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1806
FResearch Advances Series
Intracellular Calcium Homeostasis
in Cardiac Myocytes
William H. Barry, MD, and John H.B. Bridge, PhD
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C alcium homeostasis in cardiac myocytes is of
functional importance for at least three reasons. First, cardiac myocytes must achieve a
resting cytosolic calcium ion concentration ([Ca2+]) of
<200 nmol/L if the contractile elements are to relax.
With extracellular [Ca2"] at 1 mmol/L, this low [Ca2"]
must be maintained in the presence of a 5,000-fold
gradient for Ca2' across the sarcolemma. Second, coupling of excitation to contraction (E-C coupling) in
heart involves a complex interaction of membrane electric events mediated by specific ion channels in the
sarcolemma. This results in calcium influx, the release
of calcium from intracellular stores in the sarcoplasmic
reticulum (SR) via Ca2+-specific channels in the SR
membrane, and subsequent extrusion of the calcium.
This in turn is coordinated with reuptake of calcium into
the SR stores. A fundamental principle of this process is
that to maintain steady-state calcium homeostasis, the
amount of calcium entering the cell with each contraction must be extruded before the subsequent contraction. Third, the force of contraction in cardiac myocytes
is modulated by variations in the magnitude of the
calcium transient. Drugs that modify calcium homeostasis may significantly alter the force of contraction of an
individual myocyte and thus of the intact heart. In this
discussion, we review recent work that has increased our
understanding of the structure and function of the ion
channels and transport proteins that are most critically
involved in [Ca 2+]i homeostasis. We consider how they
interact during the processes of E-C coupling, contraction, and relaxation and briefly discuss how a number of
positively inotropic drugs produce an increase in force.
The reader is also referred to several other recent
reviews that deal with these issues.1-3
Control of Resting [Ca2"Ji
Various ion channels and transport proteins involved in
calcium homeostasis in the cardiac myocyte are shown
schematically in Figure 1. In the resting ventricular myocyte (Figure 1A), the [Ca2+]i is determined by a Ca2+ leak
that is compensated for by an ATP-dependent sarcolemmal Ca 2+ pump (sarcolemmal Ca2+-ATPase) and the
sarcolemmal Na+-Ca2' exchanger. This exchanger has
recently been cloned.4 The complementary DNA encodes
From the Division of Cardiology and the Nora Eccles Harrison
Cardiovascular Research and Training Institute, University of
Utah School of Medicine, Salt Lake City.
Address for correspondence: William H. Barry, MD, Division of
Cardiology, University of Utah Medical Center, 50 N. Medical
Drive, Salt Lake City, UT 84132.
Received June 19, 1992; revision accepted February 26, 1993.
a protein of 970 amino acids with a molecular mass of 108
kd. The native exchanger protein has a maximal apparent
molecular mass of about 160 kd, the discrepancy possibly
a result of glycosylation of the native protein. Hydropathy
analysis indicates that the protein can be divided into
three regions: a hydrophobic NH2 terminal portion containing six potential membrane-spanning segments; a long
hydrophilic region that is modeled as a large cytoplasmic
loop; and a hydrophobic COOH terminal portion comprising six potential membrane-spanning segments. At
this time, little is known about relations between structure
and function of this molecule. However, deletion mutants
in which the putative cytoplasmic loop has been removed
have been expressed in Xenopus oocytes. Whereas the
native molecules have a requirement for activating Ca2 ,5
these mutant exchangers lack this requirement (K Philipson, personal communication). Some information is also
available on the distribution of this exchanger. Recent
experiments investigating the binding of antibodies to the
Na+-Ca2' exchanger with immunofluorescence and immunoelectron microscopy indicate that although the exchanger is detectable over the entire myocyte surface,
antibody binding sites appear to be concentrated in the T
tubule region of the myocyte.6 The functional significance
of this arrangement is not yet clear.
The exchanger is a counterion transporter on which
three Na+ are exchanged for each Ca2+.7 This results in
positive charge movement that opposes the direction of
calcium transport. The net electrochemical force producing the exchange is given by the following equation:
A=3A/Na A/lCa
(1)
where A is the total electrochemical driving force (free
energy), 'A/.Na is the free energy available in the electrochemical gradient for Na+, and 1A/lCa is the free
energy in the electrochemical gradient for Ca2`. Thus, if
three times the energy in the inward Na+ gradient
exceeds that in the Ca2' gradient, Ca2' will be extruded.
If, on the other hand, the Na+ gradient is collapsed
relative to the Ca2' gradient, Ca2' entry and Na+
extrusion takes place. Equation 1 can be stated in a
more familiar form as
A=3ENa-2ECa-Em
(2)
where Em is membrane potential and EN. and ECa are the
equilibrium potentials for Na+ and Ca2`. If the exchange is at equilibrium, A=O, and we obtain
Eeq =3ENa-c2Ea
(3)
The reversal potential is the membrane potential at
which exchange changes direction, and in the case of the
Barry and Bridge Calcium Homeostasis in Myocytes
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Na+-Ca ` exchange, it is identical to the equilibrium
potential, Eeq (around -40 mV) in resting ventricular
cells. The Na+-Ca`+ exchanger is known to be voltage
sensitive.8 If the membrane potential is more negative
than the reversal potential, as is normally the case in the
resting myocyte, the exchanger functions in a Na+-in/
Ca`+-out ("forward") mode and thus produces calcium
extrusion. The importance of the Na+-Ca`+ exchanger
in regulating the resting level of calcium within cardiac
myocytes was demonstrated most directly by the experiments of Sheu and Fozzard,9 who measured intracellular sodium and calcium activities in cardiac Purkinje
cells with ion-sensitive microelectrodes. These experiments revealed a direct relation between [Na+]i and
[Ca'+],. Thus, increases in [Na+]i cause dissipation of
the electrochemical energy in the Na' gradient available to extrude Ca'+, resulting in an increase in [Ca`+]j.
The level of [Na+]l in myocytes is controlled largely by
Na+,K+-ATPase, and thus the Na' pump is also an
important indirect regulator of Na+-Ca2` exchange.
The activity of the Na+-Ca`+ exchanger can also be
modulated by a variety of other mechanisms. For example, the exchanger is inhibited by acidosis10 and by
severe ATP depletion.1' It appears likely that ATP
increases the affinity of the exchanger for Ca`+,12 but it
has not yet been demonstrated that protein kinasemediated phosphorylation of the exchanger has any
regulatory influence. Calmodulin is a 148-amino acid
regulatory protein involved in many Ca 2-dependent
signaling pathways.13 In some transport proteins, including the sarcolemmal calcium ATPase,14 there is an
autoinhibitory calmodulin-binding domain. Calmodulin
can increase the activity of the protein by binding to this
amino acid sequence, presumably interfering with the
interaction of this domain with other components of the
molecule and thus relieving the autoinhibition. Recently, Li et al'5 have synthesized a Na+-Ca2 exchanger
inhibitory peptide. This molecule is identical to the
calmodulin-binding sequence of the exchanger and effectively inhibits exchange.'5 It is not established, however, that this calmodulin-binding peptide sequence
within the exchanger exerts any autoinhibitory effect
under normal physiological conditions.
Another Ca2 transport system that contributes to
maintenance of the low cytosolic concentration in ventricular myocytes is the sarcolemmal calcium ATPase.
The structure and function of plasma membrane calcium ATPases have recently been reviewed by Carafoli.16 The plasmalemmal calcium ATPases are found
in most cells and use the free energy released by the
hydrolysis of one ATP to transport one Ca2 out of the
cell against its concentration gradient. General properties of the plasma membrane calcium ATPases include
a molecular mass of 134,000 kd; a Km for Ca2 of <0.5
gmol/L in the optimally activated state; and as mentioned above, stimulation by Ca2 calmodulin and possibly kinase-induced phosphorylation. In cultured cardiac myocytes, the rate at which the sarcolemmal
calcium ATPase can extrude the Ca2 from the myocyte
appears to be about 1/10 that of the Na+-Ca2' exchanger over the range of physiological cytosolic calcium concentrations.'7 Cannell18 has estimated that the
Na+-Ca'+ exchanger accounts for as much as 75% of
resting Ca'+ efflux. Thus, although the sarcolemmal
calcium ATPase probably does contribute to mainte-
1807
nance of [Ca'+], in resting myocytes, its importance in
this regard relative to the Na+-Ca'+ exchanger seems to
be minor. The intracellular Ca'+ storage sites, such as
mitochondria,'9,20 as well as calcium-binding proteins2'
can buffer short-term (seconds to minutes) changes in
calcium. Steady-state loading of these storage sites is
determined by the [Ca'+]j, which in turn is regulated
largely by transsarcolemmal calcium entry and extrusion
by Na+-Ca'+ exchange and perhaps to a lesser extent by
the sarcolemmal calcium ATPase, as discussed above.
Excitation-Contraction Coupling
In cardiac myocytes, the transition from the resting
relaxed state with low [Ca'+], to a contraction occurs
because a small quantity of Ca'+ crosses the sarcolemma and induces a much larger release of Ca2+ from
the SR. The initial events that couple excitation to
contraction are displayed in Figure 1B. This process is
initiated by depolarization of the cell membrane, which
causes opening of the voltage-gated Na+ and Ca'+
channels. The initial upstroke of the action potential in
ventricular cardiac myocytes is caused by sodium influx
via the Na+ channel, whereas the subsequent inward
current maintaining the plateau of the action potential
is caused primarily by Ca'+ influx via the L-type Ca'+
channel. Cumulative Na+ influx via the Na+ channel also
contributes to maintenance of the intracellular sodium
concentration in myocytes and thus can influence
[Ca'+]i via the Na+-Ca2' exchanger. However, it is Ca2
influx via the L-type Ca2 channel that is considered to
be of greatest importance in E-C coupling.22-25
An important pharmacological characteristic of Ca2
channels is that they contain a high-affinity receptor for
1,4-dihydropyridine ligands. These ligands can function
either as antagonists or agonists. Antagonists such as
nifedipine, when bound to channels, effectively block
their activity. Bay K 8644 is a well-known dihydropyridine receptor agonist and increases Ca2+ current carried by cardiac Ca2+ channels.26 Because dihydropyridine receptors are found in extremely high density in
skeletal muscle T tubules,27 Ca 2+ channels have been
isolated from this source. Most of our present knowledge of these channels is based on studies of the skeletal
muscle channel. The skeletal muscle channel differs
functionally from the heart channel in that it is coupled
to the SR Ca2+ release channel by a voltage sensor
mechanism.28 However, it is believed that the architecture of the two channels is essentially similar.29
The L-type calcium channel is an oligomeric complex
of five subunits. These subunits are designated a,, a2, 13,
, and &. The a2 and 8 are linked by disulfide bonds and
are encoded by the same gene.29,30 As such, they are
designated a2/8. The a, subunit seems to provide the
primary structural and functional basis for the assembled channel. For example, it contains the receptor for
at least three classes of channel antagonists, including
the dihydropyridines; it contains phosphorylation sites
for cyclic AMP (cAMP)-dependent protein kinase; and
it is known to contain the pore of the channel. It can
transmit modest quantities of current and apparently
retains the voltage sensitivity of the native channel. The
a, subunit from heart is somewhat larger than the
skeletal muscle subunit.3'
The relation between subunit organization and function is now under investigation. It appears that the
1808
Circulation Vol 87, No 6 June 1993
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FIGURE 1. Thispage and facing page. Schematic illustration ofthe sequence ofE-C coupling in an adult mammalian ventricular
myocyte. The intracellular ion transport events are shown in the main figures; the corresponding changes in membrane potential,
cytosolic [Ca2+], and contractile force (shortening) from an isolated adult rabbit ventricular myocyte loaded with the
Ca 2`-sensitive dye indo-1 stimulated at 1 Hz at 360C are shown in the insets on the left. Panel A: The processes ongoing in the
resting myocyte or at end diastole. Panel B.: The events associated with early E-C coupling before a rise in cytosolic [Ca 2+].
Barry and Bridge Calcium Homeostasis in Myocytes
1809
MOTION
[c2 ]i.LO
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MEMRANE
POTENTIALV
MOTION
STU
boo-
21m
-
MEMBRANE
POTENTIAL.mV
FIGURE 1. Continued. Panel C: Processes associated with the rise in [Ca 2`Ji and contraction. Panel D: The events associated
with a fall in [Ca 2`Ji and relaxation. See text for discussion. SR sarcoplasmic reticulum; SL, sarcolemma.
1810
Circulation Vol 87, No 6 June 1993
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various subunits of the calcium channel regulate and
enhance many of the properties exhibited by the a1
subunit. It has recently become possible to express
cardiac a, subunits in Xenopus oocytes by injecting the
appropriate messenger RNA.30 By expressing the skeletal muscle subunits a2/8 and f3 alone or in combination,
it has become possible to demonstrate interactions
between these subunits and a. The current caused by
the Ca24-channel agonist Bay K through a, alone is
small but is enlarged if a1 is coexpressed with a2/8 and
is somewhat reduced by coexpression with f3+a2/8.30
Experiments of this type reveal that the various subunits
modulate sensitivity to dihydropyridine agonists, kinetics, and voltage dependence of activation and inactivation. The broad conclusion is that all subunits are
required before the channel can fully express its native
properties, although it should be emphasized that no
combination of subunits yet reported completely mimics
the native channel.
It appears that Ca24 influx via the Na+-Ca2+ exchanger
("reverse" mode) can also occur during initial depolarization (See Figure 1B). First, theoretical considerations
(see Equation 3) indicate that with abrupt membrane
depolarization positive to the reversal potential for the
Na+-Ca2+ exchanger, Ca21 influx should occur as the
exchange reverses. This process may be further stimulated by subsarcolemmal rises in sodium concentration
caused by the sodium influx via the cardiac sodium
channel.32'33 However, whether or not this occurs during
normal E-C coupling remains controversial.m
The Ca21 that enters the cell early after depolarization via the L-type Ca21 channel (and possibly on the
Na+-Ca2' exchanger) can bind to the Ca2' release
channel of the SR (Figure 1B) and activate this channel,35 causing release of calcium from internal stores
within the SR (calcium-induced calcium release36) (Figure 1C). As the [Ca2+]i concentration subsequently
begins to rise as a result of Ca24 release from the SR,
the reversal potential for the Na4-Ca2' exchanger becomes greater than the plateau of the membrane potential because of the rise in the [Ca21]i. Thus, after an
initial period, which may be as brief as 10 msec,18 the
Na4-Ca2' exchanger begins a Na4-in/Ca24-out "forward" mode of operation and thus contributes to Ca24
efflux, as we discuss below.
The Ca24 release channel in the SR has been cloned37
and has been shown to be a 4,969-amino acid protein
with a molecular mass of 564,711 d. Experiments by
Anderson et a135 and Rousseau et a138 have demonstrated that the probability of opening of this channel is
markedly increased by exposure to micromolar concentrations of Ca24. This channel is also opened by methylxanthines such as caffeine39 and has a high affinity for
the plant alkaloid ryanodine. In low concentrations
(<10 ytmol/L), ryanodine induces an open, low-conductance configuration of the channel38; at higher concentrations, ryanodine completely blocks the SR calcium
release channel.40 The SR calcium release channel has
phosphorylation, ATP-binding, and calmodulin-binding
sites. The extent and mechanisms of its regulation are
not well understood at present, although calmodulin
appears to directly reduce the duration of single-channel opening without having an effect on single-channel
conductance.38
Low concentrations of ryanodine open the SR calcium release channel and thus deplete the SR calcium
stores. This effect has been used to examine the extent
to which calcium release from SR by the calciuminduced calcium release mechanism is involved in cardiac E-C coupling. The magnitude of the decrease in
contractile force induced by exposure to ryanodine
ranges from 10% to 90%, depending on the species
studied,41 with rat being the most sensitive. However,
this may not accurately reflect the contribution of Ca24
released from the SR to force development, because in
the presence of continued stimulation of myocardial
cells, it appears that SR Ca2' stores can be maintained
even in the presence of ryanodine.42'43 Thus, with
continued stimulation of myocytes, ryanodine does not
completely deplete the SR. In rested cells exposed to
ryanodine,43 the SR becomes completely depleted. Under these conditions, Ca 2+ influx via the Ca 2+ channel
(and perhaps on the Na4-Ca2' exchanger) causes little
or no contraction directly. Interpretation of this result is
complicated by the possibility that some of this influx of
Ca24 could be directly taken up by an empty SR and
thus prevented from binding to contractile elements.
These observations, however, suggest that most of the
Ca causing contraction in mammalian myocytes during normal E-C coupling is Ca24 released from the SR.
The primary roles of the quantitatively relatively small
Ca24 influx across the sarcolemma44 are to trigger SR
Ca24 release in mammalian myocardium and to maintain SR Ca24 loading.
There is good evidence that not all the Ca24 within
the SR is released with each beat45 and that SR Ca24
release can be "graded" by the amount of trigger Ca2+
entering the cell. The evidence indicating a central role
of Ca2+ influx via the Ca2+ channel in this process has
recently been summarized by Fabiato.46 However, a
graded mechanism requires that Ca2+ released from a
Ca2+ release channel does not open adjacent Ca24
release channels not initially opened by the trigger
Ca2+. This might occur if the rate constant of Ca2+
binding to an inactivating site of the SR Ca2+ release
channel were lower than to an activating site.47 However, studies of the behavior of SR Ca2+ release channels incorporated into lipid bilayer membranes35,40 have
failed to show evidence of Ca2+-induced inactivation of
these channels. It is also possible that diffusion in the
Ca2+ release channel environment is restricted,33 so that
Ca2+ released from an SR release channel might not
have easy access to other channels. However, Ca2+
spontaneously released from a Ca2+-overloaded SR may
induce adjacent SR Ca2+ release, resulting in a propagating "wave" of increasing [Ca2+4] and contraction.48-50
A difference in system "gain" may account for the fact
that Ca2+ released from SR does not activate adjacent
SR Ca2+ release channels under conditions of normal
E-C coupling,25 but this issue is still not satisfactorily
resolved.
The Ca2+ that is released from the SR initiates
contraction by binding to the contractile proteins (Figure 1C). In the resting state, the interaction of actin and
myosin is inhibited by the troponin-tropomyosin complex, which is bound to actin. When Ca24 binds to
troponin C, a conformational change is induced that
results
in
relief of this inhibition, with cross-bridge
interaction and contractile element
shortening.
This
Barry and Bridge Calcium Homeostasis in Myocytes
interaction can be modulated by other contractile element proteins, such as C protein and myosin light chain
2 (see Reference 51 for review). Troponin T can also
influence the affinity of troponin C for Ca2' and thus
alter the sensitivity of the contractile elements for
Ca2+.52 The affinity of troponin C for Ca2+ is also
decreased by decreased intracellular pH.53 cAMP-dependent protein kinase-induced phosphorylation of
troponin 1s4 decreases calcium sensitivity.55 Thus,
changes in the affinity of the contractile elements for
Ca2' as well as the magnitude of the Ca21 transient
achieved during a single beat can regulate force development by a myocyte.
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Relaxation of the Ventricular Myocyte
The decay of the calcium transient (Figure 1D) occurs
because of reuptake of Ca21 into the SR mediated by the
SR calcium ATPase and extrusion of Ca2+ from the
myocyte as mentioned above primarily by the Na+-Ca21
exchanger. The SR calcium ATPase calcium transport
enzyme is concentrated in the longitudinal component of
the SR.56 The cardiac SR calcium ATPase, SERCA2, is a
100-115-kd protein that has been cloned.57 The Ca21
transport characteristics of this enzyme have been well
characterized by studies of calcium uptake by SR membrane vesicles, including vesicles from human myocardium.58 The KCa for calcium transport is 300-400 nmol/L.
Thus, the SR Ca2' ATPase transport is strongly activated
in the range of physiological calcium concentrations that
occur during the normal contraction-relaxation cycle.
Regulation of the SR calcium ATPase occurs primarily by phosphorylation of phospholamban. Phospholamban is a 6,080-d protein59 that binds to and inhibits Ca21
transport by the SR calcium ATPase. When it is phosphorylated by cAMP-dependent protein kinase, this
inhibition is removed, resulting in a greater Vmax and a
lower Kca for calcium transport.60 Phosphorylation of
phospholamban can also occur via action of Ca21 calmodulin-dependent and Ca21 phospholipid-dependent
protein kinases at distinct sites.61 Dephosphorylation of
phospholamban occurs via an SR-associated type 1
phosphatase.62 After uptake by the SR calcium ATPase
in the longitudinal SR, Ca21 is bound to calsequestrin,
which is located primarily in the junctional SR. Calsequestrin is the major Ca2+-binding protein in cardiac
muscle (-50 mol Ca21 per mol protein.) Binding and
release of Ca2' by calsequestrin is thought to be important in E-C coupling, but its exact role is not
understood.63
Ca2' extrusion from the cells via the Na+-Ca2' exchanger also occurs during the declining phase of the
Ca21 transient. At steady state, a balance exists between
the amount of Ca2' entering the cell as the Ca21
current, ICa, and the amount of Ca2+ extruded by the
Na+-Ca2' exchanger.64 Measurements of the amounts
of current generated by these opposite Ca21 transport
pathways show a 2:1 relation (Figure 2). For each 2+
charge entering the cell as a Ca 2+, a 3 + charge leaves
the cell as Na+, transported with a 3 Na+:1 Ca2+
exchange stoichiometry. As previously mentioned, this
electrogenic Na+-Ca2+ exchange is sensitive to membrane potential,8 and therefore the time course with
which calcium is extruded is influenced by the trajectory
of membrane potential. It has also been established that
inward exchange current (corresponding to Ca2+ extru-
1811
sion) is linearly related to [Ca2+],.65 Thus, as the membrane potential changes during the Ca2' transient, the
trajectory of Na+-Ca2' exchange current will reflect
both the time courses of the Ca2' transient and the
membrane potential.
The striking effect of membrane potential on [Ca2+]i
can be inferred from work by Bridge et a166 on voltageclamped myocytes. Ventricular myocytes in which the
SR was disabled with caffeine were unable to relax from
a contraction (induced by an imposed voltage clamp)
until the membrane potential was restored toward resting value (Figure 3, left panel). Moreover, the rate of
relaxation depended on the membrane potential at
which relaxation occurred and was also Na+ dependent
(see Figure 2). These experiments are consistent with a
significant effect of membrane repolarization on the
rate of Ca2+ extrusion via a voltage-sensitive Na+-Ca2+
exchange. There is not a great deal of information on
the relation between Na+-Ca2+ exchange and voltage at
various [Ca2+]i levels. Recent results indicate that at
elevated [Ca 2+], the relation between exchange current
and voltage is steep and is sigmoid67 (see Figure 3, right
panel). Miura and Kimura68 have obtained similar
results. We may infer from these current-voltage relations that membrane repolarization will have an activating effect on Na+-Ca2+ exchange. It should be appreciated that under certain circumstances, the inactivating
effect of a declining Ca2+ could be offset by the activating effect of membrane repolarization if the membrane
potential and [Ca2+]i transient coincide appropriately.
In particular, this is likely to occur if membrane repolarization is rapid during a slowly declining [Ca2+1]
transient. (see Figure 1D).
Competition between Ca 2+ extrusion on the Na+Ca2+ exchanger and Ca2+ uptake by the SR69 is physiologically important because calcium that is taken up by
the SR is available (after transport to SR Ca2' release
sites where it is stored, bound to calsequestrin) to
enhance the Ca21 transient during a subsequent beat
and thus can contribute to increased force development.
Under steady-state conditions, the amount of Ca2+
entering the cell equals that extruded from the cell, and
the amount released by the SR equals that sequestered
by the SR. If an abrupt change in this balance of fluxes
takes place, then the calcium content of the SR will be
affected. It is perhaps worth considering, therefore, how
competition between the SR Ca21 pump and the Na+Ca2+ exchange might be regulated by membrane potential. Let us imagine that some intervention abruptly
shortens the action potential, so that the steep phase of
repolarization occurs earlier during the calcium transient. Because repolarization increases forward exchange earlier during the [Ca 2+]i transient, more Ca21
will be extruded than on a beat during which action
potential duration was not reduced. Thus, the SR
calcium content would be reduced at least by the
additional calcium extruded. An action potential that
was suddenly prolonged relative to the calcium transient
would have the opposite effect, i.e., Ca2+ extrusion
would be suppressed by membrane depolarization.
Results reported by Spurgeon et a170 indicate that in
rat ventricular myocytes, initial repolarization does occur early during the calcium transient. Thus, one would
expect Na'-Ca 2+ exchange to be activated and the
resulting inward current to contribute to the plateau of
Circulation Vol 87, No 6 June 1993
1812
145maM
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0
160
-
120
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80
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40
80
160
120
200
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0
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I
sec
FIGURE 2. Tracings and graph demonstrating the quantitative relation between the amount of Ca2+ entering the cell as the
calcium current ICa and the amount subsequently extruded by the Nai-Ca 2+ exchanger, INa-Ca. In the leftpanel, an inward calcium
current
(initial portion of trace A)
is elicited
by
a
voltage clamp pulse from
-40 to 0 mVin the absence
of external
sodium
(Nao).
This is associated with a contraction, trace D. On repolarization of the voltage clamp to -40 mV (lower trace), there is no
relaxation in the absence ofNa0. However, when Na0 is abruptly increased to 145 mmolIL (upper trace), there is onset of relaxation
(trace E) associated with an inward current caused by electrogenic Na+-Ca 2+ exchange (trace B and with an expanded scale, trace
C). The right panel shows the relation of the integrals of ICa and INa Ca from 11 experiments obtained in a similar protocol. The
solid lines indicate the expected relations for a stoichiometry of 4 or 3 Na+ :1 Ca 2+. The results indicate that for the expected
stoichiometry of 3:1, the amount of Ca 2+ entering as ICa is balanced by the amount of Ca2+ extruded by Nai-Ca 2+ exchange.
Reprinted with permission.66
before significant repolarization had taken place, one
might expect a reeverse exchange (Ca2' influx) during
the late plateau of the action potential. Thus, agents
that prolong the cardiac action potential might cause
calcium overload by both impairing Ca2' extrusion and
the rat action potential, as demonstrated by Schouten
and Ter Keurs.71 Conversely, if the Ca transient were
short compared with the cardiac action potential duration, different effects might be predicted. For example,
were the SR to pump [Ca"2]i down to diastolic levels
mV
0
-150 -100
-20
mY
-80
-40
-50
0
50
100
0.0
]
-0.1 <
2
2
s*c
-0.2
FIGURE 3. Left panel: Tracings showing voltage clamp pulses (top trace) and corresponding shortening records obtained in
isolated guinea pig ventricular myocytes. In these experiments, in which the sarcoplasmic reticulum was disabled by exposure to
10 mmolIL caffeine, the extent of relaxation (and presumably decline in [Ca 2+J]) was strongly dependent on membrane voltage.
Right panel: Graph showing the dependence of INa-Ca on membrane voltage in clamped guinea pig myocytes in which [Ca 2`Ji was
elevated in the absence of Na0 by rapid pacing during exposure to 1 gumol/L ryanodine. Na0 was then rapidly applied at different
voltages to activate the Naf-Ca2+ exchange current, INa-Ca. Note the strong dependence of INaCa on membrane voltage over the
range of potentials shown in the left panel. Reprinted with permission.64
Barry and Bridge Calcium Homeostasis in Myocytes
increasing Ca2` influx via Na+-Ca2` exchange. Calcium
overload may cause spontaneous oscillatory release of
Ca2` from SR,48 resulting in delayed afterdepolarizations and "triggered" arrhythmias in the intact heart.
This mechanism may contribute to arrhythmias seen in
the context of a prolonged QT interval caused by
antiarrhythmic drug therapy and perhaps in the
long-QT syndrome.
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Inotropic Drug Effects on Ca2' Homeostasis
In the context of the above discussion, we may now
briefly consider some of the mechanisms of action of
inotropic drugs. The most commonly used of these is
digitalis. The cardiac glycosides bind to the a-subunit of
the Na+,K+-ATPase and inhibit the translocation of
Na' and K+.72 Thus, partial inhibition of the sodium
pump in the sarcolemma of a myocyte by cardiac
glycosides causes a slight rise in intracellular sodium973,74 and a slight fall in intracellular potassium. An
increase in intracellular Na' concentration would be
expected to increase the influx of calcium on the Na+Ca2' exchanger initially during the action potential75
and to impair extrusion of Ca2' by Na+-Ca2+ exchange
subsequently during the Ca21 transient. Both these
effects would be expected to cause the observed increase in diastolic [Ca 2+1]9 and loading of the SR with
Ca2+ ,76 providing more calcium available for Ca2+induced Ca2' release and thus an enhanced calcium
transient and a positive inotropic effect. A slight prolongation of membrane action potential observed in
some studies during inhibition of the Na' pump74 might
also be expected to impair calcium extrusion by Na+Ca2' exchange and contribute to an inotropic effect.
Catecholamines bind to 8-receptors in the cardiac
cell membrane, activating a guanine nucleotide-binding
protein (G protein), GQ. This in turn causes stimulation
of adenylate cyclase and a resulting increase in the
production of cAMP, which causes activation of cAMPdependent protein kinases.77 As mentioned above, this
kinase causes phosphorylation of the a, subunit of the
calcium channel, which increases calcium influx, and of
phospholamban, which increases calcium uptake by the
SR. These effects produce both an increase in the
"trigger" Ca2+ entering the cell and an increase in
releasable SR calcium content. In spite of a marked
increase in calcium influx via the calcium channel, the
cell is able to maintain its ability to relax (positive
lusitropic effect) because of the stimulation of the SR
calcium ATPase and shortening of the duration of the
action potential. The shortening of the duration of the
action potential and the corresponding enhanced extrusion of calcium by the Na4-Ca2+ exchanger may explain
why progressive calcium overload, with elevation of
diastolic [Ca2+],, does not usually occur in myocytes
stimulated with catecholamines. In fact, end-diastolic
[Ca24], may actually decline in myocytes exposed to
isoproterenol.70 The increased Ca2+ influx per beat can
be extruded more effectively by the Na+-Ca2+ exchanger because of the shorter duration of the action
potential, which in turn results from a cAMP-induced
increase in IK and IC.78,79
Some inotropic agents, including milrinone and amrinone, exert a positive inotropic effect by inhibiting
phosphodiesterase, the enzyme that breaks down
cAMP. Cyclic nucleotide phosphodiesterases differ with
1813
respect to drug sensitivity and substrate specificity,80
and the extent to which both cAMP-dependent protein
kinases and phosphodiesterases are physically located
adjacent to their particular substrates is currently under
investigation.8' The phosphodiesterase inhibitors also
are potent vasodilators, and thus the beneficial hemodynamic effects of these agents are a result of both
positive inotropy and afterload reduction.
Some inotropic agents increase the sensitivity of the
contractile elements to calcium. Endothelin82,83 and angiotensin II84 can exert a potent inotropic effect in
cardiac myocytes by inducing an intracellular alkalosis,
and this change in intracellular pH sensitizes the contractile elements to calcium. The mechanisms of other
agents that appear to sensitize contractile elements to
calcium84-86 and thus produce an increase in force development without a corresponding increase in the magnitude of the calcium transient are less well understood.
These agents may bind to the contractile element troponin-tropomyosin complex and, by a direct effect on these
thin-filament regulatory proteins, alter the calcium sensitivity of the myofilaments. These agents have generated
considerable interest because increased Ca24 loading of
the SR is not a component of their action, and thus a
positive inotropic effect may be achieved without danger
of Ca2' overload-induced arrhythmias.
Summary
Calcium homeostasis in cardiac myocytes results from
the integrated function of transsarcolemmal Ca 2+ influx
and efflux pathways modulated by membrane potential
and from intracellular Ca24 uptake and release caused
predominantly by SR function. These processes can be
importantly altered in different disease states as well as
by pharmacological agents, and the resulting changes in
systolic and diastolic [Ca24], can cause clinically significant alterations in contraction and relaxation of the
heart. It may be anticipated that a rapid increase in our
understanding of the pathophysiology of Ca24 homeostasis in cardiac myocytes will be forthcoming as the
powerful new tools of molecular and structural biology
are used to investigate the regulation of Ca 2+ transport
systems.
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KE-Y WORDs * calcium * myocytes * Research Advances Series
Intracellular calcium homeostasis in cardiac myocytes.
W H Barry and J H Bridge
Circulation. 1993;87:1806-1815
doi: 10.1161/01.CIR.87.6.1806
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