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153
Brief Review
Myocardial Contractile Function
During Ischemia and Hypoxia
D.G. Allen and C.H. Orchard
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Introduction
Scope of Review
Myocardial ischemia is one of the most common
causes of serious illness and early death in developed
societies. Furthermore, a variety of surgical procedures necessitate limited periods of ischemia. The reduced blood flow, which characterizes ischemia, deprives the heart of substrates and allows metabolites to
accumulate. These changes lead to decreased tension
production and to the appearance of arrhythmias. For
these reasons, the effects of ischemia on myocardial
cell function are the subject of great interest and active
investigation. In the past decade, it has become possible to measure intracellular concentrations of many of
the crucial ions and metabolites within the myocardial
cell. The way in which these intracellular constituents
modify contractile function has also become clearer
from studies of their effects on the isolated components
of the cardiac cell.
In this review, we consider how the application of
these new experimental techniques has contributed to
our understanding of myocardial contractile function
during ischemia and hypoxia; electrophysiological aspects are discussed only in so far as they affect contraction. Arrhythmias are not considered. Many earlier
reviews overlap parts of this review and are recommended for the reader who requires more background
information. Metabolic aspects have been reviewed by
Neely and Morgan' and by Gibbs. 2 Electrophysiological aspects have been considered by Carmeliet3 and by
Janse and Kleber.4 Ionic changes and more particularly
the role of calcium have been described by Nayler et
al,5 Poole-Wilson,6 and Jennings and Steenbergen.7
The first section of the review describes briefly the
main changes in mechanical performance of a heart
subjected to global ischemia. The second and third
sections describe the major metabolic and ionic
changes that occur during ischemia and hypoxia and
consider how these changes are brought about. In the
final three sections the metabolic and ionic changes
that may underlie the altered mechanical performance
observed during ischemia are considered.
From the Department of Physiology, University College London, London, and the Department of Physiology, University of
Leeds, Leeds, United Kingdom.
Address for reprints: Dr. D.G. Allen, Department of Physiology,
University College London, Gower Street, London WC1E 6BT,
United Kingdom.
Received May 10, 1986; accepted October 6, 1986.
Mechanical Changes During Ischemia
The effects of ischemia on pressure development by
the heart are now well recognized.68-9 When a beating
heart is subjected to global ischemia the developed
pressure declines rapidly over several minutes (acute
ischemic failure). After 10-20 minutes developed
pressure is small or absent and a gradual rise in diastolic pressure occurs (ischemic contracture). After about
1 hour of complete ischemia, histological and biochemical evidence of cell damage becomes apparent
(e.g., contraction bands, swollen mitochondria, and
leakage of intracellular enzymes into the extracellular
space). l0 If perfusion of the heart is restarted before or
during the early part of the ischemic contracture, recovery of developed pressure is virtually complete.
However, if reperfusion is started when the contracture
is well established, then developed pressure fails to
recover and cell damage is accelerated, leading to massive enzyme release (reperfusion damage). 6 "
Ischemia vs. Hypoxia
Ischemia is difficult to study experimentally because it is not possible to apply drugs to the heart or to
change the extracellular ionic composition during ischemia. In addition, for preparations of cardiac tissue
that have no blood supply, e.g., isolated cells, there is
no direct experimental equivalent to ischemia. For
these reasons, many studies have maintained perfusion
of the tissue but removed all or most of the O2 (anoxia
or hypoxia) or used agents that inhibit oxidative phosphorylation, e.g., cyanide (CN). However, ischemia
involves both inadequate supply of substrates, notably
oxygen and glucose, and inadequate removal of products of metabolism, notably lactate, H + and K + .
In an attempt to mimic ischemia more closely, hypoxia has frequently been combined with procedures
that inhibit anaerobic glycolysis. Under these conditions, all the main mechanical features of ischemia can
be observed. Thus, in anoxia, when glycolysis can
continue, the developed pressure of a heart falls to
about one-third of normal but can then continue at this
level for at least 30 minutes. 1213 However, in anoxia,
after glycolysis has been prevented, the developed
pressure declines rapidly and completely, a response
similar to that observed in ischemia.8 It has also been
shown that an hypoxic contracture14 and a reoxygenation paradox 6 " can be elicited in perfused preparations
subjected to hypoxia with glycolysis prevented and
154
Circulation Research
have properties comparable to the equivalent phenomena in ischemia. The similarity between mechanical
responses in ischemia and in hypoxia with glycolytic
inhibition suggests that it is the prevention of adenosine triphosphate (ATP) production and/or its metabolic consequences that lead to the mechanical features of
ischemia; accumulation of the products of metabolism
no doubt modifies the mechanical response but does
not seem to be essential for their occurrence. Thus,
throughout this review, while the aim is to explain the
cellular changes during ischemia and their consequences for myocardial contraction, there will be extensive use of observations made in perfused tissues
but with oxidative phosphorylation and anaerobic glycolysis blocked.
Vol 60, No 2, February 1987
Metabolic Consequences of Ischemia and Hypoxia
Changes in Phosphorus-Containing Metabolites
Both ischemia and hypoxia reduce the oxygen supply to the myocardial cells and reduce the rate of ATP
production by oxidative phosphorylation. If there were
no other compensatory mechanisms, the concentration
of ATP would fall at a rate that is the difference between the rate of production and consumption of ATP.
In fact, however, [ATP] remains relatively constant
during the first few minutes of hypoxia while phosphocreatine concentration ([PCr]) falls and inorganic
phosphate concentration ([Pi]) rises.1315"17 These
changes can be understood with the help of the model
shown in Figure 1, which is a development of that
described by Carlson and Wilkie.18 This model shows
30
-70
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60
Phosphorus
metabolite
ATP
Affinity
20
50
concentration
(kJ/mol)
40
(mM)
30
10 -
20
Pi hydrolysed
30
(mM)
FIGURE I. Model of concentrations of PCr, ATP, ADP, AMP, and Pi in the presence of creatine kinase and myokinase as ATP is
hydrotyzed. The thermodynamic affinity (free energy change) of ATP hydrolysis is also shown. The following reactions were assumed
to occur:
ATP -* ADP + Pi
(1)
PCr + ADP ^ ATP + Cr
(2)
2ADP ±^ ATP + AMP
(3)
Starting concentrations were [ATP] = 7 mM, [PCr] = 25 mM, [ADP] = [AMP] = [Cr] = [Pi] = 0. This model is described by the
following equations that were solved for various values of [Pi].
Total adenine = 7 mM = [ATP] + [ADP] + [AMP]
(4)
Total creatine = 25 mM = [PCr] + [Cr]
(5)
Total phosphate = 46 mM = [PCr] + [Pi] + 3[ATP] + 2[ADP] + [AMP]
(6)
[ATP][Cr]
= 200
[ADP][PCr]
(7)
[ADP]2
= 1
[ATP][AMP]
(8)
Equilibrium constants for the reaction catalyzed by creatine kinase (7) and myokinase (8) were taken from Lawson and Veech.21
Hydrolysis ofATP was regarded as irreversible. The reactions were assumed to occur at pH 7.0, and changes in pH were ignored. The
5 equations (4—8) can be reduced to a cubic equation that was solved iterativety. The results are similar (over the relevant range) to
those of Carlson and Wilkie18 but very different from those ofKubler and Katz.8 No details of the method of calculation are given in
Kubler and Katz and we do not know the reason for the discrepancy.
AATP was calculated using the equation given in the text (page 156), with — AG0 = 30 Idlmol. The arrow represents the approximate
concentration of P metabolites in aerobic perfused hearts at low or moderate workloads.
Note that, as plotted, the abscissa has units of moles of Pi hydrotyzed. However, during a period when there is net consumption of
ATP in excess of production, the figure can be used to indicate the way in which P metabolites will change as a function of time.
Allen and Orchard
Contractile Function in Ischemia and Hypoxia
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how the concentrations of ATP, PCr, Pi, adenosine
diphosphate (ADP), and adenosine monophosphate
(AMP) will change as ATP is hydrolyzed, assuming
that creatine kinase and myokinase are present in sufficient concentrations to maintain the reactions catalyzed by them at equilibrium.1920
Under normal aerobic working conditions, P-metabolites should be similar to those near the left of the
diagram as indicated by the arrow, i.e., [PCr] 2-3
times greater than [ATP], [Pi] less than [ATP], and
[ADP] very low." (Note that total ADP is much higher
than myoplasmic [ADP] because of binding to myosin;
see discussion in Dawson and Wilkie.19) Under these
conditions, it is thought that the low concentrations of
Pi and ADP are capable of stimulating ATP production
by the mitochondria at a rate equal to consumption (for
discussion, see Hansford22). It is worth noting from
Figure 1 that a 20% fall in [PCr] from the normal
cellular level is associated with a 200% increase in
[ADP] and a 160% increase in [Pi]. These substantial
changes in [ADP] and [Pi], coupled with the sensitivity
of mitochondrial ATP production to [ADP] and [Pi],
explain how large increases in ATP consumption,
which accompany an increased work load, can occur
with only a small fall in [PCr] and with no detectable
change in [ATP]. 2324 During ischemia or anoxia, ATP
production by oxidative phosphorylation falls to zero
and P-metabolite concentrations move towards the
right of the diagram and continue to move to the right
until either ATP production can be increased, e.g., by
increased anaerobic glycolysis, and/or ATP consumption can be decreased, e.g., by a fall in developed
tension. Many studies have now shown that the general
features of this model are correct and that the maintenance of [ATP] while [PCr] falls and [Pi] rises results
from the resynthesis of ATP from PCr catalyzed by
creatine kinase. 1723 It has been suggested in the past
that the relative constancy of [ATP] while [PCr] falls is
evidence of "compartmentation" of ATP production, 82627 but this model shows that it is due simply to
buffering by PCr in the presence of creatine kinase.
Although this model is helpful in understanding the
P-metabolite changes when ATP consumption exceeds
production, other processes occur in ischemia and anoxia that complicate the situation. The effect of these
will be to change both the end point at which the ATP
consumption and production are again equal and the Pmetabolite concentrations present at this new steady
state. These processes include the following: 1) Anaerobic glycolysis is accelerated twentyfold within the
first minute of ischemia or hypoxia1516 and at this rate
0.1-0.2 mM/sec of ATP will be produced. During
ischemia this high rate of glycolysis is not sustained
and decreases despite the presence of glycogen
stores.28 The mechanism of this reduction is not established although both intracellular acidosis and lactate
accumulation are thought to play important roles.29
During hypoxia, glycolysis continues at a high rate. If
glucose is removed from the perfusate, glycolysis will
continue until the available glycogen is consumed.30 2)
Tension falls rapidly during ischemia and hypoxia so
155
that the major component of ATP consumption may
fall. 3) AMP is further degraded to inosine monophosphate (IMP) and adenosine, which are eventually lost
from the cellular pool. These processes take place over
30-60 minutes (for review see Jennings and Steenbergen7). 4) Changes in pH (see later section) will modify
the equilibrium constant for creatine kinase and myokinase and lead to small changes in the equilibrium
P-metabolite concentrations.
However, there are studies that do not seem to support this model, even allowing for the complications
described above. For instance, Doorey and Barry,31 in
studies of cultured embryonic chick ventricular cells,
have shown that changes in [ATP] occur very rapidly
(10-20 seconds) after application of CN and precede
changes in [PCr]. However, in this preparation the
ratio of PCr/ATP under control conditions was only
0.7 compared with 2-3 in most studies on intact adult
hearts. The reason for this unusually poor initial metabolic status is not clear but examination of Figure 1
shows that for such starting conditions a rapid early
decline in ATP, which is more or less proportional to
the decline in PCr, is the expected result on the basis of
this model.
Compartmentation
Mammalian hearts contain approximately 109 muscle cells. Inevitably, the metabolic status of these cells
will vary due to local variations in work load, blood
supply, etc. For instance, differences in metabolic levels between endocardial and epicardial layers of the
left ventricle are well recognized.32 Similarly, in many
isolated heart preparations the left side of the heart will
experience a greater work load than the right either
because the left ventricle is stretched by a balloon or
because, in the working heart, it is contracting against
a load and performing external work. These regional
variations represent one kind of compartmentation of
metabolite levels in the heart.
Within the cardiac cell there are also well-defined
compartments surrounded by internal membranes, notably the sarcoplasmic reticulum and the inner mitochondrial matrix. These compartments are known to
have different ionic and metabolic concentrations from
the myoplasm.33'34 This is one reason why both biochemical and NMR measurements of metabolites that
represent spatially averaged results from whole hearts
need careful interpretation.
In recent years, there has been much interest in a
third kind of compartmentation, which arises because
ATP is synthesized mainly in the mitochondria but
utilized mainly in the myofibrils. In cardiac muscles,
the myofibrils are 1-2 /urn in diameter and are surrounded by columns of mitochondria. Concentration
gradients must therefore exist across the myofibrils.
The magnitude of this kind of "myoplasmic compartmentation" can be calculated in the steady state using
the diffusion equation35 that describes diffusion into a
cylinder with consumption uniformly distributed
across it. Assuming an external [ATP] of 7 mM, ATP
consumption of 1 mM/sec in a beating heart,2 ATP
156
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diffusion coefficient of 1 X 10~4 mmVsec36 and a
myofibrillar diameter of 2 ^im, then in the steady state
the [ATP] in the center of the myofibril is 6.9975 mM.
A larger gradient will exist transiently during systole
when ATP consumption is higher, but the time-averaged gradient will be close to the above value. On this
model, ADP must diffuse in the opposite direction and
because its concentration outside of the myofibril is
lower, perhaps 10 /xM, the required concentration at
the center is 12.5 /i.M. However, even these small
gradients are an overestimate because the enzyme creatine kinase will hold [ATP] and [ADP] more nearly
constant and produce gradients of [PCr], [Pi], and
[Cr]. This leads to the situation of facilitated diffusion
that has been recently analyzed in detail by Meyer et
al.37 Their calculations show that a very large proportion of the high-energy phosphate flux is in fact carried
by PCr and that the gradients of [PCr] are even smaller
than those calculated above for [ATP],
It should be stressed that there is no direct evidence
for any substantial myoplasmic compartmentation and
the theoretical considerations above suggest that it
should be small. Nevertheless, myoplasmic compartmentation has been invoked to explain a number of
puzzling phenomena. For instance, as discussed on
pages 159-162, acute hypoxic failure has been attributed to myofibrillar compartmentation though subsequently other explanations have emerged. (For further
discussion of phenomena attributed to myoplasmic
compartmentation, see discussion in Meyer et al.37)
Further progress in this area will occur only if direct
evidence can be obtained demonstrating the presence
and magnitude of this type of compartmentation.
[ATP] vs. AATP
There is general agreement in the literature that
some consequence of ATP depletion plays a crucial
role in the development of the effects of ischemia on
cell function. ATP has at least two roles in any energetic process which it fuels. In the first place, ATP must
bind to a site on the enzyme for which it is the substrate. Even when there is more than one site with
different affinities, this role can be quantified by the
apparent K ^ i.e., the concentration of ATP that leads
to half maximum saturation of the process under consideration. In the second place, the hydrolysis of ATP
can drive a reaction in the forward direction only if the
free energy change of hydrolysis of ATP is greater than
the free energy required for the process under consideration. The free energy change of ATP hydrolysis is a
differential quantity whose magnitude depends on the
advancement of the hydrolysis reaction. Its nomenclature in the literature is confused. It is commonly designated AGATP, although recently dG/de has been used to
indicate its differential nature; however, both of these
are negative quantities for ATP hydrolysis and can be
misleading. We have therefore chosen an alternative
form known as (thermodynamic) affinity or AATP,
which has the advantage of being a positive quantity
for spontaneous reactions such as ATP hydrolysis.38
The relation between these quantities is
Circulation Research
Vol 60, No 2, February 1987
AATP = -AG A T P = - d G / d e
= -AG° A 1 P + RT In [ATP]/([ADP] X [Pi])
where AG°ATP is the standard free energy change of
ATP hydrolysis under specified conditions of pH,
[Mg 2+ ], etc., and R and T have their usual meanings.
Note that the ability of ATP to bind to a site depends
principally on [ATP] whereas the (thermodynamic)
affinity depends on [ADP] and [Pi] in addition to
[ATP]. Figure 1 shows that while [ATP] is held essentially constant during the early part of metabolic decline, [ADP] and [Pi] rise considerably so that AATP
falls substantially over this period. Table 1 lists values
of K^ taken from the literature for various ATP-dependent processes in the myocardial cell. Table 2 shows
calculations of the work per ATP consumed in the
operation of various ion pumps in the myocardial cell.
It is worth noting that the K^ for all the pumps and for
rigor tension development are less than 0.2 mM and
much lower than the normal level of 6-8 mM. An
important point that emerges from Figure 1 and Tables
1 and 2 is that operation of the various ion pumps is
likely to be affected by the reduction of AATP before
[ATP] falls below the Km of any of the pumps. The real
situation may be more complex than this analysis suggests if ADP competes with ATP for the binding site,
since under these circumstances the apparent K,,, of
ATP for its binding site will decline during metabolic
depletion.
Ionic Consequences of Ischemia and Hypoxia
In this section we consider the ions Ca 2+ , Na + , H + ,
and K + : each has a major role in cellular function and
the regulation of contraction. Furthermore, these ions
are known to change their intracellular concentration
during anoxia or ischemia and these changes are
thought to be involved in the functional consequences
of anoxia or ischemia. Ca 2 + , Na + , and H + exist in the
myoplasm at concentrations below electrochemical
equilibrium so that active (energy consuming) processes are required to extrude them from the cell.
Thus, on first principles, one would expect the intracellular concentrations of these ions to increase towards electrochemical equilibrium as the pathways for
ATP production are blocked. Intracellular [K + ] is
higher than electrochemical equilibrium so that it tends
Table 1. ATP Concentrations at Which Various Processes
Occur at Half-maximal Rates
ATP
Process
concentration
Reference
SR Ca pump
0.18 mM
Shigekawa et al 39
Na-K pump*
0.1
Glynn and Karlish40
SM Ca pump
0.03 mM
Carom and Carafoli41*
Rigor attachment of
crossbridget
0.05 mM
Fabiato and Fabiato42
mM
SR = sarcoplasmic reticulum, SM = surface membrane.
•Study on red blood cells containing choline chloride.
tThe number of rigor crossbridges attached is decreased at higher
concentrations and increased at lower concentrations.
Allen and Orchard
Table 2. Calculated Work Performed by Ion Pumps per
Mole of ATP Hydrotyzed*
Work
Assumptions
SR Ca pump
43 Id/mole
2 Ca pumped/ATP hydrolyzed
Zero potential across SR
SR [Ca] = 1 mM, myoplasmic
[Ca] = 0.2 /iM
Na-K pump
44 kJ/mole
3 Na and 2K pumped/ATP
hydrolyzed
- 8 0 mV across SM
[Na] 0 = 140 mM, [Na], = 10
mM
[K] o = 5 mM, [K], = 120 mM
SM Ca pump
39 kJ/mole
1 Ca pumped/ATP hydrolyzed
- 80 mV across SM
[Ca] 0 = 2 m M , [Ca], = 0,
Pump
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Contractile Function in Ischemia and Hypoxia
•Reproduced with permission from Eisner et al. 43
SR = sarcoplasmic reticulum, SM = surface membrane.
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to leak passively from the cell, and active processes are
required to pump it back to the myoplasm.
Intracellular Calcium
Calcium is highly compartmentalized within the
cardiac cell, and it remains difficult to obtain reliable
estimates of its concentration in the various cellular
compartments. Measurements of total cell calcium are
technically easier and were the earliest measurements
to be made in ischemia. Total cell calcium does not
change dramatically during the first hour of ischemia.4445 Similarly, measurements of isotopic calcium
fluxes during hypoxia have shown only very small
changes over the first 30 minutes though by 60 minutes
a small increase in influx becomes apparent.46 Although these global measurements of calcium show
little change, it has long been suspected that distribution of calcium between compartments is altered.
Thus, understanding the role of calcium in ischemia
requires knowledge of the concentration of calcium in
the various cellular compartments, particularly the
myoplasm in which the free calcium concentration
([Ca2+],) controls contraction and influences many other cellular processes. In the rest of this section, we
review briefly what is known about the distribution and
control of calcium in the various cellular compartments. Details of measurements of [Ca 2+ ], during various phases of hypoxia will be considered in later
sections.
Total cell calcium is in the range of 1-3 mmol/kg
wet weight while the [Ca 2+ ], during diastole is only
0.1-0.2 fiM. The main cellular compartments in
which calcium is stored are 1) in the myoplasm, 2) in
the sarcoplasmic reticulum, 3) in the mitochondria,
and 4) bound to membrane phospholipids and to proteins in the myoplasm.
2+
THE MYOPLASM. The resting [Ca ], is maintained at
its low level by a variety of "pumps" that remove
calcium either to the extracellular space or to one of the
internal stores. In the steady state, calcium pumped to
the extracellular space must balance the inward calcium leak across the surface membrane. Calcium is
pumped to the extracellular space by one of two parallel systems: 1) the Na-Ca exchanger, which uses the
energy of the Na + gradient to extrude calcium,4748 and
2) the surface membrane calcium pump, which uses
ATP directly.41 The Na-Ca exchange is thought to
have a relatively high capacity but low affinity while
the calcium pump has a low capacity but high affinity.49 Since [Na + ], is likely to rise during ischemia4450 a
decrease in calcium efflux by the Na-Ca exchange
would be expected; however the properties of the
Na-Ca exchanger are not yet clear so that the contribution of the Na-Ca exchanger to calcium efflux during
ischemia remains uncertain (see pages 163-165).
THE SARCOPLASMIC RETICULUM. The
sudden
in-
crease in [Ca 2+ ], that initiates contraction (the calcium
transient) is thought to be caused principally by the
relase of calcium stored in the sarcoplasmic reticulum,5152 although the fraction of the activator calcium
coming from this source probably varies between species.53 The amount of calcium in the sarcoplasmic reticulum will depend on [Ca 2+ ], and on the Ca-pump in
the sarcoplasmic reticulum, whose properties are relatively well defined.54 However, if the free energy of
hydrolysis of ATP decreases below a critical level (see
Table 2), the ability of the sarcoplasmic reticulum to
take up calcium may be compromised. In this case, the
calcium transient would be expected to decrease and
diastolic [Ca 2+ ], would be expected to increase, unless
another mechanism, such as the Na-Ca exchanger or
the mitochondria, can continue to remove calcium
from the myoplasm.
THE MITOCHONDRIA. Mitochondria constitute about
one-third of the myocardial cell volume and have a
very large capacity for calcium (possibly as high as
4-10 mmol/1 cell volume49). The inner mitochondrial
membrane has a Na-Ca exchange mechanism, a Na-H
exchange mechanism, and a calcium uniporter. The
mitochondria have the potential, therefore, to play a
substantial role in the calcium metabolism of the cell.
It has been suggested that an increase of myoplasmic
[H + ], such as occurs during hypoxia and ischemia,
may release calcium from the mitochondria.55 However, it now appears that the mitochondria contain little
calcium under normal conditions,49 so that even if it
was all released it would have a relatively small effect
on [Ca 2+ ]j. Nevertheless, under conditions of prolonged ischemia, the mitochondria may become loaded with calcium.56 This has led to speculation that
calcium loading of the mitochondria and the resulting
uncoupling of oxidative phosphorylation might be a
factor in ischemic cell damage. However, loss of respiratory control over oxidative phosphorylation can occur in the absence of calcium loading of mitochondria,37 so that metabolic consequences of anoxia do not
appear to be a direct consequence of mitochondrial
calcium loading (for further discussion, see
Williams58).
BINDING TO PROTEINS AND MEMBRANE PHOSPHOLIP-
IDS. Troponin is probably the most quantitatively important myoplasmic Ca 2+ buffer. Other ions in the
myoplasm, particularly H + , may compete with Ca 2+
158
for binding sites.39 Such competition will move the
concentration-effect curve for calcium on troponin to
the right, so that a given concentration of Ca2+ will
have less effect, e.g., less tension will be developed
for a given calcium release, but more calcium will
remain free in the myoplasm.
The role of membrane-bound calcium in excitationcontraction coupling remains controversial.60-61 Calcium from this source may be released by the action
potential and contribute to contraction; alternatively,
these sites may simply act as passive buffers.
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Intracellular Sodium
The free myoplasmic concentration of Na + ([Na+]i)
is close to 10 mM while the total cellular sodium is
20-30 mmol/kg cells (see discussion in Ellis62). Efflux
of sodium depends principally on the Na pump, which
ejects 3 Na + in exchange for 2 K + for the hydrolysis of
one ATP molecule. Current knowledge about the Na
pump in cardiac muscle is summarized by Eisner.63
Influx of sodium into the cell can occur by various
routes: 1) voltage-dependent channels, such as the fast
Na channel, and 2) exchangers, such as the Na-H
exchange and Na-Ca exchange. These exchangers are
potentially reversible, the direction of operation depending on the gradients for each ion, the stoichiometry, and the membrane potential.48 Under normal resting conditions, Na + will enter the cell under the
influence of the electrochemical gradient for Na + and
either H + or Ca 2+ will be ejected from the myoplasm,
but under other conditions, e.g., Na pump inhibition
(see Dietmer & Ellis64), the Na-Ca exchange may reverse and allow Ca 2+ entry in exchange for Na + efflux.
Small changes in [Na + ], have no direct effect on the
contractile proteins, but changes in [Na + ], affect
[Ca 2+ ], by means of Na-Ca exchange. Thus, when
[Na + ], rises, the inward gradient for Na + is reduced
and Na + influx and Ca 2+ efflux on the Na-Ca exchange are both reduced. Consequently, calcium rises
in the myoplasm and is taken up into the sarcoplasmic
reticulum store. This leads to larger calcium release
and stronger contractions. The net effect is that developed tension shows a very steep dependence on
[Na + ],, M and agents such as the cardiac glycosides,
which block the Na pump, lead to increased [Na + ],,
[Ca 2+ ],, and developed tension.66-67
Activity of the Na pump may also be inhibited if
[ATP], falls below the Km (0.1 mM, Table 1) or if AATP
falls below the minimal level of energy required for
maintenance of ,the electrochemical gradients (44 kJ/
mol under aerobic conditions, Table 2). Inspection of
Figure 1 suggests that the latter may occur first, though
changes in intracellular K and membrane potential during ischemia complicate the issue. Total sodium has
been shown to rise both during prolonged (60 minutes)
ischemia and hypoxia.44>68 Although there is some
compartmentation of sodium, this probably means that
[Na + ], should also rise. There are now several studies
of myoplasmic [Na + ] that examine this issue. A study
by Kleber69 on isolated, ischemic hearts showed that
[Na + ], was unchanged or fell slightly after 15 minutes
Circulation Research
Vol 60, No 2, February 1987
of ischemia. The investigator suggested that a rise of
[Na + ]| due to reduced activity of the Na pump might
have been offset by the reduced influx associated with
depolarization.70-71 This interpretation has been supported by more recent work72 in which papillary muscles were exposed to conditions designed to mimic
ischemia (hypoxia, raised Ko, extracellular acidosis,
and glucose removal). When all these conditions were
combined, [Na + ], changed little over 12 minutes.
However, when [K + ] o was maintained constant but the
other conditions imposed, [Na"1"], showed a small rise
over 12 minutes. A study by Guarnieri50 on hypoxic
but perfused papillary muscles (in which [K + ] o will
hardly change and depolarization should be relatively
small) also showed a rise of [Na + ],, provided glucose
was absent from the perfusate. In addition, Guarnieri
showed that over the same time, [Ca 2+ ], did not rise,
suggesting that Na-Ca exchange may be inhibited during this period (see pages 163-165).
Further studies of the changes in [Na + ], in ischemia
and hypoxia are needed and are of particular importance for assessing the contribution that Na-Ca exchange makes to calcium regulation.
Intracellular pH
For an extracellular pH of 7.4, the intracellular pH
(pH,) at electrochemical equilibrium would be about
6.1. Many studies have shown that pH, is about 7.0, so
some active pump must be ejecting protons. Several
mechanisms seem to contribute to pH regulation, including a Na-H exchange that is mainly active in the
presence of an acid load73-74 and a HCO3"/C1" exchange
that is mainly active under alkaline conditions.75
When interventions lead to changes in pH,, the various pump mechanisms lead to recoveries of pH, with
time courses of 10-20 minutes. It follows that over
periods of a few minutes, such as the time course of
acute ischemic failure, the effects of the pump are
small and the changes in pH, reflect net production/
consumption of H + in the presence of intracellular
buffers. A number of metabolic reactions involve H + ;
these include:
ATP->ADP + Pi + nH +
PCr + n H + - > C r + Pi
glucose—* 2 lactate + nH+
n = 0.8
n = 0.4
n = 2.0
Estimates of the stoichiometry of H + production
under physiological conditions of pH, [Mg 2+ ], etc. are
shown for each reaction (for discussion see Gevers76
and Wilkie77). NAD + H + -*• NADH + is ignored
because the total amounts of these substances are so
small (less than 1 mM77). The intracellular buffering
power of ventricular tissue is around 70 mM/pH unit,78
so that provided the net change in the above metabolites is known it is possible to calculate the expected
changes in pH, and compare them with the measured
changes. During ischemia or hypoxia, PCr breakdown
is rapid; complete breakdown from the normal level of
20 mM would be expected to give a maximum alkalosis of 0.11 pH units (20 x 0.4/70). Allen et al13 have
observed a transient alkalosis of 0.1 pH unit in the first
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1-2 minutes after hypoxia. When anaerobic glycolysis
can occur, the initial alkalosis is rapidly overwhelmed
by a much larger acidosis associated with lactate production. For instance, if all glycogen stores (20-50
mM glucose units, depending on species79) were converted to lactate, it would lead to an acidosis of
0.6-1.4 pH units. In ischemia, when lactate cannot
leave the heart, intracellular acidosis of 1.0 unit has
been observed.80 In hypoxia when perfusion continues,
the acidosis is much smaller because lactate leaves the
cardiac cells at a moderate rate. In addition, as expected, if glycolysis is prevented, acidosis is abolished.13
Over longer periods, such as occur in ischemic contractures, the situation is much more complex since the
mitochondria and surface membrane H pumps will be
involved and changes in [Na + ], and membrane potential may affect both passive fluxes of H + and the H
pumps. In ischemia, the acidosis associated with lactate accumulation will dominate the measured pH,77;
with continuing perfusion during hypoxia the effects of
ionic and membrane potential changes on the H +
fluxes and pumps may well dominate the regulation of
pH,, but this situation has not yet been investigated or
analyzed.
Intracellular and Extracellular Potassium
The onset of ischemia or hypoxia leads to an increase in potassium efflux from myocardial cells so
that intracellular [K + ] tends to fall and extracellular
[K + ] tends to rise (for review, see Kleber81). Recent
work suggests that the mechanism of this increased
efflux is linked to the additional metabolic anions associated with anaerobic metabolism.82 In ischemia, potassium cannot leave the extracellular space, and its
concentration rises over about 10 minutes to 10-15
mM83; in hypoxia, potassium is carried away by the
perfusate so that extracellular concentration changes
will be small and transient. Increased [K + ] o leads to
depolarization that has a crucial role in slowing conduction and the production of arrhythmias.84
Possible Mechanisms Underlying
Acute Ischemic Failure
A striking feature of acute ischemic failure is its
rapidity. A substantial decline in developed tension is
apparent after one minute of ischemia, and the decline
is generally complete within 5-10 minutes.85 In this
section, the mechanisms that may underlie this rapid
decline of tension are considered. The consensus of a
wide range of studies (for reviews, see Kubler and
Spieckermann'6 and Gibbs2) is that [ATP]; falls by only
a small amount during this period. This fact has led
some groups to speculate that there may be compartmentation of ATP within the myoplasm, with [ATP]
falling to much lower levels in some critical region of
the cell, 826 but an alternative conclusion is that the
decline in tension is not directly related to [ATP],. In
this context, it is worth noting from Figure 1 that at the
time when [ATP] is little changed, there are substantial
falls in [PCr] and AATP and rises in [ADP] and [Pi]; any
of these might conceivably inhibit tension production.
159
Part of the very rapid phase of the decline of developed pressure in ischemia probably has a mechanical
origin. This phase of decline is synchronous with the
fall in perfusion pressure and is usually attributed to
reduced stiffness and stretch associated with the fall of
perfusion pressure in the vascular system.86 This component will of course be absent from preparations that
are made hypoxic while perfusion continues.
Current understanding of the control of tension production in cardiac muscle51 suggests that it can be conveniently considered under three headings: the action
potential, the amount of calcium delivered into the
myoplasm, and the properties of the myofibrils.
The Action Potential
Not only is the action potential the trigger for contraction, but its amplitude and duration exert some
control over the magnitude of contraction. In particular, many workers (for review, see Morad and Goldman87) have shown that developed tension is a function
of the duration and amplitude of the action potential (or
of a voltage clamp pulse of comparable magnitude).
Changes in action potential duration and amplitude
have long been known to occur in ischemia and hypoxia (for review, see Carmeliet3) and in this section,
we consider the contribution such changes make to the
sudden fall of developed tension in ischemia or
hypoxia.
Action potentials have been measured in ischemic
tissue.8889 The changes observed are variable between
different studies (see discussion in Downar et a P ) and
include a slowing of the rate of rise of the action potential and of conduction velocity, and a reduction in the
duration and amplitude of the action potential. When
these changes are pronounced, as in the study of Downar et al, they lead to a failure of conduction within
10-15 minutes. Detailed studies of the mechanisms
that underlie the above changes in the action potential
have generally used perfused, isolated preparations so
that the various components of ischemia can be
separated.
The extracellular potassium accumulation of ischemia has been noted above and leads to depolarization
of the membrane potential and a small shortening of
the action potential (see Carmeliet3 for discussion of
the mechanism involved). However, such changes in
[K + ] o lead to only a small reduction in developed tension in isolated cardiac preparations,90 so that the
membrane potential changes associated with increased
[K + ] o probably make only a small contribution to the
rapid decline of tension in ischemia.
McDonald and MacLeod68 studied action potentials
under a variety of hypoxic conditions. In general, they
found substantial decreases in action potential duration
but only small depolarizations of the resting membrane
potential. With 5 mM glucose present in the perfusate,
the action potential duration during anoxia fell to about
40% of control over 60 minutes. However, over the 5
minutes in which the rapid fall of tension occurred, the
reduction in action potential duration was less than 5%
and seems unlikely to have had a major effect on ten-
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sion production. Thus, the rapid fall in tension that
occurs during anoxia, when glycolysis is accelerated,
does not seem to be attributable to changes in action
potential duration.
When anoxia is produced in muscles perfused with
substrate-free solutions, a much greater shortening of
the action potential occurs.68 Only recently, however,
has the question been studied of whether this shortening occurs sufficiently rapidly to account for the decline of developed tension. Recent work suggests that
when glycolysis has been prevented, either by glycogen depletion91 or by 2-deoxyglucose treatment,92 anoxia leads to a shortening of the action potential duration to < 20% control within 2-3 minutes and this was
coincident with a rapid fall of tension to 0-5% of
control. Such a dramatic shortening of the action potential would be expected to lead to reduced calcium
release from the sarcoplasmic reticulum and could explain the reduced calcium transients reported under
these conditions.93
A number of possible mechanisms for the decline in
action potential duration were discussed by Carmeliet.3 The plateau of the action potential represents a
balance between small inward and small outward currents so that a decrease in the inward current or an
increase in the outward current would lead to shortening of the action potential. Early work showed that
CN, dinitrophenol,** or intracellular acidosis95 led to a
reduction of the inward Ca 2+ current. Both the shortening of the action potential and the reduced inward
current have now been observed in single cells and can
be triggered by a fall in [ATP], and reversed by injection of ATP into the cell.96 However, the contribution
of reduced inward Ca 2+ current to the shortening of the
action potential is still uncertain. Reasons include: 1)
Recent investigations in single cells show that the Ca2+
current is briefer in single cells than measurements in
multicellular preparations suggested,97-98 so that the
contribution of the Ca 2+ current to the plateau of the
action potential may be small. 2) Experiments by
Vleugels et al99 suggest that increases in outward K+
currents are quantitatively more important than reductions in inward current. 3) The timecourse of the decrease in [ATP], or pH, in hypoxia may not be sufficiently fast to account for the shortening of the action
potential by means of their known effects on the Ca2+
current.
A mechanism that may explain the increased outward K + currents seen in hypoxia99 is the recently
described K + channel that is activated when [ATP],
falls.100 The Km for the suppression of this current was
found to be 0.1 mM in excised patches'00 and 0.5 mM
in perfused whole cells.101 It is not yet clear what the
effective Km in undisturbed cells might be, but it appears that significant increases in K + conductance are
unlikely until [ATP], has fallen below 1-2 mM. In
most situations where direct measurements have been
made, [ATP], does not fall to this level at the time
when the rapid decline of tension occurs and when
rapid shortening of the action potential may be important. Simultaneous measurements of action potential
Circulation Research
Vol 60, No 2, February 1987
duration, developed tension, and [ATP], are needed to
resolve this point.
In summary, substantial changes in action potential
duration and magnitude can occur rapidly in ischemia
and hypoxia with glycolysis inhibited. While the
mechanism of these changes is not yet well understood, it is likely that they contribute to early contractile failure.
Ca2+ Delivery to Contractile Proteins
The tension developed in each contraction of the heart
depends mainly on the amount of Ca2+ delivered to the
myoplasm (see pages 157-158). Changes in this Ca2+
delivery are a major source of the variations in tension
production that are so characteristic of the heart. For this
reason, and because of the known reduction in the Ca2+
current under hypoxic conditions,94102 it has often been
suggested that there might be a reduction in Ca2+ delivery during ischemia and hypoxia and that this might
contribute to the rapid decline of tension.
Unfortunately, at present there is no direct way of
measuring Ca 2+ delivery to contractile proteins. Developed tension is not a satisfactory indicator because
the properties of the myofibrils can also change (see
next section). The myoplasmic [Ca 2+ ] during contraction (the Ca2+ transient) is the nearest approximation
available at present. If both the calcium transients and
the tension are measured simultaneously it is possible
to distinguish, at least qualitatively, between changes
in Ca2+ delivery, which change both the Ca 2+ transient
and the developed tension in a parallel fashion, and
changes in apparent sensitivity to [Ca 2+ ],, which lead
to [Ca 2+ ], and developed tension changing in opposite
directions.
There are at present no studies of the Ca 2+ transients
in ischemic cardiac tissue, but Allen and Orchard93
have observed the effects of anoxia, both in the presence and the absence of glycolysis, on the Ca 2+ transients of ferret ventricular muscle. With glycolysis
intact, the amplitude of the Ca 2+ transient was unaffected by preventing oxidative phosphorylation, by either anoxia or cyanide, despite the fact that developed
tension fell to 30% of control. This result makes it
unlikely that there are major changes in the Ca 2+ delivery to the contractile proteins; it suggests instead that
either the ability of the contractile proteins to produce
tension or their sensitivity to calcium has been
reduced.
However, when glycolysis was prevented, anoxia
led to a quite different result. Under these conditions,
the amplitude of the Ca 2+ transients declined rapidly
with a time course that was comparable to the decline
of developed tension. Thus, failure of Ca 2+ delivery to
the contractile proteins is apparently one of the causes
of the decline of tension. The mechanism of this failure
of Ca2+ delivery has not been conclusively determined;
it is not known, for instance, whether there is Ca2+
stored in the sarcoplasmic reticulum but the release
mechanism has failed or whether sarcoplasmic reticulum Ca pump has failed and there is less calcium stored
and available for release. At present, the authors think
Alien and Orchard
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that the most likely explanation is that changes in the
action potential described in the previous section lead
to a failure of the calcium release mechanism. An
alternative possibility is suggested by the work of
Smith et al.103 They have identified an ATP-activated
Ca channel in isolated sarcoplasmic reticulum. Obviously, if this channel is involved in Ca2+ release, it
must normally be inhibited by some other mechanism
except during activation. However, when [ATP]! falls
to low levels, the channel may be inhibited and Ca2+
release prevented.
Two other possibilities for the failure of Ca 2+ release in anoxia with glycolysis prevented have been
experimentally tested and rejected. Allen et al13 measured the [ATP], by NMR under similar conditions and
showed that [ATP], at the time of the rapid fall of
tension was close to normal and certainly much greater
than the apparent Km of the sarcoplasmic reticulum CaATPase. They also calculated AATP to test the suggestions of Kammermeir et al104 and Allen and Orchard93
that free energy had fallen below the level that would
allow the sarcoplasmic reticulum Ca pump to operate.
These experiments did not support this hypothesis because the fall in AATP was similar in hypoxia with
glycolysis present and in hypoxia with glycolysis absent despite the fact that in the former case the Ca2+
transients were little affected, while in the latter case
the Ca2+ transients were abolished.
The Properties of Myofibrils
Studies with skinned cardiac muscle preparations
have identified a range of interventions that affect either the sensitivity of the myofibrils to calcium or the
maximum Ca-activated tension (for review, see
Rupp105). Such changes can occur by a variety of
mechanisms. 1) Covalent modification of the contractile or regulatory proteins, the best known example of
which is phosphorylation of the inhibitory subunit of
troponin,106 which leads to a reduction in the affinity of
troponin for calcium. The actomyosin ATPase activity
of myofibrils extracted from ischemic hearts has similar Ca sensitivity to that of myofibrils from normal
hearts,107 making this mechanism unlikely. 2) Competition between another cation and calcium for the troponin binding site, which should lead to a parallel shift
in the pCa-tension response curves, i.e., a change in
apparent sensitivity to Ca 2+ with no change in the
maximum Ca-activated tension. It is now established
that at least part of the inhibitory effects of acidosis on
tension production is caused by this mechanism.59 3)
Ions or metabolites may affect one of the other contractile proteins, leading to changes in apparent sensitivity
or maximum Ca-activated tension. The effects of ATP
and Pi on the contractile proteins are probably of this
variety.
In this account, we will mention only those interventions that seem to be of importance in ischemia.
P H . In 1969, Katz and Hecht106 suggested that an
intracellular acidosis was responsible for the decrease
of tension observed during ischemia. This was an attractive hypothesis since myocardial anoxia was
161
known to lead to accelerated glycolysis and increased
lactic acid production" and hence, presumably, to a
decrease of pH,. It had been known since 1880 that
acid solutions led to a decrease in the amount of tension, developed by the heart,109 and recent studies110
show that much of this effect can be explained by the
effects of intracellular acidosis on the myofibrils.
Qualitatively, therefore, it appeared possible that a
decrease in pHj was responsible for the decreased tension observed during ischemia. This hypothesis received further support when nuclear magnetic resonance (NMR) studies showed that pH, did indeed
decrease when intact, working heart muscle was made
ischemic or hypoxic.80
However, it was also necessary to show that the
decrease of pH: was both large enough and fast enough
to account for the decrease of tension. In an attempt to
answer this point, several groups have used 3IP NMR
to monitor pH, in isolated hearts during hypoxia and
ischemia. Jacobus et al 1 " showed that pH, and tension
both decreased monophasically during ischemia.
However, the decrease in mechanical performance
during ischemia was greater than could be accounted
for by the acidosis. Studies of hypoxia have also
shown that the contribution of acidosis to the decline of
tension is small. Allen et al13 showed that when glycolysis was present, hypoxia led initially to an alkalosis
(see pages 158—159 for discussion) followed after several
minutes by an acidosis. Since developed pressure fell
more or less monotonically, the contribution of pH
changes to the tension change was clearly small. Furthermore, when glycolysis was prevented, hypoxia led to a
rapid fall in developed tension but without any acidosis.l3
In conclusion, there is a decrease of pH, during
ischemia and hypoxia that undoubtedly makes some
contribution to the observed decrease in tension. However, it seems unlikely that it is the major cause of
tension decline, the development of acidosis being too
small and too slow to explain the decline of tension.
PHOSPHATE COMPOUNDS. Reducing [ATP] (in reality, MgATP2~) has a variety of effects on the tension
development of skinned cardiac muscle.42 Maximum
Ca-activated tension increases slightly (at least down
to 0.3 mM) and Ca sensitivity also increases. Note that
these effects will result in increased developed tension
when [ATP] falls from its normal myoplasmic level to
submillimolar levels. The rigor-promoting effects of
low [ATP] will be considered in the next section.
PCr and ADP have very small direct effects on tension development in skinned fibers."2
Inorganic phosphate (Pi) has, however, recently
been shown to have major effects on skinned cardiac
fibers, and it seems likely that the substantial increases
in [Pi] that occur in ischemia and hypoxia exert a major
depressant effect on tension development. Herzig and
Ruegg" 3 showed that Pi exerted a pronounced inhibitory effect on maximum Ca-activated tension.
Kentish"2 has shown that, in addition, Pi leads to a
reduction in Ca sensitivity. Both effects are large. For
instance, if the normal [Pi] is 1-3 mM and it increases
to 20 mM during ischemia or hypoxia, maximum Ca-
162
activated tension will be reduced to about 50% control.
The effect on Ca sensitivity leads to a further reduction, so that developed tension would be about 20% of
the original control. Not only is this effect large, it is
also rapid in onset. Figure 1 and many experimental
results have shown that [Pi] starts to rise as soon as
ATP consumption exceeds ATP production and well
before there is a significant change in [ATP].
Although Pi depresses tension production, it does
not seem to reduce ATPase activity of the myofibrils. " 4 Thus, it may be misleading to assume that ATP
consumption by the heart during ischemia or anoxia is
reduced in proportion to the decline in tension
production.
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Summary
The factors responsible for the rapid fall of developed tension in ischemia and hypoxia fall into three
categories.
METABOLIC FACTORS. These are probably the most
important contributors to early ischemic and hypoxic
failure. The earliest effect arises from the inhibitory
effects of Pi on the contractile proteins. Subsequently,
the acidosis, particularly in ischemia in which retention of lactate causes a very large fall in pH, leads to a
further reduction in tension.
MECHANICAL FACTORS. The fall in perfusion pressure during ischemia leads to reduced muscle stretch
and tension development with a time course similar to
the fall in perfusion pressure.
ACTIVATION FACTORS. Rapid shortening of the action potential duration and reduced calcium release
have both been observed in hypoxia with glycolysis
inhibited and probably contribute to the rapid decline
of tension under these conditions. A similar mechanism may operate in ischemia.
Possible Mechanisms Underlying
Ischemic Contracture
The contracture that develops in ischemia or in hypoxia with glycolysis prevented has been attributed to
two mechanisms.
RISE IN [ C A 2 + ] , . Evidence for this possibility was the
small rise in total calcium measured over this period.43
Skinned fiber experiments at normal [ATP] suggest
that [Ca 2+ ], would need to rise to 5-10 (xM to explain
the observed developed tension.
FALL IN [ATP], LEADING TO RIGOR CROSSBRUXJE FORMATION. Skinned fiber experiments suggest that at the
normal resting [Ca 2+ ],, [ATP], would have to fall to 0.1
mM or below before rigor development became significant. Although discussed as if they were independent, there is evidence that the two conditions may
interact with each other. Thus, Bremel and Weber" 3
showed that under conditions that led to rigor attachment, the binding constant of troponin for calcium was
increased, so that only a small rise in [Ca 2+ ], from the
normal resting level might lead to considerable Cadependent tension production. Fabiato and Fabiato42
showed, for instance, that 0.1 fiM [Ca 2+ ], which at
normal [ATP] does not cause any tension develop-
Circulation Research
Vol 60, No 2, February 1987
ment, led to 10% of Ca-activated tension when [ATP]
was lowered to 0.3 mM.
[Co1*],
[Ca2+]j has now been measured by a variety of techniques in cardiac cells exposed to hypoxic conditions.
There is general agreement that metabolic depletion
leads to increases in [Ca 2+ ],, but the conditions that
lead to this rise and the magnitude and time course of
the rise show considerable variability. A report by
Dahl and Isenberg," 6 who used Ca 2+ -sensitive microelectrodes to measure [Ca 2+ ] : in Purkinje fibers, has
been quoted as demonstrating a large and rapid rise in
[Ca 2+ ], on metabolic inhibition. In fact, the investigators used dinitrophenol (DNP), a proton ionophore,
that eliminates the proton gradient in mitochondria.
This agent prevents ATP production by the mitochondria, which may then actually consume ATP, so that
the decline in [ATP]j may be larger than when oxidative phosphorylation is prevented by CN or anoxia.
Dinitrophenol will also allow any calcium stored in the
mitochondria to be released. In "fresh" preparations,
stored for less than 3 hours, application of DNP led to a
slow rise in [Ca 2+ ]; that reached 10 /AM in 20-30
minutes. Older preparations showed much faster rises,
reaching 10 /u,M in 2-4 minutes. It is not clear from
these experiments whether the increases in [Ca2+]j are
due to the decline in [ATP]; and consequent inhibition
of Ca pumps or to the release of calcium stored in
mitochondria. In either case, the result is probably not
what would be expected in ischemia or anoxia.
Snowdowne et al" 7 also showed a very rapid rise in
[Ca 2+ ], on metabolic inhibition. These workers first
isolated cells by a collagenase method and then loaded
them with aequorin, using an osmotic shock technique. Large numbers of such cells were then exposed
to either hypoxia in the absence of external glucose or
to FCCP, another proton ionophore. Either treatment
produced rapid and reversible increases in [Ca 2+ ] : .
There are two reasons to doubt whether this rapid rise
in [Ca2+]i is representative of healthy cells under these
conditions. First, the metabolic status of these cells is
unknown but is likely to be poor since collagenase
isolation is known to lead to loss of P-metabolites.34
The subsequent osmotic lysis of cells seems likely to
lead to further deterioration. Second, about 30% of the
loaded cells were already rounded up and these cells,
which are already damaged, may have contributed disproportionately to the recorded increases in [Ca2*],.
In contrast, there are studies from several laboratories indicating that metabolic inhibition leads to rises in
[Ca 2+ ], after a delay of only 20-40 minutes and that a
contracture of intact preparations or rounding up of
isolated cells precedes the rise in [Ca 2+ ],. Allen and
Orchard93 showed that during short exposures to hypoxia after inhibition of glycolysis, a substantial contracture could develop with no significant increase in
resting [Ca 2+ ],. Subsequent studies with more complete inhibition of glycolysis confirmed this finding"8
and showed that, at about the time that the contracture
was close to maximal, the resting [Ca 2+ ], started to
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rise. Similar findings have been reported by Cobbold
and Bourne, 1 " who used single isolated ventricular
cells microinjected with aequorin. They showed that
during hypoxia with glycolytic inhibition, single cells
took 10-20 minutes to show signs of contracture, and
only 5-10 minutes later were increases in [Ca 2+ ], recorded. As mentioned earlier, studies by Guamieri50
using ion-sensitive electrodes showed that [Ca 2+ ] : did
not change in 20 minutes of hypoxia in the absence of
glucose, despite the fact that [Na + ], rose (for discussion, see pages 163-165).
We suggest that the disparities in the time required
for [Ca 2+ ], to rise on hypoxia reflect differences in the
initial metabolic status of the preparations, although
further tests of this point will require simultaneous
measurements of [Ca 2+ ], and metabolic status during
ischemia and hypoxia. All the results cited are consistent with the following conclusions. When [ATP],, or
perhaps AATP, falls below a certain level, [Ca 2+ ], rises
quite rapidly, presumably because of failure of the
various Ca pumps that remove calcium from the myoplasm. [ATP]j does not fall below this level in cardiac
cells with oxidative phosphorylation blocked, provided anaerobic glycolysis can continue. When glycolysis is blocked in addition to oxidative phosphorylation, [ATP], will, of course, eventually fall below this
level, but the threshold for rigor production seems to
occur at a significantly higher level than that necessary
to block Ca pumps and lead to elevated [Ca 2+ ],.
[ATP],
There are a number of experimental observations
that suggest that ischemic and hypoxic contractures are
due to the attachment of rigor crossbridges, precipitated by the low [ATP],. Thus, interventions that lead to
greater tension production and therefore increased
ATP consumption lead to enhanced rigor production. u
The stiffness of muscle in ischemic or hypoxic contracture increases considerably above that associated with
a normal contraction, consistent with rigor bridge attachment.120 Heat measurements have shown that the
heat/tension ratio is similar in a twitch and a K + contracture but smaller in hypoxic contractures.'21 Since
ATP consumption associated with crossbridge cycling
is the major cause of heat production, these findings
suggest that the crossbridges in hypoxic contractures
are turning over at a slower rate, as expected for rigor
crossbridges.
[ATP], has been measured in contractures by both
biochemical and NMR methods, and the consensus is
that it is at very low levels during contractures. 133O8° It
is difficult, however, to prove quantitatively that
[ATP], has fallen to the level shown in skinned fiber
studies to cause rigor for two main reasons: 1) Present
NMR and analytical methods cannot measure myoplasmic levels of ATP with confidence in the range of
0.1 mM. 2) Problems of tissue homogeneity exist in
both intact preparations and in the large numbers of
isolated cells that are required for such measurements.
For instance, in NMR studies on whole hearts, contractures can sometimes be recorded from the left ven-
163
tricle at a time when the NMR signal from the whole
heart suggests a mean [ATP], of several mM.13 This
may mean that in the nonworking right ventricle,
[ATP], is close to normal while in the working left
ventricle it has declined to levels capable of producing
rigor.
A different explanation for the possible appearance
of rigor at [ATP], higher than 0.1 mM is suggested by
the work of Miller and Smith122 on skinned cardiac
muscle. They showed that in the presence of an elevated [ADP] the rigor tension at any given [ATP] was
increased.
Summary
The present weight of evidence supports the idea
that ischemic and hypoxic contractures are produced
by low ATP, leading to rigor. Further reduction in
ATP leads to a rise in [Ca 2+ ], that may precipitate other
cell damage (see next section) but is not itself the
initiating cause of the contracture.
Reperfusion Damage
Interruption of coronary flow to a region of the myocardium for more than about 1 hour leads to permanent
damage. Return of coronary flow is essential to survival of the myocardium, but it was realized in the early
1960s that reperfusion can also accelerate the appearance of myocardial damage.123 Early studies of this
subject were reviewed by Hearse," who drew attention
to the similarities between the calcium paradox and
reperfusion damage. (The term calcium paradox describes the cell damage observed when extracellular
Ca2+ is returned to the perfusate after a period of Cafree perfusion.) In both situations, there is a large,
rapid increase in total cell calcium, a contracture develops, and there is eventual cellular damage with release of intracellular enzymes. It is now accepted that
calcium accumulation precipitates many kinds of muscle damage. The evidence for this statement and the
mechanism by which increased Ca 2+ leads to cell damage have been extensively reviewed (e.g., Jennings
and Steenberg,7 Nayler et al,9 and Poole-Wilson6) and
will not be repeated here. Instead, discussion will be
focused on two issues — the mechanism of the Ca 2+
increase and the ionic interactions that may occur during reperfusion.
Mechanism of Calcium Influx
The mechanism of Ca 2+ accumulation in the calcium paradox is now well established. During a period
of low Ca2+ perfusion, [Na], rises64; thus when Ca 2+ is
returned to the extracellular space, operation of the NaCa exchanger will lead to increased Na + efflux, accompanied by an increased Ca2+ influx leading to Ca 2+
accumulation. If this mechanism represents the main
route for Ca2+ influx, then both Ca 2+ accumulation and
tissue damage should depend on the level of [Na + ], that
exists at the time when Ca 2+ is returned to the perfusate. Such a correlation was demonstrated by RuanoArroyo et al.124 What has recently become clear is that
the mechanism by which Na + enters the cell during the
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low Ca 2+ perfusion is not primarily by the Na channel
or by Na-Ca exchange, but via the Ca channels,125
which become highly permeable to Na + when the external Ca2+ is very low.126 In accordance with this
theory, Ca-channel blocking drugs exert a protective
effect if applied during the period of Ca-free perfusion
but not when external Ca 2+ is replaced.125 Thus, it
seems that the Ca 2+ influx that triggers damage after a
period of Ca-free perfusion is mediated by the Na-Ca
exchanger and is caused by the elevation of [Na + ],.
Clearly, the same sequence of events cannot operate
in reperfusion or reoxygenation damage because external Ca2+ is present, though in ischemia the amount of
extracellular Ca 2+ is limited and could conceivably
limit Ca 2+ influx during prolonged ischemia. However, it has been established that Ca 2+ influx does
increase dramatically on reperfusion or reoxygenation
and that this influx is not simply due to a nonspecific
increase in cell permeability nor does it occur via Ca
channels.46 The idea that the influx occurs via Na-Ca
exchange was proposed by Grinwald.l27 Isolated hearts
were subjected to ischemia and reperfusion after maneuvers designed to raise or lower [Na + ],. The increase
in total Ca2+ on reperfusion was determined to be dependent on [Na + ],. This idea was confirmed and extended by Renlund et al,128 who showed that the degree
of mechanical and metabolic recovery was inversely
related to the level of [Na + ], before reperfusion but
could not be accounted for by changes in the level of
PCr or ATP before reperfusion.
As discussed earlier, there is evidence that [Na + ],
rises during ischemia, probably because the Na pump
is inhibited by low levels of ATP or reduced AATP.
However, the reason why Ca 2+ influx occurs at a rapid
rate only after reperfusion or reoxygenation when elevated [Na + ], is present both before and after reperfusion remains a mystery. The influx of Ca 2+ could still
occur by Na-Ca exchange if the exchange is inhibited
during ischemia but reactivated on reperfusion. There
are two possible mechanisms that could allow this to
happen: 1) Intracellular pH is known to inhibit Na-Ca
exchange.129 Since ischemic or hypoxic muscle is acidotic and this acidosis recovers on reperfusion128 and
reoxygenation,17 acidosis could inhibit Na-Ca exchange during ischemia and allow it to reactivate on
reperfusion. However, the fact that damage also occurs on reoxygenation after a period of anoxia without
glucose6 or a period of anoxia with glycolysis inhibited" 8 implies that intracellular acidosis is not essential
since there will be little acidosis when lactate production is prevented. 2) In squid axon, both Nao-dependent Ca 2+ efflux130 and Na,-dependent Ca +2 influx131
have been shown to be dependent on intracellular
ATP, with a K^ of around 0.2 mM. If Na-Ca exchange
in the heart has the same properties, Na-Ca exchange
will be inhibited during ischemia by the low level of
ATP and reactivated when ATP rises in reperfusion.
This mechanism could also explain why total Ca 2+ u
and [Ca2+]|118 do not rise rapidly during ischemia despite substantial rises in [Na"1"],.50
The authors believe that Na-Ca exchange is the
Circulation Research
Vol 60, No 2, February 1987
most likely route for Ca 2+ entry on reperfusion, on the
basis of present evidence. For alternative views, see
Poole-Wilson6 and Nayler et al. 9
Interactions Between Na+, Ca2*, and H+
The intracellular concentrations of Na + , Ca 2+ , and
+
H are each elevated during reperfusion, and there is
some evidence that a regenerative cycle of the sort
shown below may occur.
r t [Na*]- T
+
T [H*l
2+
1
The raised [Na ], leads to a raised [Ca ], by Na-Ca
exchange. The mechanism by which an increased
[Ca 2+ ], leads to an increased [H + ], is the least clear
though there is good experimental evidence for its existence.132 Possible mechanisms include direct competition between Ca 2+ and H + for binding sites on proteins or for entry to mitochondria. Alternatively, the
raised [Ca 2+ ], may trigger anaerobic glycolysis and
lactic acid production, either directly or indirectly, by
activating tension production and ATP consumption.
The raised [H + ], leads to increased [Na"1"], by Na-H
exchange. The important feature of this cycle is that it
shows positive feedback; once threshold levels of
some or all of the ions are exceeded, the cycle will tend
to continue until prevented by some extraneous mechanism. Thus, once a substantial rise of [Ca 2+ ], occurs, it
may continue to rise until it causes cellular damage.
Why should this potentially damaging cycle occur
during reperfusion but not under normal conditions or
during ischemia? Under normal conditions, an elevated [Ca 2+ ], is rapidly pumped down to the diastolic level
by the sarcoplasmic reticulum Ca pump, and [Na + ], is
regulated by the Na pump. In ischemia, if ATP or AATP
falls sufficiently low, both the Na pump and the SR Ca
pump will fail, but as noted above, inhibition of Na-Ca
exchange may prevent operation of the cycle. Lazdunski et al74 have pointed out that high extracellular
[H + ] occurs during ischemia, and the competition between extracellular H + and Na + inhibits the Na-H exchange; this will also tend to prevent operation of the
cycle. After a period of ischemia, then, both [Na + ],
and [H + ]; will tend to be elevated, and [Ca 2+ ], may also
be elevated if the period of ischemia is long.'18 However, inhibition of Na-Ca exchange in the above cycle
prevents these levels from rising rapidly to damaging
levels.
When reperfusion occurs, oxidative phosphorylation will restart and P-metabolites will show some recovery.30128133 However, the recovery of ATP is limited. There is loss of total adenine nucleotides, which is
related to the duration of ischemia, and ATP consumption is potentially at a very high rate because the Na
pump, the sarcoplasmic reticulum Ca pump, and the
myofibrillar ATPase are all near maximal activation.
Reactivation of the Na-Ca exchange will lead to substantial Ca 2+ influx so that the sarcoplasmic reticulum
will soon be saturated with calcium and no longer help
to keep [Ca 2+ ]; low. Activation of the Na-H exchanger
Allen and Orchard
Contractile Function in Ischemia and Hypoxia
Downloaded from http://circres.ahajournals.org/ by guest on May 8, 2017
will tend to keep [Na + ], high and maintain the Ca2+
influx. Thus, whether or not recovery can occur will
depend on whether the Na pump, in the face of reduced
[ATP], and increased [Na + ],, can lower [Na + ], to a
level that prevents the above regenerative cycle from
becoming established.
Cell damage also occurs on reoxygenation after a
period of anoxia in which glycolysis is reduced or
prevented. 6 " 8 Under these circumstances, the accumulation of intracellular and extracellular H + will be
reduced. This implies that reactivation of the Na-H
exchanger74 and Na + entry in exchange for H + are not
essential to the activation of the pathological cycle
indicated above. In the absence of anaerobic glycolysis, the fall in [ATP] in anoxia is likely to be more
rapid than in ischemia, and the consequences of a
longer period with a very low [ATP] may lead to larger
increases in [Na + ], and [Ca 2+ ],, which compensate for
the reduction of Na + entry in exchange for H + .
Summary
There is good evidence that elevated [Ca 2+ ];, produced by an influx of Ca2+ in exchange for Na + , is the
underlying pathology in reperfusion or reoxygenation
damage. Further measurements of [Na + ], and [Ca 2+ ],
during ischemia and reperfusion, coupled with information about metabolic levels, are needed to confirm
or refute this hypothesis. Contributions to cell damage
by other mechanisms, e.g., oxygen free radicals,134
certainly cannot yet be excluded.
Acknowledgments
We thank D.A. Eisner, A.C. Elliott, B.R. Jewell,
J.C. Kentish, G.L. Smith, and M. Valdeolmillos for
valuable criticisms of the manuscript.
References
1. Neely JR, Morgan HE: Relationship between carbohydrate
and lipid metabolism and the energy balance of heart muscle.
Ann Rev Physiol 1974;36:413-459
2. GibbsCL: Cardiac energetics. PhysiolRev 1978:58:174-254
3. Carmeliet E: Cardiac transmembrane potentials and metabolism. Circ Res 1978;42:577-587
4. Janse MJ, Kleber AG: Electrophysiological changes and ventricular arrhythmias in the early phase of regional myocardial
ischemia. Circ Res 1981;49:1069-1081
5. Nayler WG, Poole-Wilson PA, Williams A: Hypoxia and
calcium. J Mol Cell Cardiol 1979; 11:683-706
6. Poole-Wilson PA: The nature of myocardial damage following reoxygenation, in Parratt JR (ed): Control and Manipulation of Calcium Movement. New York, Raven Press, 1985
7. Jennings RB, Steenbergen C: Nucleotide metabolism and cellular damage in myocardial ischemia. Ann Rev Physiol
1985;47:729-749
8. Kubler W, Katz AM: Mechanism of early "pump" failure of
the ischemic heart: Possible role of adenosine triphosphate
depletion and inorganic phosphate accumulation. Am J Cardiol 1977;40:467-471
9. Nayler WG, Sturrock WJ, Panagiotopoulos S: Calcium and
myocardial ischemia, in Parratt JR (ed): Control and Manipulation of Calcium Movements. New York, Raven Press, 1985
10. Jennings RB, Reimer KA, Steenbergen C: Myocardial ischemia and reperfusion: role of calcium, in Parratt JR (ed):
Control and Manipulation of Calcium Movement. New York,
Raven Press, 1985
165
11. Hearse DJ: Reperfusion of the ischemic myocardium. J Mol
Cell Cardiol 1977;9:605-616
12. Weissler AM, Kruger FA, Baba N, Scharpell DG, Leighton
RF, Gallimore JK: Role of anaerobic metabolism in the preservation of functional capacity and structure of anoxic myocardium. J Clin Invest 1968;47:403-416
13. Allen DG, Morris PG, Orchard CH, Pirolo JS: A nuclear
magnetic resonance study of metabolism in the ferret heart
during hypoxia and inhibition of glycolysis. J Physiol (Lond)
1985;361:185-204
14. Nayler WG, Yepez CE, Poole-Wilson PA: The effect of 0adrenoceptor and Ca 2 + antagonist drugs on the hypoxia-induced increase in resting tension. Cardiovasc Res 1978;12:
666-674
15. Williamson JR: Glycolytic control mechanisms. II. Kinetics
of intermediate changes during the aerobic-anoxic transition
in perfused rat heart. J Biol Chem 1966;241:5026-5036
16. Kubler W, Spieckermann PG: Regulation of glycolysis in the
ischaemic and anoxic myocardium. J Mol Cell Cardiol
1970; 1:351-377
17. Matthews PM, Radda GK, Taylor DJ: A 3 I P n.m.r. study of
metabolism in the hypoxic perfused rat heart. Biochem Soc
Trans 1981 ;9:236-237
18. Carlson FD, Wilkie DR: Muscle physiology, Englewood
Cliffs, New Jersey, Prentice-Hall Inc, 1974
19. Dawson MJ, Wilkie DR: Muscle and brain metabolism studied by 3 'P nuclear magnetic resonance, in Baker PF (ed):
Recent Advances in Physiology. Edinburgh, Churchill Livingston, 1984
20. Veech RL, Lawson JWR, Cornell NW, Krebs HA: Cytosolic
phosphorylation potential. J Biol Chem 1979;254:6538-6547
21. LawsonJWR, Veech RL: Effects ofpH and free Mg 2 + on the
K^, of the creatine kinase reaction and other phosphate transfer reactions. J Biol Chem 1979;254:6528-6537
22. Hansford RG: Control of mitochondrial substrate oxidation.
Curr Top Bioenerget 1980; 10:217-278
23. Neely JR, Denton RM, England PJ, Randle PJ: The effects of
increased heart work on the tricarboxylic cycle and its interactions with glycolysis in the perfused rat heart. Biochem J
1972;128:147-159
24. Allen DG, Eisner DA, Morris PG, Pirolo JS, Smith GL:
Metabolic consequences of increasing intracellular calcium
and force production in perfused ferret hearts. J Physiol
(Lond) 1986;376:121-141
25. Garlick PM, Radda GK, Seeley PJ: Studies of acidosis in the
ischaemic heart by phosphorus nuclear magnetic resonance.
Biochem J 1979;184:547-554
26. Gudjbarnason S, Mathes P, Ravens KG: Functional compartmentation of ATP and creatine phosphate in heart muscle. J
Mol Cell Cardiol 1970;l:325-339
27. Opie LH: High energy phosphate compounds, in Drake-Holland AJ, Noble MIM (eds): Cardiac Metabolism. Chichester,
UK, John Wiley & Sons, 1983
28. Rovetto MJ, Whitmer JT, Neely JR: Comparison of the effects of anoxia and ischemia on carbohydrate metabolism in
isolated working rat hearts. Circ Res 1973;32:699-711
29. Rovetto MJ, Lamberton WF, Neely JR: Mechanisms of glycolytic inhibition ischemic rat hearts. Circ Res 1975;37:742751
30. Hearse DJ, Chain EB: The role of glucose in the survival and
recovery of the anoxic isolated perfused rat heart. Biochem J
1972;128:1125-1133
31. Doorey AJ, Barry WH: The effects of inhibition of oxidative
phosphorylation and glycolysis on contractility and high energy phosphate content in cultured chick heart cells. Circ Res
1983;53:192-201
32. Boerth RC, Covell JW, Seagren SC, Pool PE: High-energy
phosphate concentrations in dog myocardium during stress.
Am J Physiol 1969;216:1103-1106
33. Somlyo AV, Gonzalez-Serratos H, Shuman H, McClellan G,
Somlyo AP: Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron probe
study. J Cell Biol 1981 ;90:577-594
34. Geisbuhler T, Altschuld RA, Trewyn RW, Ansel AZ, Lamka
Circulation Research
166
35.
36.
37.
38.
39.
40.
41.
Downloaded from http://circres.ahajournals.org/ by guest on May 8, 2017
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
K, Brierley GP: Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells. Circ Res
1984;54:536-546
Crank J: The Mathematics of Diffusion, ed 2. Oxford, Oxford
University Press, 1975
Kushmerick MI, Podolsky RJ: Ionic mobility in muscle cells.
Science 1969;166:1297-1298
Meyer RA, Sweeney HL, Kushmerick MI: A simple analysis
of the "phosphocreatine shuttle." Am J Physiol 1984;246:
C365-C377
Dawson MJ, Gadian DG, Wilkie DR: Muscular fatigue investigated by nuclear magnetic resonance. Nature 1978;274:
861-866
Shigekawa M, Finegan JM, Katz AM: Calcium transport
ATPase of canine cardiac sarcoplasmic reticulum; a comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum. J Biol Chem 1976;251:6894-6900
Glynn IM, Karlish JD:"ATP hydrolysis associated with an
uncoupled sodium flux through the sodium pump: Evidence
for allosteric effects of intracellular ATP and extracellular
sodium. J Physiol (Lond) 1976;256:465-496
Caroni P, Carafoli E: An ATP-dependent Ca2+-pumping system in dog heart sarcolemma. Nature 1980;283:765-767
Fabiato A, Fabiato F: Effects of magnesium on contractile
activation of skinned cardiac cells. J Physiol (Lond) 1975;
249:497-517
Eisner DA, Allen DG, Orchard CH: The regulation of resting
calcium concentration in cardiac muscle, in Parratt JR (ed):
Control and Manipulation of Calcium Movement. London,
Raven Press, 1985, pp 65-86
Shen AC, Jennings RB: Myocardial calcium and magnesium
in acute ischemic injury. Am J Pathol 1972;67:417—440
Nayler WG, Grau A, Slade AA: A protective effect of verapamil on hypoxic heart muscle. Cardiovas Res 1976; 10:650662
Poole-Wilson PA, Harding DP, Bourdillon PDV, Tones MA:
Calcium out of control. J Mol Cell Cardiol 1984;16:175-187
Reuter H: Exchange of calcium ions in the mammalian myocardium. Circ Res 1974;34:599-605
Eisner DA, Lederer WJ: Na-Ca exchange: stoichiometry and
electrogenicity. Am J Physiol 1985;248:C189-C202
Carafoli E: The homeostasis of calcium in heart cells. J Mol
Cell Cardiol 1985;17:203-212
Guamieri TJ: Relationship between intracellular calcium activity (ofCa1) and intracellular sodium activity (aNa') during
hypoxia (abstract). Circulation 1985;72(suppl III, part II):III327
Chapman RA: Excitation-contraction coupling in cardiac
muscle. Prog Biophys Mol Biol 1979;35:l-52
Fabiato A: Calcium-induced release of calcium ions from the
cardiac sarcoplasmic reticulum. Am J Physiol 1983;245:C1C14
Bers DM: Ca influx and sarcoplasmic reticulum Ca release in
cardiac muscle activation during postrest recovery. Am J
Physiol 1985;248:H366-H381
Inesi G: Mechanism of calcium transport. Ann Rev Physiol
1985;47:573-601
Fry CH, Poole-Wilson PA: Effects of acid-base changes on
excitation-contraction coupling in guinea-pig and rabbit cardiac ventricular muscle. J Physiol (Lond) 1981;313:141-160
Henry PD, Shuchleib R, Davis J, Weiss ES, Sobel BE: Myocardial contracture and accumulation of mitochondrial calcium in ischemic rabbit heart. Am J Physiol 1977;233:H677H684
Cheung JY, Leaf A, Bonventre JV: Mitochondrial function
and intracellular calcium in anoxic cardiac myocytes. Am J
Physiol 1986;250:C18-C25
Williams A: Mitochondria, in Drake-Holland AJ, Noble
MIM (eds): Cardiac Metabolism. New York, John Wiley &
Sons, 1983
Blanchard EM, Solaro RJ: Inhibition of the activation and
troponin calcium binding of dog cardiac myofibrils by acidic
pH. Circ Res 1984;55:382-391
Langer GA: The effect of pH on cellular and membrane calcium binding and contraction of myocardium: A possible role
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
Vol 60, No 2, February 1987
for sarcolemmal phospholipid in EC coupling. Circ Res
1985;57:374-382
Wendt-Gallitelli MF, Isenberg G: Extra- and intracellular
lanthanum: Modified calcium distribution, inward currents
and contractility in guinea pig ventricular preparations.
PflugersArch 1985;4O5:31O-322
Ellis D: The effects of external cations and ouabain on the
intracellular sodium activity of sheep heart Purkinje fibres. /
Physiol (Lond) 1977;273:211-240
Eisner DA: The Na-K pump in cardiac muscle, in Fozzard
HA, Haber E, Jennings R, Katz A, Morgan H (eds): Handbook of Experimental Cardiology. New York, Raven Press,
1986
Deitroer JW, Ellis D: Changes in the intracellular sodium
activity of sheep heart Purkinje fibres produced by calcium
and other divalent cations. J Physiol (Lond) 1978;277:437453
Eisner DA, Lederer WJ, Vaughan-Jones RD: The quantitative relationship between twitch tension and intracellular sodium activity in sheep cardiac Purkinje fibers. J Physiol
1984;355:251-266
Baker PF, Blaustein MP, Hodgkin AL, Steinhardt RA: The
influence of calcium on sodium efflux in squid axons. J Phys10/(Lond) 1969;200:431^*58
Lee CO, Kang DH, Sokol JH, Lee KS: Relation between
intracellular Na ion activity and tension of sheep cardiac Purkinje fibers exposed to dihydro-ouabain. Biophys J 1980;29:
315-330
McDonald TF, MacLeod DP: Metabolism and the electrical
activity of anoxic ventricular muscle. J Physiol 1973;229:
559-582
Kleber AG: Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute
global ischaemia in isolated perfused guinea pig hearts. Circ
Res 1983;52:442-450
Eisner DA, Lederer WJ, Vaughan-Jones RD: The effects of
rubidium and membrane potential on the intracellular sodium
activity of sheep Purkinje fibres. J Physiol (Lond) 1981;317:
189-205
January CT, Fozzard HA: The effects of membrane potential,
extracellular potassium, and tetrodotoxin on the intracellular
sodium activity of sheep cardiac muscle. Circ Res 1984;54:
652-665
Wilde AAM, Kleber AG: The combined effects of hypoxia,
high K, and acidosis on the intracellular sodium activity and
resting potential in guinea pig papillary muscle. Circ Res
1986;58:249-256
Deitmer JW, Ellis D: Interactions between the regulation of
the intracellular pH and sodium activity of sheep cardiac
Purkinje fibres. J Physiol (Lond) 1980;3O4:471-488
Lazdunski M, Frelin C, Vigne P: The sodium/hydrogen exchange system in cardiac cells: Its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 1985;17:
1029-1042
Vaughan-Jones RD: Chloride-bicarbonate exchange in the
sheep cardiac Purkinje fibre, in Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions:
Proceedings. New York, Alan Liss, 1982, pp 239-252
Gevers W: Generation of protons by metabolic processes in
heart cells. J Mol Cell Cardiol 1977;9:867-874
Wilkie DR: Generation of protons by metabolic processes
other than glycolysis in muscle cells: A critical view. / Mol
Cell Cardiol 1979; 11:325-330
Ellis D, Thomas RC: Direct measurement of the intracellular
pH of mammalian cardiac muscle. J Physiol (Lond) 1976;
262:755-771
Van der Vusse GJ, Reneman RS: Glycogen and lipids (endogenous substrates), in Drake-Holland AJ, Noble MIM (eds):
Cardiac Energetics. New York, John Wiley & Sons, 1983
Bailey IA, Williams SR, Radda GK, Gadian DG: Activity of
phqsphorylase in total global ischaemia in the rat heart. BiochemJ 1981;196:171-178
Kleber AG: Extracellular potassium accumulation during
Allen and Orchard
82.
83.
84.
85.
86.
87.
Downloaded from http://circres.ahajournals.org/ by guest on May 8, 2017
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
Contractile Function in Ischemia and Hypoxia
acute myocardial ischemia. J Mol Cell Cardlol 1984;16:
389-394
Gaspardone A, Shine KI, Seabrooke SR, Poole-Wilson PA:
Potassium loss from rabbit myocardium during hypoxia: Evidence for passive efflux linked to anion extrusion. / Mol Cell
Cardiol 1986;18:389-399
Weiss J, Shine KI: Extracellular K + accumulation during
myocardial ischemia in isolated rabbit heart. Am J Physiol
1982;242:H619-H628
Harris HA, Bisteni A, Russell RA, Brigham JC, Firestone JE:
Excitatory factors in ventricular tachycardia resulting from
myocardial ischemia: Potassium a major excitant. Science
1954; 119:200-203
Shine KI, Douglas AM, Ricchiuti N: Ischemia in isolated
interventricular septa: mechanical events. Am J Physiol
1976;231:1225-1232
Arnold G, Kosche F, Miessner E, Neitzert A, Locher W: The
importance of the perfusion pressure in the coronary arteries
for the contractility and the oxygen consumption of the heart.
PflugersArch 1968;299:339-356
Morad M, Goldman Y: Excitation-contraction coupling in
heart muscle: membrane control of development of tension.
Prog Biophys Mol Biol I973;27:257-313
Kardesch M, Hogencamp CE, Bing RJ: The effect of complete ischemia on the intracellular electrical activity of the
whole mammalian heart. Circ Res 1958;6:715-720
Downar E, Janse MJ, Durrer D: The effect of acute coronary
artery occlusion on subepicardial transmembrane potentials in
the intact porcine heart. Circulation 1977;56:217-224
Kavaler F, Hyman PM, Lefkowitz RB: Positive and negative
inotropic effects of elevated extracellular potassium level on
mammalian ventricular muscle. J Gen Physiol 1972;60:351365
Allen DG, Harris MB, Smith GL: Failure of action potentials
during cyanide exposure in glycogen-depleted ferret ventricular muscle (abstract). / Physiol (Lond) 1987 (in press)
McDonald TF, Hayashi H, Ponnambalam C, Watanabe T:
Electrical activity and tension in guinea pig ventricular muscle during and after metabolic depression, in Yamada K, Katz
AM, Toyama J (eds): Cardiac Function under Ischemia and
Hypoxia. Nagoya, Japan, University of Nagoya Press, 1986
Allen DG, Orchard CH: Intracellular calcium concentration
during hypoxia and metabolic inhibition in mammalian ventricular muscle. J Physiol (Lond) 1983;339:107-122
Kohlhardt M, Kubler M: The influence of metabolic inhibitors upon the transmembrane slow inward current in the mammalian ventricular myocardium. Naunyn-Schmiedebergs
Arch Pharmacol 1975;29O:265-274
Kohlhardt M, Haap K, Figulla HR: Influence of low extracellular pH upon the calcium inward current and isometric contractile force in mammalian ventricular myocardium.
PflugersArch 1976;366:31-38
Taniguchi J, Noma A, Irisawa H: Modification of the cardiac
action potential by intracellular injection of adenosine triphosphate and related substances in guinea pig single ventricular cells. Circ Res 1983;53:131-139
Noble D: The surprising heart: a review of recent progress in
cardiac electrophysiology. J Physiol 1984;353:l-5O
Nilius B, Hess P, Lansman JB, Tsien RW: A novel type of
cardiac calcium channel in ventricular cells. Nature 1985;
316:443-446
Vleugels A, Vereecke J, Carmeliet E: Ionic currents during
hypoxia in voltage-clamped cat ventricular muscle. Circ Res
1980;47:501-508
Noma A: ATP-regulated K+ channels in cardiac muscle.
Nature 1983 ;3O5:147-148
Kakei M, Noma A, Shibasaki T: Properties of adenosinetriphosphate-regulated potassium channels in guinea-pig ventricular cells. J Physiol (Lond) 1985;363:441^62
Irisawa H, Kotcubun S: Modulation by intracellular ATP and
cyclic AMP of the slow inward current in isolated single
ventricular cells of the guinea-pig. J Physiol (Lond) 1983;
338:321-338
Smith JS, Coronado R, Meissner G: Sarcoplasmic reticulum
167
contains adenine nucleotide-activated calcium channels. Nature 1985;316:446-449
104. Kammermeier H, Schmidt P, Jungling E: Free energy change
of ATP hydrolysis: a causal factor of early hypoxic failure of
the myocardium? J'Mol Cell Cardiol 1982;14:267-277
105. Rupp H: Modulation of tension generation at the myofibrillar
level — An analysis of the effect of magnesium adenosine
triphosphate, magnesium, pH, sarcomere length and state of
phosphorylation. Basic Res Cardiol 1980;75:295-317
106. Solaro RJ, Moir AJG, Perry SV: Phosphorylation of troponin
I and the inotropic effect of adrenaline in the perfused rabbit
heart. Nature 1976^262:615-617
107. Krause SM, Hess ML: The effect of short term normothermic
global ischemia and acidosis on cardiac myofibrillar Ca-Mg
ATP-ase activity. J Mol Cell Cardiol 1985;17:523-526
108. Katz AM, Hecht HH: The early "pump" failure of the ischemic heart. Am J Med 1969;47:497-502
109. Gaskell WH: On the tonicity of the heart and blood vessels. /
Physiol (Lond) 1880;3:48-75
110. Fabiato A, Fabiato F: Effects of pH on the myofilaments and
the sarcoplasmic reticulum of skinned cells from cardiac and
skeletal muscles. J Physiol (Lond) 1978;276:233-255
111. Jacobus WE, Pores IH, Lucas SK, Kallman CH, Weisfeldt
ML, Flaherty JT: The role of intracellular pH in the control of
normal and ischemic myocardial contractility: a 31P nuclear
magnetic resonance and mass spectrometry study, in Nuccitelli R, Deamer DW (eds): Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Function, New
York, Alan R. Liss, Inc, 1982
112. Kentish JC: The effects of inorganic phosphate and creatine
phosphate on force production in skinned muscles from rat
ventricle. J Physiol (Lond) 1986;370:585-6O4
113. Herzig JW, Ruegg JC: Myocardial cross-bridge activity and
itsregulationby Ca + + , phosphate and stretch, in Riecker G,
Weber A, Goodwin J (eds): Myocardial Failure. International Boehringer Mannheim Symposium, 1977
114. Altringham JD, Johnston LA: Effects of phosphate on the
contractile properties of fast and slow muscle fibres from an
antarctic fish. J Physiol (Lond) 1985;368:491-5OO
115. Bremel RD, Weber A: Cooperation widiin actinfilamentsin
vertebrate skeletal muscle. Nature (New Biol) 1972;238:97101
116. Dahl G, Isenberg G: Decoupling of heart muscle cells: Correlation with increased cytoplasmic calcium activity and with
changes of nexus ultrastructure.yAf embr Biol 1980;53:63-75
117. Snowdowne KW, Ertel RJ, Borle AB: Measurement of cytosolic calcium with aequorin in dispersed rat ventricular cells.
J Mol Cell Cardiol 1985;17:233-241
118. Allen DG, Smith GL: Intracellular calcium in metabolically
depleted ferret ventricular muscle during exposure to cyanide
and its removal (abstract). J Physiol (Lond) 1985;369:92P
119. Cobbold PH, Bourne PK: Aequorin measurements of free
calcium in single heart cells. Nature 1984;312:444-446
120. Lewis MJ, Housmans PR, Claes VA, Brutsaert DL, Henderson AH: Myocardial stiffness during hypoxia and reoxygenation contractures. Cardiovasc Res 1980;14:339-344
121. Holubarsch C, Alpert NR, Goulette R, Mulieri LA: Heat
production during hypoxic contracture of rat myocardium.
Circ Res 1982;51:777-786
122. Miller DJ, Smith GL: The contractile behaviour of EGTA and
detergent-treated heart muscle. J Muscle Res Cell Motil
1985;6:541-567
123. Jennings RB, Sommers HM, Smyth GA, Rack HA, Linn H:
Myocardial necrosis induced by temporary occlusion of a
coronary artery in the dog. Arch Pathol 1960;70:68-78
124. Ruano-Arroyo G, Gerstenblith G, Lakatta EG: "Calcium
paradox" in the heart is modulated by cell sodium during the
calcium-free period. J Mol Cell Cardiol 1983;16:783-793
125. Chapman RA, Rodrigo GC, Tunstall J, Yates RJ, Busselen P:
Calcium paradox of the heart: A role for intracellular sodium
ions. Am J Physiol 1984;247:H874-H879
126. Hess P, Tsien RW: Mechanism of ion permeation through
calcium channels. Nature 1984;3O9:453-456
127. Grinwald PM: Calcium uptake during post-ischemic reperfu-
Circulation Research
168
128.
129.
130.
131.
sion in the isolated rat heart: Influence of extracellular sodium. J Mol Cell Cardiol 1982;14:359-365
Renlund DG, Gerstenblith G, Lakatta EG, Jacobus WE, Kailman CH, Weisfeldt ML: Reduction of post-ischemic myocardial injury without altering the ischemic insult. J Mol Cell
Cardiol 1984;16:795-801
Philipson KD, Bersohn MM, Nishimoto AY:. Effects of pH
on Na-Ca exchange in canine cardiac sarcolemmal vesicles.
Circ Res 1982;50:287-293
Baker PF, McNaughton PA: Kinetics and energetics of calcium efflux from intact squid giant axons. J Physiol (Lond)
1976:259:103-144
DiPolo R: Calcium influx in internally dialyzed squid giant
axons. J Gen Physiol 1979;73:91-113
Vol 60, No 2, February J987
132. Vaughan-Jones RD, Eisner DA, Lederer WJ: Ca2+ ions can
affect intracellular pH in mammalian cardiac muscle. Nature
1983;301:522-524
133. Neely JR, Grotyohann LW: Role of glycolytic products in
damage to ischemic myocardium. Dissociation of adenosine
triphosphate levels and recovery of function of reperfused
ischemic hearts. Circ Res 1984;55:816-824
134. Hess ML, Manson NH: Molecular oxygen: Friend or foe? The
role of oxygen free radicals in the calcium paradox and ischemia/reperfusion injury. J Mol Cell Cardiol 1984;16:969985
KEY WORDS • cardiac
ia • ischemia
muscle • contraction
• hypox-
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Myocardial contractile function during ischemia and hypoxia.
D G Allen and C H Orchard
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Circ Res. 1987;60:153-168
doi: 10.1161/01.RES.60.2.153
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