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G.L. Brown Prize Lecture
Calcium in the heart: from physiology to disease David Eisner Unit of Cardiac Physiology, University of Manchester, Manchester, UK
Experimental Physiology
New Findings r What is the topic of this review? This review considers the factors that regulate the systolic Ca transient in cardiac muscle and how disturbed Ca signalling can result in heart failure and arrhythmias. r What advances does it highlight? Sarcoplasmic Reticulum (SR) Ca content is controlled by a mechanism in which it is regulated by alterations in Ca fluxes across the surface membrane. This has important consequences for the regulation of the size of the systolic Ca transient.
Contraction of the heart results from an increase of cytoplasmic Ca2+ concentration ([Ca2+ ]i ), the so-called systolic Ca2+ transient. Most of this results from the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). In turn, the amplitude of this Ca2+ transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca2+ release. The amplitude of the Ca2+ transient is a steep function of SR Ca, resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism, in which an increase of SR Ca content increases the size of the Ca2+ transient, resulting in a decrease of Ca2+ influx on the L-type Ca2+ current and an increase of efflux through Na+ –Ca2+ exchange. Changing the activity of any of the Ca2+ -cycling proteins will change the steady-state SR Ca content. This feedback mechanism has many consequences, including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca2+ transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca2+ waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia, which is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Calcium waves occur only when the SR Ca content exceeds a threshold level. Our data show that the threshold is reduced by the RyR mutation and that the adrenergic stimulation increases SR Ca content. (Received 25 May 2014; accepted after revision 4 August 2014; first published online 15 August 2014) Corresponding author D. Eisner: Unit of Cardiac Physiology, University of Manchester, 31.18 Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, UK. Email: [email protected]
The heart before science
This article is based on the Physiological Society’s G. L. Brown Prize Lecture, given from March to May 2014 at Queen’s University Belfast, Bristol, Cambridge, Edinburgh, Glasgow, Liverpool, The Open and Southampton Universities and Imperial College London. C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
The heart is perhaps the only organ whose function we are aware of because its beat can be felt on the body surface. We know that the heart beat changes in emotion and exercise and, possibly for this reason, many poets and composers have made reference to the heart. Macbeth (1606; Act 1, Scene 3) declares: DOI: 10.1113/expphysiol.2013.077305
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I am thane of Cawdor: If good, why do I yield to that suggestion Whose horrid image doth unfix my hair And make my seated heart knock at my ribs, Against the use of nature? Present fears Are less than horrible imaginings.
In other words, Shakespeare was aware of the link between emotions and the force of the heart beat. Two centuries later (1828), in the song ‘Die Post’ from the cycle Die Winterreise, Schubert provides a perfect representation of a heart beat in the piano accompaniment to the words ‘Was dr¨angst du denn so wunderlich, mein Herz’ (Supplementary video, also available on youtube https://www.youtube.com/ watch?v=VXSJkq5Dk8o). The birth of the era of calcium and the heart
Today, we take it for granted that calcium is involved, not only in the contraction of the heart, but also in the control of the vast majority of mammalian systems. The first indication of the role of calcium came from the work of Sydney Ringer (1836–1910). The story of the discovery is a wonderful reminder that science does not always move in a straightforward way. In 1882, Ringer published the finding that a simple solution of sodium chloride is sufficient to maintain contraction of the isolated frog heart (Ringer, 1882). A year later, however, he retracted this (Ringer, 1883), stating: I discovered, that the saline solution which I had used had not been prepared with distilled water, but with pipe water supplied by the New River Water Company. As this water contains minute traces of various inorganic substances, I at once tested the action of saline solution made with distilled water and I found that I did not get the effects described in the paper referred to. It is obvious therefore that the effects I had obtained are due to some of the inorganic constituents of the pipe water.
From this serendipitous discovery came not only the realization of the biological role of calcium but, in addition, Ringer’s work characterizing the requirements for solutions to maintain the heart (and therefore other organs) in a healthy state, which allowed the introduction of replacement fluids in the clinical arena. It is also worth noting his careful use of language. His leading role in the research is emphasized by ‘I discovered’ and ‘I had used’. In contrast, ‘had not been prepared’ distances him from the error. It took almost 100 years for a direct measurement of the rise of intracellular calcium concentration ([Ca2+ ]i ) that activates contraction (the systolic Ca2+ transient). Fittingly, given the fact that Ringer had worked on the frog heart, Ca2+ measurements were first made on that species (Allen & Blinks, 1978) before data were obtained in the mammalian heart (Allen & Kurihara, 1980).
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Overview of cardiac Ca2+ signalling
There are two sources of Ca2+ for contraction in the heart, as follows. (i) Calcium enters the cell via the L-type Ca2+ channels. This is not the major source of Ca2+ , and its main role is to trigger the release of much more Ca2+ from the sarcoplasmic reticulum (SR). (ii) Calcium is released from the SR through a channel known as the ryanodine receptor (RyR). The probability that the RyR is open, and can therefore allow Ca2+ to leave the SR, is increased by an increase of both cytoplasmic and SR Ca (Meissner, 1994; Sitsapesan & Williams, 1997). The dependence on cytoplasmic Ca2+ results in Ca2+ entry via the L-type Ca2+ channel, triggering Ca2+ release in a mechanism known as calcium-induced calcium release (Fabiato, 1985). For the heart to relax, [Ca2+ ]i must be lowered to resting levels. This occurs by the following two processes: (i) Ca2+ is taken back into the SR by the SR Ca2+ -ATPase (SERCA); and (ii) it is pumped out of the cell by Na+ –Ca2+ exchange (NCX), with some contribution from the plasma membrane Ca2+ -ATPase. The activity of SERCA depends on both Ca2+ and the accessory protein phospholamban. Unphosphorylated phospholamban inhibits SERCA, and when it is phosphorylated (as occurs during β-adrenergic stimulation) this inhibition is relieved (Tada & Katz, 1982). The NCX uses the energy provided by three Na+ ions entering the cell to pump a Ca2+ ion out, thereby generating an electric current. These events are shown in Fig. 1. The following two points need to be emphasized: (i) changes in the amplitude of the systolic Ca2+ transient are the major means by which the contractility of the heart is controlled; and (ii) in the steady state, the cell must be in calcium flux balance on each beat; that is to say, the amount of Ca2+ entering the cell must equal that pumped out and the amount released from the SR must equal that taken back into the SR. Many of the results developed in the remainder of this article follow from this concept of calcium flux balance (see Eisner et al. 2000, 2009, 2013a for reviews). The relationship between SR Ca2+ content and the systolic Ca2+ transient
The experiment illustrated in Fig. 2A shows the effects on the systolic Ca2+ transient of changing the SR Ca content (Trafford et al. 1997). Before the trace began, the SR had been emptied of Ca2+ by exposure to caffeine (10 mM). At this concentration, caffeine binds to all the RyRs and makes them open, thereby releasing Ca2+ into the cytoplasm, whence it is pumped out of the cell. Caffeine was then removed and stimulation recommenced. The Ca2+ transient is initially small because the SR is virtually empty. It then increases in amplitude over a few seconds, presumably because the SR refills. Figure 2D shows the typical, supralinear dependence of the amplitude of the C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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Ca2+ transient on SR Ca content (Bassani et al. 1995; Trafford et al. 1997). On average, the amplitude of the Ca2+ transient depends on the cube of SR Ca content (Trafford et al. 2000). Several factors contribute to this steep dependence, including the fact that an increase of SR Ca will both increase the driving force for Ca2+ efflux from the SR and also increase the number of RyRs that are open. In addition, cytoplasmic Ca2+ buffers may tend to saturation such that, at higher [Ca2+ ]i , a given release of Ca2+ will result in a larger increase of free Ca2+ (D´ıaz et al. 2001). Whatever its origin, the steepness of this relationship means that even modest changes of SR Ca will have marked effects on the amplitude of the Ca2+ transient. There are two immediate consequences of this, as follows. (i) A given fractional increase of SR Ca will have a much larger fractional effect on the amplitude of the Ca2+ transient; therefore, a small increase of SR Ca, resulting, for example, from an increase of heart rate, will result in a larger increase of systolic Ca2+ . Conversely, a given fall of SR Ca content in heart failure will translate to a larger decrease of systolic Ca2+ (D´ıaz et al. 2004). (ii) The steepness of the relationship also presents the cell with a challenge. If the amplitude of the Ca2+ transient is to be maintained constant from beat to beat then the SR Ca content needs to be controlled very precisely. It is therefore important to consider how SR Ca is controlled.
The control of SR Ca2+ content
Figure 2B and C shows the changes of membrane currents accompanying the refilling of the SR. Two changes are obvious. (i) When the SR is empty, the L-type Ca2+ current inactivates more slowly and there is therefore more Ca2+ flux into the cell. This slower inactivation results from decreased Ca2+ -dependent inactivation due to the smaller Ca2+ transient (Sipido et al. 1995; Adachi-Akahane et al. 1996). (ii) The empty SR is also associated with decreased Ca2+ efflux via NCX due to decreased activation of the exchanger by the smaller Ca2+ transient. The net result of these two changes of membrane current is that an increase of systolic [Ca2+ ]i increases Ca2+ efflux and decreases Ca2+ influx (Trafford et al. 1997). This, in turn, results in a powerful negative feedback mechanism to control SR Ca2+ . Briefly, as mentioned above, an increase of SR Ca results (Fig. 2D) in an increase of the amplitude of the systolic Ca2+ transient. This will decrease Ca2+ influx and increase efflux, leading to a decrease of cell and therefore SR Ca. This feedback mechanism is responsible for the beat-to-beat regulation of SR Ca. It is worth noting that this mechanism achieves a similar result as do store-operated channels. These channels, which are particularly prominent in non-excitable cells, open in response to a decrease of endoplasmic reticulum (ER) Ca content (for review, see Parekh & Putney, 2005).
Figure 1. The events involved in excitation–contraction coupling The diagram shows the surface membrane and T-tubule of a ventricular myocyte. (1) The action potential leads to the opening of the L-type Ca2+ channel (ICa-L ) and an increase of [Ca2+ ]i near the ryanodine receptor (RyR). (2) The increase of [Ca2+ ]i makes the RyR open, releasing much more Ca2+ from the sarcoplamic reticulum (SR) and producing the systolic Ca2+ transient. (3) The Ca2+ transient decays due to the closing of RyRs and pumping of Ca2+ back into the SR by the SR Ca2+ -ATPase (SERCA). (4) Calcium is also pumped out of the cell by Na+ –Ca2+ exchange (NCX). Figure modified from Eisner et al. (2013b) with permission.
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It is worth considering why the adult cardiac ventricular myocyte does not make use of store-operated channels. I can only speculate and suggest that it may be more efficient for the myocyte to modify Ca2+ fluxes, which are already present, to serve excitation–contraction coupling, rather than adding an additional Ca2+ -entry mechanism via store-operated channels. It should also be noted that cells which make use of store-operated channels generally have much smaller Ca2+ fluxes than do cardiac myocytes. Finally, it is important to point out that changing the expression or activity of any of the main Ca-handling proteins (L-type Ca2+ channel, NCX, SERCA or RyR) will alter the set point at which SR Ca is regulated. A good example of this is provided by the effects of heart failure (HF). Much previous work has shown that the SR Ca2+ content is decreased in HF (Hobai & O’Rourke, 2001; Piacentino et al. 2003). This is probably due to the observed increase of NCX, decrease of SERCA and increased leakiness of the RyR, all of which will lead to a decrease of SR Ca content. It must, of course, be remembered that many other factors change in heart failure (see Houser et al. 2000 for review); these include structural changes in the myocyte, including the loss of transverse tubules and therefore a loss of the close apposition of L-type Ca2+ channels and RyRs (He et al. 2001; Houser, 2001; Heinzel et al. 2008; Dibb et al. 2009; Crossman et al. 2011).
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The effects of potentiating the RyR
The RyR is the largest channel protein in the cell and has several phosphorylation sites. Phosphorylation of the RyR increases its open probability (po ). It has been suggested that this contributes to the positive inotropic effects of β-adrenergic stimulation (Shan et al. 2010), although this is controversial, with others disputing the result (MacDonnell et al. 2008). See Eschenhagen (2010) for a review. The intracellular messenger cyclic-ADP ribose (Iino et al. 1997) also increases RyR po , and it has been reported that this effect increases the force of contraction of the heart (Macgregor et al. 2007). We therefore became interested in the general question of what the effects of potentiating RyR opening are on the systolic Ca2+ transient. If one is interested in the effects of phosphorylation, it would be nice to phosphorylate the RyR specifically. Unfortunately, it is impossible to do this without also phosphorylating other targets. We have therefore used low (submillimolar) concentrations of caffeine to increase the opening of the RyR (O’Neill & Eisner, 1990; Trafford et al. 1998). A typical result is shown in Fig. 3A (Trafford et al. 2000). As expected, application of caffeine increases the amplitude of the Ca2+ transient. However, this increase is not maintained, and the amplitude of the Ca2+ transient decreases back to control levels within only a few beats. This disappearance of the increase of the Ca2+ transient is a
Figure 2. Regulation of SR Ca2+ content A, refilling of the SR. Before this record began, the SR had been emptied by application of 10 mM caffeine. Caffeine was then removed and stimulation begun. The first Ca2+ transient is shown in red (a) and the last in blue (b). B, enlarged Ca2+ transients (top) and current records (bottom) of transients a and b. C, expanded records of Ca2+ influx (L-type Ca2+ current; left) and Ca2+ efflux (NCX; right). D, the steep dependence of the Ca2+ transient amplitude on SR Ca2+ content. Records redrawn from Trafford et al. (1997).
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result of the feedback regulation of SR Ca (Fig. 2). Calcium influx and efflux are shown in the middle panel of Fig. 3A. Initially, the cell is in Ca2+ flux balance; influx and efflux are equal. Sarcoplasmic reticulum Ca content is therefore controlled at a steady level (bottom trace). However, the increase of the amplitude of the Ca2+ transient produced by caffeine results in an increase of Ca2+ efflux to a level considerably higher than the influx. The cell is no longer in Ca2+ flux balance and therefore both cell and SR Ca decrease, resulting in a decrease of the amplitude of the Ca2+ transient. Eventually, the Ca2+ transient decreases back to control levels and the cell returns to Ca2+ flux balance (see Fig. 3B). A similar transient potentiation of the Ca2+ transient is observed when RyR opening is increased by butanedione monoxime (Adams et al. 1998). Conversely, decreasing RyR opening with either tetracaine (Overend et al. 1998) or acidification (Choi et al. 2000) results in a transient decrease of systolic Ca2+ . We conclude from experiments such as that illustrated in Fig. 3 that increased opening of the RyR does not contribute to the positive inotropic effects of, for example, β-adrenergic stimulation. This leaves unanswered the question as to why the RyR is phosphorylated. This work
also makes the point that the feedback regulation of SR Ca2+ content needs to be considered when interpreting manoeuvres that alter Ca2+ fluxes. Calcium and arrhythmias
Heart failure is a major killer; roughly half of deaths are gradual, due to the inability of the heart to pump enough blood, while the remainder occur suddenly from arrhythmias. A variety of factors, including both structural and electrophysiological remodelling, are responsible for the increased propensity to arrhythmias. One important factor is disturbed calcium handling (for review, see Venetucci et al. 2008 ). As discussed above, Ca2+ release from the SR is triggered by a local rise of [Ca2+ ]i produced by the L-type Ca2+ current. In normal conditions, Ca2+ release stays local as the Ca2+ is absorbed by buffers and taken back into the SR and is seen as a spatially restricted Ca2+ spark (Cheng et al. 1993). If the SR Ca content increases sufficiently then the leak of Ca2+ out of the SR exceeds the ability of the buffers, and Ca2+ diffuses to the next region of the SR, thereby setting up a propagating wave of Ca2+ -induced
Figure 3. The effects of potentiation of RyR opening on the systolic Ca2+ transient and Ca2+ fluxes A, original data. Traces show (from top to bottom): [Ca2+ ]i ; Ca2+ movement across sarcolemma on each pulse (filled symbols show Ca2+ influx on the L-type Ca2+ current and open symbols efflux via NCX); and predicted change of SR Ca2+ content. Caffeine (500 μM) was applied for the period shown. B, graph of systolic [Ca2+ ]i as a function of SR Ca2+ content. Open circles were obtained before application of caffeine, filled circles in caffeine and grey circles on wash out. The letters correspond to the points in A. C, flow diagram showing events underlying the transient behaviour seen in A. Data redrawn from Trafford et al. (2000) .
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Ca2+ release (Eisner & Valdeolmillos, 1986; Wier et al. 1987; Trafford et al. 1993, 1995; Cheng et al. 1996). The probability of occurrence of waves is a very steep function of SR Ca content, with waves occurring only above a certain threshold SR Ca content (D´ıaz et al. 1997). This steep dependence of wave occurrence on SR Ca content may result in part from the steep dependence of SR Ca2+ release on content. The arrhythmogenic consequences of these waves were first noted as an explanation of the arrhythmogenic effects of digitalis intoxication. Digitalis has been used to treat the failing heart but, as is the case for many other inotropic agents, its use is limited by a tendency to produce ventricular arrhythmias. Early work showed that this was correlated with the production of delayed after-depolarizations, which could trigger ectopic beats (Ferrier et al. 1973; Rosen et al. 1973). It is now known that the delayed after-depolarization results from a transient inward current (Lederer & Tsien, 1976), which is produced by the electrogenic NCX removing some of the Ca2+ in the wave out of the cell (Mechmann & Pott, 1986). As originally described, these Ca2+ waves occur in a state of Ca2+ overload when SR Ca2+ content is elevated to above the threshold level (Diaz et al. 1997). This accounts for the production of waves in conditions such as digitalis toxicity and on reperfusion following ischaemia. It also bedevils the use of inotropic agents which act by increasing
Figure 4. The effects of caffeine on generation of Ca2+ waves A, absence of isoprenaline (ISO). A rat ventricular myocyte was stimulated at 0.5 Hz. Caffeine (0.5 mM) was applied for the period shown. Traces show: top, [Ca2+ ]i (expressed as fluorescence normalized to resting levels); bottom, calculated SR Ca2+ content. B, presence of ISO (1 μM). Diagram reproduced from Venetucci et al. (2007), with permission.
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SR Ca content. However, more recent work has shown that Ca2+ waves can also occur if the properties of the RyR are changed such that it opens more and therefore leaks Ca2+ during diastole. This increase in leak has been suggested to contribute to arrhythmias in heart failure, possibly due to phosphorylation of the RyR (Marx et al. 2000; Pogwizd et al. 2001). It is also relevant to an inherited arrhythmia syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT). Patients with CPVT have structurally normal hearts and, at rest, their ECG is indistinguishable from control subjects. However, β-adrenergic stimulation associated with exercise or emotional stress can provoke life-threatening ventricular arrhythmias. Many of those affected have mutations in their RyRs (Priori et al. 2001; Laitinen et al. 2001) that increase the open probability (Jiang et al. 2002). We have investigated two questions relating to CPVT. (i) How does a leaky RyR lead to arrhythmias? (ii) What is the mechanism by which β-adrenergic stimulation produces Ca2+ waves and arrhythmias? In order to investigate the effects of simply increasing RyR open probability, we have, again, studied the actions of caffeine. Figure 4A shows that the application of 0.5 mM caffeine resulted in the production of three Ca2+ waves. Following this, however, no Ca2+ waves were seen. One explanation for this disappearance of Ca2+ waves is provided by the estimate of SR Ca content in the lower trace of Fig. 4A, which shows that the initial occurrence of Ca2+ waves decreases SR Ca as Ca2+ is pumped out of the cell via NCX. This will decrease SR Ca content to below the threshold for producing Ca2+ waves, thereby abolishing the waves (Venetucci et al. 2007). In hindsight, it was perhaps not surprising that simply making the RyR leaky did not result in the occurrence of Ca2+ waves in the steady state. Individuals possessing a CPVT mutation do not have arrhythmias at rest, but only during β-adrenergic stimulation. This raised the idea of repeating the experiment of adding caffeine but now in the presence of β-adrenergic stimulation. When this was done (Fig. 4B), the waves persisted in the maintained presence of caffeine. In other words, these simple experiments mimic the clinical situation. Just as the CPVT patients have leaky RyRs throughout life but develop their arrhythmias only during β-adrenergic stimulation, the experiments show that, at least in the steady state, Ca2+ waves occur only in the combined presence of leaky RyRs and β-adrenergic stimulation. The above experiments, of course, relate only to the effects of adding caffeine. We have investigated the origin of Ca2+ waves in cells in which a CPVT RyR mutation has been knocked in. This work (Fig. 5A) showed that the threshold SR Ca level required to produce a wave is lower in CPVT myocytes than in those from wild-type littermates (Kashimura et al. 2010). There are two explanations for how β-adrenergic stimulation increases the probability of occurrence of C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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Ca2+ waves in CPVT, as follows: (i) phosphorylation of the RyR increases the open probability of the RyR, thereby decreasing the threshold for Ca2+ release from the SR (Wehrens et al. 2003); or (ii) phosphorylation of phospholamban will increase SERCA activity and thence SR Ca2+ content. The experiment illustrated in Fig. 5B shows that in a CPVT myocyte, β-adrenergic stimulation increases SR Ca content. Adding caffeine still decreases SR Ca (Fig. 5B) but, given that the SR Ca starts at a higher level, even when decreased it still exceeds the threshold for Ca2+ waves. Furthermore, we have found that β-adrenergic stimulation increases the threshold for SR Ca2+ release, therefore making Ca2+ waves less likely at a given SR Ca2+ content (Kashimura et al. 2010; cf. Domeier et al. 2012). We conclude, therefore, that β-adrenergic stimulation produces Ca2+ waves by increasing SR Ca2+ content as opposed to decreasing threshold.
Therapeutic strategies against Ca2+ waves
The final question to consider is how one might intervene and inhibit arrhythmias resulting from Ca2+ -dependent waves. One approach is a general anti-arrhythmic strategy using local anaesthetics, which inhibit the Na+ current and thereby prevent ectopic action potentials. An alternative would be to abolish the underlying Ca2+ wave. In
principle, this could be achieved by inhibiting the opening of the RyR, but this strategy would also abolish the Ca2+ release responsible for systole. Rather than inhibiting Ca2+ release through the RyR, one can apply a drug which decreases the open probability and thereby increases the threshold SR Ca content required to produce a Ca2+ wave. We have done this with tetracaine, a compound which decreases the opening of the RyR (Tinker & Williams, 1993) and, as shown in Fig. 6A, increases the threshold SR Ca content required for production of Ca2+ waves (Overend et al. 1997). We found (Fig. 6B) that tetracaine abolishes Ca2+ waves (Venetucci et al. 2006). Tetracaine, however, is not suitable for clinical use in this context because it would inhibit Na+ channels in other tissues and render them inexcitable. It has, however, been shown that the related local anaesthetic flecainide suppresses arrhythmias in CPVT patients, and this effect was attributed to decreased RyR opening (Watanabe et al. 2009). The increased suitability of flecainide in comparison to tetracaine (Watanabe, 2009) has been suggested to result from the fact that flecainaide affects the RyR when it is open, whereas tetracaine acts on the closed channel (Hilliard et al., 2010). Finally, it should be noted that there is some controversy as to whether the beneficial clinical effect of flecainide is solely due to decreasing RyR opening or whether there is also a contribution from an effect on sarcolemmal Na+ channels (Liu, 2012).
Figure 5. Factors affecting the occurrence of Ca2+ waves in catecholaminergic polymorphic ventricular tachycardia A, the effects of a RyR mutation on the SR Ca2+ threshold for Ca2+ waves. The bars show mean (±SEM) values of the SR Ca2+ content required to produce a wave. The left-hand bar represents wild-type (WT) mice and the right-hand one heterozygote littermates expressing the RyR R4496C mutation. B, effects of ISO (1 μM) on SR Ca2+ content. Traces show (from top to bottom): [Ca2+ ]i ; membrane current; and integral of current to calculate SR Ca2+ content. Diagrams reproduced from Kashimura et al. (2010), with permission.
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Figure 6. Tetracaine removes Ca2+ waves A, comparison of the effects of tetracaine and caffeine on the occurrence of Ca2+ waves. The ordinate shows the probability of a Ca2+ wave occurring as a function of the SR Ca2+ content (abscissa). The dashed line shows control data; the other two lines represent the effects of the addition of caffeine (1 mM) or tetracaine (100 μM). Diagram reproduced from Venetucci et al. (2008) with permission. B, effects of tetracaine on Ca2+ waves. Traces show original records of [Ca2+ ]i . Panels show the following conditions (from left to right): control; ISO (1 μM); and ISO (1 μM) + tetracaine (100 μM). Diagram reproduced from Venetucci et al. (2006) with permission.
Conclusion
In this article, I have only been able to touch the surface of Ca2+ signalling in the heart. I have emphasized the importance of the fact that the myocyte is in calcium flux balance and the important consequences that result from this for both inotropy and arrhythmogenesis. There is much more still to be done in this field and, in a light vein, I end with the (politically incorrect) words sung by Peter Sellers and Sophia Loren (1960). Oh doctor, I’m in trouble Well, goodness gracious me! For every time a certain man Is standing next to me A flush comes to my face And my pulse begins to race It goes boom boody-boom boody-boom boodyboom . . . .
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Adams WA, Trafford AW & Eisner DA (1998). 2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes. Pflugers Arch 436, 776–781. Allen DG & Blinks JR (1978). Calcium transients in aequorin-injected frog cardiac muscle. Nature 273, 509–513. Allen DG & Kurihara S (1980). Calcium transients in mammalian ventricular muscle. Eur Heart J Suppl A, 5–15. Bassani JWM, Yuan W & Bers DM (1995). Fractional SR Ca2+ release is regulated by trigger Ca2+ and SR Ca2+ content in cardiac myocytes. Am J Physiol Cell Physiol 268, C1313–C1329. Cheng H, Lederer MR, Lederer WJ & Cannell MB (1996). Calcium sparks and [Ca2+ ]i waves in cardiac myocytes. Am J Physiol Cell Physiol 270, C148–C159. Cheng H, Lederer WJ & Cannell MB (1993). Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262, 740–744. Choi HS, Trafford AW, Orchard CH & Eisner DA (2000). The effect of acidosis on systolic Ca2+ and sarcoplasmic reticulum calcium content in isolated rat ventricular myocytes. J Physiol 529, 661–668. Crossman DJ, Ruygrok PR, Soeller C & Cannell MB (2011). Changes in the organization of excitation-contraction coupling structures in failing human heart. PLoS One 6, e17901. D´ıaz ME, Graham HK & Trafford AW (2004). Enhanced sarcolemmal Ca2+ efflux reduces sarcoplasmic reticulum Ca2+ content and systolic Ca2+ in cardiac hypertrophy. Cardiovasc Res 62, 538–547. D´ıaz ME, Trafford AW & Eisner DA (2001). The role of intracellular Ca buffers in determining the shape of the systolic Ca transient in cardiac ventricular myocytes. Pflugers Arch 442, 96–100. D´ıaz ME, Trafford AW, O’Neill SC & Eisner DA (1997). Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release. J Physiol 501, 3–16. Dibb KM, Clarke JD, Horn MA, Richards MA, Graham HK, Eisner DA & Trafford AW (2009). Characterization of an extensive transverse tubular network in sheep atrial myocytes and its depletion in heart failure. Circ Heart Fail 2, 482–489. Domeier TL, Maxwell JT & Blatter LA (2012). β-Adrenergic stimulation increases the intra-sarcoplasmic reticulum Ca2+ threshold for Ca2+ wave generation. J Physiol 590, 6093–6108. Eisner D, Bode E, Venetucci L & Trafford A (2013a). Calcium flux balance in the heart. J Mol Cell Cardiol 58, 110–117. Eisner D, Caldwell J & Trafford A (2013b). Sarcoplasmic reticulum Ca-ATPase and heart failure 20 years later. Circ Res 113, 958–961. Eisner DA, Choi HS, D´ıaz ME, O’Neill SC & Trafford AW (2000). Integrative analysis of calcium cycling in cardiac muscle. Circ Res 87, 1087–1094. Eisner DA, Kashimura T, O’Neill SC, Venetucci LA & Trafford AW (2009). What role does modulation of the ryanodine receptor play in cardiac inotropy and arrhythmogenesis? J Mol Cell Cardiol 46, 474–481. C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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Eisner DA & Valdeolmillos M (1986). A study of intracellular calcium oscillations in sheep cardiac Purkinje fibres measured at the single cell level. J Physiol 372, 539–556. Eschenhagen T (2010). Is ryanodine receptor phosphorylation key to the fight or flight response and heart failure? J Clin Invest 120, 4197–4203. Fabiato A (1985). Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85, 247–289. Ferrier GR, Saunders JH & Mendez C (1973). A cellular mechanism for the generation of ventricular arrhythmias by acetylstrophanthidin. Circ Res 32, 600–609. He J-Q, Conklin MW, Foell JD, Wolff MR, Haworth RA, Coronado R & Kamp TJ (2001). Reduction in density of transverse tubules and L-type Ca2+ channels in canine tachycardia-induced heart failure. Cardiovasc Res 49, 298–307. Heinzel FR, Bito V, Biesmans L, Wu M, Detre E, von WF, Claus P, Dymarkowski S, Maes F, Bogaert J, Rademakers F, D’hooge J & Sipido K (2008). Remodeling of T-tubules and reduced synchrony of Ca2+ release in myocytes from chronically ischemic myocardium. Circ Res 102, 338–346. Hilliard FA, Steele DS, Laver D, Yang Z, Le Marchand SJ, Chopra N, Huke S & Knollman BC (2010). Flecainide inhibits arrhythmogenic Ca2+ waves by open state block of ryanodine receptor Ca2+ release channels and reduction of Ca2+ spark. J Mol Cell Cardiol 48, 293–301. Hobai IA & O’Rourke B (2001). Decreased sarcoplasmic reticulum calcium content is responsible for defective excitation-contraction coupling in canine heart failure. Circulation 103, 1577–1584. Houser SR (2001). Reduced abundance of transverse tubules and L-type calcium channels: another cause of defective contractility in failing ventricular myocytes. Cardiovasc Res 49, 253–256. Houser SR, Piacentino V 3rd & Weisser J (2000). Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol 32, 1595–1607. Iino S, Cui Y, Galione A & Terrar DA (1997). Actions of cADP-ribose and its antagonists on contraction in guinea pig isolated ventricular myocytes: influence of temperature. Circ Res 81, 879–884. Jiang D, Xiao B, Zhang L & Chen SRW (2002). Enhanced basal activity of a cardiac Ca2+ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death. Circ Res 91, 218–225. Kashimura T, Briston SJ, Trafford AW, Napolitano C, Priori SG, Eisner DA & Venetucci LA (2010). In the RyR2R4496C mouse model of CPVT, β-adrenergic stimulation induces Ca waves by increasing SR Ca content and not by decreasing the threshold for Ca waves. Circ Res 107, 1483–1489. Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA & Kontula K (2001). Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation 103, 485–490.
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Shan J, Kushnir A, Betzenhauser MJ, Reiken S, Li J, Lehnart SE, Lindegger N, Mongillo M, Mohler PJ & Marks AR (2010). Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice. J Clin Invest 120, 4388–4398. Sipido KR, Callewaert G & Carmeliet E (1995). Inhibition and rapid recovery of Ca2+ current during Ca2+ release from sarcoplasmic reticulum in guinea pig ventricular myocytes. Circ Res 76, 102–109. Sitsapesan R & Williams AJ (1997). Regulation of current flow through ryanodine receptors by luminal Ca2+ . J Memb Biol 159, 179–185. Tada M & Katz AM (1982). Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Ann Rev Physiol 44, 401–423. Tinker A & Williams AJ (1993). Charged local anesthetics block ionic conduction in the sheep cardiac sarcoplasmic reticulum calcium release channel. Biophys J 65, 852–864. Trafford AW, D´ıaz ME & Eisner DA (1998). Stimulation of Ca-induced Ca release only transiently increases the systolic Ca transient: measurements of Ca fluxes and sarcoplasmic reticulum Ca. Cardiovasc Res 37, 710–717. Trafford AW, D´ıaz ME, Negretti N & Eisner DA (1997). Enhanced Ca2+ current and decreased Ca2+ efflux restore sarcoplasmic reticulum Ca2+ content following depletion. Circ Res 81, 477–484. Trafford AW, D´ıaz ME, Sibbring GC & Eisner DA (2000). Modulation of CICR has no maintained effect on systolic Ca2+ : simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca2+ fluxes in rat ventricular myocytes. J Physiol 522, 259–270. Trafford AW, Lipp P, O’Neill SC, Niggli E & Eisner DA (1995). Propagating calcium waves initiated by local caffeine application in rat ventricular myocytes. J Physiol 489, 319–326. Trafford AW, O’Neill SC & Eisner DA (1993). Factors affecting the propagation of locally activated systolic Ca transients in rat ventricular myocytes. Pfl¨ugers Archiv 425, 181–183. Venetucci L, Trafford AW, D´ıaz ME, O’Neill SC & Eisner DA (2006). Reducing ryanodine receptor open probability as a means to abolish spontaneous Ca2+ release and increase Ca2+ transient amplitude in adult ventricular myocytes. Circ Res 98, 1299–1305.
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Venetucci L, Trafford AW & Eisner DA (2007). Increasing ryanodine receptor open probability alone does not produce arrhythmogenic Ca2+ waves: threshold Ca2+ content is required. Circ Res 100, 105–111. Venetucci LA, Trafford AW, O’Neill SC & Eisner DA (2008). The sarcoplasmic reticulum and arrhythmogenic calcium release. Cardiovasc Res 77, 285–292. Watanabe H, Chopra N, Laver D, Hwang HS, Davies SS, Roach DE, Duff HJ, Roden DM, Wilde AAM & Knollmann BC (2009). Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in mice and humans. Nat Med 15, 380–383. Wehrens XHT, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, Sun J, Guatimosim S, Song LS, Rosemblit N, D’Armiento JM, Napolitano C, Memmi M, Priori SG, Lederer WJ & Marks AR (2003). FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell 113, 829–840. Wier WG, Cannell MB, Berlin JR, Marban E & Lederer WJ (1987). Cellular and subcellular heterogeneity of [Ca2+ ]i in single heart cells revealed by fura-2. Science 235, 325–328.
Additional Information Competing interests None declared. Funding I am grateful to the British Heart Foundation for funding. Acknowledgements I am indebted to my colleagues for their contributions to the work described in this lecture and would, in particular, like to express my appreciation of the late Stephen O’Neill as both co-worker and friend. I also thank colleagues in the host institutions for their hospitality.
Supporting information Video of GL Brown Lecture
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G.L. Brown Prize Lecture
Calcium in the heart: from physiology to disease David Eisner Unit of Cardiac Physiology, University of Manchester, Manchester, UK
Experimental Physiology
New Findings r What is the topic of this review? This review considers the factors that regulate the systolic Ca transient in cardiac muscle and how disturbed Ca signalling can result in heart failure and arrhythmias. r What advances does it highlight? Sarcoplasmic Reticulum (SR) Ca content is controlled by a mechanism in which it is regulated by alterations in Ca fluxes across the surface membrane. This has important consequences for the regulation of the size of the systolic Ca transient.
Contraction of the heart results from an increase of cytoplasmic Ca2+ concentration ([Ca2+ ]i ), the so-called systolic Ca2+ transient. Most of this results from the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). In turn, the amplitude of this Ca2+ transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca2+ release. The amplitude of the Ca2+ transient is a steep function of SR Ca, resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism, in which an increase of SR Ca content increases the size of the Ca2+ transient, resulting in a decrease of Ca2+ influx on the L-type Ca2+ current and an increase of efflux through Na+ –Ca2+ exchange. Changing the activity of any of the Ca2+ -cycling proteins will change the steady-state SR Ca content. This feedback mechanism has many consequences, including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca2+ transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca2+ waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia, which is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Calcium waves occur only when the SR Ca content exceeds a threshold level. Our data show that the threshold is reduced by the RyR mutation and that the adrenergic stimulation increases SR Ca content. (Received 25 May 2014; accepted after revision 4 August 2014; first published online 15 August 2014) Corresponding author D. Eisner: Unit of Cardiac Physiology, University of Manchester, 31.18 Core Technology Facility, 46 Grafton Street, Manchester M13 9NT, UK. Email: [email protected]
The heart before science
This article is based on the Physiological Society’s G. L. Brown Prize Lecture, given from March to May 2014 at Queen’s University Belfast, Bristol, Cambridge, Edinburgh, Glasgow, Liverpool, The Open and Southampton Universities and Imperial College London. C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
The heart is perhaps the only organ whose function we are aware of because its beat can be felt on the body surface. We know that the heart beat changes in emotion and exercise and, possibly for this reason, many poets and composers have made reference to the heart. Macbeth (1606; Act 1, Scene 3) declares: DOI: 10.1113/expphysiol.2013.077305
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I am thane of Cawdor: If good, why do I yield to that suggestion Whose horrid image doth unfix my hair And make my seated heart knock at my ribs, Against the use of nature? Present fears Are less than horrible imaginings.
In other words, Shakespeare was aware of the link between emotions and the force of the heart beat. Two centuries later (1828), in the song ‘Die Post’ from the cycle Die Winterreise, Schubert provides a perfect representation of a heart beat in the piano accompaniment to the words ‘Was dr¨angst du denn so wunderlich, mein Herz’ (Supplementary video, also available on youtube https://www.youtube.com/ watch?v=VXSJkq5Dk8o). The birth of the era of calcium and the heart
Today, we take it for granted that calcium is involved, not only in the contraction of the heart, but also in the control of the vast majority of mammalian systems. The first indication of the role of calcium came from the work of Sydney Ringer (1836–1910). The story of the discovery is a wonderful reminder that science does not always move in a straightforward way. In 1882, Ringer published the finding that a simple solution of sodium chloride is sufficient to maintain contraction of the isolated frog heart (Ringer, 1882). A year later, however, he retracted this (Ringer, 1883), stating: I discovered, that the saline solution which I had used had not been prepared with distilled water, but with pipe water supplied by the New River Water Company. As this water contains minute traces of various inorganic substances, I at once tested the action of saline solution made with distilled water and I found that I did not get the effects described in the paper referred to. It is obvious therefore that the effects I had obtained are due to some of the inorganic constituents of the pipe water.
From this serendipitous discovery came not only the realization of the biological role of calcium but, in addition, Ringer’s work characterizing the requirements for solutions to maintain the heart (and therefore other organs) in a healthy state, which allowed the introduction of replacement fluids in the clinical arena. It is also worth noting his careful use of language. His leading role in the research is emphasized by ‘I discovered’ and ‘I had used’. In contrast, ‘had not been prepared’ distances him from the error. It took almost 100 years for a direct measurement of the rise of intracellular calcium concentration ([Ca2+ ]i ) that activates contraction (the systolic Ca2+ transient). Fittingly, given the fact that Ringer had worked on the frog heart, Ca2+ measurements were first made on that species (Allen & Blinks, 1978) before data were obtained in the mammalian heart (Allen & Kurihara, 1980).
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Overview of cardiac Ca2+ signalling
There are two sources of Ca2+ for contraction in the heart, as follows. (i) Calcium enters the cell via the L-type Ca2+ channels. This is not the major source of Ca2+ , and its main role is to trigger the release of much more Ca2+ from the sarcoplasmic reticulum (SR). (ii) Calcium is released from the SR through a channel known as the ryanodine receptor (RyR). The probability that the RyR is open, and can therefore allow Ca2+ to leave the SR, is increased by an increase of both cytoplasmic and SR Ca (Meissner, 1994; Sitsapesan & Williams, 1997). The dependence on cytoplasmic Ca2+ results in Ca2+ entry via the L-type Ca2+ channel, triggering Ca2+ release in a mechanism known as calcium-induced calcium release (Fabiato, 1985). For the heart to relax, [Ca2+ ]i must be lowered to resting levels. This occurs by the following two processes: (i) Ca2+ is taken back into the SR by the SR Ca2+ -ATPase (SERCA); and (ii) it is pumped out of the cell by Na+ –Ca2+ exchange (NCX), with some contribution from the plasma membrane Ca2+ -ATPase. The activity of SERCA depends on both Ca2+ and the accessory protein phospholamban. Unphosphorylated phospholamban inhibits SERCA, and when it is phosphorylated (as occurs during β-adrenergic stimulation) this inhibition is relieved (Tada & Katz, 1982). The NCX uses the energy provided by three Na+ ions entering the cell to pump a Ca2+ ion out, thereby generating an electric current. These events are shown in Fig. 1. The following two points need to be emphasized: (i) changes in the amplitude of the systolic Ca2+ transient are the major means by which the contractility of the heart is controlled; and (ii) in the steady state, the cell must be in calcium flux balance on each beat; that is to say, the amount of Ca2+ entering the cell must equal that pumped out and the amount released from the SR must equal that taken back into the SR. Many of the results developed in the remainder of this article follow from this concept of calcium flux balance (see Eisner et al. 2000, 2009, 2013a for reviews). The relationship between SR Ca2+ content and the systolic Ca2+ transient
The experiment illustrated in Fig. 2A shows the effects on the systolic Ca2+ transient of changing the SR Ca content (Trafford et al. 1997). Before the trace began, the SR had been emptied of Ca2+ by exposure to caffeine (10 mM). At this concentration, caffeine binds to all the RyRs and makes them open, thereby releasing Ca2+ into the cytoplasm, whence it is pumped out of the cell. Caffeine was then removed and stimulation recommenced. The Ca2+ transient is initially small because the SR is virtually empty. It then increases in amplitude over a few seconds, presumably because the SR refills. Figure 2D shows the typical, supralinear dependence of the amplitude of the C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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Ca2+ transient on SR Ca content (Bassani et al. 1995; Trafford et al. 1997). On average, the amplitude of the Ca2+ transient depends on the cube of SR Ca content (Trafford et al. 2000). Several factors contribute to this steep dependence, including the fact that an increase of SR Ca will both increase the driving force for Ca2+ efflux from the SR and also increase the number of RyRs that are open. In addition, cytoplasmic Ca2+ buffers may tend to saturation such that, at higher [Ca2+ ]i , a given release of Ca2+ will result in a larger increase of free Ca2+ (D´ıaz et al. 2001). Whatever its origin, the steepness of this relationship means that even modest changes of SR Ca will have marked effects on the amplitude of the Ca2+ transient. There are two immediate consequences of this, as follows. (i) A given fractional increase of SR Ca will have a much larger fractional effect on the amplitude of the Ca2+ transient; therefore, a small increase of SR Ca, resulting, for example, from an increase of heart rate, will result in a larger increase of systolic Ca2+ . Conversely, a given fall of SR Ca content in heart failure will translate to a larger decrease of systolic Ca2+ (D´ıaz et al. 2004). (ii) The steepness of the relationship also presents the cell with a challenge. If the amplitude of the Ca2+ transient is to be maintained constant from beat to beat then the SR Ca content needs to be controlled very precisely. It is therefore important to consider how SR Ca is controlled.
The control of SR Ca2+ content
Figure 2B and C shows the changes of membrane currents accompanying the refilling of the SR. Two changes are obvious. (i) When the SR is empty, the L-type Ca2+ current inactivates more slowly and there is therefore more Ca2+ flux into the cell. This slower inactivation results from decreased Ca2+ -dependent inactivation due to the smaller Ca2+ transient (Sipido et al. 1995; Adachi-Akahane et al. 1996). (ii) The empty SR is also associated with decreased Ca2+ efflux via NCX due to decreased activation of the exchanger by the smaller Ca2+ transient. The net result of these two changes of membrane current is that an increase of systolic [Ca2+ ]i increases Ca2+ efflux and decreases Ca2+ influx (Trafford et al. 1997). This, in turn, results in a powerful negative feedback mechanism to control SR Ca2+ . Briefly, as mentioned above, an increase of SR Ca results (Fig. 2D) in an increase of the amplitude of the systolic Ca2+ transient. This will decrease Ca2+ influx and increase efflux, leading to a decrease of cell and therefore SR Ca. This feedback mechanism is responsible for the beat-to-beat regulation of SR Ca. It is worth noting that this mechanism achieves a similar result as do store-operated channels. These channels, which are particularly prominent in non-excitable cells, open in response to a decrease of endoplasmic reticulum (ER) Ca content (for review, see Parekh & Putney, 2005).
Figure 1. The events involved in excitation–contraction coupling The diagram shows the surface membrane and T-tubule of a ventricular myocyte. (1) The action potential leads to the opening of the L-type Ca2+ channel (ICa-L ) and an increase of [Ca2+ ]i near the ryanodine receptor (RyR). (2) The increase of [Ca2+ ]i makes the RyR open, releasing much more Ca2+ from the sarcoplamic reticulum (SR) and producing the systolic Ca2+ transient. (3) The Ca2+ transient decays due to the closing of RyRs and pumping of Ca2+ back into the SR by the SR Ca2+ -ATPase (SERCA). (4) Calcium is also pumped out of the cell by Na+ –Ca2+ exchange (NCX). Figure modified from Eisner et al. (2013b) with permission.
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It is worth considering why the adult cardiac ventricular myocyte does not make use of store-operated channels. I can only speculate and suggest that it may be more efficient for the myocyte to modify Ca2+ fluxes, which are already present, to serve excitation–contraction coupling, rather than adding an additional Ca2+ -entry mechanism via store-operated channels. It should also be noted that cells which make use of store-operated channels generally have much smaller Ca2+ fluxes than do cardiac myocytes. Finally, it is important to point out that changing the expression or activity of any of the main Ca-handling proteins (L-type Ca2+ channel, NCX, SERCA or RyR) will alter the set point at which SR Ca is regulated. A good example of this is provided by the effects of heart failure (HF). Much previous work has shown that the SR Ca2+ content is decreased in HF (Hobai & O’Rourke, 2001; Piacentino et al. 2003). This is probably due to the observed increase of NCX, decrease of SERCA and increased leakiness of the RyR, all of which will lead to a decrease of SR Ca content. It must, of course, be remembered that many other factors change in heart failure (see Houser et al. 2000 for review); these include structural changes in the myocyte, including the loss of transverse tubules and therefore a loss of the close apposition of L-type Ca2+ channels and RyRs (He et al. 2001; Houser, 2001; Heinzel et al. 2008; Dibb et al. 2009; Crossman et al. 2011).
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The effects of potentiating the RyR
The RyR is the largest channel protein in the cell and has several phosphorylation sites. Phosphorylation of the RyR increases its open probability (po ). It has been suggested that this contributes to the positive inotropic effects of β-adrenergic stimulation (Shan et al. 2010), although this is controversial, with others disputing the result (MacDonnell et al. 2008). See Eschenhagen (2010) for a review. The intracellular messenger cyclic-ADP ribose (Iino et al. 1997) also increases RyR po , and it has been reported that this effect increases the force of contraction of the heart (Macgregor et al. 2007). We therefore became interested in the general question of what the effects of potentiating RyR opening are on the systolic Ca2+ transient. If one is interested in the effects of phosphorylation, it would be nice to phosphorylate the RyR specifically. Unfortunately, it is impossible to do this without also phosphorylating other targets. We have therefore used low (submillimolar) concentrations of caffeine to increase the opening of the RyR (O’Neill & Eisner, 1990; Trafford et al. 1998). A typical result is shown in Fig. 3A (Trafford et al. 2000). As expected, application of caffeine increases the amplitude of the Ca2+ transient. However, this increase is not maintained, and the amplitude of the Ca2+ transient decreases back to control levels within only a few beats. This disappearance of the increase of the Ca2+ transient is a
Figure 2. Regulation of SR Ca2+ content A, refilling of the SR. Before this record began, the SR had been emptied by application of 10 mM caffeine. Caffeine was then removed and stimulation begun. The first Ca2+ transient is shown in red (a) and the last in blue (b). B, enlarged Ca2+ transients (top) and current records (bottom) of transients a and b. C, expanded records of Ca2+ influx (L-type Ca2+ current; left) and Ca2+ efflux (NCX; right). D, the steep dependence of the Ca2+ transient amplitude on SR Ca2+ content. Records redrawn from Trafford et al. (1997).
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result of the feedback regulation of SR Ca (Fig. 2). Calcium influx and efflux are shown in the middle panel of Fig. 3A. Initially, the cell is in Ca2+ flux balance; influx and efflux are equal. Sarcoplasmic reticulum Ca content is therefore controlled at a steady level (bottom trace). However, the increase of the amplitude of the Ca2+ transient produced by caffeine results in an increase of Ca2+ efflux to a level considerably higher than the influx. The cell is no longer in Ca2+ flux balance and therefore both cell and SR Ca decrease, resulting in a decrease of the amplitude of the Ca2+ transient. Eventually, the Ca2+ transient decreases back to control levels and the cell returns to Ca2+ flux balance (see Fig. 3B). A similar transient potentiation of the Ca2+ transient is observed when RyR opening is increased by butanedione monoxime (Adams et al. 1998). Conversely, decreasing RyR opening with either tetracaine (Overend et al. 1998) or acidification (Choi et al. 2000) results in a transient decrease of systolic Ca2+ . We conclude from experiments such as that illustrated in Fig. 3 that increased opening of the RyR does not contribute to the positive inotropic effects of, for example, β-adrenergic stimulation. This leaves unanswered the question as to why the RyR is phosphorylated. This work
also makes the point that the feedback regulation of SR Ca2+ content needs to be considered when interpreting manoeuvres that alter Ca2+ fluxes. Calcium and arrhythmias
Heart failure is a major killer; roughly half of deaths are gradual, due to the inability of the heart to pump enough blood, while the remainder occur suddenly from arrhythmias. A variety of factors, including both structural and electrophysiological remodelling, are responsible for the increased propensity to arrhythmias. One important factor is disturbed calcium handling (for review, see Venetucci et al. 2008 ). As discussed above, Ca2+ release from the SR is triggered by a local rise of [Ca2+ ]i produced by the L-type Ca2+ current. In normal conditions, Ca2+ release stays local as the Ca2+ is absorbed by buffers and taken back into the SR and is seen as a spatially restricted Ca2+ spark (Cheng et al. 1993). If the SR Ca content increases sufficiently then the leak of Ca2+ out of the SR exceeds the ability of the buffers, and Ca2+ diffuses to the next region of the SR, thereby setting up a propagating wave of Ca2+ -induced
Figure 3. The effects of potentiation of RyR opening on the systolic Ca2+ transient and Ca2+ fluxes A, original data. Traces show (from top to bottom): [Ca2+ ]i ; Ca2+ movement across sarcolemma on each pulse (filled symbols show Ca2+ influx on the L-type Ca2+ current and open symbols efflux via NCX); and predicted change of SR Ca2+ content. Caffeine (500 μM) was applied for the period shown. B, graph of systolic [Ca2+ ]i as a function of SR Ca2+ content. Open circles were obtained before application of caffeine, filled circles in caffeine and grey circles on wash out. The letters correspond to the points in A. C, flow diagram showing events underlying the transient behaviour seen in A. Data redrawn from Trafford et al. (2000) .
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Ca2+ release (Eisner & Valdeolmillos, 1986; Wier et al. 1987; Trafford et al. 1993, 1995; Cheng et al. 1996). The probability of occurrence of waves is a very steep function of SR Ca content, with waves occurring only above a certain threshold SR Ca content (D´ıaz et al. 1997). This steep dependence of wave occurrence on SR Ca content may result in part from the steep dependence of SR Ca2+ release on content. The arrhythmogenic consequences of these waves were first noted as an explanation of the arrhythmogenic effects of digitalis intoxication. Digitalis has been used to treat the failing heart but, as is the case for many other inotropic agents, its use is limited by a tendency to produce ventricular arrhythmias. Early work showed that this was correlated with the production of delayed after-depolarizations, which could trigger ectopic beats (Ferrier et al. 1973; Rosen et al. 1973). It is now known that the delayed after-depolarization results from a transient inward current (Lederer & Tsien, 1976), which is produced by the electrogenic NCX removing some of the Ca2+ in the wave out of the cell (Mechmann & Pott, 1986). As originally described, these Ca2+ waves occur in a state of Ca2+ overload when SR Ca2+ content is elevated to above the threshold level (Diaz et al. 1997). This accounts for the production of waves in conditions such as digitalis toxicity and on reperfusion following ischaemia. It also bedevils the use of inotropic agents which act by increasing
Figure 4. The effects of caffeine on generation of Ca2+ waves A, absence of isoprenaline (ISO). A rat ventricular myocyte was stimulated at 0.5 Hz. Caffeine (0.5 mM) was applied for the period shown. Traces show: top, [Ca2+ ]i (expressed as fluorescence normalized to resting levels); bottom, calculated SR Ca2+ content. B, presence of ISO (1 μM). Diagram reproduced from Venetucci et al. (2007), with permission.
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SR Ca content. However, more recent work has shown that Ca2+ waves can also occur if the properties of the RyR are changed such that it opens more and therefore leaks Ca2+ during diastole. This increase in leak has been suggested to contribute to arrhythmias in heart failure, possibly due to phosphorylation of the RyR (Marx et al. 2000; Pogwizd et al. 2001). It is also relevant to an inherited arrhythmia syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT). Patients with CPVT have structurally normal hearts and, at rest, their ECG is indistinguishable from control subjects. However, β-adrenergic stimulation associated with exercise or emotional stress can provoke life-threatening ventricular arrhythmias. Many of those affected have mutations in their RyRs (Priori et al. 2001; Laitinen et al. 2001) that increase the open probability (Jiang et al. 2002). We have investigated two questions relating to CPVT. (i) How does a leaky RyR lead to arrhythmias? (ii) What is the mechanism by which β-adrenergic stimulation produces Ca2+ waves and arrhythmias? In order to investigate the effects of simply increasing RyR open probability, we have, again, studied the actions of caffeine. Figure 4A shows that the application of 0.5 mM caffeine resulted in the production of three Ca2+ waves. Following this, however, no Ca2+ waves were seen. One explanation for this disappearance of Ca2+ waves is provided by the estimate of SR Ca content in the lower trace of Fig. 4A, which shows that the initial occurrence of Ca2+ waves decreases SR Ca as Ca2+ is pumped out of the cell via NCX. This will decrease SR Ca content to below the threshold for producing Ca2+ waves, thereby abolishing the waves (Venetucci et al. 2007). In hindsight, it was perhaps not surprising that simply making the RyR leaky did not result in the occurrence of Ca2+ waves in the steady state. Individuals possessing a CPVT mutation do not have arrhythmias at rest, but only during β-adrenergic stimulation. This raised the idea of repeating the experiment of adding caffeine but now in the presence of β-adrenergic stimulation. When this was done (Fig. 4B), the waves persisted in the maintained presence of caffeine. In other words, these simple experiments mimic the clinical situation. Just as the CPVT patients have leaky RyRs throughout life but develop their arrhythmias only during β-adrenergic stimulation, the experiments show that, at least in the steady state, Ca2+ waves occur only in the combined presence of leaky RyRs and β-adrenergic stimulation. The above experiments, of course, relate only to the effects of adding caffeine. We have investigated the origin of Ca2+ waves in cells in which a CPVT RyR mutation has been knocked in. This work (Fig. 5A) showed that the threshold SR Ca level required to produce a wave is lower in CPVT myocytes than in those from wild-type littermates (Kashimura et al. 2010). There are two explanations for how β-adrenergic stimulation increases the probability of occurrence of C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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Ca2+ waves in CPVT, as follows: (i) phosphorylation of the RyR increases the open probability of the RyR, thereby decreasing the threshold for Ca2+ release from the SR (Wehrens et al. 2003); or (ii) phosphorylation of phospholamban will increase SERCA activity and thence SR Ca2+ content. The experiment illustrated in Fig. 5B shows that in a CPVT myocyte, β-adrenergic stimulation increases SR Ca content. Adding caffeine still decreases SR Ca (Fig. 5B) but, given that the SR Ca starts at a higher level, even when decreased it still exceeds the threshold for Ca2+ waves. Furthermore, we have found that β-adrenergic stimulation increases the threshold for SR Ca2+ release, therefore making Ca2+ waves less likely at a given SR Ca2+ content (Kashimura et al. 2010; cf. Domeier et al. 2012). We conclude, therefore, that β-adrenergic stimulation produces Ca2+ waves by increasing SR Ca2+ content as opposed to decreasing threshold.
Therapeutic strategies against Ca2+ waves
The final question to consider is how one might intervene and inhibit arrhythmias resulting from Ca2+ -dependent waves. One approach is a general anti-arrhythmic strategy using local anaesthetics, which inhibit the Na+ current and thereby prevent ectopic action potentials. An alternative would be to abolish the underlying Ca2+ wave. In
principle, this could be achieved by inhibiting the opening of the RyR, but this strategy would also abolish the Ca2+ release responsible for systole. Rather than inhibiting Ca2+ release through the RyR, one can apply a drug which decreases the open probability and thereby increases the threshold SR Ca content required to produce a Ca2+ wave. We have done this with tetracaine, a compound which decreases the opening of the RyR (Tinker & Williams, 1993) and, as shown in Fig. 6A, increases the threshold SR Ca content required for production of Ca2+ waves (Overend et al. 1997). We found (Fig. 6B) that tetracaine abolishes Ca2+ waves (Venetucci et al. 2006). Tetracaine, however, is not suitable for clinical use in this context because it would inhibit Na+ channels in other tissues and render them inexcitable. It has, however, been shown that the related local anaesthetic flecainide suppresses arrhythmias in CPVT patients, and this effect was attributed to decreased RyR opening (Watanabe et al. 2009). The increased suitability of flecainide in comparison to tetracaine (Watanabe, 2009) has been suggested to result from the fact that flecainaide affects the RyR when it is open, whereas tetracaine acts on the closed channel (Hilliard et al., 2010). Finally, it should be noted that there is some controversy as to whether the beneficial clinical effect of flecainide is solely due to decreasing RyR opening or whether there is also a contribution from an effect on sarcolemmal Na+ channels (Liu, 2012).
Figure 5. Factors affecting the occurrence of Ca2+ waves in catecholaminergic polymorphic ventricular tachycardia A, the effects of a RyR mutation on the SR Ca2+ threshold for Ca2+ waves. The bars show mean (±SEM) values of the SR Ca2+ content required to produce a wave. The left-hand bar represents wild-type (WT) mice and the right-hand one heterozygote littermates expressing the RyR R4496C mutation. B, effects of ISO (1 μM) on SR Ca2+ content. Traces show (from top to bottom): [Ca2+ ]i ; membrane current; and integral of current to calculate SR Ca2+ content. Diagrams reproduced from Kashimura et al. (2010), with permission.
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Figure 6. Tetracaine removes Ca2+ waves A, comparison of the effects of tetracaine and caffeine on the occurrence of Ca2+ waves. The ordinate shows the probability of a Ca2+ wave occurring as a function of the SR Ca2+ content (abscissa). The dashed line shows control data; the other two lines represent the effects of the addition of caffeine (1 mM) or tetracaine (100 μM). Diagram reproduced from Venetucci et al. (2008) with permission. B, effects of tetracaine on Ca2+ waves. Traces show original records of [Ca2+ ]i . Panels show the following conditions (from left to right): control; ISO (1 μM); and ISO (1 μM) + tetracaine (100 μM). Diagram reproduced from Venetucci et al. (2006) with permission.
Conclusion
In this article, I have only been able to touch the surface of Ca2+ signalling in the heart. I have emphasized the importance of the fact that the myocyte is in calcium flux balance and the important consequences that result from this for both inotropy and arrhythmogenesis. There is much more still to be done in this field and, in a light vein, I end with the (politically incorrect) words sung by Peter Sellers and Sophia Loren (1960). Oh doctor, I’m in trouble Well, goodness gracious me! For every time a certain man Is standing next to me A flush comes to my face And my pulse begins to race It goes boom boody-boom boody-boom boodyboom . . . .
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Additional Information Competing interests None declared. Funding I am grateful to the British Heart Foundation for funding. Acknowledgements I am indebted to my colleagues for their contributions to the work described in this lecture and would, in particular, like to express my appreciation of the late Stephen O’Neill as both co-worker and friend. I also thank colleagues in the host institutions for their hospitality.
Supporting information Video of GL Brown Lecture
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