Chapter 11 STABILITY AND INSTABILITY OF RELEASE FROM THE SR

STABILITY AND INSTABILITY OF RELEASE FROM THE SR

Mary E. Díaz, Stephen C. O’Neill, Andrew W. Trafford, and David A.

Eisner

Unit of Cardiac Physiology, University of Manchester, 1.524 Stopford Building, Oxford Rd, Manchester M13 9PT, UK

INTRODUCTION

As discussed in many other chapters in this book, release of calcium during systole occurs through the ryanodine receptor (RyR) via the process of calcium-induced calcium release (CICR). On this mechanism (Fig. 11-1 A), the entry of a small amount of into the cell via the L-type current results in the release of a considerably larger amount from the sarcoplasmic reticulum (SR) (see Bers

for review). The amount of calcium released depends on at least three factors: (1) The size of the L-type current; (2) the properties of the RyR and, in particular, their sensitivity to activation by Ca; (3) the calcium content of the SR. It is changes in these factors that will be important in disease. In this article we focus on these control points and how they affect systolic In particular, we emphasize the factors that determine the stability of control. First, however, it is important to discuss how SR content is controlled.

THE CONTROL OF SR CONTENT

At its simplest, the SR content depends on the balance between the

amount released (via the RyR) and the amount taken up from the cytoplasm

(by the SR SERCA). Therefore anything which increases the

opening of the RyR would be expected to decrease SR content.

Conversely, agents that increase the rate of SERCA (by phosphorylation of the regulatory protein phospholamban)

increase SR content. However it is also clear that, while modulation of SERCA and the RyR will affect the actual level of SR reached, other factors are responsible for the fact that SR can be controlled and, as we will see later, whether or not this control is stable.

Figure 11-1. Steps involved in calcium induced calcium release and regulation of SR content. A. The diagram shows: (i) entry of via the L-type current (ii) release of from the SR through the ryanodine receptor (RyR); (iii) uptake of into the SR by the (iv) efflux of from the cell via exchange. As the amplitude of the transient increases more is pumped out of the cell (iv) and there is greater inactivation of the decreasing entry (v). The three control points described in the text ( SR content and RyR) are shown in bold. B.

Measurement of sarcolemmal fluxes during recovery of SR Caffeine had previously been added to deplete SR and then removed. Stimulation was begun at the start of the record shown. Panels show (from top to bottom): ; sarcolemmal fluxes (influx on L-type current, efflux on NCX); net gain per pulse calculated as influx - efflux; cumulative gain calculated by summing net gains per pulse. Modified from Trafford et al.

It is important to realize that SR content can only be kept at a

constant level if the entry of into the cell from the extracellular fluid

(largely via the L-type current) has exactly the same magnitude as the

efflux (largely via exchange, NCX). If the entry is larger than the

efflux then the cell and therefore the SR will gain The interaction

between the control of SR and cytoplasmic is shown in Fig. 11-1 B. In

this experiment the SR had previously been emptied of calcium by exposure to caffeine. Caffeine was removed and then, at the start of the record shown, stimulation recommenced. The transient was initially small, presumably because the SR content was low. However, over the time- course of a few beats, the transient increased in amplitude to a steady level (top panel). These changes of the amplitude of the calcium transient are accompanied by changes of membrane current. As shown in the second panel of Fig. 11-1 B, as the amplitude of the transient increases, the entry via the L-type current decreases and the efflux on NCX efflux increases.

In the steady state, efflux and influx are exactly balanced.

The lower panels of Fig. 11-1 B show that, at first there is a net influx of on each pulse and, in the steady state there is no net flux. These changes of net flux result in a calculated SR gain of (bottom panel).

This result shows that measured changes of influx and efflux account for significant changes of SR content. The decrease of entry of is due to increased inactivation of the L-type current due to the increased transient.

The increase of efflux is a result of the larger transients, thereby increasing the activation of the NCX. The fact that an increase of amplitude of the systolic transient increases efflux and decreases influx provides an important mechanism for controlling SR as follows. An increase in SR content will increase the amplitude of the transient and this, in turn, will increase efflux and decrease influx (steps iv and v respectively of the scheme of Fig. 11-1 A). These changes of membrane flux will therefore tend to lower SR back towards the initial level. While the exact level of SR reached will depend on the properties of the RyR and SERCA, the ability to maintain a steady SR content depends on this simple homeostatic mechanism whereby changes of SR content affect the amplitude of the systolic transient and hence the sarcolemmal fluxes.

CONTROL POINTS FOR REGULATING THE AMPLITUDE OF THE TRANSIENT

In the introduction we pointed out the three factors that affect the

amplitude of the transient. We will briefly consider these in turn.

The SR content

As shown in Fig. 11-1 B (an increase of SR content results in an increase of the amplitude of the systolic transient. Several studies have shown that the relationship can be steep)

and we have found that the amplitude of the transient is proportional to the cube of SR content.

This steep dependence may be due, in part, to the fact that the open probability of the RyR is increased by an increase of the content of the SR.

The SR content, in turn will depend on the balance between entry into the cell and efflux from the cell. Therefore maneuvres that decrease efflux result in overload of the SR, spontaneous release and arrhythmias.

The properties of the RyR

Agents that increase the open probability of the RyR (such as caffeine or BDM) produce a transient potentiation of the amplitude of the systolic transient.

In the steady state the amplitude of the transient in the presence of these agents is the same as in the control. The transient nature of these effects arises because the potentiation of release decreases SR content. Conversely, depressing RyR opening with either tetracaine or acidification results in only a transient decrease of the transient.

The amplitude of the L-type current

The L-type current has two roles in calcium induced calcium release.

First, it triggers release from the SR and second it contributes to

loading the cell and therefore the SR with

We have found that

changes of external concentration that have large effects on the

amplitude of the L-type current and the transient have very little

effect on the SR content. Indeed lowering external from 2.0 to 0.2

mM resulted in a small increase of SR content.

The relative lack of

effect of external on SR content reflects the fact that the increased

trigger function (which will tend to decrease SR content- like caffeine) is

balanced by the increased loading (which will increase content).

INSTABILITY AND ALTERNANS

In the previous sections of this article, we have emphasised how simple homeostatic mechanisms regulate SR However, it is a general property of such feedback mechanisms that they do not always behave in a stable manner. Indeed if there are delays in the system and the gain is too high then instabilities can result. In the case of the system shown in Fig. 11-1 A, the gain is equivalent to the change of net sarcolemmal flux divided by the initiating change of SR content. This, in turn, depends on a combination of (a) the dependence of release from the SR on the content and (b) the fraction of this released that is pumped out of the cell rather than returned into the SR. Previous modelling has shown that if relationship (a) is made steeper then instabilities result.

One can argue that the advantage of a steep dependence of release on SR content is that it provides a sensitive means whereby small changes of SR content can have large effects on the amplitude of the transient. The disadvantage of a very steep relationship is that instabilities may result.

One form of instability is that of mechanical alternans in which identical stimuli alternately produce large and small contractions (see Euler

for review), associated with alternans of the amplitude of the underlying transients.

Alternans is seen clinically in heart failure

and experimentally in ischaemia and acidosis.

One obvious question is whether the above model of alternans (couched in terms of increased feedback gain) can account for the fact that acidosis and related conditions produces alternans. Indeed it is not immediately obvious how this model can explain the fact that acidosis

or metabolic inhibition

produce alternans since both manoeuvres would be expected to decrease RyR

The question then is what effect does decreasing RyR per se have on the probability of alternans occurring?

The effects of reducing RyR on alternans

In order to investigate this question, we have investigated the effects of

specifically decreasing RyR with the local anaesthetic tetracaine. As

shown by the confocal linescans of Fig. 11-2 A, this results in a subcellular

alternans. Thus the region with the greatest release in c has little release

in d and, conversely, those regions that release in d did not do so in c. The

traces labelled as i and ii below emphasise the discordant nature of this

subcellular alternans. Fig. 11-2 B shows another line-scan in tetracaine. This

shows that there are two phases of release. The first is more or less

uniform throughout the cell whereas the second spreads as a wave through

part of the cell. The superimposed specimen traces below demonstrate that

the second phase of release reaches region i before ii and never enters region iii. Similar results were found when intracellular acidification was used to decrease RyR

These results therefore show that the large local responses in this alternans require wave propagation. This is important because wave propagation is known to occur only above a threshold SR content.

This result therefore raises the possibility that the threshold nature of wave propagation produces a steepening of the relationship between SR content and release. Wave propagation (in this case radially into the cell) has also been observed in alternans in atrial cells.

Figure 11-2. Local alternans produced by tetracaine. A. Linescans a and b were obtained

in control and the others in 50 tetracaine. In each panel a 100 ms duration depolarizing

pulse was applied from –40 to 0 mV. Traces i & ii (below) represent the fluorescence

measured at the points indicated. B. Linescan in the presence of tetracaine with (below) three

specimen fluorescence records from the points indicated. Modified from Diaz et al.

Does SR content alternate?

As described above, the hypothesis for alternans depends on a beat-to- beat alternation of SR content. However it has also been suggested that alternans may be due to beat-to-beat alternation of the release process of calcium from the SR rather than the content.

Figure 11-3. Alternation of SR content. A. Original data. The cell was stimulated with

a 10 mV depolarizing pulse (see text) resulting in alternans. Stimulation was stopped after

either a large (left) or small transient (right pulse) in order to measure SR content. Note

that the caffeine response is larger following the small transient. B. SR content is greater

before a big than a small transient but the fractional change is less than that of systolic

C. Data taken from.

D. Model. enters via the L-type current (i) inducing release

from a “coupled” RyR (ii). On the first pulse SR content is above a threshold value and

this spreads as a wave (iii) and activates release from other RyRs (iv). As a result of

consequent loss of from the cell, on the next pulse the SR content will be below

threshold and the initial release of (ii) will not be able to produce a wave. The smaller

response will therefore lead to an increase of SR content and thence alternans.

Measurement of SR content during alternans induced by tetracaine

or acidification is complicated by the subcellular heterogeneity of the

alternans. We have therefore recently developed an alternative model of

alternans. To do this we use small amplitude (10 mV) depolarizing pulses

from a resting potential of –40 mV (in the presence of elevated external

concentration, 5 mM). As shown in Fig. 11-3, this stimulation protocol

results in alternans.

This figure also shows that the SR content is

greater at the time of the large than the small transient and, therefore,

that the alternans of transient amplitude is, indeed, accompanied by

alternans of SR content. Figs. 11-3 B+C show that the rather small fractional

alternation of SR content is accompanied by a much larger change of

transient amplitude. As discussed in the original paper,

this is a

steeper dependence of transient amplitude on SR content than is seen

under control conditions. Again, this steep dependence seems to be due to

the fact that the larger transients require wave propagation and a small

change of SR content about the threshold level therefore determines

whether or not propagation occurs. A diagram of what may be happening in

alternans is shown in Fig. 11-3 D. With small depolarizing pulses, only a

small fraction of L-type channels open (i) and therefore only a small

fraction of RyRs are activated (ii). If the SR content is above the

threshold level then release will spread as a wave from the open RyR

(iii) and activate other RyRs (iv) leading to wave propagation. The

wave will decrease SR as some is pumped out of the cell thereby

decreasing SR content to below the threshold level. As a consequence wave

propagation will not occur on the next stimulus. Little will be lost and

the SR will therefore refill with to a level at which wave propagation

can occur on the next beat. Repetition of these events will thereby produce

alternans. In this model of alternans, the fact that only a small fraction of the

L-type channels are opened results in the opening of only a small fraction of

RyRs. This model can also account for the fact that agents such as acid pH

and tetracaine also produce alternans associated with wave propagation. In

this case many L-type channels will open, however the intrinsic

depressed of the RyR means that only a small number of RyRs will be

activated. In contrast, when small depolarizing pulses are used, the RyR has

a normal sensitivity but the reduced opening of the L-type channel results in

reduced RyR opening. The common factor in the two circumstances is

therefore the decreased opening of the RyR.

CONCLUDING REMARKS

As reviewed above, the SR content is a major factor determining the amplitude of the systolic transient and hence the heart beat. Since the transient affects sarcolemmal fluxes, this provides an important mechanism to control SR content. However, we suggest that excessive feedback gain may result in alternans. One circumstance that produces increased feedback gain is a decrease in the open probability of the RyR.

Here, the increased feedback gain is due to the fact that, when the RyR is decreased, only a small fraction of RyRs are initially activated and activation of the majority of RyRs depends on propagation of waves of CICR. Future work needs to address the question of whether this experimental model of alternans is relevant to clinically observed pulsus alternans. Furthermore it is as yet unclear how alternans is synchronized between cells in the heart.

ACKNOWLEDGMENTS

Work from the author’s laboratory was funded by The British Heart

Foundation.