Contents

Summary

1. Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions.

2. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans), and Ca²⁺ transient amplitude (Ca²⁺ alternans).

3. The cause of alternans is multifactorial, however alternans always originate from disturbances of the bi-directional coupling between membrane voltage (V_m) and intracellular calcium ([Ca²⁺]_i). Bi-directional coupling refers to the fact that in cardiac cells, V_m depolarization and the generation of action potentials cause the elevation of [Ca²⁺]_i that is required for contraction (a process referred to as excitation-contraction coupling). The changes of [Ca²⁺]_i on the other hand control V_m because important membrane currents are Ca²⁺-dependent.

4. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca²⁺ homeostasis. Here we review how two key factors of cardiac cellular Ca²⁺ signaling - the release of Ca²⁺ from internal stores and the capability of clearing the cytosol from Ca²⁺ after each beat - determine the conditions under which alternans occurs. The contributions from key Ca²⁺ handling proteins - surface membrane channels, ion pumps and transporters, and internal Ca²⁺ release channels - are discussed.

Introduction

Cardiac alternans refers to a condition characterized by a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. The clinical manifestations of alternans occur in many settings in which arrhythmias are also common; however, its origin can be followed to the cellular and subcellular level. Here, we will review the alternans field from the perspective of the cellular disturbances of electrical and calcium signaling which lead to the proarrhythmic condition of alternans.

Excitation-contraction coupling in cardiac muscle

Each heartbeat requires a coordinated activation of cardiac muscle cells to sustain the pump function of the heart. Excitation-contraction coupling describes the process that converts electrical activation into mechanical activity and muscle contraction. The sequence of events begins with depolarization of the surface membrane potential (V_m) by an action potential, followed by the entry of extracellular calcium through voltage-gated sarcolemmal L-type Ca²⁺ channels (also referred to as dihydropyridine receptors, DHPRs). Ca²⁺ influx triggers intracellular Ca²⁺ release by activating Ca²⁺-sensitive Ca²⁺ release channels (ryanodine receptors, RyRs) in the sarcoplasmic reticulum (SR) membrane by a mechanism termed Ca²⁺-induced Ca²⁺ release (CICR).¹ The amplified Ca²⁺ release from the SR raises intracellular [Ca²⁺] ([Ca²⁺]_i) which activates the contractile apparatus and force is produced. Relaxation of cardiac cells is dependent upon mechanisms that lower [Ca²⁺]_i through reuptake into the SR by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) and extrusion from the cell primarily via sarcolemmal sodium-calcium exchange (NCX). Reuptake of Ca²⁺ provides the necessary filling of the SR to allow sufficient Ca²⁺ for release during the next heartbeat.

Ventricular myocytes typically have a well-developed transverse (t) tubular system. The t-tubular system consists of invagination of the surface membrane that extends as a 3-dimensional network of narrow transverse and longitudinal tubules throughout the entire cell.² Approximately 30-50% of the sarcolemma exists as the t-tubular system and forms a well-connected membrane network within the cell, but contiguous with the extracellular space. DHPRs together with many other ion channels and transporters are located in the t-tubular membrane. Clusters of RyRs on the terminal cisternae of the SR membrane appose DHPRs separated only by a narrow (a few nanometers) cleft, forming a dyad of two adjacent membranes.³ The dyad is the functional unit of SR Ca²⁺ release, termed SR Ca²⁺ release unit (CRU)⁴ or couplon.⁵ Ca²⁺ sparks are considered the elementary events of Ca²⁺ signaling in cardiac cells⁶ arising from CICR at individual CRUs, and according to the ‘local control’ model of cardiac excitation-contraction coupling⁷ are recruited independently and spatially summate to produce a Ca²⁺ transient.^8,9 The well developed t-tubular network in ventricular myocytes ensures simultaneous activation of SR Ca²⁺ release throughout the entire ventricular myocyte during an action potential, resulting in spatially separate rather homogeneous Ca²⁺ transients (Figure 1A).

Figure 1. Cellular and subcellular Ca²⁺ alternans in cardiac myocytes. A,B: Spatiotemporal characteristics of Ca²⁺ transients during alternans in a atrial (\f4A\f2) and ventricular (\f4B\f2) myocyte. From top: whole cell Ca²⁺ transients, transverse confocal line scan images and subcellular [Ca²⁺]_i profiles recorded from subsarcolemmal (ss, black; corresponding to j-SR Ca²⁺ release) and central (ct, red; corresponding to nj-SR Ca²⁺ release) regions of the myocyte. Panels A and B modified from Hüser et al.¹⁰ with permission. C: Spatiotemporal characteristics of Ca²⁺ transients during alternans in an atrial myocyte where subcellular discordant or ‘out-of-phase’ alternans are present. The global [Ca²⁺]_i profile suggests no Ca²⁺ alternans, however spatially restricted profiles identify subcellular regions with no alternans coexisting with regions alternating out-of-phase.

The fundamental process of excitation-contraction coupling in atrial and ventricular cells shows similarities, but also important structural and functional differences. The t-tubular system in atrial cells is significantly less developed or even entirely absent,^10,11 although there are species differences. For example, rudimentary t­tubular structures are found in rat,¹² sheep¹³ and dog.¹⁴ The spatial vicinity to the surface membrane defines two types of SR, termed junctional (j-SR) and non-junctional (nj-SR) SR. Because of the absence of t-tubules in atrial myocytes j-SR is restricted to the cell periphery. Both j-SR and nj-SR express RyRs, and - compared to ventricular myocytes - have a higher density of IP₃ receptors.^15,16 In atrial cells, peripheral j-SR and the more centrally located nj-SR are capable of active and robust SR Ca²⁺ release, however the mechanism of activation differs. Action potential-induced membrane depolarization activates Ca²⁺ entry through L-type Ca²⁺ channels which triggers CICR from RyRs of the j-SR. Elevation of peripheral [Ca²⁺]_i propagates then via CICR in a Ca²⁺ wave-like fashion in centripetal direction by a diffusion-reaction process or a ‘fire-diffuse-fire’ mechanism (Figure 1B). As a characteristic consequence of this mode of activation and ultrastructural arrangements, Ca²⁺ release is spatially inhomogeneous^16-18 with complex subcellular [Ca²⁺]_i gradients (Figures 1B and 1C). These structural and functional differences are important for the susceptibility to spontaneous pro-arrhythmic Ca²⁺ release events (Ca²⁺ waves) and the propensity to develop cardiac alternans as will be discussed below.

Cardiac alternans

In 1872, for the first time a very interesting phenomenon, consisting of beat-to­beat oscillations in arterial pressure that occurred while the heart rate remained constant, was reported by Traube.¹⁹ This observation, called ‘pulsus alternans’ would ultimately be known as mechanical alternans. With the arrival of the electrocardiogram (ECG) similar beat-to-beat alternations of electrical activity of the heart (electrical alternans) were recorded in laboratory animals²⁰ and humans,²¹ and are typically referred to as repolarization or T-wave alternans. It was recognized early on that conditions of pulsus alternans were associated with severe cardiac pathologies and poor prognosis.²² To date, it is well established that cardiac alternans is linked to increased risk for atrial and ventricular arrhythmias and sudden cardiac death across a wide range of pathophysiological conditions, including ischemia and myocardial infarction.^16,23-29 T-wave alternans in the ECG and microvolt electrical alternans testing have become a prognostic tool for arrhythmia risk stratification and antiarrhythmic therapy.^30-32

At the cellular level, cardiac alternans is defined by cyclic, beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans), and Ca²⁺ transient amplitude (Ca²⁺ alternans) at constant stimulation frequency (Figure 2). Alternans is induced typically by rapid heart rates, however, the pacing threshold required to initiate it is influenced by a wide variety of factors and conditions^33-36 and varies among different mammalian species.^37,38 Conditions that lower the pacing threshold include hypothermia,^38-42 interference with cellular energy metabolism through inhibition of glycolysis,^10,43-45 hypocalcaemia,^38,41,46,47 disturbance of mitochondrial functions,^44,48,49 hypercapnic acidosis,^50,51 ischemia,^52-56 hypertrophy,⁵⁷ IP₃ receptor-dependent Ca²⁺ release,^58,59 and heart failure.^60-62 A shift to a higher pacing threshold for alternans has been reported in conditions of hypercalcaemia,^38,46 pharmacological sensitization of the SR Ca²⁺ release channels,⁶³ and calcium channel antagonists.^42,54 Interestingly, β-adrenergic stimulation, while generally having positive inotropic effects, can either enhance⁶⁴ or suppress^10,44,48 alternans (cf. discussion below).

Figure 2. Electrical, mechanical and Ca²⁺ alternans in cardiac myocytes. A: Simultaneous recordings of action potentials and cell shortening from a single ventricular myocyte revealing discordant electromechanical alternans. To the right, two action potentials recorded during successive small- (open circle) and large-amplitude (filled circle) shortenings are superimposed to illustrate the differences in duration and kinetics. Modified from Hüser et al.¹⁰ with permission. B: Simultaneous recordings of cytosolic ([Ca²⁺]_i; top) and intra-SR ([Ca²⁺]_SR; bottom) Ca²⁺ alternans from a single ventricular myocyte. C: Simultaneous recordings of [Ca²⁺]_i (top) and I_Ca (bottom) in voltage-clamped atrial myocytes. To the right, an overlay of I_Ca measured during a large-amplitude Ca²⁺ transient (L; blue trace) and a small-amplitude Ca²⁺ transient (S; red trace) shows that Ca²⁺ alternans are not accompanied by alternating peak I_Ca. Modified from Shrkyl et al.⁶ with permission.

Mechanism of cardiac alternans: bi-directional coupling between V_m and [Ca²⁺]_i

The plethora of studies on cardiac alternans clearly document that this proarrhythmic condition is multifactorial. Nonetheless, it is generally agreed that instabilities of the bi-directional coupling of V_m and [Ca²⁺]_i are a cucial factor for the generation of alternans. ‘Bi-directional’ coupling refers to the fact that membrane depolarization in form of an action potential is required to initiate Ca²⁺ release and to elevate [Ca²⁺]_i, however the ensuing dynamics of [Ca²⁺]_i affect V_m through the Ca²⁺­dependence of numerous membrane conductances as outlined below.⁶⁵ Consequently, the question arises as to whether alternans are either V_m or [Ca²⁺]_i driven.^35,36,66,67 As such, a classic ‘chicken or egg conundrum’ exists in the literature relating to the fact that the mechanisms responsible for alternans remain incompletely understood.^68-70

V_m→[Ca²⁺]_i coupling

V_m-driven alternans is determined by a single parameter - APD restitution. The key concept behind the paradigm of V_m-driven alternans is that APD restitution is a time-dependent process resulting from the fact that recovery from inactivation of ion currents underlying the action potential requires time (thus resulting in absolute and relative refractoriness of excitability). APD restitution is defined as the relationship between APD and diastolic interval (DI). The heart rate is inversely related to cycle length (CL), which is calculated as CL = APD + DI. When heart rate increases, the APD shortens to preserve the diastolic interval for ventricular filling. Therefore, electrical alternans is critically dependent on beat-to-beat changes in diastolic interval. V_m→[Ca²⁺]_i coupling is generally believed to be positive, i.e., a long APD is paralleled by a strong contraction and large amplitude Ca²⁺ transient. Positive coupling between APD and Ca²⁺ transient or contraction amplitude is also referred to as ‘in-phase’ or ‘concordant’ at the cellular level. ‘Negative’ V_m↔[Ca²⁺]_i coupling results in ‘discordant’ or ‘out-of-phase’ alternans at the single cell level (Figure 2A). The term ‘discordant’ is also used at the multicellular tissue level where it refers to different regions of the myocardium alternating asynchronously or ‘out-of-phase’. Such regions are separated by nodal lines⁷¹ which mark areas of highest [Ca²⁺]_i and APD gradients and become sites of origin for arrhythmias. The terminology discordant/concordant is also used at the subcellular level and describes alternans pattern of subcellular regions within a single cell (Figure 1C).^43,72,73 Alternations of the diastolic interval is critical for the availability of the L-type Ca²⁺ channel current (I_Ca,L) at a given heartbeat. A longer diastolic interval allows more time for recovery of I_Ca,L, leading to enhanced I_Ca,L, larger Ca²⁺ release and longer APD during the following beat. Now the longer APD is followed by a shorter diastolic interval, leading to less recovery of I_Ca,L with less Ca²⁺ release and shorter APD during the next beat, thus sustaining alternans.

[Ca²⁺]_i→V_m coupling

[Ca²⁺]_i→V_m coupling is determined by the fact that [Ca²⁺]_i feeds back on V_m. This occurs through the Ca²⁺-dependence of ion channels and transporters, i.e. membrane conductances that in turn also control [Ca²⁺]_i cycling. With respect to cardiac alternans, I_Ca,L and I_NCX are most important.^35,36 [Ca²⁺]_i→V_m coupling can be positive or negative depending on which of the Ca²⁺-dependent ion currents or transporters dominates. For example, a positive [Ca²⁺]_i→V_m coupling occurs when the large Ca²⁺ transient causes a prolongation of APD by potentiating the inward I_NCX (1 Ca²⁺ ion extruded in exchange to 3 Na⁺ ions) to a greater extent than reducing I_Ca,L through Ca²⁺-dependent inactivation.

Negative [Ca²⁺]_i→V_m coupling occurs when reduction of I_Ca,L dominates over increased I_NCX which ultimately results in APD shortening. Other Ca²⁺-sensitive currents (non­selective cation current, Cl⁻ current) may modulate [Ca²⁺]_i→V_m coupling, but appear to be quantitatively less important.

Two key parameters relevant to the generation of [Ca²⁺]_i-driven alternans at the cellular level are i) fractional Ca²⁺ release from the SR and SR Ca²⁺ load, and ii) the efficiency of beat-to-beat cytosolic Ca²⁺ sequestration.^35,36 Fractional release of Ca²⁺ refers to the nonlinear relationship between the end-diastolic SR Ca²⁺ content and the amount of Ca²⁺ (or % of SR Ca²⁺ content) released by CICR with each heartbeat (i.e., a larger fraction of Ca²⁺ is released at a higher SR Ca²⁺ content).⁷⁴ Ca²⁺ sequestration is a phenomenological parameter and refers to the net efficiency of cytosolic Ca²⁺ removal. It is dependent on i) the activity of SERCA to reload the SR, ii) Na⁺/Ca²⁺ exchange and plasmalemmal Ca²⁺-ATPase activity to extrude Ca²⁺ from the cell, iii) cytosolic buffering (including mitochondrial Ca²⁺ uptake), and iv) diastolic SR Ca²⁺ leak. Therefore, alternans can occur at modest SR loads and small fractional releases under conditions where Ca²⁺ sequestration is low. Alternatively at high sequestration rates, higher Ca²⁺ loads and fractional release are required to induce alternans. In general, factors increasing fractional release promote, and factors increasing Ca²⁺ sequestration efficiency protect against alternans. To illustrate, in heart failure where SERCA expression is reduced and Ca²⁺ release from the SR is increased, or during acute cardiac ischemia (where SR Ca²⁺ load is initially unaffected, but SERCA activity is diminished due to reduced ATP levels), the heart is pushed into instability due to diminished Ca²⁺ sequestration. On the other hand, under β-adrenergic stimulation SERCA activity and consequently SR Ca²⁺ uptake and load are increased, leading to enhanced fractional release that tends to promote alternans. Increased SERCA activity, however, also increases the efficiency of Ca²⁺ sequestration, resulting in protection against alternans. Whether β-adrenergic stimulation favors⁶⁴ or protects^10,44,48 against alternans and alternans-related arrhythmias depends on which β-adrenergic effects predominate.

Recently, an overarching conceptual model for cardiac alternans has been forwarded, termed ‘3R theory’.^34,75,76 The 3R theory links Ca²⁺ spark properties, i.e. the properties of Ca²⁺ release from individual CRUs, to whole-cell Ca²⁺ alternans. Ca²⁺ alternans occurs due to instabilities of the relationship of 3 critical spark properties (the ‘3 Rs’): 1) Randomness of Ca²⁺ sparks, 2) Recruitment of sparks by neighboring CRUs, and 3) Refractoriness of a CRU. An individual CRU can be in 3 different states: recovered (i.e. ready to fire), firing or refractory. The theory predicts (by numerical computations) that alternans occurs when the probability of a spontaneous primary spark is intermediate (intermediate randomness), coupling among CRUs is strong (high probability of a primary spark triggering a secondary spark from a neighboring CRU; high degree of recruitment), and a high degree of refractoriness is prevalent (i.e. the probability of a CRU not being recovered from previous release is high). This unifying theoretical framework predicts how Ca²⁺ cycling proteins and organelles (L-type Ca²⁺ channels, RyR, SERCA, NCX, Ca²⁺ buffers and mitochondria) affect the 3 Rs and SR Ca²⁺ load, and thus the prevalence of Ca²⁺ alternans. Interestingly, in the 3R framework SR Ca²⁺ load is not an explicit parameter which is consistent with our observation that Ca²⁺ alternans are not dependent on alternating end-diastolic [Ca²⁺]_SR.^10,63,77 Nonetheless, SR Ca²⁺ load is a critical factor for Ca²⁺ alternans since load determines the efficiency of the L-type Ca²⁺ current to trigger release; it controls RyR function through its luminal Ca²⁺ sensitivity and influences refractoriness of release. In the next section we will summarize the specific contributions of the major Ca²⁺ signaling proteins and organelles to alternans.

As mentioned earlier, alternans is a recognized risk factor for ventricular and atrial arrhythmias.^70,78,79 [Ca²⁺]_i→V_m coupling can be positive or negative, i.e., result in both concordant and discordant alternans. At the level of the heart, spatially discordant alternans favor re-entry, triggering ectopic beats and facilitating the onset of lethal arrhythmic events^80,81 whereas concordant alternans is considered less arrhythmogenic.⁸² At the cellular level atrial myocytes are particularly susceptible to Ca²⁺ alternans induced by pacing or metabolic inhibition. In atrial myocytes alternans is typically subcellularly inhomogeneous (Figures 1B and 1C). Subcellular inhomogeneities consist of subcellular transverse and longitudinal gradients of the degree of Ca²⁺ alternans, and subcellular regions alternating out-of-phase.^{10,16,43,45,59} These [Ca²⁺]_i gradients and inhomogeneities result from the unique structural and functional features of atrial excitation-contraction coupling and are consistent with simulation studies on the relationship between the lack of t-tubules and generation of alternans.⁸³ We demonstrated that the complex subcellular [Ca²⁺]_i inhomogeneities of atrial alternans generates a substrate for spontaneous (i.e., not electrically triggered) proarrhythmic Ca²⁺ release and represents a mechanistic link to atrial arrhythmia at the cellular level.⁴³ Of particular interest is the observation of subcellular ‘discordant’ Ca²⁺ alternans where subcellular regions alternate out-of-phase (Figure 1C). These subcellular areas are typically separated by regions where spontaneous Ca²⁺ waves originate with high probability, reminiscent of the nodal lines observed at tissue level.⁷¹

Thus, it appears that spatially discordant alternans phenomena at tissue level can be recapitulated at the cellular level.

Ca²⁺ handling proteins and organelles and their role in cardiac alternans

Although clearly a multifactorial phenomenon, consensus is emerging that electromechanical and Ca²⁺ alternans are ultimately linked to impaired [Ca²⁺]_i regulation, and [Ca²⁺]_i→V_m coupling dominates the mechanisms that are responsible for the occurrence of alternans.^68,84-86 In the following paragraphs we will address the contributions of L-type Ca²⁺ channels, SR and Ca²⁺ load, the SR Ca²⁺ release machinery (RyRs) and mitochondria to alternans.

L-type Ca²⁺ channels

Considering that I_Ca,L is the critical trigger for CICR during excitation-contraction coupling and SR Ca²⁺ release is graded with the magnitude of the current,^87,88 beat-to-beat alternation of I_Ca,L has been considered a candidate to cause alternans. A potential mechanism entails incomplete time-dependent recovery from inactivation of I_Ca,L which could lead to Ca²⁺ alternans.^89-91 This hypothesis, however would have to reconcile the observation in both atrial (Figure 2C) and ventricular myocytes that alternans can occur while peak I_Ca,L remains unchanged from one beat to the next.^{10,63,77,92-94} Furthermore, mechanical and Ca²⁺ alternans can occur in the absence of APD alternans (confirmed in patch-clamp experiments) and with constant I_Ca,L.^{10,63,92,95,96} Ca²⁺ alternans is observed even in myocytes stimulated at a high frequency during action potential voltage clamp in the absence of APD alternans.⁹⁵ Together these data suggest that I_Ca,L is unlikely paramount in the onset of alternans.

SERCA and SR Ca²⁺ load

It can be speculated that at higher pacing frequencies, limitations of SR Ca²⁺ uptake kinetics preclude adequate refilling of Ca²⁺ stores, particularly after a large Ca²⁺ transient. Consequently, the reduced filling only permits a small Ca²⁺ transient during the next beat thus resulting in Ca²⁺ alternans. This led to the suggestion that beat-to-beat alternations in end-diastolic SR Ca²⁺ load is a prerequisite for alternans,⁹³ possibly due to an instability in the feedback control of SR Ca²⁺ content.⁹⁷ However, direct and dynamic measurements of intra-SR [Ca²⁺] have shown (Figure 2B) that alternans do not require beat-to-beat alternations in SR Ca²⁺ content.^10,63,77 The role of Ca²⁺ reuptake into the SR and reestablishing Ca²⁺ load has been further investigated by enhancing SERCA activity.^98-100 Indeed, using genetic approaches to up-regulate SERCA2a (cardiac isoform) resulted in suppression of alternans.^83,100-102

RyR and restitution of SR Ca²⁺ release

Mitochondria

Concluding remarks

Cardiac alternans is an intriguing phenomenon with clinical implications to a range of cardiac pathologies, while also providing insights into the intricacies of cellular Ca²⁺ cycling in heart muscle. Although clearly a multifactorial process, the experimental, theoretical and computational data exploring electrical, Ca²⁺ and mechanical alternans indicate that dysfunctional Ca²⁺ cycling appears to be the crucial mechanistic link between the contractile dysfunction and electrical instabilities seen at the cellular level, as well as clinically in patients. Despite the complexity of cardiac Ca²⁺ signaling, recent years have seen remarkable progress towards the understanding of the phenomenon of cardiac alternans. Growing theoretical and experimental evidence emphasizes that cellular Ca²⁺ signaling - and particularly the key proteins responsible for beat-to-beat Ca²⁺ release - are at the ‘heart’ of the problem of cardiac alternans. The recognition of the central role of the cardiac Ca²⁺ release machinery for alternans will pave the way, by pharmacologically or genetically targeting these Ca²⁺ handling proteins, to develop novel therapeutic strategies for the suppression of cardiac arrhythmias.

Acknowledgments

This work was supported by an Early Career Fellowship from the National Health and Medical Research Council of Australia (JNE); and by National Institutes of Health Grants HL62231, HL80101 and HL101235, and the Leducq Foundation (LAB).

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