported that the subsarcolemmal Na+-concentration was higher than the bulk cytosol both during systole and diastole in contracting cells, indicating that the subsarcolemmal Na+ gradient in fact may be a standing gradient. However, there is also evidence that gradients for Na+ can only be transient since diffusion of Na+ is very rapid. We therefore introduce the terminology “Na+ hotspots” and “Na+ coldspots” to describe the possible short-lived existence of very small, localized microdomains (or rather nanodomains since they could be very small) in the cell, as indicated in Table 1, in which Na+ concentration may temporarily be different from that of the cytosol. In this terminology, Na+ hotspots are localized elevations of the Na+ concentration as might happen at the mouth of voltage gated Na+-channels during opening. Na+ coldspots are localized depletions of Na+, hypothetically existing at the cytosolic site of NKA as a result of temporarily increased NKA-activity. If such microdomains are present in the intact cardiomyocytes, a key question would be whether these spots are separate and linked to only one channel or one transporter, or whether two or more channels/transporters have shared access to a common spot. In the latter case a hotspot could switch to a coldspot during the cardiac cycle. Whether one of these two proposed models is true remains to be shown.

4.3. Biophysical basis for subcellular ion gradients What are their biophysical properties and how is the Na+concentration regulated in these micro- or nanodomains? Gradients for ions can be established due to slow diffusion or physical restrictions in the cell. Diffusion of ions in cytosol tends to be lower than in water [71], and slow cytosolic diffusion of Na+ has been reported [72]. Recently a much higher diffusion coefficient was found by Swietach et al. [73], and it is thus unclear whether a global restriction of diffusion can explain the existence of subcellular Na+-gradients. On the other hand, the dyad is packed with proteins, and this macromolecular crowding could cause hindrance to diffusion [74,75]. The presence of negatively charged phospholipids [68,76] in the membrane may also cause a surplus of soluble anions to accumulate. Finally, close colocalization of transport proteins so that they share a shielded volume could well exist. Macromolecular complexes of different ion transporters held in place by Ankyrins could potentially create micro- or nanodomains with limited access. For example, the Ankyrin-B-anchoring of both NCX and NKA raises the possibility that local Na+-concentrations at the cytosolic side of this complex, and not in a larger subsarcolemmal space, is responsible for controlling NCX-activity. 5. Sodium and regulation of CICR 5.1. CICR and Na+; fuzzy space and sodium hotspots The close proximity of LTCCs and RyRs which is the basis of CICR is well characterized [77], but the subcellular distribution of other transport proteins in the junctional cleft is less known. A main

Table 1 Terminology of ionic compartments. Microdomain: An identifiable small volume separated from the rest of the cytosol by a diffusion barrier that limits the rate of exchange of small molecules and ions between the two compartments. A very small microdomain (nanodomain) could for example be an “ion pocket” at the mouth of an ion channel or transporter. Hotspot: Proposed term to describe a microdomain or nanodomain where the concentration of the ion of interest is transiently higher than in the surrounding volume. Coldspot: Proposed term to describe a microdomain or nanodomain where the concentration of the ion of interest is transiently lower than in the surrounding volume. Fuzzy space: The term “fuzzy space” was originally proposed to define a functionally restricted intracellular space (microdomain) accessible to Na+ channels, NCX and the RyR. The existence of such space was postulated by Lederer et al. [27] to explain the observation that NCX could trigger CICR in the absence of L-type Ca2+ channels [67]. The term has later also been used to describe other subcellular microdomains with undefined volume, unclear biophysical basis and ion regulation. Subsarcolemmal space: The part of the cytosol which is located immediately beneath the cell membrane. Bulk cytosol: The part of the cytosol that is separated from microdomains. Dyad: The space between a t-tubule membrane and the membrane of a junctional SR where LTCCs and RyRs face each other establishing a Ca2+ release unit activated during CICR. [Ca2+] can reach very high values in this space. The dyad is a microdomain for Ca2+. Ca2+ must diffuse rapidly out of the dyad to reach the myofilaments.

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controversy in regulation of cardiac EC-coupling is whether voltage gated Na+-channels, NCX and NKA are directly involved in regulation of CICR in cardiomyocytes. This will be discussed in the following sections. 5.2. Voltage gated Na+-channels and cardiac EC-coupling Reverse mode NCX-activity is promoted by the combination of depolarization and a local Na+-increase at the cytosolic site of NCX-molecules. Thus, voltage gated Na+-channels possibly control CICR by increasing Na+-levels near the NCX, in addition to cause LTCC opening by depolarizing the cell membrane. As the absolute amount of Na+-entering the cell is minor during the AP, reversal of NCX would actually require presence of Na+-channels within the dyad and could then in theory provide enough Ca2+ to trigger CICR. Recent reports supporting this idea have used NCX-deficient cardiomyocytes to study the effects of TTX in cells with and without NCX. These studies have concluded that INa can induce reverse mode NCX and subsequently affect SR Ca2+ release and cardiac ECC by augmenting the dyadic pool of Ca2+ that triggers ryanodine receptors [78,79]. However, the role of Na+-channels and reverse mode NCX-activity in CICR has also been disputed. Notably, Silverman and coworkers found that the subsarcolemmal Na+-concentration was unaltered even after repetitive stimulation to maximize Na+-influx [69], and concluded that INa does not seem to regulate the subsarcolemmal Na+-concentrations. This study was based on measurements of the NKA-current to evaluate subsarcolemmal Na+-concentrations, assuming that NKA and voltage dependent Na+-channels access the same microdomain. As the voltage gated Na+ channels exist in several isoforms with different subcellular distribution, a possibility is that the different isoforms have different roles in controlling EC-coupling. The finding of a prolonged upstroke of the AP with no alterations in Ca2+-transients by a low dose TTX [80] did not support a role for the non-cardiac isoforms of voltage gated Na+-channels to regulate cardiac EC-coupling. These findings have recently been challenged by Torres and coworkers [81], who by an elegant approach show that TTX-sensitive INa modulates SR Ca2+ release through priming the dyadic cleft with Ca2+ and increasing the probability of RyR opening subsequent to opening of the LTCCs. A role for the TTX-sensitive channels in cardiac EC-coupling was further supported by the finding of slower conduction velocity and prolonged Ca2+ transients in cardiomyocytes with deletion of Nav1.6-channels [82]. Thus, further studies are needed to obtain the precise role of the subtypes of voltage gated Na+ channels in regulating cardiac EC-coupling. 5.3. Reverse mode NCX as mediator of CICR While LTCCs are the main mediators of SR Ca2+ release, a regulatory role of NCX in modulating CICR is indicated by the finding of a subpopulation of NCX-proteins close to the LTCC–RyR-couplon [41,42]. The “fuzzy space” was thought to be a cellular microdomain accessed by voltage gated Na+ channels, NCX and RyRs in which the ion concentration could be different from that of the bulk cytosol [67], supported by the finding that reverse mode NCX exchange can trigger CICR the presence of maximum blockade of LTCC [83]. However, under more physiological settings (rather than complete LTCC-blockade), NCX-mediated Ca2+-influx appeared insufficient to induce SR Ca2+ release [84–87]. Notably, Weber showed that INa was able to increase the subsarcolemmal Na+-concentration ~60 times more than the average Na+-concentrations, creating a Na+ hotspot corresponding to a volume of ~1.6% of the total cell volume (larger than the predicted volume for the dyadic cleft) [88]. In these rabbit ventricular cardiomyocytes, NCX could operate in reverse mode for up to 19 ms before switching to forward mode and Ca2+ extrusion [88]. In a combined experimental and modeling study, Lines et al [89] showed, by using rat cardiomyocytes

with a much shorter AP, that NCX seems to operate in reverse mode for just a few ms. Recently, the generation of cardiac specific NCX knockout-mice (NCX KO) has shed new light on NCX as a possible mediator of CICR. NCX KO-mice have a strikingly preserved cardiac function with unaltered Ca2+ transients despite reduced transsarcolemmal Ca2+ fluxes [90,91]. This strong ability of NCX KO to maintain SR Ca2+ release despite major reductions in ICa,L must be due to a much higher gain of the CICR [90,91], supporting a role for NCX to regulate CICR. Especially, the role of NCX has been suggested to be to prime the dyadic cleft with Ca2 prior to the opening of the LTCCs, increasing the amount of SR Ca2+ release per Ca2+ ion entering the cell by a LTCC [79]. Thus, altogether these studies suggest a regulatory role of NCX in controlling CICR, possibly by raising the Ca2+ concentration in the dyad prior to opening of the LTCCs. 5.4. A possible role for NKA α2-isoforms in regulating CICR? Corresponding to the high density of NKA α2-subunits in the t-tubules, the NKA α2-isoform seems to be more tightly linked to regulation of CICR than the NKA α1-isoform. James and coworkers showed that mice with low NKA α2-levels exert in vivo hypercontractility, accompanied by increased amplitude and shortened decay time of the Ca2+ transient, while cardiomyocytes from mice with low NKA α1-levels had no alterations in Ca2+ handling [92]. This initial finding has been extended by several experimental approaches [13,51,52,59,60], showing that the NKA α2-isoform seems to regulate the Na+-concentration at the cytosolic side of NCX. However, recently Rindler et al. [93] showed that conditional and cardiac specific knockouts of the NKA α2-isoform had remarkably little effect on the cardiac phenotype. This contrasts with several studies that show that the NKA α2-isoform plays a central role in regulating Ca2 +-homeostasis [92,94], although the precise mechanism for this is not resolved. Recent data show that acute inhibition of the NKA α2-isoform will increase the size of Ca2+ transients and fractional Ca2+ release despite unaltered SR Ca2+ load [60]. This occurs without notable increase in the global Na+ concentration in the cell, suggesting a NKA-regulated ionic microdomain in vicinity to the dyad to be a determinant of CICR. Interestingly, the NKA α2-isoform has been shown to be localized to membrane–SR-interactions in smooth muscle [95] cells, but the exact localization and regulatory role of the NKA α2-isoform in cardiomyocytes remain to be demonstrated. 5.5. Future perspectives It is a clear need for exact knowledge of subcellular protein localization in cardiomyocytes. To what extent are voltage gated Na+-channels and NCX present in dyads? What is the localization of NKA, especially the NKA α2-isoform, relative to the dyad and how does NKA-activity affect CICR? Are both LTCCs and NCXs coupled to RyRs? A recent study showed that longitudinal t-tubules may be formed that lack LTCCs but in which NCX seems to colocalize with RyRs [40]. Further, to determine to what extent these membrane proteins form macromolecular protein–protein complexes that allow the transporters to interact closely, possibly through sharing minute pools of Na+ and Ca2+, would increase the understanding of regulation of EC-coupling. 6. Sodium and regulation of Ca2 + extrusion 6.1. NCX–SERCA2-balance as regulator of Ca2+ homeostasis Cytosolic Ca2+ removal is performed mainly by SR Ca2+ reuptake by SERCA2 and extrusion from the cell by NCX on a beat-to-beat basis. The ratio of Ca2+ carried by SERCA2 and NCX respectively, is about ~7:3 in humans and larger animals such as rabbits [96], whereas in rats [97] and mice [98] it is closer to ~9:1. This balance seems to be dynamically

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regulated, as both NCX-KO mice and SERCA2-KO mice are able to maintain cardiac performance by adjusting sarcolemmal Ca2+-fluxes [90,91,98,99]. The SERCA2–NCX-balance also seems to be dynamically regulated during the decay of the Ca2+-transient, as Yao and coworkers showed that NCX was responsible for 13% of the Ca2+ removal in the first phase of the decay of the Ca2+-transient and 45% in the second phase in rabbit cardiomyocytes [100]. This reflects differences in transport capacity and affinity for Ca2+ of the two transporters, but also how accessible the Ca2+ binding sites are. We also speculate that due to the voltage dependency, NKA-activity will be low at low membrane potentials leading to Na+-accumulation during the diastole, thereby reducing forward mode operation of the NCX in the early phase of the Ca2+ extrusion phase. The combined Ca2+-efflux rate of SERCA2 and NCX determines the relaxation rate of the cardiomyocytes during the heartbeat as well as the diastolic Ca2+ level. The relative importance of the two proteins will be different at low and high heart rates. At low heart rates the NCX will be a main determinant of the diastolic Ca2+ level in line with its greater contribution during the tail of the Ca2+ transient. At higher heart rates SERCA2 will be dominant since the diastolic interval is shorter. Furthermore, the competition between SERCA2 and NCX for removal of Ca2+ is an important determinant of contractile strength. When SERCA2 is favored, SR Ca2+ will increase and the next contraction will be stronger, and opposite when Ca2+ removal through NCX is favored, the next contraction will be weaker. 6.2. NKA–NCX-interplay controls Ca2+ extrusion in cardiomyocytes NCX-proteins are clustered in the t-tubules and regulated by the transsarcolemmal ion gradients for Na+ and Ca2+ in addition to the membrane potential. We and other groups have shown that NKAactivation alters both reverse [13,51,94,101,102] and forward mode NCX-transport [59], indicating that NCX-currents are influenced by the level of NKA-activity. Several studies have shown that the NKA-current in quiescent cardiomyocytes declines over time after activation of the pump by adding K+ to a K+-free perfusate, possibly as a result of subsarcolemmal Na+-depletion and the generation of Na+ coldspots. This phenomenon appears in the literature within two different timeframes, one rapidly over seconds and another decline over several minutes. Semb and Sejersted [103] and Despa and Bers [104] both showed that after rapid activation of the NKA in Na+ loaded cells, there is a lag period in which the NKA is active, without a detectable decline in intracellular Na+. This is compatible with creation of Na+ coldspots at the cytosolic site of NKA since the NKA current (Ip) declines rapidly. After the initial phase Ip and intracellular Na+ declined in parallel. These observations indicate that during periods of net Na+ efflux from the cell, NKA may create a cytosolic gradient with lower Na+-concentration in the vicinity of the NKA-transporters. Recently, glutathionylation of the β1-subunit of the NKA has been shown to inhibit the pump and activation of the β3-adrenoceptor effectively relieves this inhibition [105,106]. It is possible that regulation of the pump activity by the oxidative status of cell can be an additional regulating factor to consider. 6.3. NKA α2 isoforms regulate Ca2+ extrusion in cardiomyocytes In support of the hypothesis that the NKA α2-isoform is able to regulate NCX-mediated Ca2+ extrusion through controlling the Na+ concentration in a microdomain shared with NCX, Swift and coworkers [59] showed that Ca2+ extrusion by NCX was significantly slowed following specific inhibition of the NKA α2-isoform with low dose ouabain. This reduced NCX-activity could not be explained by an increase in global Na+ as assessed by the Na+ indicator SBFI, and was probably caused by increased Na+ in microdomains. A Na+ coldspot controlled by the NKA α2-isoform could regulate Ca2+-extrusion and thus the

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NCX–SERCA2-balance at beat-to-beat-basis. A main unsolved question regarding the NCX–NKA-interaction is why the NKA α2 isoform seems to control NCX-activity better than the NKA α1-isoform independent of Na+ in bulk cytosol. It has been shown that the t-tubules contain an equal amount of the two isoforms and both seem to be functionally linked to the NCX. To understand the biophysical basis for this greater ability of NKA α2 to control NCX-fluxes would be a major step forward since it seems that the link between NKA α2 and NCX is broken in failing hearts [51]. 7. Conclusion and future perspectives Cardiac EC-coupling and contractility are under tight control by Na+ in cardiomyocytes, where cellular microdomains of both Ca2+ and Na+ seem to be involved in regulating EC-coupling. A more detailed understanding of the cellular basis especially for intracellular Na+-gradients will provide new insight into the regulation of EC-coupling. Protein targeting, protein clustering, and protein–protein interactions seem to be important for local control of Na+ and Ca2+. Another key to a more detailed understanding of the cardiac EC-coupling would be to develop new fluorescent dyes for Na+, as direct measurement of subsarcolemmal differences in Na+-concentration would be of great interest. Further, mathematical models taking the different subsarcolemmal spaces (dyadic vs. extradyadic, coldspots vs hotspots) could provide new insight in Na+-dependent regulation of cardiac EC-coupling. Disclosures None declared. References [1] Withering W. An account of the foxglove, and some of its medicinal uses: with practical remarks on dropsy and other diseases. London: G.G.J. & J. Robinson; 1785 . [2] Schatzmann HJ. Herzglycoside als Hemmstoffe für den aktiven Kalium und Natrium Transport durch die Erythrocytenmembran (Cardiac glycosides as inhibitors of active potassium and sodium transport by erythrocyte membrane). Helv Physiol Pharmacol Acta 1953;11:346–54. [3] Skou JC. The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim Biophys Acta 1957;23:394–401. [4] Skou JC. Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol Rev 1965;45:596–617. [5] Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev 1999;79:763–854. [6] Reuter H, Seitz N. The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol 1968;195:451–70. [7] Sheu S-S, Fozzard HA. Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development. J Gen Physiol 1982;80:325–51. [8] Nicoll DA, Longoni S, Philipson KD. Molecular cloning and functional expression of the cardiac sarcolemmal Na+–Ca2+ exchanger. Science 1990;250:562–5. [9] Reuter H, Henderson SA, Han T, Matsuda T, Baba A, Ross RS, et al. Knockout mice for pharmacological screening: testing the specificity of Na+–Ca2+ exchange inhibitors. Circ Res 2002;91:90–2. [10] Wasserstrom JA, Schwartz DJ, Fozzard HA. Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands exposed to cardiac glycosides. Circ Res 1983;52:697–705. [11] Ellingsen Ø, Sejersted OM, Vengen ØA, Ilebekk A. In vivo quantification of myocardial Na–K pump rate during β-adrenergic stimulation of intact pig hearts. Acta Physiol Scand 1989;135:493–503. [12] Wasserstrom JA, Aistrup GL. Digitalis: new actions for an old drug. Am J Physiol Heart Circ Physiol 2005;289:H1781–93. [13] Swift F, Tovsrud N, Enger UH, Sjaastad I, Sejersted OM. The Na+/K+-ATPase alpha2-isoform regulates cardiac contractility in rat cardiomyocytes. Cardiovasc Res 2007;75:109–17. [14] Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol 1983;245:C1–C14. [15] Lee CO, Dagostino M. Effect of strophanthidin on intracellular Na ion activity and twitch tension of constantly driven canine cardiac Purkinje fibers. Biophys J 1982;40:185–98. [16] Pieske B, Maier LS, Piacentino III V, Weisser J, Hasenfuss G, Houser S. Rate dependence of [Na+]i and contractility in nonfailing and failing human myocardium. Circulation 2002;106:447–53. [17] Bers DM, Barry WH, Despa S. Intracellular Na+ regulation in cardiac myocytes. Cardiovasc Res 2003;57:897–912.

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