Download The control of sarcoplasmic reticulum Ca content in cardiac muscle

Cell Calcium 38 (2005) 391–396
The control of sarcoplasmic reticulum Ca content in cardiac muscle
M.E. Dı́az b , H.K. Graham a , S.C. O’Neill a , A.W. Trafford a , D.A. Eisner a,∗
b
a Unit of Cardiac Physiology, University of Manchester, 1.524 Stopford Building, Oxford Rd, Manchester M13 9PT, UK
Veterinary Biomedical Sciences, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK
Received 20 June 2005; accepted 28 June 2005
Abstract
Most of the calcium that activates contraction in the heart comes from the sarcoplasmic reticulum (SR) and it is therefore essential to control
the SR Ca content. SR Ca content reflects the balance between uptake (via the SR Ca-ATPase, SERCA) and release, largely via the ryanodine
receptor (RyR). Unwanted changes of SR Ca are prevented because, for example, an increase of SR Ca content increases the amplitude of
the systolic Ca transient and this, in turn, results in increased loss of Ca from and decreased Ca entry into the cell thereby restoring cell and
SR Ca towards control levels. We discuss the parameters that affect the steady level of SR Ca and how these may change in heart failure.
Finally, we discuss disordered Ca regulation with particular emphasis on the condition of alternans where successive heartbeats alternate in
amplitude. This behaviour can be explained by excessive feedback gain in the processes controlling SR Ca.
© 2005 Elsevier Ltd. All rights reserved.
Keywords: Cardiac muscle; Sarcoplasmic reticulum; SERCA
1. Introduction
As reviewed in several other articles in this Special Issue,
many cellular functions are regulated by changes of intracellular Ca. In many cases, this calcium is released from an
intracellular store. Of these intracellular stores, the best characterized is undoubtedly the endoplasmic reticulum (ER) and
its counterpart in muscle the sarcoplasmic reticulum (SR).
In cardiac muscle, the bulk of the calcium that activates
contraction comes from the SR. Release occurs via the process of Ca-induced Ca release (CICR). The depolarisation
that occurs during the plateau of the action potential results
in the opening of sarcolemmal L-type Ca channels. These
channels are placed very close to the SR and the resulting
local increase of [Ca2+ ]i triggers the opening of the SR Ca
release channel known as the ryanodine receptor (RyR). The
amount of Ca released from the SR depends not only on
the trigger but also on SR Ca content and there is a steep
(approximately cubic) dependence of Ca release on SR con∗
Corresponding author. Tel.: +44 161 275 2702; fax: +44 161 275 2703.
E-mail address: [email protected] (D.A. Eisner).
0143-4160/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ceca.2005.06.017
tent [1–3]. This means that the ability to control the amplitude
of the systolic Ca transient requires precise control of SR
content.
2. Control of SR Ca in a resting cell
A state of rest is, of course, not a natural state for the heart
which beats through life. It is, however, relevant to other cell
types where Ca release occurs less regularly and, in the context of the heart, can also be instructive. At rest, the SR Ca
content depends on the balance between Ca uptake by the SR
Ca-ATPase (SERCA) and efflux of Ca through channels. The
activity of SERCA depends on various factors: (1) the cytoplasmic Ca concentration—the higher this is the greater is the
activity of SERCA; (2) the SR Ca content—an increase of SR
Ca increases the gradient against which SERCA must pump
and therefore slows its rate [4]. In some cell types, this inhibition by intra-SR Ca has been shown to be at least as important
as activation by cytoplasmic Ca in determining the overall rate
of Ca pumping into the SR [5]; (3) SERCA activity is also
controlled by the natural inhibitory protein phospholamban
[6]. Phosphorylation of phospholamban relieves this inhibi-
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M.E. Dı́az et al. / Cell Calcium 38 (2005) 391–396
tion and this is the basis of enhanced Ca uptake into the SR
under the influence of beta adrenergic stimulation.
As far as Ca efflux from the SR is concerned, a major pathway is through the RyR [7]. Since the RyR open probability
is increased by cytoplasmic Ca concentration, then the open
probability will be low under resting conditions. It is not,
however, zero as shown by the occurrence of Ca “sparks”
at rest [8]. The open probability of the RyR is not solely
controlled by [Ca2+ ]i . The RyR can be phosphorylated and
this affects opening [9,10]. Indeed, it has been suggested that
“hyperphosphorylation” in heart failure leads to a decrease
of SR content [11], but this is controversial ([12] and see
also later). There have also been reports of IP3 receptors in
cardiac muscle [13,14] but, at least in ventricular muscle (as
opposed, for example to the atrium) these are thought to be
of less significance than the RyR. Another potential pathway
for Ca efflux is by reversal of SERCA [15]. Finally, there may
be other “leak” channels [16].
3. What limits the maximum SR Ca content?
There are several factors that may limit the maximum
SR Ca content: (1) the leak may be so small that SERCA
comes into equilibrium such that the free energy of the SR
Ca gradient exactly balances that available from ATP hydrolysis. If this is the case then there will be no net flux of Ca
through SERCA and the forward (uptake) reaction will be
exactly balanced by reverse (efflux through SERCA) [15];
(2) a steady state may be reached at which the outward leak
of Ca from the SR balances Ca uptake on SERCA. This explanation is favoured by the observation that application of the
local anaesthetic tetracaine that decreases the open probability of the RyR can produce a large increase of SR Ca content
[17,18]; (3) another factor limiting SR Ca content is that when
this content reaches a threshold level [19] then spontaneous
release of Ca that propagates as a wave of CICR along a cell
is observed [20]. This behaviour may result from the fact that
an increase of Ca concentration inside the SR increases the
open probability of the RyR [21,22].
4. Regulation of SR Ca content in stimulated cells
Analysis of the control of Ca under stimulated conditions
is complicated by the fact that on each beat Ca enters the cell
via the L-type current and is pumped out of the cell (largely
on Na–Ca exchange, NCX). The resulting changes of [Ca2+ ]i
will then influence SR content. How then is the SR content
controlled?
Control arises because: (1) an increase of SR Ca increases
the amplitude of the systolic Ca transient; and (2) the
increased Ca transient results in loss of Ca from the cell and
therefore from the SR. This effect of Ca transient amplitude
on cell Ca arises due to two factors. First, the Ca transient
accelerates inactivation of the L-type Ca current and there-
fore the larger the Ca transient, the faster the inactivation of
Ca entry and therefore the smaller the Ca entry. Second, an
increase in the amplitude of the Ca transient increases the
efflux of Ca from the cell as NCX is more strongly activated.
The net result is therefore that an increase in the amplitude
of the Ca transient leads to a net loss of Ca from the cell.
The combination of the two factors results in a simple feedback loop in which an undesired change of SR Ca content is
compensated for by changes of sarcolemmal fluxes [2,23,24].
This feedback scheme provides a means to control SR Ca
content. It plays an analogous role to that carried out by store
dependent Ca entry in non excitable cells [25,26]. It should
be noted that other factors will determine the exact value of
the steady state level of SR content. We will consider some
of these factors below.
4.1. Effects of RyR open probability
An increase of RyR open probability will decrease SR Ca
content. An example of this is shown in Fig. 1. Here a low
concentration of caffeine was applied to increase the open
probability of the RyR. This results in an increase of the
systolic Ca transient (Fig. 1A). However, as shown in the
specimen records of Fig. 1E and F, this increases Ca efflux
such that efflux is now greater than influx (Fig. 1B). As a
result of this flux imbalance SR content decreases (Fig. 1C).
4.2. Effects of Ca entry into the cell
At first sight, one might expect that an increase of Ca
entry into the cell would increase SR Ca content. That this is
not the case is shown by the data reproduced in Fig. 2. In this
experiment, the Ca influx was altered by changing external Ca
concentration ([Ca2+ ]o ). As expected (upper trace of Fig. 2B),
a given change of external Ca resulted in a roughly proportional change of the amplitude of the systolic Ca transient. In
contrast, SR Ca content (lower graph of Fig. 2B) was much
less sensitive to [Ca2+ ]o . Indeed, the only significant change
of SR content was that as [Ca2+ ]o was decreased from 1.0 to
0.2 mM, there was an increase of SR content. The explanation of this unexpected result is indicated in Fig. 2C. There
are two effects of increasing the Ca influx: (1) an increase in
the opening of the RyR. This will result in an increase in the
fraction of SR Ca that is released and therefore in the amplitude of the Ca transient. This effect alone is equivalent to
that of the low concentration of caffeine shown in Fig. 2 and,
alone, would result in a transient increase of the amplitude
of the systolic Ca transient accompanied by a decreased SR
content; (2) the other effect is to load the cell and therefore
the SR with Ca. This effect by itself would increase the SR
content. The net effect on SR content depends on which of
the two is a stronger effect [27].
We have argued previously [27,28] that the fact that large
changes of Ca current have little effect on the SR content is
physiologically important. It means that an abrupt increase
of the L-type Ca current will produce an immediate increase
M.E. Dı́az et al. / Cell Calcium 38 (2005) 391–396
393
Fig. 1. The effects of potentiating RyR opening on SR and cytoplasmic Ca. (A) The effects of 500 !M caffeine on systolic [Ca2+ ]i . The inset shows that the
systolic Ca transients in control (a) and in the steady state in caffeine (c) are indistinguishable. (B) Measured Ca influx on L-type Ca current (!) and efflux
("). (C) Calculated change of SR Ca. (D and E) Specimen Ca transients and current for control (a) and peak in caffeine (b) responses in A. (F) Calculated Ca
fluxes. These are changes of total cell Ca. Modified from [3].
of the amplitude of the systolic Ca transient without the delay
that would be required if SR Ca had to increase. It also
means that other manoeuvres that change Ca influx will not be
expected to affect SR content. Thus, we have recently found
that ventricular myocytes from older sheep have larger Ca
transients and L-type Ca currents than do those from younger
Fig. 2. The effects of changing Ca influx on SR and cytoplasmic Ca. (A)
Effects of changing external Ca to the levels shown. (B) Average values of
systolic (top) and SR Ca (bottom). (C) Schematic showing assumed mechanism. (1) Raising external Ca increases ICa . (2) This increases RyR opening
which leads to an increase of systolic [Ca2+ ]i (4) but would also deplete SR
Ca. However, this is balanced by increased uptake of Ca into the SR via
SERCA (3). The combined effect is no change of SR Ca (5). Modified from
[27].
animals. Interestingly, the SR Ca contents are identical in the
young and old animals [29]. This lack of effect on the SR
content of changes of L-type current is exactly what would
be predicted from the above work.
5. SR function in heart disease
As discussed previously, the main source of Ca2+ that activates contraction in the heart is the SR and the amplitude of
the systolic Ca2+ transient is approximately proportional to
the cube of the SR Ca2+ content [3]. Thus, in disease situations where cardiac contractility is reduced, any form of SR
dysfunction should have profound effects on contractility. To
this end reductions in SERCA mediated Ca2+ uptake into SR
vesicles have been demonstrated in models of cardiac disease
[30]. More recently, direct quantitative analysis of changes
in SR Ca2+ content (Fig. 3A) has been performed [31–33].
Fig. 3B shows typical mean data obtained using such quantitative methods; here SR Ca2+ content is reduced by 20% in
the hypertrophied myocardium. Given the steep dependence
of the systolic Ca2+ transient on SR Ca2+ content, this apparently modest reduction in SR Ca2+ content exactly accounts
for the greater than 50% fall in the amplitude of the systolic
Ca2+ transient in this study [32].
5.1. What reduces SR Ca2+ content in heart disease?
To produce the reduction of SR Ca2+ content noted above
in heart disease requires that the normally tightly controlled
balance between sarcolemmal Ca2+ entry and efflux through-
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Fig. 3. The effects of left ventricular hypertrophy (LVH) produced by aortic coarctation on Na–Ca exchange (NCX) and SR content. (A) The SR Ca content
(as assessed by the caffeine-evoked increase of [Ca2+ ]i ) is decreased in LVH yet the NCX current is increased. (B) Mean data of the effects of LVH on SR
content. (C) Typical plot of NCX as a function of [Ca2+ ]i showing the increased steepness in LVH. (D) In LVH, the ability of the SR to remove Ca from the
cytoplasm is decreased but that of the NCX is increased. Modified from [32].
out the contractile cycle becomes unbalanced in favour of
Ca2+ removal. Fig. 3C shows that in the diseased heart, the
Na+ –Ca2+ exchanger generates a greater inward current for
a given change in [Ca2+ ]i thereby providing evidence that
the sarcolemma now has a greater role in removing Ca2+
from the cytosol. Further evidence of a shift away from SR
mediated Ca2+ uptake to sarcolemmal mediated Ca2+ extrusion is also provided in Fig. 3D. Here, the SR and Na+ –Ca2+
exchange dependent rates of Ca2+ removal have been quantitatively assessed and in the diseased hearts show that SERCA
mediated Ca2+ uptake is reduced and, in line with the data of
Fig. 3C, that Na+ –Ca2+ exchange mediated Ca2+ removal is
enhanced. Overall, quantitative analysis of sarcolemmal and
SR Ca2+ fluxes in this study showed that the greater reliance
on sarcolemmal mediated Ca2+ efflux, which occurred without any change in sarcolemmal Ca2+ entry via the L-type
Ca2+ channel, accounted for nearly all of the fall in SR Ca2+
content and thus ultimately the lower contractility that was
observed.
In addition to these effects on Ca removal mechanisms,
it has also been suggested that an increased diastolic leak of
calcium from the SR contributes to decreasing SR Ca content [10,11,34]. The relative contribution of this mechanism
versus effects on Ca removal remains to be elucidated [12].
6. Unstable control of calcium
As reviewed above, it appears that the SR Ca content is normally regulated by a negative feedback mechanism whereby
changes of SR content affect the amplitude of the Ca tran-
sient and thence Ca fluxes across the surface membrane. Such
feedback systems do not always operate in a stable manner.
One example of instability is a phenomenon known as “alternans” where the amplitude of the contraction and underlying
Ca transient alternate from beat to beat. Modelling studies
have suggested that such alternans could arise if the feedback gain is too high. In other words, a given change of SR
Ca content results in a very large change of Ca efflux from
the cell and this could arise because Ca release from the SR
is a very steep function of SR Ca content [23,35,36].
Ca alternans can be produced reliably using small depolarizing voltage clamp pulses that activate only a small fraction
of the L-type Ca channels [37]. As shown in Fig. 4A, this
results in a marked alternans. The larger responses in the
alternans result from propagation of Ca waves from sites of
initial release. This involvement of Ca waves in the larger
responses is of interest given the fact that Ca waves only
occur above a threshold level of SR Ca content [19]. This
threshold like behaviour will result in an even steeper than
normal dependence of Ca release on SR content. Briefly, the
small depolarizing pulses will result in the activation of a
small number of L-type Ca channels and therefore a small
number of RyRs. If, locally, the SR Ca content is above the
threshold for wave propagation, then Ca waves will spread
out from these initiating sites and a large response will be
observed. This large response will produce Ca efflux from
the cell thereby decreasing SR Ca content such that on the
next stimulus the SR content is below threshold and no waves
result and the SR consequently refills. Fig. 4C shows that the
expected changes of SR Ca content can indeed be observed.
It is these modest changes of SR Ca content that are respon-
M.E. Dı́az et al. / Cell Calcium 38 (2005) 391–396
395
Fig. 4. Calcium alternans. (A) Three consecutive linescans in response to a depolarizing pulse from −40 to −20 mV are shown. The first and third responses are
large and the second small. (B) SR Ca content measured at the time of the big and small Ca transients. (C) Mean amplitudes of the big and small Ca transients.
Modified from [37].
sible for the much larger fractional changes of systolic Ca
(Fig. 4B).
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