DEPRESSANT EFFECTS OF ISCHEMIC BLOOD/Downar, Janse, Durrer
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25.
Catheterization of the heart in man with the use of a flow-directed
balloon-tipped catheter. N Engl J Med 283: 447, 1970
Haldane JS, Priestly JB: The regulation of the lung-ventilation. J Physiol
(Lond) 32: 225, 1904
Campbell EJ, Howell JB: Simple rapid methods of estimating arterial
and mixed venous Pco2. Br Med J 1: 458, 1960
Collier CR: Determination of mixed venous CO2 tensions by rebreathing.
J Appl Physiol 9: 25, 1956
Comroe JH, Forster RE, Dubois AB, Briscoe WA, Carlsen E: The Lung.
Chicago, Year Book Medical Publishers, 1955, pp 104-106
McHardy G: The relationship between the differences in pressure and
content of carbon dioxide in arterial and venous blood. Clin Sci 32: 299,
1967
Killip T, Kimball JT: Treatment of myocardial infarction in a coronary
care unit. Am J Cardiol 20: 457, 1967
Zeidfard E, Silverman M, Godfrey S: Reproducibility of indirect (CO2)
Fick method for calculation of cardiac output. J Appl Physiol 33: 141,
1972
Sharp JT, Griffith GT, Bunnell IL, Greene DG: Ventilatory mechanics in
pulmonary edema in man. J Clin Invest 37: 111, 1958
455
26. Woodward E, Burchell HB, Wood E: Dilution curves associated with
valvular regurgitation. Mayo Clin Proc 32: 518, 1957
27. Levinson GE, Carleton RA, Abelmann WH: Assessment of mitral
regurgitation by indicator dilution: An analysis of the determinants of the
abnormal dilution curve. Am Heart J 58: 873, 1959
28. Smith BC: Measurement of valvular insufficiency. In Dye Curves: The
Theory and Practice of Indicator Dilution, edited by Bloomfield D.
Baltimore, University Park Press, 1974, ch 9
29. Larson CP, Severinghaus JW: Postural variations in dead space and CO2
gradients breathing air and 02. J Appl Physiol 17: 417, 1962
30. West JB: Regional differences in gas exchange in the lung of erect man. J
Appl Physiol 17: 893, 1962
31. Godfrey S, Wolf E: An evaluation of rebreathing methods for measuring
mixed venous Pc02 during exercise. Clin Sci 42: 345, 1972
32. Asmussen E, Nielsen M: Physiological dead space and alveolar gas
pressures at rest and during muscular exercise. Acta Physiol Scand 38: 1,
1956
33. Lundin G, Thomson D: Mixed venous CO2 tension determined by a CO2
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63: 55, 1965
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The Effect of "Ischemic" Blood
on Transmembrane Potentials
of Normal Porcine Ventricular Myocardium
EUGENE DOWNAR, M.D., MICHIEL J. JANSE, M.D.,
AND DIRK DURRER, M.D.
SUMMARY "Ischemic" blood was obtained in pigs from a local
coronary vein on release of coronary artery occlusion. The effects of
this blood on transmembrane potentials of muscle strips taken from
the same heart were compared with control blood. Whereas action
potentials remained stable in control blood, ischemic blood collected
after more than 15 minutes of coronary occlusion produced shortening of action potential duration, reduction of resting potential, upstroke velocity and amplitude, then postrepolarization refractoriness
and finally unresponsiveness. Ischemic blood collected after shorter
periods of coronary occlusion produced only mild effects (shortening
of action potential and postrepolarization refractoriness).
These effects of ischemic blood could not be attributed to increased
potassium concentration even in combination with acidosis, hypoxia
and hypoglycemia. It appears that during ischemia unidentified factors are released which have potent depressant effects on the excitability of even normal myocardium.
THERE HAVE BEEN MANY REPORTS on the nature
of biochemical changes in coronary venous blood which occur in response to myocardial ischemia.5-1 Changes in coronary venous potassium levels and their possible relationship
to ventricular arrhythmias have attracted particular attention5-1 since Harris first implicated potassium as a major excitant of ventricular arrhythmias in myocardial ischemia.12
Despite such interest, both the mode of onset of early ventricular arrhythmias and their relationship to altered biochemical parameters remain matters for conjecture. This
situation exists in part because there is no satisfactory model
which enables study of ischemic changes in the intracellular
electrophysiology of ventricular myocardium.
In an attempt to gain insight into the possible nature of
such intracellular changes and their relationship to some of
the altered biochemical parameters produced by ischemia,
we examined the effects of coronary venous blood draining
an ischemic region on action potentials recorded from an
isolated strip of normal ventricular myocardium.
From the Department of Cardiology and Clinical Physiology, University
Hospital Wilhelmina Gasthuis, Amsterdam and the Interuniversity Cardiology Institute, The Netherlands.
Dr. Downar is a Senior Research Fellow of the Ontario Heart Foundation,
Canada. His present address is Cardiovascular Unit, Toronto General
Hospital, Toronto, Canada.
Received May 27, 1976; revision accepted October 14, 1976.
Methods
Thirty-two pigs weighing 20 to 25 kg were anesthetized by
intravenous injection of sodium pentobarbital (20 mg/kg).
Under artificial respiration the chest was opened by a midsternal incision and a pericardial cradle was constructed. A
full-thickness piece of right ventricular wall from the outflow tract region was removed and the defect closed by a
purse-string suture. The preparation was trimmed to about
0.5 cm2 in area and 3 mm thickness. It was mounted endocardial side up in a 2 ml tissue bath superfused with running
oxygenated modified Tyrode solution at a rate of 2 ml/min.
The composition of the solution, in mEq/L, was: Na+ 156.5,
K+ 4.7, Ca++ 1.5, Mg++ 0.7, H2PO0 0.5, HCO3 28.0, Cl137.0, and glucose 20 mM/L. Temperature was maintained
constant at 37°C ± 0.5°. The preparation was stimulated
via a bipolar silver electrode, insulated except at the tips, at
a basic cycle length of 700 msec, using rectangular current
pulses with a duration of 2 msec and an intensity of
456
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2 X diastolic threshold strength. Premature stimuli of
4 X diastolic threshold strength could be applied after every
eighth basic stimulus, at any desired interval with msec
precision. Transmembrane potentials were recorded using
conventional glass microelectrodes having a resistance of 10
to 20 megohm, and high impedance capacity-compensated
amplifiers. The preparation was kept in running Tyrode
solution for 30 minutes of equilibration before the next stage
of the procedure. Meanwhile, the distal part of the great cardiac vein was cannulated with an 18 G polyethylene catheter.
Ligatures were placed around the left anterior descending
coronary artery, distal to the first diagonal branch. A 2 ml
sample of coronary venous blood was taken from the
catheter, using a heparinized syringe, and was introduced
into the tissue bath after removal of the Tyrode solution. In
some cases a 4 ml sample was collected, half of which was
used for biochemical analysis. The blood remained stagnant
in the bath up to 20 minutes. To prevent evaporation, the
surface was covered with a thin layer of paraffin oil. Action
potentials remained stable within the first 15 minutes in incubation. Thereafter some shortening of action potential
duration occurred, while the general configuration remained
essentially unchanged. For these reasons, control action
potentials were recorded in the first 12 minutes of incubation. Having established that action potentials from a given
preparation remained stable during the control period, and
after determining the refractory period duration with
premature test stimuli, the blood was removed and the
preparation maintained in running Tyrode solution for at
least 15 minutes. During this period, coronary artery occlusion was applied for 5 to 40 minutes. After release of coronary artery occlusion, the first 2 ml of coronary venous
blood which appeared were collected for the tissue bath. The
first 4 ml were collected when biochemical analysis was
planned. The time taken to collect these samples varied from
5 to 15 seconds, taking less when there was some residual
flow from the coronary venous catheter during the period of
occlusion.
Biochemical analysis included the determination of
potassium and sodium concentrations by flame photometry,
calcium and magnesium concentrations titrometrically with
EDTA, glucose by 0-toluidine method, lactic acid concentrations spectrophotometrically, pH with a glass electrode
(Astrup), PO2 polarographically with a Clarke type electrode and pCO2 tonometrically. On several occasions the occurrence of ventricular fibrillation either during the period of
coronary artery occlusion or on release necessitated defibrillation. In these instances no venous samples were taken
until control circumstances were again established. Action
potentials were recorded during the first 12 minutes of incubation in this blood, which we will refer to as "ischemic"
blood. After this period of observation, the ischemic blood
was removed and in most cases used for potassium determinations. The tissue was again superfused with running
Tyrode solution until action potentials returned to control
configuration. The preparation could then be used for
further observations either during incubation in a second
sample of ischemic blood, or in artificially abnormalized
coronary venous blood for comparison. Normal coronary
venous blood was abnormalized to simulate ischemic blood
by raising the potassium concentration, adding lactic acid to
produce acidosis and equilibrating with nitrogen to produce
hypoxia.
VOL 55, No 3, MARCH 1977
CIRCULATION
I( 5.3
pH 6.73
glucose 2.3
pO2 34
mO-
0
mV50L
9'
control
0-
71
11_
FIGURE 1. Transmembrane potentials from the endocardial surface of an isolated muscle strip takenfrom the right ventricular outflow tract of a pig heart. The figures show the changes which occur
during the first 12 minutes of incubation in ischemic blood. Within
12 minutes of incubation, the preparation became unresponsive to
stimulation, even when using currents of 20 mA. Potassium and
glucose (mM/L), pH and pO2 in mm Hg of ischemic blood are indicated.
Results
Effects on Action Potential Configuration and Refractory Period
Typical changes in action potential configuration pro-
duced by ischemic blood are shown in figure 1. These
changes initially included a gradual shortening of action
potential duration, a loss of upstroke velocity and the
appearance of a notch in the upstroke suggesting the separation of the action potential into two components. Finally,
only a small local response is left. Throughout these changes
there is a gradual loss of resting membrane potential, until
at levels of about -50 mV the tissue becomes virtually unresponsive. Unresponsiveness developed by a fairly gradual
diminution of the action potential as described, or by a
period of alternation of action potential amplitude leading
to 2:1 responses to the basic stimulation which then faded
out (fig. 2 top). At this stage large amplitude responses could
be re-evoked for one or two beats by an increase in stimulation strength. Within a few beats, however, thresholds rose
=
Ii
ImA
1- 3
3 mA
~Ii
6 mA
mA .\.E._ .t
3-6 mA
FIGURE 2. Separation of action potential into two components
during incubation of preparation in ischemic blood (two different
experiments). Top panel) Alternation in amplitude followed by 2:1
responses in which the second component is absent every second
beat. Lower panel) Transient re-establishment of large amplitude
responses by increasing the stimulating current from I to 3 mA and
from 3 to 6 mA. Basic cycle length of stimulation in both experiments is 700 msec.
DEPRESSANT EFFECTS OF ISCHEMIC BLOOD/Downar, Janse, Durrer
457
[K+] 5.9
mV50[
X
,j
control
Si S2
ischemic blood
mnec
200
[KI
7.4
300,
0
260
,<
6min
SIS2 10
220
0
I"I X- -* action potential
duration
180
efactory period
_
.-J
-j
8 min
S1S2 170
140I
100
L
8/Y2min
S1S2
380
'Kx--K
, a
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1 2 3 4 5 0
7'
8 9
min
FIGURE 3. Postrepolarization refractoriness (two different experiments). Left panel) Basic and earliest induced
potentials. In control blood, time course of recovery of excitability closely follows time course of
repolarization (S1S2 200 msec). After six minutes of incubation in ischemic blood the action potential has shortened and
the shortest successful S1S2 is 160 msec. After eight minutes, the test pulse given after 170 msec only gives rise to a small
response, and after eight and one-half minutes the refractory period lags considerably behind completion of repolarization. Right panel) Graph from another experiment indicating the changes in action potential duration and refractory
period (ordinate) during incubation in an ischemic blood sample (abscissa).
premature action
rapidly, leading finally to inexcitability (fig. 2 bottom).
Removal of the ischemic blood followed by superfusion of
the preparation with flowing Tyrode's solution always
restored action potentials to control configuration. Five
minutes of superfusion was usually sufficient to reverse unresponsiveness. On a few occasions a longer period was
necessary to restore normal action potentials, but this never
exceeded 20 minutes.
Estimates of refractory period duration were made
throughout the period of incubation by using test stimuli of
4 X diastolic threshold intensity, applied within 2 to 3 mm
from the recording site after every eighth basic stimulus, and
determining the shortest interval at which the test stimulus
elicited a response. In the initial stages of incubation with
ischemic blood, the time course of recovery of excitability
closely followed repolarization, and the refractory period
shortened commensurate with the action potential duration.
Subsequently, however, despite continued shortening of action potential duration, the refractory period began to
prolong, resulting in the development of postrepolarization
refractoriness (fig. 3). The refractory period in some instances exceeded the control value and went on to be hundreds of msec longer than the action potential, even encroaching on the next basic stimulus at cycle lengths of 700
msec. In these final stages, the exact determination of refractory period duration became impossible because diastolic
threshold level began rising rapidly to high values. As these
changes in excitability developed there was a partial separation of the action potential into two components. This
separation was exacerbated by the application of premature
stimuli. As can be seen in figure 4, the premature stimulus
evoked only a relatively fast, short spike. Its effect on the
next basic response, however, was to prevent the occurrence
of the second component, which only reappeared in the sec-
ond basic stimulus. In other instances, a similar premature
beat could prevent a response to the next basic stimulus
altogether (fig. 4 bottom).
The Effect of Duration of Ischemia
The intensity of the effects described above could be
related to the duration of the ischemic period. As shown in
figure 5, ischemic blood collected after a period of occlusion
of five minutes only produced shortening of the action potential and some degree of postrepolarization refractoriness.
Similar effects were seen when blood was taken from the
A
L)
w
~
~
~
LK
,V_
B
control
SiS2 155
5'isch. bl.
S1S2
170
6' isch. bi.
S1S2 200
_
FIGURE 4. Effects of induced premature beats on subsequent basic
beats in experiments A and B. Basic cycle length 700 msec. In A,
after 10 minutes of incubation in ischemic blood, test stimulus
evokes only a short, relatively fast spike, as does the next basic
stimulus. The second component reappears only at the second basic
stimulus. In B, aftersix minutes of incubation in ischemic blood, the
test pulse results in a very small response and prevents the response
to the next basic stimulus altogether.
VOL 55, No 3, MARCH 1977
CIRCULATION
458
control
S1iS2 229
.0
.
-W.
5min occl.
S1-S2 215
I
10
min
occl-
(15 min)
(15 min)
(10
~Sl- S2 240
-
15
min occl.
5~1-
min)
(7Y2 min)
310
2
~~~30 min
occl.
(9
min)
S1- S2 350
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
FIGURE 5. Effects of duration of coronary artery occlusion. Action potentials and earliest induced premature responses during immersion of preparation in control blood and in ischemic blood
collected after different periods of coronary artery occlusion.
Numbers in brackets indicate minutes of incubation in the different
blood samples. Ischemic blood collected after five and ten minutes
of occlusion causes mild effects (shortening of action potential and
postrepolarization refractoriness). Potent depressant effects are
seen in blood collected after occlusion of 15 minutes and longer.
vein after 10 minutes of coronary artery occlusion,
although these occurred after a shorter period of incubation.
The preparation became virtually unresponsive after 7½/2
minutes of incubation in blood collected after a 15 minute
period of occlusion, and the same effects were produced by
same
blood sampled after 30 minutes of ischemia. Longer periods
of up to one hour of occlusion did not produce more marked
effects, suggesting that the factors responsible were released
in the first 15 to 30 minutes of ischemia. Periods of occlusion
of five minutes or so usually produced only the mild effects
shown in figure 5. On occasion, however, five minutes of
coronary artery occlusion produced more marked effects
resulting in very short, low amplitude responses.
The depressant effect of ischemic blood was not confined
to the first few milliliters to emerge on release of occlusion.
In several instances a second sample of ischemic blood was
collected two minutes after release, during the hyperemic
phase, and compared with the initial sample. Such
hyperemic samples were able to produce the same effects as
the ischemic samples, but took longer.
Altered Biochemical Parameters
Table 1 shows the results of biochemical determinations
in 29 ischemic blood samples, all of which produced unresponsiveness. The numbers in brackets indicate corresponding values of nonischemic coronary venous blood. Because
of the limited quantity available of each sample, it was not
possible to determine more than a few parameters in each
sample. Potassium was determined in every case because a
sufficiently high concentration could account for the observed effects. Using Tyrode solutions with different potassium concentrations, we found that [K+] had to be raised to
14 to 15 mEq/L in order to produce unresponsiveness in a
similar way to that of ischemic blood. Figure 6 shows in
histogram form the potassium concentrations, and where
available the corresponding pH of ischemic samples which
led to unresponsiveness and those which produced mild
TABLE 1. Results of Biochemical Determinations in 29 Ischemic Blood Samples
K+
Expt
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
(mM/L)
8.6
7.1
7.5
16.2
6.5
7.2
8.1
10.4
9.2
5.6
8.3
6.6
8.0
8.6
7.4
13.0
7.5
9.7
7.8
7.1
12.2
6.9
4.6
5.3
8.1
4.8
5.0
7.0
Glucose
(mM/L)
pH
pO2
(mm Hg)
PC02
(mm Hg)
Ca++
(mM/L)
Mg++
(mM/L)
Miscellaneous
7.25
7.60
7.02
7.39
6.83
1.8 (2.2)
1.2 (1.09)
1.3 (1.09)
(4.7)
(3.6)
(4.0)
(3.7)
(4.4)
(4.4)
(4.4)
(4.0)
(4.0)
(4.0)
4.3
4.1
5.4
1.9
(6.3)
2.2
3.1
2.3
1.4
4.0
3.5
2.4
(5.3)
(4.4)
(4.4)
(4.4)
(4.2)
(4.2)
(4.2)
(5.0)
(6.4)
(5.5)
2.8 (5.3)
7.16 (7.55)
6.89 (7.55)
6.85 (7.55)
17 (38)
22 (38)
24 (38)
7.33 (7.69)
24 (21)
7.04 (7.50)
6.70 (7.54)
6.86 (7.54)
7.18 (7.46)
6.73 (7.46)
6.93 (6.47)
7.26 (7.48)
7.34 (7.48)
6.90 (7.48)
29 (29)
18 (29)
22 (22)
34 (22)
27 (22)
20 (21)
32 (27)
44 (27)
1.82 (1.7)
1.65 (1.7)
1.69 (1.69)
1.69 (1.69)
1.86 (1.69)
130 (29)
88 (29)
60 (38)
127 (38)
88 (38)
a4 (32)
32 (32)
105 (32)
0.73
0.82
0.65
0.70
0.68
(0.68)
(0.68)
(0.64)
(0.64)
(0.64)
lactate 3.7 (1.99) mM/L
lactate 4.64 (1.99)
lactate 4.88 (1.99)
DEPRESSANT EFFECTS OF ISCHEMIC BLOOD/Downar, Janse, Durrer
5.
ire 6, the majority of ischemic samples which
iild effects had potassium levels between 5 and 7
whereas samples which produced unresponsiveness
uently had K+ concentrations between 7 and 9
Although there was a trend for ischemic blood
o produce the most marked effects at higher K+
.tions, the fact that unresponsiveness was produced
ncentrations of about 7 mEq/L, and on occasion
wvels within the limits of normal, suggested that
was not the only factor responsible for the
effects.
nsidered whether a combination of a moderately
potassium concentration with alterations in other
rs might account for the effect of ischemic blood.
in table 1 most ischemic samples had a low pH,
the scatter was wide and the values ranged from
.60. In all cases where control pH was measured
ted alkalosis in combination with a low pCO2, inhat the animal was in respiratory alkalosis due to
lation. There was no definite trend for the lowest
)e associated with the lowest K+ concentrations
.01
7.22
4-
mild effects
686 696
668
3-
2-
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
6.93
1-
3
4
I~~~
6
1
16
9
8
1'1
1'2
II
11m 7
113 14 15 [6
[K+] mEq/L
FIGURE 6. Histograms indicating K+ concentrations of ischemic
blood samples (abscissa) causing mild effects (action potential
shortening and postrepolarization refractoriness) and unresponsiveness during first 12 minutes of incubation of the preparation in
the sample. Where available, pH of ischemic sample is indicated in
the appropriate /K+J range. Ordinate: number of experiments.
effects. The mild effects were defined as shortening of action
potential duration, and the development of postrepolarization refractoriness but no terminal unresponsiveness, or the
development of double components within the 12 minutes of
incubation.
Nonischemic coronary venous samples had potassium
concentrations between 3 and 5 mEq/L. As can be seen
nples had a low p02 (range 17 to 44) and often a
)2 (range 32 to 130). Ca++ and Mg++ concenvere not significantly altered in ischemic blood.
glucose concentrations tended to be low (range
r to evaluate the effect of low glucose levels, 5 mM
was added to three ischemic blood samples which
iced unresponsiveness (experiments 27, 28 and 29).
Ecient time had elapsed for the preparation to
uring superfusion with Tyrode solution from the inin the original ischemic blood sample, the high
amples were tested. These samples, in which gluoentration was now 8.3, 7.8 and 8.3 mM/L, rer, still produced unresponsiveness in exactly the
and after the same period of incubation as did the
,ample.
ABNORMAL
ISCHEMIC BLOOD
10' occlusion
control -
.
12'V--
6'L
P\-
I
t3mA
[K] 7.5 meq/l
BLOOD
control
K
5
8
459
t 6mA
J
>
r\
v
t4O pA
K* 8.2 meq/l
pH 6.83
P02 32 mm Hg
FIGURE 7. Effects of ischemic blood and abnormal blood with similar K+ concentration, pH and PO2. The abnormal
blood causes action potential shortening and postrepolarization refractoriness but there is no increase in stimulus requirement, no separation of the action potential into two components and no unresponsiveness as in the case of ischemic blood.
Numbers indicate minutes of incubation of the preparation in the blood sample.
CI RCULATION
460
In order to test whether a combination of elevated K+,
low pH and low pO2 could account for the effects of ischemic
blood, normal coronary venous blood was rendered abnormal in six experiments by raising lactic acid to a concentration of about 25 mM/L in order to produce a pH of about
6.9, and by equilibrating with a gas mixture of N2 (1400 ml),
CO2 (1 10 ml) and air +5% CO2 (350 ml) to achieve a pO2 of
about 25 mm Hg and a CO2 of about 45 mm Hg. The
potassium concentration in these blood samples was raised
to three different levels: about 8, 12 and 16 mEq/L. The abnormal blood with a K+ concentration of about 8 mEq/L
did not produce the effect of ischemic blood with a similar
K+ level (fig. 7).
Abnormal samples with K+ concentrations of 12.9 and
13.0 mEq/L only produced mild effects, whereas samples
with K+ levels of 13.6, 15.8 and 16.0 mEq/L produced unresponsiveness. Thus, the effects of ischemic blood cannot be
ascribed to even the combined effects of acidosis, hypoxia,
elevated lactate and hyperkalemia in ischemic blood.
Downloaded from http://circ.ahajournals.org/ by guest on June 15, 2017
Discussion
Our findings demonstrate that coronary venous blood
draining an ischemic area can produce striking electrophysiologic changes in healthy myocardium. These changes
range in severity from shortening of action potential duration, the development of postrepolarization refractoriness,
loss of upstroke velocity, loss of resting potential, the fractionation of the action potential into two components, increase in diastolic threshold and finally total unresponsiveness.
All of the effects observed could be caused by a sufficiently
elevated extracellular potassium concentration, as was
shown by the experiments with Tyrode solutions containing
16 mEq/L of K+. In a perfused preparation of the pig's
moderator band, Gettes and Surawicz" reported that
potassium concentration of 10 to 12 mEq/L produced in
both muscle and Purkinje cells shortening of action potential
duration and refractory period, and decrease of its
magnitude and maximal diastolic potential. However, at
these K+ levels, their preparations were far from being unresponsive. K+ concentrations in the ischemic blood samples
were usually 7 to 8 mEq/L. In our experiments such K+ concentrations in Tyrode or blood produce only mild changes
such as action potential shortening, slight loss of dV/dt and
some degree of postrepolarization refractoriness.
The possibility of this mild degree of hyperkalemia having
potent depressant effects when combined with acidosis,
hypoxia and hypoglycemia was considered. Each of these
derangements alone has significant effects on the action
potentials of in vitro heart preparations. Acidosis has been
reported to lengthen action potentials in Purkinje fibers,14
frog ventricular strips'5 and rabbit atria."6 It also lengthened
ventricular action potentials in perfused guinea pig hearts
but shortened ventricular action potentials in perfused rat
hearts." Using CO2-induced acidosis, Coraboeuf et al.'8
produced depolarization in some canine Purkinje fibers and
repetitive humps in the repolarization phase. We do not feel
that acidosis played an important role in producing the
ischemic effect for two reasons.
1) The depressant effect was seen once during alkalosis
VOL 55, No 3, MARCH 1977
and four times when there was little or no acidosis (pH 7.6,
7.34, 7.33, 7.26, 7.25) as a consequence of a respiratory
alkalosis from overventilating the animals.
2) Our abnormal bloods with a lactic acid-induced pH of
6.9 failed to have even a mild ischemic effect.
Anoxia has long been known to reduce action potential
duration and amplitude'9 but it has little effect on the resting
membrane potential.20 Our ischemic blood samples had a
major effect on the resting potential and in seven of 12 instances had a PO2 at least as high as the control coronary
venous blood which had no depressant effects. The effects of
hypoxia on action potential configuration can be prevented
by high glucose concentrations and can be exaggerated in
glucose-free solutions.2" 22 Although glucose levels in
ischemic blood were always lower than the controls, the
reduction was not always very marked and the depressant
effects could not be prevented by elevating glucose above
control values.
It is possible that the ischemic blood induced abnormalities in ATP production which resulted in suppression of
electrogenic pumps. The electrogenic sodium pump maintains the resting potential of anoxic ventricular muscle at a
more negative level than can be accounted for by the ionic
distribution of potassium across the myocardial membrane.23 Inhibition of the sodium pump by cooling and ouabain results in a reduction in the resting potential to levels
predicted by the potassium gradient. Ischemic blood, unlike
anoxia, rapidly produced severe depolarization at [K+]
levels that were usually only slightly elevated above normal
(on thret occasions it even remained within the upper limit
of normal). It seems unlikely therefore that suppression of
an electrogenic pump could account for one of the main
features of the ischemic blood effect. Reduced ATP levels
may have been responsible in part for the observed shortening in action potential duration.2"
In view of the fact that the effects of ischemic blood cannot be accounted for by the combination of hyperkalemia,
acidosis, hypoxia and hypoglycemia, the possibility arises
that an additional factor or factors are present in ischemic
blood, that also have depressant effects on the electrical
behavior of normal ventricular myocardium. As yet we have
no indication as to which of the many metabolites released
by ischemic myocardium may be implicated, alone or in
combination.
The appearance of two-component action potentials in the
ischemic blood may have been due to a true separation of
fast and slow components as a result of depolarization. If so,
the slow component was the first to be inhibited at a time
when the remnant of the faster component was able to persist for some considerable time longer. However, at a stage
prior to total unresponsiveness, it was possible to evoke the
second component by either increasing the stimulus strength
or introducing a sufficiently long diastolic pause. Also, the
amplitude of this second component varied with stimulus
strength. These are characteristics of the secondary depolarizations, described by Carmeliet and Vereecke25 and the
slow responses of Cranefield et al.26 observed in partially depolarized Purkinje fibers under the influence of epinephrine.
We were unable for technical reasons to measure catecholamine levels of our ischemic blood samples. If they were
sufficiently elevated then the second slower component that
we observed prior to the onset of unresponsiveness should
DEPRESSANT EFFECTS OF ISCHEMIC BLOOD/Downar, Janse, Durrer
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have persisted at the terminal resting potential of about -55
mV. This did not occur and we never saw the spontaneous
emergence of typical slow responses after the tissue had
become inexcitable. The addition of epinephrine and calcium gluconate on the one occasion it was tried did evoke
such responses with a 35 mV positive overshoot - a feature
we never saw as part of the ischemic blood effect. In considering the nature of the two components of these action
potentials, however, it must be stated that we cannot exclude
as a cause electrotonic interaction between tissue close to the
stimulating electrode and more remote tissue at a time when
regenerative conduction was at the point of failing. However, as far as it was possible to tell by using a second exploring microelectrode, the changes observed were representative of the electrical activity throughout the small piece of
tissue. It is well known that refractory period duration is
shortened in ischemic heart muscle.27 More recently,
however, it has been reported that although refractory
period duration shortens in the initial phase of myocardial
ischemia, it lengthens again in later stages.28 In Purkinje
fibers, isolated some hours after coronary occlusion, El
Sherif et al." found that the recovery of excitability outlasted action potential duration, and in later papers the
phrase "postrepolarization refractoriness" was used to
describe this phenomenon in the ischemic canine His
bundle.30 The very marked postrepolarization refractoriness
observed by us in healthy myocardium exposed to ischemic
blood may simply be secondary to loss of resting potential,
which is known to retard recovery from inactivation of the
fast current;3' or it may be an expression of structural
derangement of the cell membrane. In favor of the first
possibility is the fact the postrepolarization refractoriness
could be evoked by superfusion with Tyrode containing 15
mEq/L of K+.
If our observations on the occurrence of postrepolarization refractoriness can be applied to ischemic myocardium in the intact heart, they have important implications.
First, estimates of refractory period duration based upon
measurements of Q-T intervals of local electrograms or action potential duration may be grossly misleading. Second,
the extent of heterogeneity of recovery times, a key determinant of ventricular fibrillation threshold, could be greater
than previously supposed.32 It is likely that at a certain
critical stage of ischemia recovery times range from the abnormally short values of mildly ischemic border cells to
those more ischemic cells on the verge of unresponsiveness
that require the entire diastolic interval. The vulnerable
period would indeed extend throughout diastole as has
recently been suggested.33
The K+ concentration that we found in ischemic blood
was higher than reported in the literature. Values found by
Harris et al.12 did not exceed 25 mg % (6.4 mEq/L).
The highest value found by Thomas et al.9 in the local coronary vein draining an ischemic area in the dog's heart was
6 mEq/L. However, these values were found during coronary occlusion, indicating that there was still venous flow
during the occlusion, presumably because of collateral flow
or because the vein also was draining normally perfused
myocardium. This implies that the actual interstitial K+
level might have been higher. In order to get a better reflection of ischemic interstitial fluid, we required that there
should be little or no local venous flow during the period of
461
coronary artery occlusion - a requirement met more reliably in porcine than canine hearts, as we found in preliminary experiments.
We attempted to standardize ischemic samples by collecting the first 2 ml to appear on release of coronary occlusion. However, since it took a variable time to collect these 2
ml, an inevitable and variable dilution factor probably was
present. To some extent this may have accounted for the
observed variations in K+ concentrations and potency of the
depressant properties of the ischemic blood. In this regard it
is of interest to emphasize that blood collected two minutes
after release of occlusion still had potent depressant properties, albeit to a lesser extent than blood collected immediately on release. This is also a further argument that the
effects were not solely determined by K+ since K+ in the
hyperemic phase was only slightly above control values.
Some questions may be raised about the fact that our
observations were made on tissue immersed in stagnant
blood. It should be stressed that before each observation in
ischemic blood, the preparation was monitored in stagnant
normal coronary venous blood and could on every occasion
be maintained in a stable condition;as far as action potential
configuration and excitability were concerned for a period of
at least 15 minutes. The effects of ischemic blood took
variable periods of time to develop; sometimes a maximal
effect was seen after two minutes of incubation, other times
it took 12 to 15 minutes. In order to stay within safe limits,
our observations were always limited to the first 12 minutes
of incubation. A second factor which might have influenced
the time course of events was the depth of the cell from
which recordings were made. We always tried to record
from the most superficial cells to minimize differences due to
concentration gradients. Most of our observations were on
the subendocardial layers of the right ventricular outflow
tract. This introduces some uncertainty as to the exact
morphological nature of the cells impaled. However, on a
number of occasions the corresponding epicardial surface
was used and the same phenomena were observed. Even if
some of the cells impaled were subendocardial Purkinje
fibers, their response to ischemic blood did not differ
qualitatively from that of ventricular myocardial cells.
In conclusion, ischemic myocardium releases agents
which have a potent depressant effect on the electrical activity of normal myocardium. Although potassium contributes to these effects, it cannot completely account for
them, not even in combination with acidosis, hypoxia,
hypoglycemia and elevated lactate. The nature of the other
depressing agents remains to be determined as well as the
way in which they mediate their effects.
The extent to which our observations reflect the actual
changes which occur during ischemia in the intact heart may
well be questioned. However, recently we have been able to
record action potentials directly from ischemic regions of
the intact heart and have observed precisely similar
changes.34
The fact that our observations were made on normal myocardium raised the interesting possibility of the existence of
nonischemic cells on the periphery of the ischemic area,
which functionally behave as though they were ischemic.
Such cells would respond not only to changes in perfusion
but also to changes in the pattern of coronary venous
drainage.
462
CIRCULATION
Acknowledgment
The authors wish to thank Sara Tasseron and Dr. J. W. T. Fiolet for invaluable technical assistance.
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The effect of "ischemic" blood on transmembrane potentials of normal porcine
ventricular myocardium.
E Downar, M J Janse and D Durrer
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Circulation. 1977;55:455-462
doi: 10.1161/01.CIR.55.3.455
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