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Contractility, Metabolism and Pharmacological
Reactions of Isolated Gas-Perfused Cat Hearts
By Lloyd P. Gobel, Ivan Bihler, and Peter E. Dresel
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ABSTRACT
Cat hearts were perfused through the aorta with substrate-free Krebs solution
for 5 min, then with 5% CO2-95% O 3 saturated with water vapor at 37.5°C.
Heart rate decreased rapidly but contractility was maintained at pre-gas levels
for 3 to 4 hours, decreasing to 25% at 10 hours. Driving the preparations at 168
beats/min did not appreciably change the time course of change in contractility.
The hearts developed more tension at all resting tensions and were able to perform more work than hearts perfused with substrate-free Krebs solution, but their
work capacity was less than that of hearts perfused with glucose-Krebs solution.
They reacted normally to sympathomimetic amines and blocking agents. They
were very sensitive to increases in perfusion pressure, developing completely reversible arrhythmias at pressures 80 to 150$ above the control (60 mm Hg).
Most hearts developed contractile altemans which usually was not accompanied
by electrical alternation. Glycogen, lactate, pyruvate content decreased rapidly,
the first to a residual level and the others to undetectable levels.
ADDITIONAL KEY WORDS
glycogen loss
heart rate
mechanical alternation
• As early as 1902, Magnus (1) maintained
a beating cat heart for 1 hour while perfusing it with gaseous oxygen. Burns et al. (2)
repeated this experiment and described the
relationship between the perfusing pressure
and the flow of gas through the coronary arteries. Sabiston et al. (3) and Talbert et al.
(4) described a technique for gas perfusion
with which electrical, but not mechanical,
activity could be maintained for periods approaching 8 hours.
The liquid media used to perfuse the isoFrom the Department of Pharmacology and Therapeutics, University of Manitoba, Faculty of Medicine,
Winnipeg, Manitoba, Canada.
This work was aided by grants from the Medical
Research Council of Canada, the American Heart
Association and the Muscular Dystrophy Association
of Canada.
A preliminary report was presented to the Fed. Am.
Soc. Expd. Biol. in April 1965. (Federation Proc.
24: 365, 1965.)
Dr. Gabel's work was supported by a U. S. Public
Health Service Training Grant (T.I. CM 1054) from
the Division of General Medical Sciences. His present
address is Wamer-Lambert Research Institute, Morris
Plains, New Jersey.
Dr. Bihler is a Medical Research Associate of the
Medical Research Council of Canada.
Accepted for publication September 16, 1966.
CircuUtion Rtinrcb.
Vol. XIX. Novembtr
1966
prolonged survival
Starling relationship
pressure arrhythmias
lated heart perform several functions:
(A) maintenance of a constant temperature
and ionic environment, (B) supply of oxygen,
(C) supply of energy-yielding substrates, and
(D) removal of metabolic end products. Perfusion with a gaseous medium may maintain
a constant temperature and a supply of oxygen, but is obviously incapable of supplying
or removing metabolites other than CO2.
It is well documented (5-7) that the isolated heart fails rapidly when perfused with
a substrate-free liquid medium. This paper
describes a procedure for gas perfusion of
the isolated cat heart which allows contractility
to remain stable for periods exceeding 3 hours,
and it compares gas- and liquid-perfused
hearts with respect to contractility, performance of work and response to pharmacological agents.
Methods
PERFUSION SYSTEM
We were unable to maintain contractility using
the procedures of Burns et al. (2) or of Sabiston et al. (3) primarily because of difficulties in
regulating the temperature of the gaseous perfusate. An apparatus was constructed which permitted both close temperature control of the
891
892
GABEL, BIHLER, DRESEL
0LA3S
WOOL
HUMIDIFIER I
—
RESISTANCE
WIRE
no v
TEMPERATURE
CONTROL
83
TELETHERMOMETER
j
«v :
TRAP
1
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3
<
TT %
;
FAN
f
,
m
L 1
TRANSDUCER
—
:
, t
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FIGURE 1
Apparatus for gas and liquid perfusion. For explanation see text.
humidified gas mixture at various flow rates and
rapid switching from liquid to gas perfusion (Fig.
1). The heart was perfused by the Langendorff
technique from a cannula with four side arms
close to its tip. Two thermistor probes (YSI Models 402 and 403) extended directly into the perfusate flowing through side arm A. One was used
to monitor temperature, the other functioned in
conjunction with a temperature regulator (YSI
Model 63) which controlled the flow of current
through a heating coil placed around the glass
tube leading to the cannula. This coil served
only as a final temperature adjustment (see below) and maintained the temperature of both
liquid and gaseous perfusates at 37.5 ± 0.4°C.
During gas perfusion, side arm B was connected
by a stopcock (S-3) and a trap to a gas pressure
regulator (PR) constructed by immersing a glass
tube into a column of water to a depdi corresponding to 60 mm Hg. The trap collected liquid
perfusate contained in the system at the time of
change from liquid to gas perfusion. Side arm C
was connected to a Statham P 23D pressure
transducer (PT). A thin plastic tube used for
injection of drugs passed through side arm D and
terminated in the cannula close to its point of
insertion into the aorta.
Liquid perfusates of different composition were
stored in reservoirs placed level with the heart,
pressurized to 60 mm Hg with 95% O2 and 5%
CO2. The liquid passed through a water-jacketed
spiral condenser which served as a heat exchanger, and then through stopcocks S-l and S-2 before reaching the cannula. Stopcock S-l selected
either reservoir; stopcock S-2 selected either gas
or liquid perfusate.
The 95% O2 and 5% CO2 mixture for perfusion
was preheated in a water-jacketed condenser
and was then humidified in a water-jacketed gas
scrubbing bottle containing distilled water. It
then passed through glass wool which removed
water droplets and was led to stopcock S-2.
The heart was enclosed in a water-jacketed
Lucite box. High humidity was maintained in
the chamber by a fan which circulated air past
several vertical strips of moist filter paper. A funnel under the heart passed through the bottom
of the box for recovery of liquid perfusate. The
water circulating through all jacketed components
was supplied from a constant-temperature bath
at40.0±0.5°C.
In those experiments in which liquid perfusate
was used exclusively, the perfusion system was
changed to allow recirculation of the perfusate.
Our apparatus was similar to that of Bleehen
and Fisher (8) except that the perfusate was
recirculated by a pump instead of an air lift and
the pressure was supplied by the equilibrating
gas instead of gravity. The total volume of liquid
in our system was 200 to 250 ml.
Circulation Resurcb, Vol. XIX, Novtmbtr 1966
893
THE GAS-PERFUSED CAT HEART
PREPARATION OF THE HEART
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Kittens of either sex weighing 0.4 to 1.2 kg
were fed a diet of meat, fish and eggs ad libitum
for at least 2 days before use. The animals were
killed by a blow on the head and the hearts removed and placed in a beaker of cold Krebs-Henseleit-bicarbonate solution. Anticoagulants were
not used because of their possible effects on
tissue metabolism (9).
The hearts were cleaned of extraneous tissue
and a small slit was made in the walls of each
ventricle. The aorta was then cannulated and the
heart connected to the perfusion apparatus. A
5-min period of liquid perfusion preceded gas
perfusion in all experiments. Ten seconds of gas
perfusion was used to clear the vasculature before weighing hearts which had last been perfused with liquid. The liquid perfusate was substrate-free Krebs-Henseleit solution gassed with
95* O2 and 5% CO.,; it contained NaCl, 118.0
IDM; KC1, 4.7 mM; KH2PO4) 1.2 mM; CaCl2 •
2H.O, 2.5 mM; MgSO4, 2.4 mM and NaHCO,,,
26.6 mM. Glucose (2.0 g/liter) was added only
for the experiments in which work capacity was
determined.
MEASUREMENTS
Isometric contractility was measured with a
Grass FT-03 force-displacement transducer attached to a string sewn to the ventricular apex;
resting tension was defined as the diastolic tension exerted on the apex. Isotonic contractility
and external work were measured electronically
with an apparatus in which work was estimated
from the lifting of imposed weights attached to
the apex of the ventricle (10).
Heart rates were determined from the recordings of contractile force. Some hearts were driven
electrically through small clip electrodes placed
on the right atrial appendage and ventricular
apex. Suprathreshold rectangular pulses (4 to
8 v) of 5 msec duration were supplied by a
Grass SD-5 stimulator. Electrograms were obtained using electrodes placed in the same
positions as the stimulating electrodes. Recordings were made with a Grass polygraph.
Analytical Mcthodi
Only the ventricles, cut below the fat pad
surrounding the circumflex artery, were used for
analysis. They were blotted with tissue paper
and then dropped into liquid nitrogen. The frozen tissue was placed in steel test tubes previously cooled with liquid nitrogen and powdered
by percussion. The powder was mixed by stirring
and stored at — 20°C. Aliquots of tissue powder
were weighed rapidly and analyzed as follows:
Water content. This was calculated from the
weight loss after drying to constant weight at
80°C in vacuo. This value was used to calculate
CircmUiio* Restrcb, Vol. XIX, Novemttr 1966
the dry weight of aliquots used for other analyses.
Glycogen and total carbohydrate. Samples
were hydrolyzed in 501 NaOH and glycogen
precipitated with cold 80* methanol. Other
samples were homogenized in 5% trichloracetic
acid to extract total tissue carbohydrate. The
glycogen precipitate and the trichloroacetic acid
extract were analyzed spectrophotometrically by
the sulfuric acid method of Kemp and Kits Von
Hejningen (11). Results are expressed as glucose
equivalents.
Lactate and pyruvate. Cold 656 perchloric acid
extracts were analyzed by enzymatic methods
(12, 13). The reagents were obtained from
Sigma Chemical Co., St. Louis, Mo.
Results are expressed as micromoles per gram
dry weight. Data for each group of hearts are
expressed as means ± their standard errors. The
significance of differences between groups was
estimated by Student's t test.
DRUGS USED
Stock solutions of epinephrine bitartrate, norepinephrine bitartrate, isoproterenol hydrochloride, pronethalol hydrochloride and cocaine hydrochloride containing 10 mg/ml of the bases were
made in acidified 0.9% NaCl, and further dilutions made with the same vehicle on the day of
each experiment; 0.05 to 0.1 ml of the appropriate
dilution was made to 1 ml with Krebs-Henseleit solution immediately before administration.
In later experiments it was found that more reproducible results were obtained if the drugs
were injected in a volume of 5 ml.
Results
CONTRACTILITY AND HEART RATE
Isometric contractile force and heart rate
at a constant resting tension of 10 g were
measured in two groups of hearts. The first
was perfused for 2 hours with recirculating
substrate-free Krebs-Henseleit solution (6
hearts). The second group (4 hearts) was
perfused with gas for 10 hours after 5 min
of perfusion with liquid. Contractile force
and rate at the end of 5 min of liquid perfusion were taken as the initial (control) values in both groups.
Figures 2A and 2B show that the changes in
contractility and rate resulting from long periods of perfusion were strikingly different under the two conditions. Figure 2A shows
that the rate of liquid-perfused hearts declined throughout the 2-hour period of the
experiments, reaching 73 ± 6* of the initial
894
GABEL, BIHLER, DRESEL
value of 163 ± 8 beats/min at the end of the
experiments. The rates of gas-perfused hearts
fell rapidly during the first hour to a value of
43 ± 3* of initial but declined only slowly
during the remaining 9 hours. The contractile
force of liquid-perfused hearts declined continuously throughout the experiment (Fig.
2B). It was 50* of the initial value after 80
min and 10* of initial after 120 min. The contractility of gas-perfused hearts increased significantly during the first 20 min and remained
above the initial level for 100 min. No statistically significant fall below the initial level
was observed before the fourth hour of gas
perfusion. The contractile force continued to
decline slowly, reaching a value of 50* of initial at 7 hours and of 25* of initial after 10
hours, at which time the experiments were
terminated. A third group (4 hearts) was perfused with gas for 7 hours while being driven
at a rate (168 beats/min) close to the initial
rate of liquid-perfused hearts. The contractility
of these hearts was only slightly less than that
of the other gas-perfused group. The increase
in contractile force above the initial value,
of 75 min duration, was not statistically sig-
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HEART RATE
3
4
HOURS
3
6
PERFU90N
OF
10
PERCENT
OF
CONTROL
123'
CONTRACTILE
FORCE
IOO'
73'
OAS
SO-
25'
1M/MIN
3
4
HOURS
3
OF
6
10
PERFU9ON
FIGURE 2
Top, time course of change in heart rate during perfusion with gas and with substrate-free Krebs
solution. Bottom, time course of isometric contractile force; same hearts as above. Additional
group labeled 168 beats/min was driven electrically at that rate. Bars show SE; 4 hearts/group.
CircmUiion Research, Vol. XIX, Novunbtr
1966
THE GAS-PERFUSED CAT HEART
895
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nificant; 50* of control was reached after 5
hours and 25% of control after 7 hours.
In agreement with the observations of Cattell and Gold (14) and Luisada and Weiss
(15), we observed that hearts maintained on
liquid tended to shorten gradually throughout
the period of perfusion and it was necessary
to adjust the force-displacement transducer in
order to maintain the 10 g resting tension.
This did not occur in gas-perfused hearts.
When gas perfusion was started after 2 hours
of liquid perfusion, diastolic tension decreased
to the control within 3 min but contractile
force increased only 20*.
Contractions of alternate strength (contractile alternans) were observed in 90* of more
than 50 hearts perfused with gas. This usually
began during the first 15 min. The difference
between alternate contractions, very small initially, increased during the next 45 min and
then remained constant for the remainder of
the experiment. Contractility during alternans
is expressed as the arithmetic mean of the
alternating beats. The curve relating contractility to time for any 1 heart shows a slight
dip of less than 5 min duration immediately
after onset of contractile alternans, followed by
a continuing increase in average contractility.
This is not seen in the composite curve (Fig.
2B) because of variations in the time at which
alternation began.
Electrograms were obtained from 37 hearts
with contractile alternans. No electrical alternans was seen in 27 of these (73*). The
weaker beat in each of these hearts began
only after relaxation from the stronger beat
was complete (Fig. 3B). The weaker beat in
each of the 10 hearts which did show electrical
alternans began before relaxation was complete (Fig. 3A).
Many experimentally induced arrhythmias
are affected by changes in perfusion pressure (16, 17). We observed that increasing
the perfusion pressure from 60 to more than
100 mm Hg caused multifocal ventricular
arrhythmias which continued until the pressure was lowered (Fig. 4). In 6 gas-perfused
hearts the mean pressure threshold for the
initiation of the arrhythmia was 124 ± 9 mm
Hg, whereas it was 187 ± 12 mm Hg in 6
liquid-perfused hearts. The contractility and
electrical activity of the latter group did not
return to control conditions when the pressure was lowered. Gas-perfused hearts did
not show these irreversible changes. The pressure threshold for arrhythmia production could
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I SEC
FIGURE 3
Contractile alternans during gas perfusion. The upper tracing is an electrogram; lower tracing
shows isometric contractile force. In A, electrical alternans accompanies mechanical alternans.
In B, recording from another heart shows mechanical alternans only.
CmuUlicm Rumrcb, Vol. XIX, Noptnktr 1966
896
GABEL, BIHLER, DRESEL
PERFUSION
mm
PRESSURE!!
Hg
FIGURE 4
Response of a gas-perfused heart to increased perfusion pressure. Electrogram recorded with
electrodes on right atrial appendage and on ventricular apex. The arrows indicate threshold
pressure (120 mm Hg) for induction of arrhythmia and for return to sinus rhythm on decreasing
the perfusion pressure.
50Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
43'
40
35
I-
30
'
KREBS
o
r
a
is
10
6
RESTING
10
13
TENSION
20
g/g
23
30
35
h«ort wt.
FIGURE 5
Relationship between resting tension and isometric
developed tension in hearts perfused first with
Krebs solution and then with gas. Heart rate was 180
beats/min. Regression lines calculated by method of
least squares.
be determined repeatedly in the same heart
and was found to remain remarkably constant. The atria did not appear to be affected
by the increases in perfusion pressure.
STARLING'S RELATIONSHIP
Five hearts, driven electrically at 180 beats/
min, were perfused with liquid for a 10-min
period. The effect of graded increases in resting tension on isometric developed tensions
was determined. Gas perfusion was then begun and the procedure repeated as soon as
contractile force became stable (5 to 15 min).
Figure 5 shows the relationship of resting
tension to developed tension, both expressed
in terms of heart weight. This method of expressing the tensions was used because the
more commonly used units (grams per square
millimeter) were obviously impractical in
whole hearts. The tension developed at any
given resting tension was greater during gas
perfusion than during liquid perfusion. Imposed resting tensions were not allowed to exceed 35 g/g heart because preliminary experiments showed that excessive tensions cause
irreversible changes in contractility.
We have compared the external work done
by hearts perfused with gas, with substratefree Krebs solution and with Krebs to which
2 g/liter glucose had been added. Four hearts,
driven at 180 beats/min, were perfused initially with Krebs plus glucose, and successively heavier weights suspended from the
apex. Work and power output were recorded
electronically. The time required to estimate
work with the series of 5 weights was less
than 10 min. The hearts were then perfused with substrate-free Krebs solution and
the procedure repeated. A second determination during Krebs plus glucose perfusion was
done in order to ensure that the preparation
had not been altered by the substrate-free
CircuUlicm Research, Vol
XIX, Novtmber
1966
897
THE GAS-PERFUSED CAT HEART
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medium. The procedure was then repeated
during gas perfusion. Figure 6A shows a
characteristic series of curves obtained in 1
heart. Except at the smallest and the largest
loads used, power output during gas perfusion
was approximately 1.5 times that during substrate-free liquid perfusion, whereas power
output in the presence of glucose was more
than 3 times that in its absence. The results
from the entire group of preparations, which
differed from each other in weight, have been
combined by expressing the power output in
terms of the maximum power obtained during
perfusion with Rrebs plus glucose. Figure 6B
shows these "normalized" curves to have the
same quantitative relationship to each other.
METABOLIC STUDIES
We have studied the tissue content of various metabolites during gas and substrate-free
liquid perfusion. A 10-g resting tension was
applied to the hearts during these studies and
contractility was recorded to ensure that the
preparations were similar to those used in
the physiological studies. The experiments
were terminated by quickly cutting off the
ventricles during continued gas perfusion, blotting them and freezing in liquid nitrogen.
The control group of hearts was treated in
the same manner within 30 sec of removal
from the animals. The results are summarized
in Table 1.
Table 1 shows that the water content of
the control group was higher than that of
hearts perfused for a brief period, presumably
because their vasculature had not been cleared
of fluid. The increase in tissue water due to
prolonged perfusion with liquid is well known.
Tissue water did not decrease during prolonged gas perfusion, which indicates that
adequate humidification of the perfusate and
of the environment was achieved in our system.
Tissue glycogen content decreased rapidly
during the initial 5 min of liquid perfusion
and did not change greatly when liquid perfusion was continued for 2 hours. Gas perfusion for 5 min after 5 min of liquid perfusion resulted in a further significant decrease
in glycogen content to a level which remained
stable for the next 10 hours. Nonglycogen
carbohydrate levels changed in a manner
similar to that of glycogen. The short period
of gas perfusion produced a similar rapid
further fall in the levels of lactate and pyruvate which, however, continued so that their
levels in individual hearts were no longer
measurable.
PHARMACOLOGICAL STUDIES
We determined the response of oxygenperfused hearts to a number of drugs which
were administered in 1 or 5 ml Krebs solution.
It was essential that the solution be administered into the perfusion cannula rapidly
enough to pass through the coronary vessels
as a single bolus. Slow injection caused
the formation of multiple gas-liquid inter-
TABLE 1
Effect of Perfusions on Water and Carbohydrate Contents
Water
content
Perfusion
Control
(0 time)
Krebs, 5 min
Gas, 5 min
Gas, 3 hours
Gas, 10 hours
Krebs, 2 hours
%
Glycogen
/tmole«»/f
Other carbohydrates
/imole»»/t
80.9 ± 1.5
72.4 ± 3.8
47.9 ± 4.6
95.7 ± 7.6
0.16 ± 0.02
76.6 ± 0.4f
76.1 ± 0.3
76.3 ± 0.4
76.8 ± 0.5
84.3 ± 1.9t
23.8 ± 2.0f
13.8 ± O.lt
13.4 ± 0.5
13.6 ± 0.1
19.6 ± 0.7
19.3 ± 1.9f
15.6 ± 1.5
9.2 ± 0.9f
10.0 ± 0.6
33.2±4.1t
10.6 ± 2.5t
0.1*
17.4 ± 1.8t
9.2 ± 1.9f
0.08 ± O.Olf
<0.03
<0.03
<0.03
<0.03
Lactate
fimola/g
—
Pyruvate
>imoles/g
Means ± SE, four hearts per group; results calculated per gram dry weight of tissue.
*As glucose equivalents.
tSignificantly different (P < 0.05) from preceding group and control.
^Pooled sample.
CircuUtio» Rtstrcb,
Vol. XIX, Novtmktr 1966
898
GABEL, BIHLER, DRESEL
KIBS OLUCOSf
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KftftS
20
LOAD
B
/
30
35
•
PBtCBtTOf
MJUQMUM
rovm
KM-I
KKBS OWCOSE
•0-
60-
40
KIllS
0
5
1
LOAD
0
g/
a
1
5
hnH wt.
2
0
2
5
3
0
3
5
FIGURE 6
Top, effect of various loads on the power output of 1 heart perfused with Krebs plus glucose,
substrate-free Krebs solution, and gas. Heart rate was 180 beats/min. Bottom "normalized?'
curves for 4 hearts treated as above. Power output of each heart is expressed as percent of
maximum obtained during perfusion with solution containing glucose.
CircmUiUm Kutrcb,
Vol. XIX. Novtmbtr 1966
THE GAS-PERFUSED CAT HEART
899
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faces which would not pass through the heart
at the usual perfusion pressure.
Injections of Krebs solution resulted in a
transient (less than 20 sec) increase in contractility of approximately 10*. There was no
change in heart rate. Epinephxine and norepinephrine (0.1 to 0.5 fig) increased contractility and rate by 50 to 150*. Dose-response
curves were determined for isoproterenol in
4 hearts. This amine was 10 times more potent than epinephxine, a dose of 0.05 \ig,
producing 50% of maximal increase in contractility. The durations of action of these sympathomimetics were not increased over those
produced by similar injections into liquidperfused hearts. This is probably due to the
rapid decomposition of the amines at the high
oxygen tensions, although uptake into tissue
storage sites cannot be discounted.
The stimulus voltage of driven preparations
was increased to 10 X threshold in order to determine the response of the gas-perfused heart
to release of endogenous catecholamines (18).
Figure 7A shows the positive inotropic response to this procedure. The magnitude and
duration of the response were potentiated
50 V
by the prior administration of 1 yxg of cocaine (Fig. 7B). Pronethalol (1 to 10 /ng),
administered after washing out the cocaine
by brief perfusion with Krebs solution blocked
this response (Fig. 7C). Figure 7D shows
that prior treatment of an animal with reserpine makes the gas-perfused heart unresponsive to increases in driving voltage.
Discussion
The question arises whether the capillary
circulation is perfused by the gas mixture or
whether the heart remains viable because oxygen passing through the open left ventricular
cavity diffuses into the tissue. We cite
the following evidence against the latter possibility :
(A) Although we can observe the escape
of gas from the slit in the free wall of the
left ventricle, the resistance of the system
provides an argument against this being the
only route of egress of gas. In a number of
experiments the aortic valve opened suddenly, apparently spontaneously; this resulted in a precipitous fall in perfusion pressure and a decrease of 80 to 100* in the force
5V
A
50V
B
C
D
5OV
50 V
5V
FIGURE 7
Effect of drugs on the contractile response of gas-perfused hearts to catecholamines released
by supramaximal electrical stimulation. Heart rate was 180 beats/min. A = Control; B = after
cocaine (1/ig); C = cocaine washed out, treated with pronethalol (lfLg); D — as in A, but heart
obtained from an animal treated with reserpine 24 hours previously.
Circulation Rtsurch, Vol. XIX, Novtmitr 1966
5V
900
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of contraction of the heart. These preparations
could often be restored by gentle manipulation of the left ventricle. Contractility returned
to normal together with the return of perfusion pressure. We have also found that
slitting of the ventricle is a convenient, rather
than an essential, aspect of the preparation.
(B) Cardiogreen dye or diluted india ink
(5 ml) were injected through the aortic cannula into several hearts after 2 to 6 hours
of gas perfusion. This volume of solution
passes through the hearts in 20 to 30 sec.
Dissection showed that most or all of the
muscle mass had been stained. The exact distribution of dye was somewhat variable,
however, small portions of the free walls of
the ventricles sometimes showing only light
pink coloration.
(C) Although our preparation has maintained contractility for periods longer than
those reported by other workers, it is not
unique. Dog hearts have been kept alive in
vitro and in situ either by orthograde or by
retrograde perfusion of the coronary vasculature with gaseous oxygen. Camishion et al.
(19) have shown that tissue oxygen tensions,
measured with embedded oxygen electrodes
during retrograde perfusion, exceed those observed during perfusion with blood. Talbert
et al. (4) showed that gas flows of 700 to
1,500 ml/min at pressures of 20 to 50 mm
Hg are required during retrograde perfusion.
Sabiston et al. (3) measured flows of 300 to
1,000 ml/min at 80 to 120 mm Hg during
orthograde perfusion. These flows were observed under conditions where gas escape was
impossible. We conclude that gas will flow
through the coronary vasculature at reasonable pressures if perfusion with a saline
medium precedes gas perfusion and the formation of bubbles is studiously avoided. However, none of the studies, including our own,
presents definitive evidence that gas flow occurs through capillary beds.
Gas-perfused hearts beat more strongly,
perform more work and fail much more slowly than do hearts perfused with substrate-free
Krebs solution. There are several possible
reasons for this. It is immediately apparent
GABEL, BIHLER, DRESEL
that the oxygen content, but not necessarily
the Po-j of the perfusing medium, is greatly increased during gas perfusion. Lentini (20)
reported that the optimal oxygen tension for
isolated heart tissue is 700 mm Hg, a partial
pressure exceeded in our liquid perfusate.
Fisher and Williamson (21) and Opie et al.
(22) showed that the oxygen content of liquid
perfusates is sufficient to support metabolism
in hypodynamic preparations. This may not
be true in hearts which are performing work.
Blinks and Koch-Weser (23) concluded that
the oxygen carrying capacity of liquid perfusates is not sufficient to permit maximal
working capacity. Our experiments support
their contention by showing that gas perfusion allowed a small increase in work
capacity. No definite conclusion can be
reached until a method is found to measure
the oxygen consumption of hearts perfused
with gas. However, maximal work production
during oxygen perfusion was only 60$ of that
obtained when glucose was added to the
liquid medium, showing clearly that the major
determinant of work capacity is not oxygen
content. This confirms the experiments of
Neely et al. (24). The possibility that high
oxygen may exert a direct pharmacological
effect on cardiac contractility cannot be eliminated.
Another reason for better performance could
have been a qualitative or quantitative change
in the pattern of substrate utilization. Both
gas- and liquid-perfused hearts lost most of
their carbohydrate stores within 15 min of isolation, indicating that there are no gross
changes in the rate of metabolism of these
stores. By analogy with the observations of
Shipp et al. (25), it would appear probable
that gas-perfused hearts derive the energy
for contraction from the oxidation of lipids.
We are now studying the lipid metabolism of
these preparations.
The better performance of gas-perfused
hearts may be related to the unique nature
of this preparation, in which the extracellular
fluid does not equilibrate with a circulating
fluid but comes to at least partial equilibrium
with the intracellular space. It is possible
CirctUtiw Rutmrd, Vol. XIX, Novmttr
1966
901
THE GAS-PERFUSED CAT HEART
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that the accumulation in the extracellular fluid
of nonvolatile substances lost from the intracellular space may help to maintain contractility.
Adenosine and its phosphorylated derivatives, certain lipids, and metabolic cofactors
such as nicotinamide have all been shown
to be lost into the perfusing medium (6,
26, 27). These substances are all known to
have, in higher concentrations, positive inotropic effects on the heart (28, 29, 30). Catecholamines also are lost in the perfusing medium (31), but our experiments showing that
injected catecholamines act for only brief
periods and the observation that a beta-receptor blocking agent has no negative inotropic effect, appear to eliminate catecholamines
as essential for maintained contractility. Preliminary experiments have shown that a new
steady-state level of extracellular sodium and
potassium ions is achieved within 2 hours
of beginning gas perfusion. We have not as
yet eliminated the possibility that the increase in contractility during this period is
due to the limitation on the net inward diffusion of sodium or outward diffusion of
potassium ions. We believe that the observed
ability of the gas-perfused heart to maintain
contractility for long periods may result in an
interesting approach to the problem of cardiac
failure.
The gas-perfused heart may lend itself to a
variety of other specialized studies of cardiac
function. Drugs or other materials may be
administered with the assurance that they
and their metabolites are not being removed
from the preparation by the perfusate. Quantitative recovery of administered vehicle allows estimation of the material retained in
the tissue. We cannot be sure, however, that
gas-perfused tissue handles drugs in exactly
the same manner as do tissues bathed or
perfused \vith liquid.
The absence of substrates in liquid perfusates lowers the threshold for induction of
arrhythmia in isolated hearts (32). The gasperfused heart has a lower threshold than the
substrate-free, liquid-perfused heart for the
arrhythmia induced by increasing the perfusion pressure. Pressure must exert its effect
CircmUtio* Riswcb,
Vol. XIX, Novmitr
1966
via the coronary vasculature, since both
ventricular cavities are open to the atmosphere. This supports the work of Mainwood
(33) and of Panisset et al. (17). Our preparation is of particular interest because pressureinduced changes in rhythm may be reproduced
at will and occur at a constant threshold pressure. It may be of interest to study the effects
of vasoactive versus cardioactive drugs on
these arrhythmias.
The preparation may also be useful in the
study of mechanical alternation which is not
secondary to arrhythmia or to the action of
pharmacological agents. It should be noted
especially that alternation occurred in our
preparations during the phase of increasing
total contractility and was maintained
throughout the remainder of the experiment.
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OrcuUium Rfjtrcb,
Vol. XIX, Novmbv
1966
Contractility, Metabolism and Pharmacological Reactions of Isolated Gas-Perfused Cat
Hearts
LLOYD P. GABEL, IVAN BIHLER and PETER E. DRESEL
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Circ Res. 1966;19:891-902
doi: 10.1161/01.RES.19.5.891
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