Damage-induced arrhythmias: reversal of excitation–contraction coupling

Time for primary review 16 days

1 Introduction

Sustained or self-terminating ventricular arrhythmias commonly arise as the oscillatory response of the mechanisms involved in re-entry to a premature beat in non-uniform working myocardium or conduction tissue. Non-uniformity of conduction in the human heart itself is often a result of myocardial ischemia or infarction due to coronary artery disease. Ischemic non-uniformity may further be aggravated by cardiac remodelling during development of congestive heart failure, thereby, providing a possible mechanism substrate for fatal arrhythmias which often cause the demise of patients with congestive heart failure [1, 2]. Most discussions of these arrhythmias emphasize the importance of electrical non-uniformity. Nevertheless, it is evident that myocardium that has been rendered non-uniform will also exhibit variations of mechanical stresses and strains from cell to cell as well as differences among cells in excitation contraction coupling, in addition to electrical non-uniformity.

Too little is known about the role played by non-uniform myocardial stress and strain distributions and by non-uniform excitation contraction coupling in mechanisms underlying premature beats that initiate an arrhythmia. Still, this knowledge is essential to the choice of treatment aimed at prevention of a premature beat versus treatment aimed at suppression of re-entry. An especially clinically important scenario in which arrhythmias occur is that of ischemic damage of myocardium. It is generally accepted that acute ischemic damage of myocardium leads after 20–30 min to Ca²⁺ overload of the damaged cells and their neighbours [3]. The arrhythmias of ischemic myocardium that occur in this phase may well be related to the Ca²⁺ overload [4]. Hence, it is conceivable that damage-induced premature beats may initiate re-entry arrhythmias and that abnormal cellular Ca²⁺ transport may play a crucial role in the initiation of the premature beat. The purpose of this paper is to review evidence for this concept. In particular, we will consider the evidence in support of a mechanism in which both non-uniform contraction and increased Ca²⁺ load of cells adjacent to acutely damaged cells are essential in the ‘spontaneous’ generation of Ca²⁺ transients during the relaxation phase of the electrically driven twitch. A novel aspect of this concept is that these Ca²⁺ transients induce propagating Ca²⁺ waves, which travel into the adjacent normal myocardium and cause after-depolarizations, which in turn may cause premature beats. We assume that the mechanism of initiation of the propagating Ca²⁺ waves involves feedback of rapid length or force changes to binding of Ca²⁺ ions to the contractile filaments. Hence, consideration of these mechanisms is of interest in the context of this issue of Cardiovascular Research.

2 After-contractions and after-depolarizations and Ca²⁺ transients

It has been known for half a century [5, 6], that two types of oscillations in membrane potential may follow an action potential of cardiac muscle: (1) early after-depolarizations and (2) delayed after-depolarizations (DAD). Widespread interest in DAD resulted in the early 70's from the observation that these after-depolarizations could be caused by digitalis intoxication [7–12]. Also, triggered arrhythmias appeared to result from DAD emphasizing their potential clinical importance [13]. A clue to their mechanism was provided by the observation that after-depolarizations in Purkinje fibers and the accompanying after-contractions occur when the muscle was ‘loaded’ with Ca²⁺. Both initiation after the last stimulated twitch and the time course of these transients accelerated if [Ca²⁺]₀ or the stimulation rate was increased. Digitalis appeared to accelerate the after-depolarizations even more than high [Ca²⁺]₀ and a high stimulus rate [14, 15].

Like in any arrhythmia, the action potentials that triggered arrhythmias both result from the previous impulse and lead to subsequent impulse generation. It has been shown that the DADs are based on a spontaneous increase in intracellular [Ca²⁺] ([Ca²⁺]_i) leading to a transient inward current on the one hand and to activation of the contractile filaments on the other hand [16, 17]. Kass et al. have proposed that a [Ca²⁺]_i transient assumed to be due to ‘spontaneous’ Ca²⁺ release from the sarcoplasmic reticulum (SR) leads to a transient inward current [17]. Hence, a sufficiently large Ca²⁺ load of the SR would create an unstable state where the spontaneous Ca²⁺ release may become so large that the resulting transient inward current depolarizes the cells enough to trigger a new action potential, which perpetuates itself as a triggered arrhythmia [13].

3 Spontaneous Ca²⁺ release from the SR and Ca²⁺ waves in myocytes

Spontaneous Ca²⁺ release from the sarcoplasmic reticulum has been well-documented in isolated enzymatically dispersed cardiac cells using confocal microscopy. These events range from Ca²⁺ release from single Ca²⁺ channels in the SR (Ryanodine receptors (RyR)) to Ca²⁺ release by clusters of RyR in response to Ca²⁺ entering via the L-type Ca²⁺ channels called sparks [18]. Ca²⁺ sparks may also trigger each other and cause Ca²⁺ waves as is suggested by the observation that Ca²⁺ sparks lead the wave front of Ca²⁺ waves [19]. The arrival of Ca²⁺-sensitive fluorescent dyes such as Fura 2, Indo 1, and Fluo 3 has made it possible to record images of the regional increase in [Ca²⁺]_i that elicit regional contractile activity. Clearly, isolated cardiac myocytes have proven to be an important tool to study spontaneous Ca²⁺-release events and their consequences for contractile activity providing insights into phenomena such as after-contractions, arrhythmias, and depression of systolic and diastolic function in the state of [Ca²⁺]_i overload [20]. While spontaneous activity is most easily observed in rat myocytes, essentially the same is seen in myocytes from other mammals under conditions in which [Ca²⁺]_i is forced to an excess [21]. We will focus here on regional Ca²⁺ transients, which have been shown to appear as ‘Ca²⁺ waves’ that propagate along a myocyte [21–29]. Fig. 1 shows a Ca²⁺ wave after an action potential induced a synchronous Ca²⁺ transient in a myocyte accompanied by an after-contraction and a DAD. In this case, the spontaneous Ca²⁺ wave emerged at the center of the myocyte and spread in both directions concomitant with a DAD and a spontaneous contraction [30]. Typically, the interval between the last stimulation and the onset of the first Ca²⁺ wave shortens and the probability of multiple foci of Ca²⁺ waves increases when the stimulus frequency or [Ca²⁺]₀ is increased [31]. When Ca²⁺ waves arise simultaneously from multiple foci, the resultant sarcolemmal depolarization is augmented to a level that can induce an action potential [32]. These observations are consistent with the concept that during a DAD the increase in [Ca²⁺]_i causes a transient inward current arising from electrogenic Na⁺/Ca²⁺ exchange and Ca²⁺-activated non-selective cation channels [17].

Fig. 1

Spatial changes in fluorescence signals during a delayed after-depolarization (DAD), caused by a slowly propagating Ca²⁺ wave in a cardiac myocyte. Top panel shows membrane potential, middle shows spatio-temporal changes in Fura 2 fluorescence signals, and bottom shows cell length. Middle: Focal fluorescence transients emerged spontaneously at the center of the myocyte and spread in both directions after fluorescence transients were elicited by an action potential. Propagating patterns of fluorescence transients seemed to be ‘waves’. It should be noted that spontaneous fluorescence transients were occurring concomitant with a DAD in top (arrow) and a spontaneous contraction in bottom, ST, electrical stimulation. Modified from Ref. [30].

Ca²⁺ waves usually start at one end of myocytes, where one might envisage gap junctions, but sometimes multiple and variable foci of Ca²⁺ waves can been observed [29]. When a Ca²⁺ wave begins in a focus within a myocyte, it spreads at equal velocity in all directions, so that the observer gets the impression of a wave spreading in a pond [23]. Because such a wave will reach the sides of a cylindrical cell first, it spreads subsequently in both directions at equal speed while the wave front becomes flatter (Fig. 1). In addition to linear propagation, Ca²⁺ waves may also propagate in spirals spinning around intracellular organelles [26]. Sometimes Ca²⁺ waves emerge, upon initiation at the end of a myocyte, as a dome-like region of spontaneously elevated [Ca²⁺]_i (300 nM) approximately 20 μm in diameter, and propagate as a localized 10 μm wide band of elevated [Ca²⁺]_i[23, 28]. Amplitude and width of Ca²⁺ waves are fairly constant during propagation [23–25]and their velocity of propagation is typically about 100 μm/s in unstimulated cells [21–26, 28, 33]. However, it has been observed that the propagation velocity can be increased transiently up to about 1 mm/s after rapid electrical stimulation (Miura, unpublished observations).

Ca²⁺ waves may start in multiple sites in a myocyte as is shown in Fig. 2. As a result they may appear to collide while travelling through the cell. Also, waves may be present in the cell at the moment that the cell is electrically excited. The behaviour of the waves under these circumstances is quite revealing about the process that generates them. When Ca²⁺ waves start from two or more foci within a myocyte, the waves appear to collide without augmentation of the [Ca²⁺]_i. After the collision, the [Ca²⁺]_i declines without evidence of further propagation of the waves demonstrating refractoriness of the propagation mechanism (the left part of Fig. 2) [25–27]. Fig. 2 further illustrates that Ca²⁺ waves are the consequence of a process with a refractory period. The figure shows that if the experimenter elicits an action potential during the propagation of a Ca²⁺ wave, the amplitude of action potential-induced Ca²⁺ transients and the accompanying twitch are reduced by the preceding Ca²⁺ wave (the middle part of Fig. 2). The decrease of the Ca²⁺ wave induced by the action potential is more pronounced if the interval with the preceding Ca²⁺ transient is shorter [34]. It appears that the resultant twitch of the myocyte recovers with a time course similar to that of the mechanical restitution curve, i.e. full recovery as attained at room temperature after ∼1200–2000 ms [35]. This is suggestive, but indirect, evidence that the spontaneous contraction and twitch generation share the same mechanisms involved in intracellular Ca²⁺ cycling [36].

Fig. 2

[Ca²⁺] transients are followed by refractoriness of Ca²⁺ release by the SR. The figure shows membrane potential (upper panel), spatial and temporal changes in fura-2 fluorescence intensities (middle panel), and cell length (lower panel). When a [Ca²⁺] wave (F(t)W) precedes a Ca²⁺ transient induced by an action potential (F(t)AP), the latter transient is smallest at the site where the wave has just arrived. Conversely, the wave stops as a result of the occurrence of the action potential. F(t)W, fluorescence transients due to calcium wave; F(t)AP, fluorescence transients induced by an action potential; Ex 380, excitation wavelength at 380 nm; ST, electrical stimulation. Modified from Ref. [34].

Ca²⁺ waves occur usually randomly with a frequency that may vary from less than 0.1 to 5–6 Hz, although remarkably stable intervals between spontaneous Ca²⁺ waves can be observed [29]. In an individual cell the frequency increases monotonically with increased SR Ca²⁺ loading [33]as does the number of foci of Ca²⁺ waves [31]. When two or more foci with different frequencies periodically generate Ca²⁺ waves within a myocyte, a Ca²⁺ wave originating from fastest focus can reset wave generation at other foci and dominate activation of the whole myocyte [29].

4 Triggered propagated contractions in cardiac muscle

Events related to spontaneous Ca²⁺ release by the SR similar to those in isolated myocytes have been observed using confocal microscopy under physiological conditions in intact cardiac muscle [37]. However, Ca²⁺ waves occur infrequently and propagate at low velocities (∼30 μm/s) and over limited distances (<10 μm) in intact cardiac muscle under physiological conditions (37°C and normal free [Ca²⁺]₀). Specific requirements, such as lowering of the temperature and/or increase of [Ca²⁺]₀, have to be met in order to provoke significant spontaneous activity in myocardium. Even then, spontaneous Ca²⁺ release manifests itself only as Ca²⁺ waves that occur inside cells and which rarely (<20%) move from cell to cell. Only when cardiac muscle is damaged locally, such as by microelectrode impalement or dissection procedures, do we see in and near the damaged region the initiation of Ca²⁺ waves that propagate in a coordinated fashion into adjacent tissue [38]. Several observations suggest that after-contractions in multicellular preparations occur as the combined result of the mechanical effects and elevated cellular Ca²⁺ levels owing to regional damage and may give rise to premature beats as well as triggered arrhythmias. Unique aspects of the after-contractions that arise in damaged regions of cardiac muscle are that they appear to be initiated by stretch and release of the damaged region during the regular twitch and that they propagate into neighbouring myocardium. Most of the observations that will be discussed here have been made on isolated rat ventricular and human atrial trabeculae. These studies have suggested that the after-contractions may be caused by Ca²⁺ release, first by the myofilaments and, then, by the SR in the cells near the damaged region. This local release of Ca²⁺ in the damaged region causes a Ca²⁺ transient that is conducted into adjacent muscle by the combination of Ca²⁺ diffusion and Ca²⁺-induced Ca²⁺ release. The propagating [Ca²⁺]_i transient is accompanied by a local contraction, that we have denoted as triggered propagated contraction (TPC) [32, 39]. Fig. 3 shows a typical example of a TPC. The [Ca²⁺]_i transient underlying the TPC also causes a depolarization of the cell membrane which may reach threshold and become responsible for action potential generation [38]. Damage-induced after-contractions may, therefore, serve as the mechanism that couples regional damage with the initiation of premature beats and arrhythmias in the adjacent myocardium. TPCs appeared to arise in the ends of trabeculae [32, 39], which are easily damaged by dissection and mounting of the muscle. The damage by dissection and mounting of the muscle is worrisome for studies of muscle mechanics, but provides a means to induce and control the extent of the damage and to study the consequences of acute injury on myocardium.

Fig. 3

Panel A: Sarcomere length (SL) recordings at five different sites (each 300 μm apart) along a 2.94-mm long trabecula during a TPC with a propagation velocity of 1.4 mm/s. The interval between peak sarcomere shortening due to the TPC (vertical dashed lines) was constant from site to site, indicating that propagation velocity remained constant along the preparation. F=force. [Ca²⁺]₀ 1.0 mM, temperature 21°C. Initial sarcomere length varied less than 0.05 μm between the sites of measurement. Modified from Ref. [41]. Panel B: Schematic drawing of a muscle in which TPCs started from a centrally located damaged region (marked by the asterisk) near two cut side branches. The bottom panel shows the sarcomere length (SL) recordings at three sites of the preparation, illustrating that the TPC propagated away from the damaged region in both directions. Modified from Ref. [41].

5 Triggered propagated contractions: damage and the initiating event

TPCs arise invariably in damaged regions of cardiac muscle, as is shown in Fig. 3. Damage of a cardiac cell causes loss of integrity of the cell membrane and allows a Ca²⁺ entry into damaged cells which in its turn will induce Ca²⁺ overload and asynchronous spontaneous activity [32]. A consequence of this effect of damage is that Ca²⁺ will diffuse via gap junctions into adjacent cells in the border zone where cells still have an intact sarcolemma. Entry of Ca²⁺ into the border zone continues, as long as the gap junctions remain open [40], which depends on the cell's environmental conditions including temperature [41]. Spontaneous activity in the damaged zone is random; hence, the accompanying Ca²⁺ transients are small and do not propagate through the muscle, but can cause Ca²⁺ overload and spontaneous activity in border zone. This process continues until the [Ca²⁺] gradient between cells is too small or until gap junctions close [32]. The existence of spontaneous contractions increases resting tension and decreases twitch force [42, 43]. Thus, the damaged cells and the border zone may be weaker during the twitch than the central region of trabeculae. So, during twitch contraction the central region of the trabeculae stretches the damaged region. During the rapid relaxation phase of the twitch the stretched damaged region shortens suddenly. Stretch or quick release of damaged ends of trabeculae during the electrically driven twitch trigger TPCs [39]may provide an explanation for the triggering mechanism. Support for this contention comes from the observation that elimination of the stretch of the damaged region and border zone during the twitch and hence elimination of the quick release during the relaxation phase prevents the induction of TPCs. Fig. 4 shows an example of such an experiment with servo control of the after-load on the muscle. When the after-load is reduced, stretch of the damaged areas decreases but sarcomere shortening in the center of the trabeculae increases, while the TPC is delayed.

Fig. 4

Sarcomere length (SL) and force (F) recordings of the last of the electrically stimulated twitches and a TPC at different after-loads in one muscle. [Ca²⁺]₀ 2.50 mM. Initial sarcomere length 2.15 μm. All sarcomere length recordings were made at one site of the preparation. Both sarcomere length and force tracings were artificially shifted on the vertical axis. A decrease in after-load delayed the initiation of the TPC significantly. Modified from Ref. [39].

It appeared that reduction of the after-load below twenty percent of the isometric force eliminated the TPCs completely; an effect that was immediately reversed by forcing the muscle to contract again against a high after-load. At the same time, it was clear that as long as a TPC was observed, the propagation velocity (V_prop) was not influenced by the manipulation of the after-load. So, apparently the probability of triggering a TPC depends on the degree of stretch of the damaged area during the twitch or on the shortening during the relaxation phase of the twitch. Several previous studies have suggested a similar coupling between the mechanical events during the twitch and membrane electrophysiology. First, studies of both skeletal and of cardiac muscle have shown that a quick release of the muscle during contraction causes rapid release of Ca²⁺ ions from contractile filaments [44]. Second, Kaufmann et al. [45]have shown that a quick release of the muscle by the experimenter could induce a propagated action potential and a contraction. M. Lab and collaborators have provided the link between these observations by showing that quick releases can induce an intracellular [Ca²⁺] transient accompanied by an after depolarization [46].

6 Propagation of TPCs

Fig. 3 showed a characteristic TPC in a rat cardiac trabecula. The displacement of the TPC occurs at a V_prop which varies in these preparations at room temperature from 0.1 to 15 mm/s [32]and correlated tightly with the amplitude of the twitch preceding the TPC suggesting that the Ca²⁺ load of the SR dictates V_prop[41]. In contrast, sarcomere stretch, which increases twitch force for any level of loading of the SR, did not increase V_prop[39]. Studies of the effects of interventions such as varied [Ca²⁺]₀, Ca²⁺ channel agonists and antagonists also support the idea that the Ca²⁺ load of the SR is the main determinant of V_prop[47]. On the other hand, interventions, which cause a leak of Ca²⁺ from the SR (caffeine and ryanodine), increase V_prop, suggesting that V_prop also depends on the diastolic cytosolic Ca²⁺ level [47]. The rate of initiation of TPCs is tightly correlated with V_prop when the Ca²⁺ load of the SR is modulated suggesting that the triggering process and the propagation process share closely related mechanisms.

7 Mechanisms underlying initiation and propagation of Ca²⁺ waves and TPCs

7.1 Initiation of Ca²⁺ waves in myocytes

Fabiato's work on the properties of cardiac SR has provided a potential explanation for spontaneous Ca²⁺ release in isolated myocytes. He observed that in mechanically skinned cardiac cells in which the SR was intact, excessive Ca²⁺ loading of the SR caused spontaneous Ca²⁺ release [48]. The mechanism for increased probability of opening of the SR–Ca²⁺ channel when the SR is heavily loaded with Ca²⁺ is still uncertain, but suggests that the channel is directly or indirectly sensitive to the lumenal [Ca²⁺] of the SR. Recently the probable site of the Ca²⁺ sensor of the SR–Ca²⁺ channel has been identified [49]. The localization of the Ca² sensor in (glutamate 3885) the transmembrane domain of the channel would make it suitable as a sensor of both lumenal and of cytosolic [Ca²⁺]. Intact cells with a high SR–Ca²⁺ load show similar phenomena [31, 42]. Hence, the oscillatory character of a triggered arrhythmia in myocardium with a high cellular Ca²⁺ load may be due to further increase of Ca²⁺ entry into the cells during the action potentials of the arrhythmia causing even more Ca²⁺ loading of the SR. Consequently, as soon as the release process has recovered after an electrically induced Ca²⁺ release, the overloaded SR again releases a fraction of its Ca²⁺. The requirement that the Ca²⁺ release mechanism has to recover first [50]would explain the presence of a delay between after-contractions and after-depolarizations to the preceding twitch. A small degree of Ca²⁺ depletion of the SR may play an additional role in this process [51].

7.2 Initiation of TPCs in multicellular preparations

The observation that the TPCs always start shortly after the rapid shortening of the damaged areas suggests that it is actually the shortening during relaxation that initiates a TPC. Housman's [44]and others' observation [52–55]that rapid shortening of a contracting muscle causes release of Ca²⁺ ions from the myofilaments provides a candidate mechanism for initiation of TPCs. Ca²⁺ ions that dissociate from the contractile filaments due to the quick release of the damaged areas during relaxation could initiate a TPC if Ca²⁺-induced Ca²⁺ release has recovered sufficiently [35]to allow amplification of the initial Ca²⁺ transient in the damaged region and/or the border zone.

7.3 Propagation of Ca²⁺ waves in myocytes

The observation that Ca²⁺ waves travel at a constant velocity and with constant amplitude through isolated myocytes and through multicellular preparations is an important clue about the mechanism of propagation. Diffusion of Ca²⁺ alone would clearly be too slow by at least 2–3 orders of magnitude and would be accompanied by a decline of the observed wave amplitude. On the other hand the propagation of electrical activity is much faster, 1 m/s for the action potential in ventricular myocardium. Also, electrotonic conduction is too fast to be compatible with the observed values for V_prop. The mechanism involved in the propagation of Ca²⁺ waves in cells has been proposed which consists of diffusion of Ca²⁺ due to the local increase of [Ca²⁺]_i and subsequent Ca²⁺-induced Ca²⁺ release from adjacent SR [56, 57]. Recently, the use of fluorescence imaging and confocal microscopy has revealed the presence of several spontaneous ‘Ca²⁺ sparks’ near the site of initiation of a Ca²⁺ wave and a ‘macro-spark’ at the point of initiation [58]. This observation suggests that these sparks may act as a trigger for Ca²⁺ waves. The transition from non-propagating sparks to waves is possibly caused by an increase in sensitivity of the SR Ca²⁺-release channel for activation by cytosolic Ca²⁺ as a consequence of the greater amount of SR Ca²⁺ loading [58]. Although these observations have provided a reasonable framework for explanation of propagated Ca²⁺ waves, the model is still only a working model and many questions remain unanswered. For example it has been noted that in myocytes without Ca²⁺ overload a local increase in [Ca²⁺]_i using ‘caged’ Ca²⁺ does not propagate [59], and that a Ca²⁺ wave induced by local application of caffeine decreases in both amplitude and velocity as it propagates along the cell [60].

7.4 Propagation of TPCs

TPCs propagate with a constant velocity along a trabecula. Like in isolated myocytes, this wave-like character is thought to be consistent with a model of Ca²⁺-induced Ca²⁺ release from the SR mediated by Ca²⁺ diffusion along its concentration gradient to adjacent sarcomeres and adjacent cells [31, 32, 38, 39, 61–63]. This model is supported by work on saponin-skinned muscle fibers which also exhibit propagating local contractions after rapid local exposure of the fiber to a Ca²⁺-containing solution suggesting that the SR, but not the cell membrane, is essential for the phenomenon [38]. The observation that neither initiation of TPCs nor their propagation is affected by Gd³⁺ ions suggests that stretch-activated channels play little or no role in the initiation or propagation of damage-induced TPCs [64]. This is consistent with the hypothesis that the TPCs depend on intracellular organelles. As was shown by the lack of effects of varied after-load and varied sarcomere length on V_prop, it is unlikely that stretch of the myofibrils is essential to the propagation process.

The characteristics of Ca²⁺ waves are quite similar to those of TPCs in trabeculae. In addition, neither spontaneous activity in single myocytes nor TPCs in trabeculae require an intact sarcolemma and both are abolished by agents that interfere with SR Ca²⁺ loading or release [65]. On the other hand, at first glance, a striking difference between them is the propagation velocity. The velocity of Ca²⁺ waves in unstimulated cells is about ten times lower than V_prop. However, it is important to realize that TPCs are generated in cardiac muscle preparations at a short interval after the twitch so that their properties are affected by residual binding of Ca²⁺ to intracellular ligands. This contrasts the situation in myocytes where the moment of appearance of Ca²⁺ waves following the twitch is both random and usually later. Hence, elimination of Ca²⁺ from these ligands later during diastole, when the Ca²⁺ extrusion processes have done their work would cause slowing of the propagation velocity [32].

We have investigated which parameters of Ca²⁺ diffusion and Ca²⁺-induced Ca²⁺ release are required for the high propagation velocity of TPCs in muscles by modeling the behaviour of a myofibril accompanied by its SR during a sudden focal Ca²⁺ release [66]in a way represented in Fig. 5.

Fig. 5

Diagram of the excitation–contraction coupling system in the cardiac cell, as well as its role during TPCs. The left panel shows the events during the twitch. During the action potential a large transient Ca²⁺ influx enters the cells followed by a maintained component of the slow inward current (dashed line). Ca²⁺ entry does not lead directly to force development as the Ca²⁺ that enters is rapidly bound to binding sites on the SR. The rapid influx of Ca²⁺ via the T-tubuli is thought to induce release of Ca²⁺ from a release compartment in the SR, by triggering opening of Ca²⁺ channels in the terminal cisternae, thus activating the contractile filaments to contract. Rapid relaxation follows because the cytosolic Ca²⁺ is sequestered rapidly in an uptake compartment of the SR and partly extruded through the cell membrane by the Na⁺/Ca²⁺ exchanger and by the low capacity high affinity Ca²⁺ pump. This process loads the SR. It is important to note that the process of Na⁺/Ca²⁺ exchange is electrogenic so that Ca²⁺ extrusion through the exchanger leads to a depolarizing current. The middle panel shows the events near a damaged region during triggering of the TPC. Rapid shortening of this region occurs during relaxation of the twitch following stretch by the normal, and therefore stronger myocardium, during contraction. This rapid release of the sarcomeres leads to dissociation of Ca²⁺ from the contractile filaments during the relaxation phase. The SR has recovered enough to respond to the increase in [Ca²⁺]_i by Ca²⁺-induced Ca²⁺ release, leading to an after-contraction. The resultant elevation of [Ca²⁺]_i also causes diffusion of Ca²⁺ to adjacent sarcomeres. The right panel shows that the arrival of diffusing Ca²⁺ after release in the damaged region leads to Ca²⁺-induced Ca²⁺ release by the SR in the adjacent sarcomeres. Ca²⁺ diffuses again into the next sarcomere, while causing a local contraction as well as a delayed after- depolarization (DAD) due to electrogenic Na⁺/Ca²⁺ exchange and activation of Ca²⁺-sensitive non-selective channels in the sarcolemma. Diffusion of Ca²⁺ along its gradient maintains the propagation of the TPC.

The method of solution involved writing the diffusion equation as a difference equation in the spatial coordinates. The resultant coupled ordinary differential equations in time with banded coefficients were solved using Gear's 6th order predictor–corrector algorithm for stiff equations with reflective boundaries. It appeared that Ca²⁺ transients propagate through the cytosol (as is illustrated in Fig. 6) at a rate modified by binding to troponin and calmodulin and sequestration by the SR, as well as by the rate of Ca²⁺ release from adjacent release sites of the SR. V_prop appeared to increase in the model from 0.1 to 15 mm/s owing to the combined effects of a rise of: (i) the diastolic [Ca²⁺]_i; (ii) the rate of Ca²⁺ release and; (iii) the amount of Ca²⁺ released by the SR [66]. This combination of changes in Ca²⁺ levels in the cytosol and in the SR would be expected to result from loading of cardiac cells with Ca²⁺ during repetitive stimulation as well as due to exposure to high [Ca²⁺]₀ or Ca²⁺ agonists [32].

Fig. 6

The Ca²⁺ transients as a function of position along the preparation at different times. The propagating nature of the Ca²⁺ wave is evident from the figures. Opening of the Ca²⁺ channel in the SR was assumed to follow an exponential time course (time constant 1/k_rel); after 2/k_rel the channel was assumed to close with the same time constant, so that for k_rel=0.1 ms⁻¹ the open time of the channel would be 0.5 ms [Ca²⁺]_diast is the diastolic Ca²⁺ concentration. V_max is the maximal rate of Ca²⁺ extrusion ions from the cytosol. K_D is the threshold for Ca²⁺ release from the SR. Modified from Ref. [66].

Important to the viability of the proposed model of TPCs was a high conductance of the Ca²⁺ channel in the SR. In addition, the expected open time for the Ca²⁺ channel in the SR would have to be in the order of 0.5 ms, in order to allow for the highest V_prop. These properties of the SR Ca²⁺ channels seemed exceedingly rapid at the time of formulation of the model. Nowadays, reports on the kinetics of RyR in bilayers [49]and on extremely fast Ca²⁺ transients such as sparks recorded using confocal microscopy and fast and sensitive fluorescent Ca²⁺ dyes such as Fluo 3 [67], suggest that this assumption may not have been excessive. Nevertheless, it is clear that our understanding of TPCs requires further study of Ca²⁺ transients at a high spatial and temporal resolution.

The model of a myofibril together with SR over a length of 50 sarcomeres predicted the propagation velocities observed in muscles accurately. This accuracy is somewhat surprising because in the real muscle the Ca²⁺ transient has to travel not only from sarcomere to sarcomere but also from cell to cell. The high propagation velocity suggests that the barrier for Ca²⁺ diffusion imposed by gap junctions between cells is minor compared to the other parameters in the model such as Ca²⁺ binding to ligands in the cell and Ca²⁺ extrusion and sequestration processes. We tested the importance of gap junctions to the properties of the TPCs by exposing the trabeculae to the gap junction blockers heptanol and octanol. Although these compounds like many drugs have probably numerous side effects, their main effect is assumed to be a reduction of the open frequency of gap junctions. Exposure of the muscles to these alcohols showed a unique effect on TPCs that we have not encountered with other drugs, i.e.: the rate of initiation and V_prop decreased dramatically with only a small decrease in twitch force [68]. This suggests that closure of gap junctions reduces the rate of initiation and V_prop by reducing the effective rate of diffusion from cell to cell.

The model suggested that the rate of propagation of TPCs should be reduced by active extrusion of Ca²⁺ from the cytosol [66]. Therefore, we tested whether the rate of propagation of contractions that can be induced in skinned fibers would be equally fast as those in intact trabeculae. Skinned trabeculae were exposed to a solution, which allowed loading of the SR. Subsequently, Ca²⁺-induced Ca²⁺ release was induced by using a microelectrode in order to squirt a small amount of Ca²⁺-containing solution onto the fiber. The rapid local exposure to high Ca²⁺ led to a local contraction followed by a propagated contraction which travelled at a velocity ranging from ∼50 to 300 μm/s. We did not succeed in creating conditions under which the contraction travelled faster than 300 μm/s, irrespective of whether we enhanced or reduced the SR load with Ca²⁺ or whether we restricted Ca²⁺ diffusion to the bathing medium by placing the fibers in silicon oil. It is possible that a crucial condition for rapid propagation is an elevated [Ca²⁺] concentration around the myofibrils so that more cytosolic Ca²⁺ binding sites are occupied; this possibility remains to be tested.

7.5 Propagated Ca²⁺ release induces premature beats

Whenever a TPC arises, it is accompanied by a depolarization. These depolarizations are remarkably similar to DADs, albeit that their duration varied widely. It appears that the duration of the depolarizations correlated exactly with the time during which the TPCs travel through the trabeculae. This observation is consistent with electrotonic conduction along muscles that act like a cable with a length constant of several millimetres [38]. Also the amplitude of the after-depolarizations correlated exactly with the amplitude of the TPCs [38]. This is shown in Fig. 7.

Fig. 7

Parallel changes in the characteristics of TPC and DAD following two (middle panel) and 15 (bottom panel) conditioning stimuli. The top panel represents a trabecula with its ventricular end positioned in a cradle that was attached to a force transducer and the valvular side attached to a hook. Sarcomere length and membrane potential were monitored at two sites along the preparation (X and Y). The records in the middle and bottom panel show force (F), sarcomere length (SL), and membrane potential (V) of the last stimulated twitch and a subsequent TPC and DAD in a representative muscle. The preparation hyperpolarized slightly during stimulation, accounting for the depolarizing drift upon which DADs occurred. At the two measuring sites, membrane potential tracings were virtually identical. With an increase in the number of conditioning stimuli from two to 15, TPC propagation velocity increased from 2.4 to 9.5 mm/s. Furthermore, TPC force and DAD amplitude increased, while TPC latency, DAD latency, and duration of both TPC force and DAD decreased. Following 15 stimuli, a second TPC and DAD occurred. [Ca²⁺]₀ 1.0 mM, temperature 20.8°C, resting membrane potential −68 and −64 mV at sites X and Y respectively. Muscle length 2.23 mm; initial sarcomere length 2.10 μm. From Ref. [38].

The tight correspondence between the time course of TPCs and those of the depolarizations suggests that the depolarization is elicited by a Ca²⁺ dependent current, which exists as long as the [Ca²⁺]_i transient persists, as has been proposed by Kass and Tsien [17]. In the small trabeculae that we have used for these studies this depolarization can be recorded over a distance of a few millimetres without much decrement due to electrotonic conduction following the cable properties of these fibers [38]. This assumption could be verified experimentally by interrupting the propagation process of the TPC by locally heating the muscle over a few hundred micrometres length by a few degrees as is shown in Fig. 8.

Fig. 8

Effect of local heating of the muscle on a TPC and DAD. The top panel schematically represents the muscle with free right ventricular wall attachment, positioned in the cradle of a force transducer (left), and valvular side (right). TPCs propagated in a direction opposite to the superfusing flow. Measurements of sarcomere length were made both proximal (X) and distal (Y) to the hearing wire; membrane potential was only monitored at Y. The middle panel shows, during the control phase, recordings of sarcomere length (SL) at both X and Y, force (F), and membrane potential (V) of the last stimulated twitch and a TPC and DAD, with the local contraction in X preceding that in Y, indicative of the propagating character of the TPC. Resting membrane potential, −64 mV. [Ca²⁺]₀ 1.25 mM; temperature 19.8°C. TPC propagation velocity 3.0 mm/s. During local heating (bottom panel), a local contraction due to a TPC still occurred at site X, but propagation of the TPC was interrupted at the heating wire and no unstimulated contraction was visible at Y, despite a local depolarization. Note the accelerated relaxation of the last stimulated twitch and the decreased latency of both TPC and DAD. The decrease in distance of TPC propagation is responsible for the decrease in TPC duration and amplitude. From Ref. [38].

Local heating of the muscle caused the TPC to stop at the site of heating. In contrast, the concomitant depolarization could still be measured at a distance of about 1 mm distal of the heating site [38]again as a result of electrotonic conduction of the DAD for which the current generators are located in the region with elevated [Ca²⁺]_i. This observation clearly indicates that the depolarization cannot be the source of the TPC but must be induced by the TPC. The effect of local heating makes it also unlikely that TPCs are induced as a result of a linear gradient of Ca²⁺ overload along the muscle from a maximum in the damaged region to a minimum at the other end of the muscle. Such a gradient could potentially cause apparent propagation of a contraction if Ca²⁺ overload-induced Ca²⁺ release occurred along the muscle at a latency which is small in the damaged region and increased linearly toward the other end of the muscle.

8 Premature beats and triggered arrhythmias resulting from TPCs

TPCs always can be noticed directly following damage to the muscle. This is usually evident shortly after-mounting a muscle in an experimental setup. The acute Ca²⁺ load to the damaged cells and their neighbours is then apparently so large that virtually every electrically paced beat is followed by TPCs. In that case, we observed that the TPC was accompanied by a DAD, which was sufficiently large to elicit an action potential with twitch as shown in Fig. 9A. As discussed above, the action potential triggered by the first TPC may add so much Ca²⁺ to the cell that a triggered arrhythmia starts (Fig. 9B). Triggered arrhythmias indeed occur in the damaged muscle when the Ca²⁺ load of the SR is large. We have observed these triggered arrhythmias at room temperature during the first hour after damage to the muscle has occurred. In such a case, the full-blown arrhythmia is usually preceded by the repeated occurrence of single premature beats. At 37°C, the time span over which these damage-related events occur is much shorter and the TPCs, which cause the premature beats, disappear in 10 min or less [69]. Under those conditions it is likely that their occurrence is limited by rapid closure of gap junctions as a result of persistently elevated Ca²⁺ levels in the damaged cells. In addition, the pH in these cells may be low due to the enormous metabolic load resulting from intense ion movement across their membranes or across membranes of adjacent cells. The lowered pH may promote gap junction closure

Fig. 9

Spontaneous contractions cause development of twitches and arrhythmias. Panel A shows an example of the development of a spontaneous twitch that is triggered by a propagating after-contraction. Note the acute increase of the rate of rise of force during the development of the first after-contraction (arrow), and also the similarity of the time course of the subsequent twitch and that of the electrically elicited twitch. Modified from Ref. [32]. Panel B shows force (F) and membrane potential (V) recordings during a train of conditioning stimuli (ending at the arrow) and a subsequent triggered arrhythmia. Note the initial slow upstroke in both force and membrane potential of triggered twitches, suggestive of an underlying TPC and DAD. The triggered arrhythmia terminated spontaneously with an increase in the interval between triggered beats, followed by a TPC and DAD. Temperature 20.4°C; [Ca²⁺]₀ 2.25 mM. Resting membrane potential, −71 mV. Modified from Ref. [41].

These observations make it likely that arrhythmia initiating premature beats in acutely damaged myocardium may result from a triggered intracellular Ca²⁺ transient in the damaged region. The resulting [Ca²⁺]_i transient propagates into adjacent myocardium and induces DADs and TPCs. If the [Ca²⁺]_i transient is large enough it will lead to action potential formation. Clearly, damage initiates a chain of subcellular events leading to oscillations of [Ca²⁺]_i that may cause macroscopic arrhythmias in the damaged heart. Few studies have addressed possible pharmacological interventions aimed at this source of premature beats. In our hands, it was so far possible to reduce the chance of development of TPCs without simultaneously causing a negative inotropic effect using R56865, a Na⁺ and Ca²⁺ overload inhibitor [70], or using the gap junction blockers octanol and heptanol [68].

It is clear that many mechanisms involved in the generation of TPCs and subsequently arrhythmias require further study. The mechanisms involved in the speed at which these contractions travel need further study in order to resolve how they can propagate at a speed, which is uncommon for those who are used to observing propagation of Ca²⁺ waves in single myocytes. The role of the sarcolemma deserves particular attention as the assumption that depolarization of the membrane does not play a role (see Fig. 5) needs to be tested. Furthermore, the influence of gap junctions on the propagation process needs to be evaluated. Finally, the TPCs discussed here have been found in situations where it was obvious that cardiac muscle had been damaged mechanically. Whether injury results in similar mechanisms to those, which result from ischemia or other causes of Ca²⁺ overload, needs to be tested. Lastly, the authors hope that this review will be a stimulus to others to study whether this mechanism underlying arrhythmias in vitro may play a role in the human heart.

Acknowledgements

This work was supported by grants from the Janssen Research Foundation and the Alberta Heart and Stroke Foundation. Dr. H.E.D.J. ter Keurs is a Medical Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR).