Download Influence of Temperature on Development of Rigor Mortis in Dog

Influence of Temperature on Development of
Rigor Mortis in Dog Hearts
By Hctnsjoerg Kolder, M.D., Alan D. Horres, Ph.D., and
Gerhard A. Brecher, M.D., Ph.D.
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• The effects of temperature on the development of rigor mortis in cardiac muscle
have never been investigated systematically.
Brecher and Kissen1 found that pressure-volume characteristics of non-beating, submerged
dog ventricles remained constant for at least
30 minutes following circulatory arrest at
35°C. Using similar methods, Griggs et al.2
found this period to be approximately 80 minutes at 36°C and 300 minutes at 5°C. These
and other related studies of rigor mortis3"9
were not primarily directed at the effects of
varying temperature under controlled conditions. We have therefore adapted the technique
of Brecher and Kissen1 to permit systematic
investigation of tlie effects of temperature on
the onset and development of rigor mortis in
dog hearts. This study provides a background
for future investigations in which the time
limits for development of rigor mortis must
be known.
Methods
The principle of the method is to establish an
"elastic equilibrium" of the ventricles b}7 submerging the heart in Ringer's solution. The state of
elastic equilibrium of a ventricle is characterized
by zero transmural pressure. Under this condition
there is always a certain volume of fluid in the
freshly excised ventricle. Upon reducing the intraventricular volume, the transmural pressure becomes negative and upon fluid addition it becomes
positive. This method lends itself to pressurevolume studies in which any stretch of the ventricular walls can be avoided. Since such stretch
might affect the development of rigor mortis, half
of the experiments were done with negative transmural pressures only and compared with the others
From the Department of Physiology, Division of
Basic Health Sciences, Schools of Medicine, Dentistry,
and Nursing, Emory University, Atlanta, Georgia.
Supported by the Life Insurance Medical Research
Fund, TJ. S. Public Health Service Grant H-3796,
and the Georgia Heart Association.
Received for publication March 11, 1963.
218
in which both negative and positive transmural
pressures were produced.
Fifty-five mongrel dogs of various ages were
used, 37 for studies on left ventricles, 18 for
studies on right ventricles. Preceding these experiments, most of the animals had been employed in
other studies which did not involve the cardiovascular system. The animals were anesthetized intravenously with sodium pentobarbital (30 mg/kg
of body weight). Six minutes after the injection
they were rapidly exsanguinated from a femoral
artery. Four minutes after the beginning of exsanguination the removal of the heart from the
pericardial sac was completed. The aorta and pulmonary artery were severed 5 cm from their origin
and the systemic and pulmonary veins were divided
at their entrance into the atria. The hearts were
then rinsed in Ringer's solution and the aorta and
pulmonary artery stumps were ligated. The experimental arrangement is shown in figure 1.
A Tygon cannula (inner diameter 10 mm) was
placed in the atrioventrieular orifice and secured
by a ligature around the atrioventrieular groove.
The hearts were submerged in Ringer's solution in
a constant temperature bath. The bath could be
adjusted to temperatures between 1°C and 50°C
( ± 0.25). The composition of the buffered Ringer's
solution used was NaCl 0.9 g, KC1 0.03 g, CaCl2
anhyd. 0.026 g, 3\TaHCO3 0.055 g, water to 100 ml2
The ventricular cannula was connected via three
meters of Tygon tubing (inner diameter 9 mm,
walls 2.5 mm), to a burette, graduated in 0.2 nil.
Half of the Tygon tubing was immersed in the
bath to minimize temperature fluctuations in the
heart when fluid was added. A side tube with a
stopcock permitted equalization of the fluid levels
in the bath and burette. Operations were completed
within four minutes after removal of the heart.
The pressure-volume relationships were investigated in 29 experiments in which the intraventrieular pressures covered only a range below atmospheric pressure. The pressure, and thereby the
volume within the ventricle, was reduced stepwise
by lowering the burette. The pressure differences
between the surface of the bath and meniscus of
the burette (i.e., transmural ventricular pressure)
were read in millimeters of water. Volume increases in the burette represented corresponding
volume decreases in the ventricle. A steady state,
Circulation Research, Volume XIII, September 196S
219
RIGOR MORTIS IN HEARTS
ml
cm
6min
Vlll/I
'
0-AP-
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FIGURE 1
FIGURE 2
Experimental arrangement for measuring the pressure-volume relationships of excised hearts. Details in text.
Pressure-volume relationships of an excised left
ventricle submerged in Singer's solution ai 35°C,
and their changes with time after excision (dog,
12 hg).
generally reached in less than 15 seconds, was established before each reading. The lowest intraventrieular pressure applied was 30 mm Hg
subatmospherie. In 26 experiments the pressurevolume relationships were determined with positive transmural pressures up to 50 mm Hg. In
these experiments volume reductions in the burette
represented volume increases in ventricle. In all
experiments three series of control measurements
were made in rapid succession immediately after
immersion of the heart. Thereafter each series of
readings was repeated every five minutes for experiments above 34° C, every 15 minutes for experiments between 26°C and 34°C, every 30 minutes
for experiments between 11°C and 25°C, and
every 60 minutes for experiments between 1°C
and 10°C. Between each series of readings the
stopcock of the side tube was opened to avoid
undue stretching of the ventricular walls.
The fluid uptake of hearts submerged in Ringer's
solution was measured in 13 additional experiments. Following gentle blotting, the heart was
weighed immediately after removal from the animal and every hour thereafter. Hearts were kept
at constant temperature for the following number
of hours: 5°C: 70 hr; 10°C: 65 hr; 20°C: 22 hr;
30°C: 23 hr; 40°C: 19 hr; 45°C: 11 hr.
there was a progressive stiffening of the ventricular walls as indicated by a decrease in
volume changes in relation to the pressure
changes. After two hours the pressure-volume
curve became almost linear. It remained so
until the beginning of autolysis, which was
characterized by increased tissue fragility
leading to rupture of the wall when pressure
was applied. Some hysteresis is evident in all
curves. At approximately 30 mm Hg subatmospheric and 50 mm Hg above atmospheric
pressure respectively, the volume changes resulting from further decreases or increases in
pressure were negligible. In order to prevent
undue deformation of the ventricular walls,
these pressure limits were not exceeded.
Figure 3 shows the pressure-volume curves
from a heart at 10°C. The relationship remained unaltered between the first measurement (at four minutes) and subsequent measurements (up to three hours) after removal
of the heart. The distensibility of the ventricle decreased progressively between three
and thirteen hours. After thirteen hours the
pressure-volume curve remained constant and
S-shaped until autolysis began. This is in contrast to the almost linear relationship which
was eventually attained in. experiments done
at temperatures above 34 °C.
Results
Figure 2 shows the typical pressure-volume
relationships of a dog heart submerged in
Ringer's solution at 35°C. This pressure-volume curve remained unchanged for 30 minutes after removal of the heart. Thereafter,
Circulation Research, Volume XIII. September 19CS
220
HOLDER, HORRES, BRECHER
AV +
TEMPERATURE
50-
RIGOR MORTIS
°C
-KICCT
ONSET
STEADY
40
o
D
•
"•
LEFT
RIGHT
LEFT
RIGHT
30-
AP-
AP \
15
20-
3OmmHg45
EQUILIBRIUM
h 10 STATE
10—i—
20
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AVFIGURE 3
Pressure-volume relationships of an excised left
ventricle submerged in Ringer's solution at 10°C,
• and their changes with time after excision (dog,
12.5 kg).
At all temperatures (1°C to 50°C) the pressure-volume relationships were the same before rigor started. This was deduced from
the almost equal slopes at the inversion points
of all curves. Thus, before the beginning of
rigor, the distensibility of the heart (AV/AP)
averaged 3.5 ml/rain Hg around the inversion
point, irrespective of temperature.
It was often found that during the second
or third series of pressure-volume measurements a larger volume could be accommodated
in the ventricle, in its elastic equilibrium
state, than during the first series of measurements. This phenomenon, called "primary
dilatation" by Rothberger,8 was observed in
21 of 25 experiments on left ventricles and
11 of 18 experiments on right ventricles. In
those ventricles exhibiting primary dilatation,
an average of 14% more fluid could be accommodated during the second or third series of
measurements than during the first.
Figure 4 shows the relationship of time and
temperature to the development of rigor mortis. The criterion for the onset of rigor was
the beginning of a change in the pressureA7olume curve of the heart, disregarding the
primary dilatation. The criterion for the
2
4
6
8
10
12 hr
TIME
FIGURE 4
Time required for the development of rigor mortis
of dog ventricles as a function of temperature.
steady state of rigor was the constancy of the
pressure-volume relationship in three consecutive measurements after progressive stiffening
had occurred. At temperatures above 40°C
the onset of rigor occurred within 20 minutes
after removal of the heart and the steady state
of rigor was reached within one hour. As the
temperature was lowered, the time for onset
of the rigor and for attaining the steady state
was proportionately prolonged. At 10°C the
onset averaged five hours while the steady
state was attained after twelve hours. Apparently there was no systematic difference
between right and left ventricles regarding
the development of rigor mortis.
From figure 5 it is evident that the weight
gain of hearts submerged in Ringer's solution
was 5% ± 1.8 SD within five hours irrespective
of the temperature of the Ringer's solution.
Discussion
These experiments demonstrate that in the
myocardium the onset of rigor mortis as well
as the attainment of maximal stiffening is
temperature dependent. The time-temperature
relationship was found to be sufficiently reproducible to allow useful predictions of the
development of rigor in the dog heart.
For some types of investigations it is important to know how much time is available
Circulation Research, Volume XIII, September 19GS
221
RIGOR MORTIS IN HEARTS
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for making pressure-volume determinations in
ventricles before changes in stiffness of the
ventricular •wall begin, e.g., for studies on the
contribution of elastic forces to ventricular
diastolic filling.1 The present experiments
furnish necessary information concerning the
period at each temperature during which such
measurements can be made. There is also evidence that from 1°C to 50 °C the temperature
itself does not alter the pressure-volume relations before rigor begins. Based on this information it would be justified to make repeated pressure-volume determinations on dog
hearts for four hours at 10°C, but only for
25 minutes at 37 °C.
It must be recognized that the onset of
death of an individual heart muscle fiber is
indeterminable by means of presently available techniques. For lack of better criteria,
the present experiments made use of overall
elastic changes to define arbitrarily the onset
of rigor mortis. Similar considerations pertain to the endpoint in the development of
rigor mortis, i.e., the time at which no further
increase in the ventricular wall stiffness
occurs.
From the curves in figure 4 it can be deduced that the physicochemical processes4'1011
responsible for rigor mortis are slowed by low
temperatures and accelerated by high temperatures. This not only concerns the onset
of stiffening but also the progression of the
stiffening to its maximum, as illustrated by
divergence of the broken and solid lines in
figure 4. In fact, at 1°C the processes which
caused stiffening never reached their maximal
development, because autolysis began before
the maximum was attained. This suggests that
processes responsible for the onset of autolysis
are not slowed at the same rate of cooling as
are the rigor inducing events.
The results of the present experiments
agree in principle with findings reported by
Griggs et al.2 However, there are differences
in the methods used. Griggs et al.2 emptied
the heart, added measured volumes to the ventricle, and recorded the intraventricuiar pressures, which always exceeded atmospheric
pressure. This would result in a stretch of
Circulation Research, Volume XIII, September 196S
INCREASE
IN WEIGHT
5^
_—-—
-
.
—
i
5
i
3
0
i II
9 8G
5- 13
ib
2b
3'0
40
50
60
70 hr
TIME
FIGURE 5
Weight gain of 13 dog hearts submerged in
Ringer's solution at temperatures ranging from
5°C to 45°C. The numerals refer to the total number of experiments in which measurements tuere
made over the given time.
the ventricular muscle that might affect the
time course of rigor mortis development. This
possibility was pointed out by Mosso and Pagliani7 in objection to Rothberger's work.s
Since in the present experiments the onset of
rigor was the same irrespective of the A'entricular volume, the stretching as such seems
to have no measurable effect.
The final shape of the pressure-volume
curve at maximum rigor depended upon the
temperature. Only in experiments in which
the hearts were exposed to temperatures above
34°C did the pressure-volume curve become
approximately linear. Below this temperature, the curve remained S-shaped even until
autolysis began. This difference in the final
shape of the curves might be explained by the
phenomenon of "heat eontracture" superimposed upon rigor mortis at temperatures
above 34°C, as postulated by Morita.12
Among the numerous factors responsible
for the physical behavior of tissues (Remington13), the elasticity of a quiescent heart in
Ringer's solution might be affected by fluid
uptake. Since the weight gain of the heart
amounted to 5% within five hours and the
pressure-volume relationships did not change
during this time, the effect of fluid uptake
appeared to be negligible.
222
HOLDER, HORRES, BRECHER
The phenomenon of "primary dilatation"
of all types of muscle has been described by
many investigators.3-7i n~15 Some of them ascribed it to an artifact resulting from stretch
of the muscle.7-1S The present experiments
show that deformation caused by either
stretch or compression may induce primary
dilatation. However, it cannot be decided
from the presently available evidence whether
primary dilatation is caused by a relaxation
of "elastic elements" or by lengthening of
''plastic elements."16"20
Serial pressure-volume studies in the excised
canine heart. Am. J. Physiol. 198: 336, 1960.
3. ECKSTEIN, A.:
4.
5.
6.
7.
Summary
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The effect of temperature (1°C to 50°C)
on the onset and maximal development of
rigor mortis in dog hearts was studied systematically under static conditions. Excised
hearts were submerged in Ringer's solution
so that the ventricle could assume an elastic
equilibrium state. This was characterized by
zero transmural pressure and the presence of
a certain volume in the ventricle. Pressurevolume curves were obtained in 29 experiments by removal as well as by addition of
fluid. The pressure-volume relations were the
same at all temperatures prior to the onset of
rigor mortis. The criterion for the beginning
of rigor mortis was the first change in pressure-volume relations and for maximum rigor
the constancy of the attained relations. The
shortest interval between cessation of the circulation and the beginning of rigor mortis was
25 minutes at 37°C, and three hours at 10°C.
These measurements provide data defining the
minimal period during which pressure-volume
determinations can be made without post
mortem alterations. The phenomenon of "primary dilatation"' was observed irrespective of
a withdrawal or an addition of fluid to the
ventricle. The weight gain of the quiescent
ventricle in Ringer's solution was determined
until autolysis began and was found to be 5%
during the first five hours at all temperatures
ranging from 5°C to 45°C.
8.
Die
Totenstarre
des
Herzens.
Pfliigers Arch. Ges. Physiol. 181: 184, 1920.
LAWRIE, R. A.: The onset of rigor mortis in
various muscles of the draught horse. J.
Physiol. 121: 275, 1953.
MACWILLIAM, J. A.: Rigor mortis in the heart
and the state of the cardiac cavities after
death. J. Physiol. 27: 336, 1901-02.
MEIROWSKY, E.: Jfeue Untersuchungen fiber die
Todtenstarre quergestreifter und glatter Muskeln. Pfliigers Arch. Ges. Physiol. 78: 64, 1899.
Mosso, A., AND PAOLIANI, L.: TJeber die postmortalen Formveranderungen des Herzens.
Pfliigers Arch. Ges Physiol. 101: 191, 1904.
ROTHBERGKR, C. J.: ITeber die postmortalen Formveranclerungen des Herzens. Pfliigers Arch. Ges.
Physiol. 99: 385, 1903.
9. EOTHBERGER, C. J . : Zur Frage der postmortalen
Formveranderungen des Herzens. Pfliigers
Arch. Ges. Physiol. 104: 402, 1904.
10.
BATE-SMITH, E. C, AND BENDALL, J. R.: Fac-
tors determining the time course of rigor
mortis. J. Physiol. 110: 47, 1949.
11. FLETCHER, W. M.: Lactic acid formation, survival
respiration and rigor mortis in mammalian
muscle. J. Physiol. 47: 361, 1913-14.
12. MORITA, G.: "Totenstarre"- und Warmestarre-
Versuche an glatten Muskeln von Kalt- und
Warmbliitern. Pfliigers Arch. Ges. Physiol. 205:
108, 1924.
13. REMINGTON, J. W.: Tissue Elasticity. Washington, American Physiological Society, 1957.
14. MANGOLD, E.: TJeber die postmortale Erregbarkeit quergestreifter Warmbliitermuskeln. Pfliigers Arch. Ges. Physiol. 96: 498, 1903.
15. POTONIE, H.: 3S"eues iiber die Totenstarre glatter
Muskulatur. (Versuche am Hiiliner- und Taubenmagen.) Pfliigers Arch. Ges. Physiol. 209:
395, 1925.
16. GEHL, H.,
GRAF, K.,
AND KRAMER, K.:
Das
Druckvolumdiagramm des Kaltbltiterherzens.
Die Bedeutung des plastisehen Elements fur
die Herzmeehanik. Pfliigers Arch. Ges. Physiol.
261: 270, 1955.
17. LUNDIN, G.: Mechanical properties of cardiac
muscle. Acta Physiol. Scand. 7: Suppl. 20, 1944.
18. REICHEL, H.: Das plastische und elastische Ele-
ment des Herzmuskels. (Versuche am Herzmuskelstreifen der Schildkrote.) Pfliigers Arch.
Ges. Physiol. 257: 202, 1953.
19. RUSHMER, R. F., AND THAL, N.: Factors influenc-
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Circulation Research, Volume XIII, September 196S
Influence of Temperature on Development of Rigor Mortis in Dog Hearts
HANSIOERG KOLDER, ALAN D. HORRES and GERHARD A. BRECHER
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Circ Res. 1963;13:218-222
doi: 10.1161/01.RES.13.3.218
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