Cardiac Excitation-Contraction Coupling

Abstract

This chapter will provide you with an understanding of the regulation of Ca²⁺ in the myocardium, its physiological implication as well as its role in orchestrating myocardial contraction. The chapter explores the processes of excitation-contraction coupling (ECC) and calcium-induced calcium release (CICR) whilst appreciating the relevance of ECC in pathology and in engineering heart tissue.

FormalPara What You Will Learn in This Chapter

This chapter will provide you with an understanding of the regulation of Ca²⁺ in the myocardium, its physiological implication as well as its role in orchestrating myocardial contraction. The chapter explores the processes of excitation-contraction coupling (ECC) and calcium-induced calcium release (CICR) whilst appreciating the relevance of ECC in pathology and in engineering heart tissue.

FormalPara Learning Objectives

• Understand the molecular mechanism that underlie calcium-induced calcium release

• Appreciate and assess the different theories for the calcium-induced calcium release termination process

• Be able to discuss the issues with calcium handling in induced pluripotent stem cells

6.1 Introduction to Excitation-Contraction Coupling

Of the array of ions involved in the workings of the heart, calcium (Ca²⁺) is perhaps the most important [1]. During the cardiac action potential, Ca²⁺ entry through the sarcolemmal Ca²⁺ channels stimulates Ca²⁺ release from the sarcoplasmic reticulum (SR), causing a rise in cytosolic Ca²⁺ and the subsequent activation of troponin on myofilaments (see ► Chap. 10), resulting in the development of force to eject blood out of the ventricles [2]. The process that links myocyte electrical excitation to contraction is known as excitation-contraction coupling (ECC). Appreciating ECC is crucial as it forms the basis of physiology, is dysregulated in almost all pathology and acts as a marker of the robustness of novel experimental cardiac models such as stem cell-derived cardiomyocytes [1, 3,4,5].

In each heartbeat, the cytoplasmic Ca²⁺ concentration of a healthy cardiomyocyte (CM) oscillates from ≅100 nM to 1 μM [6]. Precise Ca²⁺ regulation is a matter of life and death, and improper cytoplasmic Ca²⁺ rise and/or removal can lead to defective systole and diastole, respectively (known as systolic and diastolic dysfunction).

6.2 Ca²⁺ Influx

During the ventricular action potential, influx of Ca²⁺ from the extracellular to subsarcolemmal space generates a Ca²⁺ current, known as I_Ca, which triggers Ca²⁺ release from the SR, mediated by SR-release channels known as ryanodine receptors (RyRs). I_Ca occurs in two main ways [5]:

• Voltage-sensitive sarcolemmal Ca²⁺ channels (LTCCs)

• Na⁺-Ca²⁺ exchanger (NCX)

6.2.1 L-Type Ca²⁺ Channels (LTCCs)

LTCCs are activated by the initial membrane potential (V_m) depolarisation cause by the opening of voltage-gated Na⁺ channels [1]. Following LTCC opening, deactivation occurs by both time-dependent, V_m-dependent, and cytosolic calcium ([Ca²⁺]_i)-dependent mechanisms [5]. The V_m-dependent inactivation of the LTCCs can be demonstrated by administering depolarisation pulses and measuring LTCC inactivation kinetics [8]. Similarly, increasing [Ca²⁺]_i accelerates the inactivation rate of LTCCs, suggesting the precense of a negative-feedback system that prevents excess Ca²⁺ influx [7]. This is observed when Ca²⁺ is replaced with Ba²⁺ and the LTCC inactivation rate decelerates as Ca²⁺-dependent inactivation is minimised [5].

Enzymatic and non-enzymatic mechanisms have been proposed to explain Ca²⁺-dependent inactivation. In the former, dephosphorylation of the LTCC by Ca²⁺-activated phosphatases deactivates the channel [7]. In the latter, a Ca²⁺-calmodulin complex on the -COOH terminal of the α1 subunit of the LTCC binds Ca²⁺ when local [Ca²⁺]_i increases, altering the channel’s conformation and thus inactivating it [5, 7] (◘ Fig. 6.1).

Fig. 6.1

Ensemble currents demonstrating Ca²⁺-dependent LTCC inactivation in planar lipid bilayer experiments; a, b, and c show currents with 10 μM-, 20 nM-, and 15 μM-[Ca²⁺] respectively. Increasing concentrations of [Ca²⁺] accelerate the rate of LTCC inactivation. (Image from [7])

6.2.2 Na⁺-Ca²⁺ Exchanger (NCX)

The second contributor of Ca²⁺ influx during the action potential is the Na⁺-Ca²⁺ exchanger (NCX): a counter-transport system that operates by exchanging 3 Na⁺ for 1 Ca²⁺. This net movement of positive charge in the direction of Na⁺ makes NCX electrogenic (i.e. it generates current). Typically, NCX moves Na⁺ ions in whilst Ca²⁺ is effluxed out of the cell – known as the ‘forward mode’ and producing an inward current. NCX can also function in ‘reverse mode’, loading the cell with Ca²⁺ whilst Na⁺ ions are effluxed out of the cell (outward current). This can be summarised mathematically in a few equations:

$$ {E}_{\mathrm{rev}}={E}_{\mathrm{NCX}}=3{E}_{\mathrm{Na}}-2{E}_{\mathrm{Ca}} $$

(6.1)

$$ 3\left({E}_{\mathrm{Na}}-{V}_{\mathrm{m}}\right)>2\left({E}_{\mathrm{Ca}}-{V}_{\mathrm{m}}\right) $$

(6.2)

$$ {V}_{\mathrm{m}}<{E}_{\mathrm{NCX}} $$

(6.3)

Let’s work through those:

• 1. E_rev or E_NCX is the reversal potential of NCX – that is, the V_m at which NCX will switch from ‘forward’ to ‘reverse’ mode. As the equation shows, this depends on the individual equilibrium potentials of Na⁺ and Ca²⁺. This is exactly the same as the reversal potential of an ion channel, meaning that the current produced by NCX (I_NCX) when the membrane potential is equal to the reversal potential (V_m = E_NCX) is zero – there is no net movement of charge through the sarcolemma.

• 2. This equation shows the thermodynamic basis for the transport that governs NCX, suggesting that when the energy for the inward movement of three Na⁺ ions exceeds the energy for the inward movement of one Ca²⁺ ion, Na⁺ influx and Ca²⁺ efflux are favoured (i.e. ‘forward mode’). Conversely, if 3(E_Na − V_m) < 2(E_Ca − V_m), then the reverse is thermodynamically favoured, and Ca²⁺ influx occurs [5].

• 3. This is a rearranged form of Eq. 6.2, demonstrating that when V_m is more negative than E_NCX, the exchanger functions in forward mode, and vice versa (◘ Fig. 6.2). That is:

  • V_m < E_NCX – NCX operates in forward mode

  • V_m > E_NCX – NCX operates in reverse mode

Fig. 6.2

Mode of operation of NCX: (a, b) V_m shown as E_m on graph, intracellular Ca²⁺ ([Ca²⁺]_i) and E_NCX, with a low intracellular Na⁺ ([Na⁺]_i) and b high [Na⁺]_i. Notice that with higher [Na⁺]_i, V_m > E_NCX for a greater period of time, meaning NCX functions in reverse mode for longer, promoting higher [Ca²⁺]_i. c Driving force corresponds to the thermodynamic drive, which determines the mode of operation of NCX according to Eq. 6.2. When V_m − E_NCX > 0 there is Ca²⁺ influx due to the transporter operating in reverse mode. d Current generated by NCX (I_NCX). Notice that I_NCX > 0 (i.e. outward) when V_m > E_NCX and I_NCX < 0 (i.e. inward) when V_m < E_NCX. Also, notice that when E_NCX = V_m, I_NCX = 0. (I_NCX = I_NA/Ca)(Image from [5])

Ultimately, whether NCX promotes Ca²⁺ influx or efflux depends on its mode of operation, determined by (a) V_m, (b) E_Na, and (c) E_Ca. Thus, although intuitively it is sensible (and typically correct) to proclaim that when the subsarcolemmal Ca²⁺ is high NCX will favour Ca²⁺ extrusion, the mode of operation is not merely a function of Ca²⁺ (and by extension its equilibrium potential), but also E_Na and V_m. Evidently, the ability of NCX to operate bidirectionally makes it a pivotal player in Ca²⁺ homeostasis.

6.3 Ca²⁺ Efflux

The dissociation of Ca²⁺ from troponin on myofilaments allows relaxation to take place. For this to happen, Ca²⁺ must be removed from the cytoplasm. This occurs via four mechanisms:

• 1. Sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA)

• 2. NCX (forward mode)

• 3. Sarcolemmal Ca²⁺-ATPase

• 4. Mitochondrial Ca²⁺ transporters (into mitochondria)

6.3.1 SERCA Protein

Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a protein pump concentrated on the longitudinal component of the SR, transporting Ca²⁺ from the cytoplasm to the SR lumen [9]. It has three different isoforms (SERCA1, 2 and 3), with SERCA2a expressed abundantly in the heart [10]. The transport reaction involves multiple steps, beginning with the binding of 2 Ca²⁺ ions and 1 ATP molecule on the pump’s cytoplasmic side, in addition to phosphorylation. This triggers conformational alterations that facilitate the release of Ca²⁺ into the SR lumen, and H⁺ into the cytoplasm [11]. Perhaps counterintuitively then, relaxation, and not merely contraction, is energy dependent [12].

In general, the ATP concentration required to saturate SERCA is 1000× fold lower than the cytoplasmic ATP of a healthy CM at any given time, meaning that except in the energy-starved heart (e.g. failing, dysrhythmic), lack of ATP is not the rate-limiting factor for Ca²⁺ removal [12]. However, ATP can also allosterically modulate SERCA activity via a lower affinity binding site on the pump, such that in ischaemia it is the lack of this allosteric effect, rather than of ATP available for hydrolysis, that may disrupt relaxation kinetics [5, 12].

The main SERCA2a activity regulator is a homopentameric protein known as phospholamban (PLN) [5, 13]. When PLN is dephosphorylated, it tonically inhibits SERCA by increasing its K_m(Ca²⁺), meaning more Ca²⁺ is required to attain the same Ca²⁺ transport rate [14]. When phosphorylated (e.g. in response to adrenergic stimulation), this tonic inhibition is lifted, enhancing SERCA affinity for Ca²⁺ by decreasing its K_m(Ca²⁺), thus accelerating Ca²⁺ SR sequestration and relaxation.

PLN phosphorylation is at least in part accountable for the positive lusitropic effects observed in the presence of adrenergic stimulation. Phosphorylation of PLN has been demonstrated in three sites including (a) serine-16, (b) threonine-17 and (c) serine-10 by cAMP-dependent protein kinase A, Ca²⁺/Calmodulin protein kinase II and Ca²⁺-activated protein kinase C, respectively [14]. Dephosphorylation of PLN by SR-associated phosphatases restores PLN’s tonic inhibition [14].

Sarcolipin (SLN) (a PLN homologue) is another SERCA regulator, albeit less well understood. It has been suggested that when SLN is co-expressed with PLN, SERCA2a Ca²⁺ affinity decreases more than with PLN alone (◘ Fig. 6.3) [15, 16]. This may be due to the increased concentration of active (inhibitory) PLN monomers in the presence of SLN [17]. In particular, PLN is present either in homopentameric or monomeric form, the latter of which exhibits increased inhibitory activity [15]. When SLN is co-expressed, it holds PLN in a monomeric form (preventing it from polymerizing into pentamers), enhancing its inhibitory activity and further decreasing SERCA Ca²⁺ uptake [15]. However, SLN has also been proposed to inhibit SERCA by a mechanism independent of PLN, as seen with SLN overexpression in PLN(^−/−) (null mutants), which display impaired contractility, altered Ca²⁺ handling and relaxation [17, 18].

Fig. 6.3

Rate of Ca²⁺ uptake in HEK-293 cells expressing SERCA2a alone, SERCA2a and PLN, SERCA2a and SLN, SERCA2a and PLN and SLN. Note that the Ca²⁺ uptake rate is considerably decreased in the latter. (Image from [16])

6.3.2 NCX

We previously considered the different modes of operation of NCX, demonstrating that when V_m < E_NCX, NCX extrudes Ca²⁺. In general, SERCA and NCX are the two main transporters responsible for relaxation, and at any one time are in the state of constant competition for Ca²⁺. This has important physiological implications and can be demonstrated by considering Ca²⁺ efflux in different species. For example, after a period of rest (i.e. no electrical stimulation), rabbit cardiac preparations show a decline in the amplitude of the first post-rest contraction, termed rest decay [19, 20] (◘ Fig. 6.4).

Fig. 6.4

Rest decay and rest-potentiation in rabbits and rats, respectively. After a period of electrical rest for 100 s, the first contraction after rest is decreased to 75% of contraction during steady state in rabbits. In contrast, rats exhibit post-rest potentiation, increasing their contraction amplitude to approximately 140% after a period of electrical rest. Notice that thermodynamic inhibition of NCX in rabbits (0Na, 0Ca) converts the post-rest decay into post-rest potentiation. Similarly, applying a 0Ca²⁺ extracellular solution in rats favours Ca²⁺ extrusion by NCX and converts the typical post-rest potentiation into post-rest decay (NT = Normal Tyrode’s solution). (Image from [19])

In contrast, rat CMs have an increased contraction amplitude after a period of rest – known as post-rest potentiation [19]. Such phenomena are explained by the dynamic relationship of Ca²⁺ extruders. In both rabbits and rats during rest, Ca²⁺ leaks from the SR due to the random openings of RyRs (see Ca²⁺ sparks, below). Ca²⁺ is then subjected to two opposing forces – SERCA and NCX. In rabbits, NCX moves Ca²⁺ out of the cell, progressively decreasing the SR Ca²⁺ content, which results in less Ca²⁺ available for myofilament activation and a diminished contraction post-rest [5, 19, 20]. In contrast, rats have high intracellular Na⁺ (resulting in V_m > E_NCX), slow NCX, and fast SERCA transport rates, the summation of which leads to increased SR Ca²⁺ content during rest and a potentiated contraction post-rest [19].

6.3.3 Other Extrusion Mechanisms

When caffeine is applied on cardiac preparations Ca²⁺ stored in the SR is released. If NCX and SERCA are blocked, the rate of cytoplasmic Ca²⁺ removal is significantly slowed but not completely abolished [21].

This is because in addition to NCX and SERCA, Ca²⁺ removal also occurs by sarcolemmal Ca²⁺-ATPase and the mitochondrial Ca²⁺ uniporter (MCU). Sarcolemmal Ca²⁺-ATPase utilises ATP to efflux Ca²⁺ out of the cell whilst MCUs facilitate the flux of Ca²⁺ in mitochondria down a large electrochemical gradient [22, 23]. With the exception of a few species (e.g. ferret), the contribution of these in removing Ca²⁺ on a beat-to-beat basis is marginal when compared to NCX or SERCA [24]. As such, they are known as slow extruders.

However, their role in maintaining CM health and function is anything but marginal. For instance, increased mitochondrial Ca²⁺ loading activates energy production (e.g. via ATP synthase) allowing CMs to cope with increased energy demands, yet prolonged periods of elevated intracellular Ca²⁺ can trigger mitochondrial dysfunction and acute CM death [25]. Ultimately, the rate of Ca²⁺ removal from the CM cytosol can be quantified to highlight the rate of removal by each transporter, such that the total rate of Ca²⁺ removal is equal to:

$$ \frac{d\left[\mathrm{Ca}\right]t}{dt}={J}_{\mathrm{SERCA}}+{J}_{\mathrm{NCX}}+{J}_{\mathrm{slow}} $$

(6.4)

where J is the rate of removal of extrusion components, each governed by a nonlinear function dependent on Ca²⁺ concentration [5].

6.4 Excitation-Contraction Coupling

I_Ca generated during the AP enhances Ca²⁺ release from the SR via an RyR-mediated process termed calcium-induced calcium release (CICR) that forms the basis of ECC [26]. Ryanodine receptors (RyRs) are Ca²⁺ release channels embedded on the SR membrane, which open in response to cytosolic Ca²⁺ [1]. There are three different RyR isoforms, with RyR2 being mainly expressed in the heart [27]. Particularly, RyRs are found in discrete groups on the junctional SR (i.e. the part of the SR in close proximity to the sarcolemma), establishing functional Ca²⁺-release units known as couplons [1]. Ca²⁺ influx causes multiple couplons to open, resulting in the release of Ca²⁺ from the SR and the development of the Ca²⁺-transient.

Each couplon consists of approximately 100 RyRs closely apposed to approximately 10–25 sarcolemmal LTCCs, forming the cardiac dyad and separated by a nanometres-wide cleft known as the dyadic space (or cleft) [2].

The close proximity between RyRs and LTCCs in the dyad is made possible by deep invaginations of the sarcolemma known as t-tubules (TT) and ensures efficient coupling between I_Ca and SR-Ca²⁺ release [1, 2, 6]. Specifically, one LTCC opening within a cardiac dyad is sufficient to trigger SR-Ca²⁺ release from a whole couplon, meaning that the ≅10–25 LTCCs for ≅100 RyRs ensures a safety margin for triggering SR-Ca²⁺ release [26,27,28]. In addition to the role of cytosolic Ca²⁺ in triggering Ca²⁺ release via RyRs, SR luminal Ca²⁺ also plays a pivotal role in SR-Ca²⁺ release [29]. For instance, increased SR Ca²⁺ content can stimulate Ca²⁺ release, whilst RyR2 activity is diminished as luminal Ca²⁺ decreases [29].

6.4.1 Calcium Sparks

In 1993, Cheng et al. used fluorescent Ca²⁺ indicators and laser scanning confocal microscopy to describe the concept of Ca²⁺ sparks for the first time [26]. Ca²⁺ sparks are microscopic elevations of cytoplasmic Ca²⁺ reflecting the synchronous opening of a cluster of RyRs [1]. They occur by either of two mechanisms described below:

• 1. Stochastic openings of RyRs [26,27,28,29,30]. The open probability of a single or a small number of RyRs can randomly become non-zero, triggering nano-elevations of the resting [Ca²⁺]_i to about 170 nM [26]. This is important, as in pathological states associated with Ca²⁺ overload (supranormal SR Ca²⁺-content), spontaneous SR Ca²⁺ release events can cause spark-induced spark-release, leading to high [Ca²⁺]_i (“macrosparks”, ≅ 500 nM) and the successive development of dysrhythmogenic waves [26, 28].

• 2. Evoked by I_Ca that raises local subsarcolemmal Ca²⁺ and activates RyRs [26, 30]. During the cardiac action potential, I_Ca evokes multiple Ca²⁺ sparks by stochastically activating clusters of RyRs. The spatiotemporal summation of ≅10⁴ individual Ca²⁺ sparks results in the production of the seeming spatially uniform Ca²⁺ transient [28].

6.4.2 Calcium-Induced Calcium Release

The idea that the Ca²⁺ transient consists of many individual ‘atomic’ subevents (i.e. Ca²⁺ sparks) revolutionised the ECC paradigm. Previously, many models of cardiac CICR assumed a common pool of cytosolic Ca²⁺, consisting of homogenously distributed Ca²⁺ from I_Ca and SR Ca²⁺ release, with the former controlling the latter [31].

According to that model, when I_Ca stimulates SR Ca²⁺ release and the pool begins to fill with Ca²⁺, a positive feedback loop is established in which Ca²⁺ released from the SR triggers more SR-Ca²⁺ release [31]. This makes SR-Ca²⁺ release an all-or-none response such that once the SR-Ca²⁺ release process commences, CICR is expected to evolve autonomously, irrespective of sarcolemmal Ca²⁺ influx [32].

However, experimental evidence does not support the notion that SR-Ca²⁺ release becomes autonomous. Instead, it is accepted that the magnitude of SR Ca²⁺-release is a function of I_Ca [31, 33]. As I_Ca is primarily carried by the LTCCs, SR Ca²⁺ release is dependent on membrane potential [34]. If I_Ca is abruptly terminated by depolarisation above the LTCC reversal potential, SR Ca²⁺ release is also terminated [31, 35]. Therefore, SR Ca²⁺ release is graded – meaning it is a function of Ca²⁺ influx through LTCCs (I_Ca) (i.e. dependent on duration and magnitude of Ca²⁺ entry) [31, 36]. This is depicted in ◘ Fig. 6.5, which shows the typical characteristic bell-shaped voltage dependency of (a) I_Ca and (b) cell shortening (reflecting Ca²⁺ transient magnitude). Such a graded response is not in accordance with common pool models, which would be expected to cause an ‘all-or-none’ SR-Ca²⁺ release.

Fig. 6.5

Bell shaped LTCC current (I_Ca) in response to increasing membrane potential (voltage-clamp). Notice bell-shaped relationship of cell-shortening as well, reflecting bell-shaped Ca²⁺ transient amplitude (peaking at approximately 10 V)

To explain this gradation, Stern et al. proposed the local control theory of ECC, whereby Ca²⁺ sensed by RyRs is not the same as the average cytoplasmic [Ca²⁺]_i [33]. In particular, the opening of LTCCs causes a very high and rapid local rise of [Ca²⁺] within a cardiac dyad to >10 μM [33]. This activates RyRs within a couplon, causing SR-Ca²⁺ release to further elevate local [Ca²⁺]_i [33].

It is also proposed that the sensitivity of RyRs to Ca²⁺ is much less than the ambient cytosolic Ca²⁺, preventing an ‘all-or-none’ regenerative calcium release [33]. Therefore, although CICR may be regenerative within an individual couplon (i.e. Ca²⁺ released by one RyR in a couplon triggering Ca²⁺ release by other RyRs in the same couplon – a positive feedback loop), Ca²⁺ released from one couplon does not spread in sufficiently high amounts to trigger Ca²⁺ release from neighbouring couplons [36].

These Ca²⁺ release events triggered by individual stochastic openings of LTCCs are in essence Ca²⁺ sparks, the spatial and temporal summation of which leads to the whole-cell Ca²⁺ transient [30]. Gradation of Ca²⁺ transient then occurs by the stochastic recruitment of more or less Ca²⁺ sparks according to the membrane potential (and by extension I_Ca) [34]. Ultimately, the distinction between common pool and local control models of ECC is highlighted by the fact that in the latter, elementary Ca²⁺-sparks are recruited not by the mean [Ca²⁺]_i in the cell, but rather by the amount of Ca²⁺ flowing through the sarcolemmal LTCC, elevating local [Ca²⁺]_i in the cardiac cleft nanodomain [37] (◘ Fig. 6.6).

Fig. 6.6

Close apposition of sarcolemmal LTCCs and RyRs on the junctional SR promote efficient coupling. During an AP, I_Ca increases [Ca²⁺]_i in the cardiac dyadic cleft, evoking Ca²⁺ sparks via RyR activation. Local [Ca²⁺]_i in the cardiac dyadic cleft is much higher than [Ca²⁺] in the bulk cytosolic space, allowing for RyR activation in couplons despite their low Ca²⁺ sensitivity

6.5 What We Don’t Know

6.5.1 Ca²⁺ Spark Theories Interlude

But what determines whether a Ca²⁺ spark will actually be evoked? Santana et al. proposed that the probability of triggering a Ca²⁺ spark is dependent upon the square of the local Ca²⁺ concentration in the nanodomain, and that opening of a single LTCC is sufficient for this to happen [30, 38] – that is:

$$ P\left(\mathrm{spark}\right)={\left(\mathrm{local}\left[{\mathrm{Ca}}^{2+}\right]\right)}^2 $$

Appreciating the role of local [Ca²⁺] in triggering a Ca²⁺ spark is critical, as alterations in the microarchitecture of the ECC apparatus (e.g. in the geometric arrangement of LTCCs and RyRs) seen with pathology, can affect the P(spark), leading to Ca²⁺ handling abnormalities with implications for cardiac contractility [30].

By far the most accepted mechanism of CICR is the RyR-mediated release of SR Ca²⁺, triggered by the influx of Ca²⁺ through the LTCCs [1]. Yet, NCX has been postulated to be involved in CICR [39]. In isolated CMs, LeBlanc et al. blocked LTCC Ca²⁺ influx using nisoldepine, demonstrating that voltage-clamp depolarisations caused an initial rapid inward current, followed by a rise in [Ca²⁺]_i, both of which were abolished with application of tetrodotoxin (TTX, a Na⁺ channel inhibitor), suggesting that the observed Ca²⁺ transient was Na⁺ channel-dependent [39]. Following further experiments, they concluded that the initial depolarisation upstroke due to the inward Na⁺ current, coupled with the increasingly positive V_m, promotes transient ‘reverse mode’ in NCX operation, leading to Ca²⁺ influx and providing the trigger for CICR [9, 39].

Another example comes from NCX^−/− isolated ventricular myocytes. These cells display normal ECC, however in the presence of heavy Ca²⁺ buffering (minimising the effect of Ca²⁺ influx from LTCCs), reduced coupling efficiency is observed vs. wild-type CMs. This suggests there is an increased proportion of couplons failing to activate during the AP in the NCX^−/− myocytes compared to wild type [40]. Accordingly, Goldhaber et al. proposed that NCX has a role in maintaining coupling during depolarisation by priming the dyadic space with a subthreshold amount of Ca²⁺, meaning only a small amount of further Ca²⁺ from LTCCs is required to trigger CICR [40]. Others have remained sceptical of the role of NCX in ECC [41, 42], as (a) Na⁺ channels may be excluded from the dyadic cleft, and (b) NCX as a transporter (and not an ion channel) is notably slower than LTCCs, meaning that when both co-exist, CICR is dominated by the latter [1, 43].

6.5.2 CICR Termination – Stopping the Domino Effect

We have seen that couplons are separated from each other and that according to the local control theory of ECC, RyR Ca²⁺ sensitivity is low enough so that Ca²⁺ released from one couplon does not trigger Ca²⁺ release from neighbouring couplons. Yet, as Ca²⁺ is both the cause and effect of the release, SR-Ca²⁺ release should still be inherently regenerative within a couplon [5]. However, with approximately 50% (i.e. not the whole amount) of SR Ca²⁺ released in each contraction, what terminates the release of Ca²⁺ from the SR?

Proposed mechanisms include stochastic attrition, ryanodine receptor inactivation, adaption and local depletion of Ca²⁺ in the SR [1, 36]. Despite the number of approaches to explain CICR termination, a single unifying mechanism does not exist, with a weighted combination of the different theories likely responsible.

Stochastic Attrition

Proposed by Stern et al. and suggests that random simultaneous closure of RyRs in a couplon could abruptly break the positive feedback cycle, halting the regenerative nature of CICR [36, 44]. As RyR are stochastically oscillating between closed and open states, there is always a chance that all channels close at the same time, degrading the local Ca²⁺ gradient [36]. The probability of this happening is dependent upon (a) the probability of the RyR being open (P_o), (b) the average time they remain open (τ_ο) and (c) the number of RyR in a couplon (n) [36]. Generally, it can be shown as P_o, τ_ο and n increase, the probability of simultaneous, stochastic closure of all RyRs drops exponentially [1, 36]. Thus, although stochastic attrition is a mechanism that is always present, it is rather improbable that it singlehandedly terminates the CICR of a highly active calcium synapse [36].

Ryanodine Receptor Inactivation

A Ca²⁺-induced inactivation mechanism was originally proposed by Fabiato, who used skinned myocytes to suggest that binding of Ca²⁺ to a high-affinity site on RyR inactivated the channel, stopping the Ca²⁺ release process [45]. Therefore, and perhaps conveniently, much like Ca²⁺-dependent inactivation of LTCCs, Ca²⁺-dependent inactivation of RyR may terminate CICR. In reality however, with most experiments being performed in planar bilayers and never validated in intact cells, this mechanism’s contribution to SR-Ca²⁺ release termination remains unclear [36, 45].

Adaption

RyRs relax to a lower P_o after activation. Strictly speaking, adaption is not RyR inactivation per se as the channels can be reactivated with exposure to higher [Ca²⁺]_i [1, 45].

Depletion of Local SR Luminal Ca²⁺

Opening of RyRs could lead to depletion of SR lumincal Ca²⁺ near the channel, resulting in either the flux of Ca²⁺ out of the SR to become zero (i.e. there is no more Ca²⁺ to be released), halting the positive feedback loop and ending CICR; or, causing the probability of RyR opening (in part determined by luminal Ca²⁺) to be decreased such that no more Ca²⁺ is released [36]. As cytosolic Ca²⁺ is constantly being pumped into the SR, typically at a different location than the one releasing Ca²⁺, the validity of this mechanism is dictated by (a) the rate of Ca²⁺ reuptake and (b) the rate of diffusion of Ca²⁺ from the ‘uptake SR compartment’ to the ‘SR release compartment’ [36].

6.6 Where We’re Heading

6.6.1 ECC as a Measure of Cardiac Maturity

Stemcell-derived cardiomyocytes hold significant therapeutic promise for the treatment of diseased myocardium. These are stem cells that have been differentiated in to cardiomyocytes. A problem that has beset this field is the immaturity of such cells, in particular the absence of adult-like functional and morphological cardiomyocyte characteristics [46]. Components of the ECC seem particularly affected, with stem cell-derived cardiomyocytes having few or even no t-tubules, which can at least in part explain the abnormal Ca²⁺ dynamics and considerably weaker contractile force amplitudes than those developed by the adult myocardium (e.g. human ventricular strips twitch tension is ≅44 mN/mm² vs. 0.08 mN/mm² in human pluripotent stem cell-derived cardiomyocytes – a 550-fold decrease) [46, 47].

In cell therapy, stem cell-derived cardiomyocytes are transplanted onto the injured myocardium in an effort to increase the contractile ability of the heart. Lack of robust ECC apparatus questions whether beneficial effects (if any) of such approaches reflect the addition of force-generating cardiomyocytes or merely the release of nurturing paracrine mediators from these cells [48].

Furthermore, transplantation of immature cells that beat asynchronously, have improper Ca²⁺ homeostasis, and/or are comparatively weaker to human cardiomyocytes may increase the dysrhythmogenic risk and by extension the safety of such approaches [4].

6.6.2 ECC in Pathology

Abnormal ECC underlies many pathological processes. In the failing heart, characterized by an inability to maintain a cardiac output sufficient to meet the metabolizing needs of the body [49, 50], disrupted geometrical arrangements of the ECC components may compromise the fidelity of ECC [51, 52]. For example, among the most well-known phenotypical changes seen in failing cardiomyocytes is the loss of t-tubules [53, 46]. Such morphological changes can disrupt the tight coupling between the LTCC and RyRs, and diminish the ability of a given I_Ca to trigger a Ca²⁺ spark, (and by extension a Ca²⁺ transient), hampering contractile performance [4, 52, 53]. Ingenious computational models have shown that for any spatial arrangement of LTCC and RyRs, there is an optimal amount of Ca²⁺ influx required to maximally activate RyRs [31]. If that becomes suboptimal (e.g. excessive Ca²⁺ influx which only minimally activates RyRs) in pathology then renormalizing this relationship between Ca²⁺ influx and RyR response with drugs or interventions may be of therapeutic benefit by enhancing contraction while simultaneously minimizing supranormal Ca²⁺ influx. This is important as excess Ca²⁺ influx raises the probabilities of Ca²⁺-induced pathological states (e.g. Ca²⁺ overload-induced arrhythmias & activation of Ca²⁺ mediated pathological hypertrophy signalling pathways).

Take-Home Message

• During ventricular action potentials, Ca²⁺ influx from the extracellular to subsarcolemmal space generates a Ca²⁺ current, I_Ca, which initiates Ca²⁺ release from the sarcoplasmic reticulum via a process known as calcium-induced calcium release.

• I_Ca is generated by two main mechanisms: voltage-sensitive sarcolemmal Ca²⁺ channels (LTCCs) and Na⁺/Ca²⁺ exchangers (NCX).

• Calcium-induced calcium release is mediated by ryanodine receptors interacting in complex microdomains with LTCCs.

• The therapeutic usefulness of transplanting stem cell-derived cardiomyocyte onto injured myocardium is hindered in part by the deficient Ca²⁺ handling exhibited by these cells.