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lACC Vol. 5. No.5
May 1985:IOA-15A
Cellular Mechanism of Action of Cardiac Glycosides
HARRY A. FOZZARD, MD, FACC, MICHAEL F. SHEETS, MD
Chicago. Illinois
It has long been known that cardiac glycosides can inhibit the membrane sodium-potassium (Na + -K +) pump,
raising intracellular Na", However, at clinical concentrations of cardiac g1ycosides, a change in intracellular
Na + that correlates with a change in cardiac contraction
has been very difficult to demonstrate. The recent use
of Na + -sensitive microelectrodes in the experimental
laboratory has made intracellular Na + measurements
possible. A doubling of contraction strength in vitro is
associated with a change of only approximately 1 mM
intracellular Na + • Another membrane transport system,
William Withering in 1785 described the use of digitalis in
his book, An Account of the Foxglove and Some of Its
Medical Uses: With Practical Remarks on Dropsy and Other
Diseases. Since that publication, digitalis preparations have
been used medically for the control of supraventricular tachycardias and treatment of congestive heart failure. Even
though the most frequent use of digitalis is to increase the
strength of cardiac contraction, only recently has the possible mechanism of its action been elucidated. However,
there still exists some controversy as to all the actions of
digitalis on the heart. In this article we will discuss in detail
the generally accepted mechanism of positive inotropic action of digitalis, and we will comment only briefly on the
controversies.
Another article in this series on cardiac glycosides (1)
discusses the interaction of the transmembrane SOdium-potassium (Na + -K +) pump and digitalis. Although this interaction is complex, it is clear that in some doses digitalis
inhibits the Na" -K+ pump (2), producing an increase in
the level of intracellular sodium activity a~a' But if intracellular sodium ion concentration (Na +) increases during
the clinical use of digitalis, how does it lead to increased
contraction strength? We conclude that a~a is increased during clinical use of digitalis, and that the increase of a~a acts
to load the cardiac cell with calcium through an effect on
From the Cardiac Electrophysiology Laboratory, the Departments of
Medicine and the Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois.
Address for reprints: Harry A. Fozzard, MD, Box 249, 5841 South
Maryland Avenue, Chicago, Illinois 60637.
© 1985 by the American College of Cardiology
the Na + _Ca2 + exchange system, exchanges extracellular
Na + for intracellular Ca 2 + • If this system is responsible
for regulating intracellular Ca2+, then it would be very
sensitive to the transmembrane Na + concentration gradient. This influence of intracellular Na + on Na + -Ca2+
exchange is thought to be the cellular basis of the positive
inotropic action of digitalis. However, a number of issues
remain unresolved, such as the extent of Na + -K + pump
inhibition by the level of cardiac glycoside achieved
clinically.
(J Am Coil CardioI1985;5:10A-15A)
the membrane Na + -Ca2+ exchange system. The increase
in cell calcium ion (Ca2 +) then leads to greater contraction
strength.
In this discussion, we review briefly the underlying physiologic processes thought to control cardiac contraction
strength and the possible methods of action of digitalis on
these mechanisms. We then address the question whether
a~a is increased during clinical action of digitalis. Next, we
offer a description of the Na + -Ca2 + exchange hypothesis
and its experimental support. Finally, we identify some of
the unresolved questions regarding the cellular metabolic
actions of cardiac glycosides.
Mechanism of Contraction
The normal sequence of contraction in a cardiac cell can
be divided into three components: 1) the action potential
that triggers release of Ca2+ into the cytoplasm, 2) the
sarcoplasmic reticulum that stores Ca 2 + in the cell, and 3)
the contractile proteins that use Ca2 + and adenosine triphosphate (ATP) to generate force. It will become apparent
that the two major issues to be addressed are control of
cytoplasmic Ca 2 + and sensitivity of the contractile proteins
to Ca 2 + .
The action potential and calcium current. Cardiac
contraction is initiated by an action potential. This is a
depolarization of the surface membrane of the cell for 200
to 400 ms from its level at rest near - 80 mV. The action
potential in atrial and ventricular muscle is generated by
movement of Na + into the cell down a steep electrochemical
0735-1097/85/$3.30
FOZZARD AND SHEETS
MECHANISM OF DIGITALIS ACTION
JACC Vol. 5, No.5
May 1985:lOA-15A
gradient, through membrane Na + channels, which cause a
fast inward Na + current (INa)' The consequent depolarization has two effects: it transiently opens Ca z+ channels,
letting some Ca z+ into the cell (Ica), and it slowly opens
K + channels, which are responsible for the final return to
resting membrane potential.
The Na + electrochemical gradient will be an important
concept later in the discussion of Ca' + metabolism, so it
will be described in greater detail. The extracellular concentration of Na + is 150 to 160 mM. At the ionic strength
of physiologic solutions, this is about 115 mM Na + activity
(a~.). The intracellular activity of Na + (a~.) is 6 to 7 mM.
This low activity is maintained by the membrane Na + -K +
pump (3). The remainder of the intracellular cation is K + ,
and the cell resting potential (Y r ) is about - 80 mY. The
total Na + electrochemical gradient is the sum of the chemical and electrical gradients acting on Na +. The easiest way
to estimate the electrochemical gradient is to measure the
difference between EN. and Y" where EN. is the membrane
potential at which the chemical and electrical gradients acting on Na + would be equal, described by the Nernst equation:
RT
a~.
EN. = -In.....,-.
F
al-l.
The total gradient favoring Na + entry (EN. - Yr ) is about
160 mY. A similar estimate can be made for Ca2+ . Cytoplasmic Ca2+ is low (around 100 oM) and z (Ec• - Yr ) is
about 400 mY (where z = 2 is the valance of Ca z +) (4).
The sarcoplasmic reticulum. The action potential,
probably acting through its Ic., triggers the release of Caz +
from its store in the sarcoplasmic reticulum. The mechanism
by which Ic• triggers release of Ca" + from the sarcoplasmic
reticulum is not entirely clear. It has been studied in skinned
cardiac fibers (cells from which the surface membrane has
been mechanically or chemically removed) by Fabiato (5).
He was able to simulate the increase in Ca? + concentration
to be expected from Ic• and to show a proportional release
of Ca2+ from the sarcoplasmic reticulum. Recently, it has
been possible to measure the transient increase in ~. by its
interaction with the injected bioluminescent molecule aequorin (6). These studies show that a normal action potential
can trigger release of Caz + to cytoplasmic levels as high as
10 p.,M, which is also the level that produces maximal contraction (5).
The sarcoplasmic reticulum is also an important factor
in relaxation by pumping Caz + from the cytoplasm back
into the sarcoplasmic reticulum space. This is accomplished
by an ATP-dependent calcium pump, which can lower
~. back to its level at rest.
The contractile proteins. The transient increase in cytoplasmic Ca? + allows for the interaction of Caz + with the
contractile proteins. These proteins are a very complex system for translating chemical energy into mechanical energy,
and they can be described only briefly here. The two prin-
lIA
cipal components are actin and myosin, which are organized
into separate filaments. When these filaments interact with
each other, they cause movement of one filament relative
to the other, or they produce force proportional to the interaction. In the relaxed state, the actin and myosin do not
interact because the regulatory proteins tropomyosin and
troponin are present. The interaction is initiated by binding
of Ca' + to a site on troponin, which removes the inhibition
by tropomyosin. ATP is used during the interaction cycle,
and in heart muscle it is consumed at a rate largely determined by the force developed.
Calcium balance. It is perhaps obvious from the preceding discussion that the central factor in control of cardiac
contraction is the cell metabolism of Ca2+. We described
Ca z + entry through the fast inward Ca z + current (Ic.), and
its cyclical release and uptake by the sarcoplasmic reticulum. However, for the cell to maintain a steady state, there
must be some means for removing Caz + from the cell at a
rate equal to its entry. Two mechanisms for Caz + efflux
have been identified. One is the cell surface membrane
Caz + -pump, similar to (but not identical with) the sarcoplasmic reticulum calcium pump. Its transport capacity seems
to be rather small, and its role is not yet clear (7). The other
mechanism is a Na + -CaZ+ exchange system in the surface
membrane (8). This system uses the energy of the Na +
gradient to move two to three Na + ions into the cell in
exchange for movement of one CaZ+ ion out of the cell.
We will discuss this large capacity system later.
Possible Mechanisms of Digitalis Action
The normal contraction cycle is initiated by an action
potential. This permits a small Ca2+ current into the cell,
which triggers release of a large amount of Caz + from the
sarcoplasmic reticulum. Cytoplasmic Ca2+ transiently rises
from 100 oM to 10 p.,M and binds to troponin. The inhibition
of the actin-myosin interaction is removed and the two types
of filaments interact, using ATP. CaZ+ then dissociates from
troponin and is taken back up into the sarcoplasmic reticulum, leading to relaxation. The amount of force developed
by the muscle normally depends on: I) the size of Ic.; 2)
the amount of Caz + stored in and released from the sarcoplasmic reticulum; and 3) the sensitivity of the contractile
proteins to CaZ +. For digitalis to strengthen cardiac contraction, it must influence one or more of these mechanisms.
The calcium current question. One of the most effective ways to increase contraction strength is to increase the
inward calcium current (lc.). This is the mechanism of the
positive inotropic effect of beta-adrenergic agonists (9). Activation of the beta-receptor initiates a series of intracellular
events, including an increase in cyclic adenosine monophosphate (cAMP) that results in phosphorylation of membrane components. Probably the Caz + channel itself is phosphorylated, altering its kinetic properties and increasing Ic•
12A
FOZZARD AND SHEETS
MECHANISM OF DIGITALIS ACTION
(10,11). This increased lea is clearly the mechanism of
increasing the contraction strength .
Efforts to show a similar increase in lea by cardiac glycosides have had mixed results (12). Recently, Marban and
Tsien (13) showed that there is a small effect of digitalis
on lea, that may follow rather than lead an increase in
cytoplasmic Ca 2 + . It seems unlikely that a change in lea is
the explanation of the clinical effect of digitalis . However,
it remains possible that effects on lea may playa role in the
alteration of the action potential duration and the QT interval
(14).
Digitalis could affect lea indirectly if it caused release of
catecholamines from cardiac nerves (15). At this point, the
evidence in favor of this mechanism for the positive inotropic effect of digitalis is not substantial, but it could be a
factor in digitalis-induced arrhythmias.
Intracellular calcium. Measurement of the amount of
Ca 2 + in the cell that is available for contraction has been
technically difficult, and no method is free from criticism .
However, most chemical studies indicate that coincident
with the positive inotropic effect of digitalis, there is an
increase in the " rapidly exchangeable Ca2+ pool" (16),
often with an increase in both Ca2+ influx and Ca2+ efflux.
The aequorin method for monitoring transient changes in
intracellular calcium activity (ab) has clearly shown that
digitalis causes an increase in the Ca 2 + transient (6,17).
Measurement of the diastolic level of Ca 2 + with aequorin
has been much more difficult , but definite elevations can be
seen with toxic doses of digitalis (6). Calcium-sensitive
microelectrode measurements do not accurately reflect the
transient changes in Ca 2 + during the contraction, but they
are sensitive enough to measure levels at rest. Sheu and
Fozzard (4) and Lee and Dagostino (18) showed that ab
does increase in an amount roughly proportional to the positive inotropic effect. Recently, Sheu et al. (19) used a new
intracellular Ca 2 + optical indicator called "Quin 2" and
also found an increase in diastolic ~a'
Calcium sensitivity of the contractile proteins. A change
in sensitivity of the contractile proteins to Ca 2 + has been
shown for caffeine (20,21). However, Fabiato and Fabiato
(22) exposed skinned cardiac fibers to cardiac glycoside and
saw no effect on contraction. This probably rules out a direct
effect of the cardiac glycoside on the contractile proteins,
although it is possible to imagine some long-term effects
that could not be seen in the short-term experiments of
Fabiato and Fabiato.
Is Intracellular Sodium Increased?
Although large doses of cardiac glycoside. which would
be expected to be toxic, are always associated with increased
intracellular levels of Na + regardless of the measurement
method, it has been very difficult to measure small intracellular sodium activity (a~a) changes at more therapeutic
JACC Vol. 5. No.5
May 1985:IOA-15A
levels. Chemical methods for measuring intracellular Na +
are both destructive and insensitive . Unidirectional isotopic
fluxes are more sensitive. An example of such a flux experiment in intact dogs receiving long-term doses of digoxin
is the study by Somberg et al. (23). They found that a 25%
reduction of active Rb + uptake (Na + -K + pump inhibition)
was associated with a 20% increase in contraction strength,
measured by dP/dt max'
Sodium-sensitive microelectrode measurements. A
more precise and sensitive method for measuring intracellular sodium activity (a~a) directly is by use of Na + -sensitive
microelectrodes. They have the advantage of permitting
continuous monitoring of a~a with an accuracy of about 0.2
tuM during drug exposure, but the disadvantage is that they
can only be used for in vitro experiments. Ellis (24) was
the first to apply this technique to the effects of cardiac
glycosides, showing dose-dependent increases in a~a from
control levels of 6 to 7 tuM to maximal levels of 25 to 35
tuM. He failed to measure tension changes to correlate with
the changes in a~a' Subsequently two other laboratories
(18,25) have simultaneously measured a~a and tension changes
in response to cardiac glycoside exposure. These studies
showed that an increase of 1 to 2 tuM in the physiologic
range of a~a is sufficient to double contraction strength,
which is remarkable sensitivity.
There are several questions derived from these in vitro
experiments. Is the increased contraction strength seen after
exposure to cardiac glycoside caused by the increase in
a~a, or are these events the result of two separate actions
of the drug? Second , is the dose of drug at which the two
effects occur in vitro applicable to clinical action?
Other interventions that change a~a' Another experimental approach used to determine whether the change in
intracellular sodium activity (a~a) is causal of the increased
contraction strength is to see whether equivalent changes in
a~a produced by other means have the same effect on contraction . Intracellular sodium activity could be altered by
other interventions that inhibit the Na + -K + pump or by
varying the passive leak of Na + into the cell . Both approaches have been explored.
Low extracellular K + also inhibits the Na + -K + pump
and increases a~a' Eisner et al. (26) examined the relation
between a~a and tension during the increase in a~a seen on
exposure to low K + and found a close relation between
increases in a~a and increases in tension, similar to that seen
with cardiac glycoside. These experiments were expanded
by 1m and Lee (27) by inclusion of other interventions that
increased a~a'
The other approach employed by 1m and Lee (27) to
decrease a~a was to reduce Na + entry into the cell by tetrodotoxin or high extracellular K + . (High extracellular K +
probably does not lower a~a by stimulating the pump , but
by reducing Na + entry [28].) As was expected if a~a was
an important determinant of contraction strength, lowering
lACC Vol. 5, No.5
May 1985:lOA-15A
a~a reduced contraction strength by the expected amount.
Several antiarrhythmic drugs decrease INa during the upstroke of the action potential, and they also have negative
inotropic actions. Fozzard and Wasserstrom (29) found that
lidocaine and encainide both decrease a~a and tension as
expected.
Another method to increase a~a of heart muscle is to
change the stimulation rate. Each action potential upstroke
is associated with some Na + entry, and more action potentials should load the cell with more Na + . Cohen et al. (30)
found that increasing the stimulation rate in the physiologic
range caused substantial increases in a~a, and associated
with these increases were increases in contraction strength.
Subsequently, Lado et al. (31) showed that these stimulation-induced increases in a~a were associated with increases
.
i
m a<::a.
The dosage problem. A major worry with these in vitro
experiments is that the dose of cardiac glycoside used is
large compared with what is thought to be the therapeutic
dose in human subjects. Efforts to study lower doses of
digitalis in vitro have either failed to show increases in
aNa, or in some cases have even shown decreases (24,32,33).
We have also occasionally seen a decrease in aNa with low
doses of cardiac glycoside, but it has been impossible to
achieve reproducibly. Recently, Sheu et al. (34) reported
that this occasional event is associated with a decreased
contraction, rather than a stronger one. At this point in its
investigation, it seems likely that the decrease in aNa may
be a laboratory artifact, because there is no clinical evidence
of a negative inotropic effect of digitalis at low doses.
How does a small increase in intracellular sodium
activity increase contraction? One of the major objections
to accepting inhibition of the Na +-K + pump as the principal
therapeutic action of digitalis has been the lack of any clear
explanation for how a small increase in aNa could increase
contraction so much. Indeed, the recent direct measurement
that the increase in a~a with low doses of cardiac glycoside
is only 0.5 to I ruM underlines this concern. Either the
contractile system is very sensitive to Na + or there is some
indirect effect of Na " on Ca 2+ metabolism (35).
The latter possibility, an effect of Na + on Ca 2 + metabolism, has received strong support in the last few years.
The support involves a mechanism called Na + -Ca 2+ exchange, and it provides a possible means for the small increase in aNa to have a large effect on cellular Ca 2 ". We
will first describe Na +-Ca 2 + exchange, and then discuss
the evidence that it plays a role in digitalis action.
The Na" -Ca'" exchange system. Sodium-calcium exchange was discovered in heart muscle by Reuter and Seitz
(36) while they were looking for a calcium pump in cardiac
cell membranes. They found that isotopic Ca 2+ fluxes were
strikingly affected by extracellular Na + and proposed that
Ca 2+ efflux occurred in exchange for Na + influx. This
mechanism is called exchange because it requires Na + to
FOZZARD AND SHEETS
MECHANISM OF DIGITALIS ACTION
13A
enter the cell through the same transporter that moves Cal +
from the cell. Because the electrochemical energy gradient
for Ca2+ is strongly in favor of Cal + entry, a great deal of
energy is needed to move Cal + outward against the gradient.
The energy in the Na +-Ca l+ exchange system is derived
froin Na + moving down its gradient (ENa - Vr ) . The most
thorough investigation of Na +-Cal + exchange has been
achieved using a special nerve cell, the intracellularly perfused squid giant axon (37). These studies show that probably three Na + ions must move into the cell to move one
Cal + ion out of the cell. Studies in the squid axon also have
shown that the exchange is sensitive to the membrane potential, probably because unequal charges are moved across
the cell membrane (3 ions in to 2 ions out). It has also been
possible to isolate this exchange system in fragmented cardiac membrane vesicles and to show that it is one transport
system which does not consume ATP (38). Although the
stoichiometry of the exchange is not exactly known, and
may not be exactly 3: I, we will discuss qualitatively how
the system works using that coupling ratio as our example.
One possible description of the Na +-Cal + exchange system is that it is a protein in the cell membrane with one site
for Ca2+ and three sites for Na ". When the sites are filled,
the carrier will transport the Na + ions one way across the
membrane and the Cal + ion the other way. Also, when all
the carrier sites are full, there is one net positive charge on
it, making it responsive to the membrane potential. (For
completeness, it should be pointed out that the carrier presumably can move Cal + in exchange for another Cal +, or
three Na + in exchange for three Na + . We are not concerned
with this self-exchange for our discussion because it would
not affect contraction, but it is an important source of error
for isotopic studies of the exchange system.) Because the
outside solution has a high concentration of Na +, the outside
sites are easily saturated with that ion, so the transporter
usually operates to move Na + in and Ca 2 + out. The specific
equation for the equilibrium relation is:
where Eca and E Na are the membrane potentials at which
the chemical and electrical gradients are equal for calcium
and sodium, respectively, and n is the coupling ratio that
we said is probably 3. Insertion of the appropriate extracellular and intracellular values Into this equation gives the
prediction that an increase of aNa by I ruM above the physiologic range will double the level of aCa at rest.
Experimental support for the role of Na + -CaZ+ exchange. The prediction of this change in aCa with cardiac
glycoside was confirmed by Sheu and Fozzard (4) and Lee
and Dagostino (18). The implication of this Na+-Ca l+ exchange hypothesis is that contraction should be very sensitive to a~a, which we have already indicated seems to be
true. One missing link in the explanation is how the (diastolic) level at rest of Cal + has such a large effect on Con-
14A
FOZZARD AND SHEETS
MECHANISM OF DIGITALIS ACTION
traction. We previously indicated that the Ca 2 + used for
activation of contraction comes from the sarcoplasmic reticulum. Either the release of Ca2+ from sarcoplasmic reticulum or the amount stored in it must be a sensitive function of a::a'Further study is needed to resolve this question.
Digitalis Toxicity
Clinical use of cardiac glycosides is sometimes associated
with serious supraventricular or ventricular tachyarrhythmias, which are thought to be secondary to transient depolarizations (also called delayed afterdepolarizations) that
become sufficiently large to reach threshold and result in
ectopic heart beats (39,40).
Kass et al. (41) studied the relation of transient depolarizations in cardiac glycoside-treated calf Purkinje fibers
under voltage clamp. They found that transient depolarizations result from a oscillatory release of Ca 2 + from an
intracellular store (probably the sarcoplasmic reticulum),
which causes both aftercontractions and a transient inward
current carried by Na + ions. The oscillatory release of Ca 2 +
is probably promoted by digitalis indirectly by increasing
intracellular Ca 2 + . These transient depolarizations can also
be produced experimentally by other interventions that increase a::a, such as when extracellular Ca2 + is increased.
A special membrane channel has been found in cardiac cells
that is permeable to Na + and is opened by increased aCa
(42).
Role of potassium. By understanding the relation among
digitalis, the inhibition of the Na + -K + pump and the resultant effects of increased intracellular Ca2 + , other clinical
consequences of digitalis therapy may be better understood.
The role of increasing serum K + to treat digitalis toxicity
is predicted because the increased extracellular K + increases
the Na + -K + pump activity that has been inhibited by cardiac
glycosides (apparently because of competitive binding). The
resulting stimulation of the Na + -K + pump decreases a~a,
decreases a::a through the Na + -Ca 2 + exchange and reduces
the Ca 2 + overload of the cell that caused the transient depolarizations and the tachyarrhythmias.
Similarly, the reason that patients who are treated with
digitalis and also have hypokalemia (frequently from concomitant diuretic therapy) more readily develop digitalisinduced arrhythmias becomes obvious. The Na + -K + pump
is already partially inhibited by the glycoside and hypokalemia further inhibits the pump, causing intracellular Ca2+
overload with transient depolarizations and cardiac
arrhythmias.
Action of antiarrhythmic agents. The efficacy of antiarrhythmic therapy such as lidocaine on digitalis-induced
arrhythmias may also be partially understood through the
relation of a~a, a::a and transient depolarizations. Cohen et
al. (30) and January and Fozzard (28) have shown that
a~a increases in sheep Purkinje fibers with increasing stim-
lACC Vol. 5, No.5
May 1985:IOA-15A
ulation rates. This increase in a~a is absent after treatment
of the cells with tetrodotoxin (a specific inhibitor of the fast
Na + channel), indicating that the increase in a~a results
from Na + ions traveling through the Na + channel. The
antiarrhythmic drug lidocaine has been shown to decrease
INa and therefore a~a' This has been demonstrated in sheep
Purkinje fibers (29,43). Furthermore, lidocaine has been
shown to also decrease both the magnitude of transient inward current and delayed aftercontraction accompanying
transient depolarizations induced by K + -free extracellular
solutions (43). However, the actions of antiarrhythmic drugs
in treating digitalis-induced arrhythmias probably involve
more than an effect on aNa, including a shift of the threshold
potential for action potentials to more positive values, possible changes in membrane resistance and possible effects
of drugs on intracellular organelles.
Role of increased vascular tone. Finally, digitalis therapy has been associated with mesenteric ischemia and infarction in some patients. Again, though our understanding
of cardiac glycosides and their effect on a~a, a::a and contractility, it is expected that digitalis may increase vascular
tone, and occasionally in a patient this may be associated
with clinical vascular insufficiency.
Conclusions
We have discussed the role of the level of intracellular sodium activity (a~a) in regulating a::a through the
Na + -Ca 2 + exchange system and the consequent effects on
cardiac contraction strength. Cardiac glycosides have been
shown to increase aNa by inhibition of the Na + -K + pump,
and we have outlined the evidence relating the increase in
aNa to the increased contraction strength. The same action
that produces the positive inotropic effect may also be important in the pathophysiology of digitalis toxicity; understanding this mechanism is useful in developing effective
treatment.
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MECH ANISM OF DIGITALIS ACTION
15A
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