Download Inhibition of Protein Synthesis in Cardiac Hypertrophy and its

Inhibition of Protein Synthesis in Cardiac
Hypertrophy and its Relation to
Myocardial Failure
By Volker Ziihlke, M.D., Wolfgang du Mesnil de Rochemont, M.D.,
Sigmundur Gudbjarnason, Ph.D., and Richard J. Bing, M.D.
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• It has been demonstrated that a heart
subjected to functional strain, imposed by increased outflow resistance, passes through a
series of metabolic alterations, characterized
by changes in energy production and myocardial protein synthesis. Under such circumstances an isometric type of hyperfunction is
produced with the development of cardiac
hypertrophy. During the first or "damage"
stage of hyperfunction, which occurs immediately after severe outflow obstruction protein
synthesis is increased; during the second stage
of relatively stable hyperfunction the degree of
protein synthesis approaches normal levels,
while during the third stage, which is characterized functionally by gradual exhaustion,
protein synthesis is inhibited. It was postulated that the primary inhibition of protein
synthesis interferes with the renewal of the
energy-producing and contractile structures of
the myocardium. This results in a disturbance
in the processes of energy production and
utilization with a decrease in the contractile
capacity of the myocardium.1- Previous experiments in this laboratory3'4
have shown that in cardiac hypertrophy protein synthesis is increased but there is no
increase in myocardial protein turnover rate.
It is the purpose of this study to investigate
the relationship between cardiac hypertrophy
From the Department of Medicine, Wayne State
University School of Medicine, Detroit, Michigan.
Supported by Grant HE-05043 from the U. S. Public Health Service, the Michigan Heart Association,
Medical Research Fund, the Burroughs-Wellcome
Fund, American Medical Association-Education and
Research Foundation, and the John A. Hartford
Foundation.
Accepted for publication November 12, 1965.
558
and cardiac failure, especially with reference
to the protective effect of cardiac hypertrophy
on the development of cardiac failure. The
aim was to produce heart failure by inhibiting
protein synthesis during the first stage of
experimentally produced cardiac hypertrophy,
thus demonstrating the importance of increased protein synthesis as a protective
adaptation against failure. In addition, the
question of altered turnover rate of myocardial
protein will be examined.
Methods
Experiments were performed on 121 rabbits
(New Zealand white albino) of either sex, with
an average weight of 1.94 kg. The rabbits were
kept in individual cages and received Rockland
rabbit diet* and water ad libitum. Cardiac hypertrophy was produced in 50 animals, anesthetized with sodium pentobarbital (Nembutal, 25
mg/kg body weight) injected into an ear vein, by
placing an aluminum clamp with adjustable diameter around the ascending aorta, approximately
2 mm above the origin of the coronary arteries.5-7 After measuring the external diameter of
the aorta, its lumen was reduced to no more
than 60% of its original external diameter. Respiration was maintained during the operation by
means of an intratracheal cannula connected to
a respiratory pump. The operative mortality was
28%. To estimate the severity of the aortic stenosis, the blood pressure gradient across the aortic valve and the stenosis was optically recorded
in the majority of animals. Left ventricular end
diastolic pressures were recorded in all animals
prior to sacrifice by means of direct puncture of
the left ventricle. End diastolic pressure of more
than 10 mm Hg was considered to be a significant
indicator for heart failure. Additional evidence for
this was the presence of pleural effusion, ascites,
*Teklad Inc., Monmouth, Illinois. Composition of
diet: crude protein, 17% min.; crude fat, 1.5% min.;
crude fiber, 16% max.
Circulation Research, Vol. XVIII, May 1966
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
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hepatic engorgement, and pulmonary congestion.
The presence of cardiac hypertrophy was ascertained by measurement of the ratios of left or
right ventricular weights (left ventricle included
the septum) to total body weight in grams of
heart muscle to kilograms of body weight (g/kg).
This ratio, in normal rabbits, and for the left ventricle, was 1.41 ± 0.05, and for the right ventricle
0.39 ±0.03 (g/kg; ± S E ; N = 28). In the presence of hypertrophy the ratio rose to 2.32 ± 0.25
for the left and to 0.48 ± 0.05 for the right ventricle (g/kg; ± SE; N = 8). This increase in the
ratio was significant for the left ventricle (P <
0.001) but not significant for the right ( P <
0.2). Further evidence of cardiac hypertrophy
was provided by the thickness of the hypertrophied left ventricular wall which was almost
twice normal. Histological examination of the
hypertrophied myocardium showed increased size
of cells. Nuclei and cytoplasm were both enlarged
with the nuclei becoming shorter and thicker.
The animals were divided into four main
groups:
Group I
: rabbits on regular diet,
la : control rabbits.
Ib : sham operated rabbits.
Ic : rabbits with aortic stenosis.
Group II : rabbits on regular diet and treatment with actinomycin D.
Ha : control rabbits.
lib : sham operated rabbits,
l i d : rabbits with aortic stenosis, 6 to 24
hr postop.
Group IIC2 : rabbits with aortic stenosis, 2 to 4
days postop.
Group III : rabbits on regular diet, with aortic
stenosis and treatment with puromycin.
Group IV : rabbits on protein-free diet.
IVa : control rabbits.
IVb : sham operated rabbits.
IVc : rabbits with aortic stenosis, 1 to 4
days postop.
IVd : rabbits with aortic stenosis, 6
months postop., only last 6 weeks
on protein-free diet.
Group Ic consisted of animals with aortic stenosis. They were maintained on a regular diet
(N = 18) and were sacrificed at various time
intervals up to four days after the operation. In
group IIci, and IIcj, animals with aortic stenosis
( N = 1 4 ) were fed a regular diet and were
treated with actinomycin D* dissolved in sterile,
pyrogen-free saline to a final concentration of
24 /ig/ml, the pH being adjusted to 7.4. In
this form 120 /[/.g/kg actinomycin D were injected
•Kindly supplied by Merck, Sharpe and Dohme,
Research Laboratories, Rahway, New Jersey.
Circulation Research, Vol. XVlll,
May 1966
559
intraperitoneally daily and the animals were also
sacrificed at various time intervals up to four days.
Group III included animals with aortic stenosis
(N = 7). They were fed a regular diet and treated with puromycin dihydrochloride* dissolved in
sterile, pyrogen-free saline to a final concentration
of 6 mg/ml, pH 7.4. The dosage of puromycin
was 30 mg/kg injected intraperitoneally at 0,
16, and 18 hours postoperatively; the last two
doses at 20 and 22 hours after operation were
given intravenously. All animals in this group
were sacrificed 24 hours after production of aortic
stenosis.
Group IVc consisted of animals (N = 8) that
were kept on a protein-free diett for two to three
weeks prior to the production of aortic stenosis.
They were maintained on this diet after operation
and were sacrificed at various time intervals from
24 hours to 4 days postoperatively. In group IVd
(N = 3) long term experiments were performed.
These rabbits were kept on a regular diet for
four months after the operation and then were
fed protein-free diet for an additional six weeks.
Each of the main groups, except for that receiving puromycin, contained two control groups
(a and b). These animals were treated identically,
except that in group a no operation was performed whereas in group b the animals were
subjected to a sham operation consisting of
thoracotomy, pericardiectomy and dissection of
the ascending aorta.
Glycine-2-C14* (specific activity 37.65 mc/millimole) was used in all experiments. Dosage was
30 fic/kg body weight injected intravenously
four hours prior to death. Food was withheld 18
hours before the injection of glycine. In a fifth
and separate investigation, emphasis was placed
on the rate of disappearance of radioactivity
from myocardial protein in rabbits with regular
diet, in comparison to those with protein-free
diet (N = 36). These animals were not operated
upon, and were sacrificed from 1 to 25 days after
injection of glycine-2-C''i.
The heart and specimens of skeletal muscle
were removed rapidly and washed with isotonic
NaCl solution. Right and left ventricles were
separated from the atria at the level of the annulous fibrosus. The right ventricle was then dissected carefully from the septum and weighed.
The remaining specimen (left ventricle and septum) was also weighed; then the left ventricle
*Purchased through Nutritional Biochemicals Corporation, Cleveland, Ohio.
fPurchased through General Biochemicals, Chagrin Falls, Ohio. Ingredients: starch, 70%; vegetable
oil, 10.0%; salt mix., 4.0%; nonnutritive fiber, 15.0%;
cod liver oil (vitamins A and D), 1.0%.
^Tracer Laboratory, Waltham, Massachusetts.
ZOHLKE, DU MESNIL DE ROCHEMONT, GUDBJARNASON, BING
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was dissected from the septum, which was discarded. The specimens of skeletal muscle, right
and left ventricles, were frozen in liquid nitrogen
and used separately for the biochemical determinations.
Protein was isolated by a modified method of
Schmidt and Thannhauser.8 The acid soluble compounds, containing free glycine were removed
from the homogenate by repeated extractions
with 0.3 N perchloric acid which precipitates protein, lipids and nucleic acids. The nucleic acids
were extracted with 5% trichloroaceric acid at
70°C for 20 minutes. The lipids were removed
from the residue with ethanol, ethanol-chloroform, ethanol-ether and finally with ether. The
protein was then washed with Tris buffer (pH
7.3), water, acetone, and ether, and dried.
The radioactivity of the protein was determined
in a toluene gel scintillator. This consists of 4 g
PPO (2,5-diphenyloxazole) and 100 mg POPOP
(l,4-bis-2(5-pheny!oxazole)-benzene) in one liter
of toluene. To this scintillator was added 4%
thixotropic gel (Packard). The dried protein was
powdered in a mortar and 10 mg was weighed
and suspended in the scintillator. The efficiency
as determined with an internal standard was 62%.
It is understood that the radioactivity of the
protein is due not only to incorporated glycine2-C* but also to serine as well as other amino
acids, which are synthesized from glycine. To
identify more clearly, specific changes in incorporation of glycine-2-CJ< into heart muscle protein, the skeletal muscle was used as reference
organ.8' * The extent of incorporation of glycine2-C'* into myocardial protein was related to the
incorporation into skeletal muscle protein as calculated by the ratio, henceforth referred to as
relative incorporation:
counts/min/mg heart muscle protein
counts/min/mg skeletal muscle protein
Results
Figure 1 illustrates the typical blood pressure gradient across the aorta before and after
production of the aortic stenosis in a rabbit.
The development of heart failure resulted in
an elevated left ventricular end diastolic pressure (fig. 2). Further evidence for heart
failure was the presence of pleural effusion
and ascites.
Figure 3 demonstrates the relationship
between the ratio of left ventricular to total
body weight and left ventricular pressures in
unoperated animals with regular diet, unoperated animals with protein-free diet, and in the
related groups of animals with aortic stenosis
LV
^
(after)
Aorta
(after)
Pre-
Aorta (after)
Po»t-
itenotle
stenotic
FIGURE 1
Pressure tracing of left ventricle and aorta before and
after aortic constriction. A moderate gradient in
pressure between the left ventricular systolic and
aortic systolic pressure is seen following operation.
mmHg
160
120
80
40
0
FIGURE 2
a: Left ventricular pressure in one rabbit four vionths
after aortic constriction with regular diet, b: After
additional six weeks on protein free diet. The elevated
end diastolic pressure is seen in b.
respectively. There was a significant increase
of the ratio of left ventricular to total body
weight (P<0.005) as well as in the left
ventricular pressure (P < 0.02), after production of aortic stenosis in animals on a regular
diet. A comparison of these parameters within
the groups maintained on a regular and
protein-free diet is not valid, since differences
in body weight of animals with different diets
were too great.9'10 After production of aortic
stenosis, left ventricular pressures increased
in animals with regular diet and in those with
protein-free diet to the same degree (16 to
18 mm Hg). Table 1 shows results that compare the incorporation of glycine-2-CJ* into
left and right ventricular muscle protein as
well as the relative specific activities in protein
from animals of the various groups.
CircmUtiot, Rtsurcb,
Vol. XVIII, Mmj 1966
561
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
0 Left Ventricular Pressure
D Ratio: Heart weight/Body weight
^150
E
LLJ
Q
O
CD
CO
ffi 100
$ 1.0
I 50
EC
LU
x0.5
o
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U cc
0
CONTROL
2 4 - 9 6 hrs
6 weeks
HYPERTROPHY
REGULAR DIET
CONTROL
24-96 hrs
HYPERTROPHY
PROTEIN-FREE DIET
FIGURE 3
Comparison of left ventricular pressure (mm Hg) and the ratio, heart weight (left ventricle) to
body weight (g/kg) between control animals and animals with aortic stenosis for groups on
regular and proteins-free diet. It may be seen that in animals with aortic stenosis, maintained on
regular or protein-free diet, the left ventricular pressure increases as does also the ratio, heart
weight/body weight.
H Left ventricle
Right ventricle
Sham Operoted
250
D Skeletal muscle
Hypertrophy
r
2 25
2 00
1 75
1 50
1 25
1 00
75
50
25
0
Control = 100%
1
Reg diet
Reg. diet Protein-free
treated
diet
with
Actinomycin D
Sham Operated = 100%
1
Reg. diet
Reg diet Protein-free
treated
diet
with
Actinomycin D
6 - 2 4 hrs
post-op
I
f
Control = 100%
Reg diet
Reg diet Protein-free
treated
diet
with
Actinomycin D
6 - 2 4 hrs
post-op
FIGURE 4
Comparison of per cent activity of heart and skeletal muscle protein between the different
groups. For each group the specific activity of protein in sham operated animals was related
to values of unoperated controls (= 100%) (left third of figure). Specific activity of protein
in animals with aortic stenosis was related to values of sham operated (= 100%) (middle third)
and unoperated controls (= 100%) (right third of figure).
Circulation Research, Vol. XVIII, May 1966
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
90
£.
^
*LV: left ventricle. RV: right ventricle. SK: skeletal muscle.
t ± standard error of the mean.
>
XVI
"*
11
993 ± 77
N= 8
IVd: Aortic stenosis, 6 months after operation,
only last 6 weeks on protein-free diet.
!•
1017 ±
1213 ± 142
1344 ± 111
IVc: Aortic stenosis, 1 to 4 days postoperatively.
I
N=3
1221 ± 119
1042 ± 88
1
IVb: Sham operation.
94
904 ± 94
913 ±
1254 ± 147
951 ±
1093 ±: 79
718 ± 26
929 ± 102
Protein-Free Diet
Croup IV:
122
149
1007 ±
1333 ± 112
64
676 ±
counts/min/10 mg protein
Specific activity of protein
Right
ventricle
IVa: Control, without operation.
£
Regular Diet, with Aortic Stenosis and Treatment
with Puromycin, 24 hr Postoperatively.
N= 7
Group III:
961 ±
1641 ± 255
He.,: Aortic stenosis, 2 to 4 days postoperatively.
N= 6
983 ±
lie,: Aortic stenosis, 6 to 24 hr postoperatively.
95
944 ± 69
98
1472 ±
lib: Sham operation.
110
1007 ±
767 ± 81t
764 ± 58
Regular Diet and Treatment with Actinomycin D
Regular Diet
Control, without operation.
N = 10
Sham operation.
N= 8
Aortic stenosis, 1 to 4 days postoperatively.
N = 18
Left
ventricle
Ila: Control, without operation.
Group 11:
Ic:
Ib:
la:
Group I:
Diet, treatment, and numbers
of rabbits in each group
TABLE 1
Incorporation of Glycine-2-CIJ< into Myocardial Protein arid Skeletal Muscle Protein
9
16
2.3 ±:0.7
4.8 ±
5.3 ±:1.0
253 ± 49
423 ± 155
9.9 ± 1.6
8.4 ± 0.9
123 ± 18
2.4 ± 0.8
1.1
8.4 ± 1.1
11.0 ± 3.5
8.7 ± 1.1
19
20
107 ±
82 ±
11.8 ± 3.0
14.1 ± 1.3
67 ±
18.4 ± 1.5
30
124 ±
89 ± 12
1.4
13.8 ± 0.9
7.2 ±
14.8 ± 1.5
1.6
7
7.6 ±
12.1 ± 1.3
13.4 ± 1.1
8.8 ± 1.7
19
10.5 ±
10.5 ± 0.7
1.1
5.3 ± 0.9
RV/SK
6.0 ± 0.9
LV/SK
Relative incorporation*
7.6 ±:1.3
100 ±
110 ± 33
96 ±
127 ±
Skeletal
muscle
a
m
z
IO
30
o
z
<n
c
o
in
8X
m
JO
F
o
m
i/i
C
m
X
c
N
562
563
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
ANIMALS ON REGULAR DIET (GROUP I, TABLE 1)
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Sham operations (Group Ib, table 1) resulted in an increased incorporation of glycine2-C'* into heart muscle protein. This increase
was significant only for the right ventricle
(P<0.05) (table 1 and fig. 4). The changes
in the uptake in the left ventricular and skeletal muscle protein were not significant (for
left ventricle P<0.1; for skeletal muscle P
<1.0). The relative incorporation into right
and left ventricular protein increased slightly
(table 1).
Table 1, Group Ic, illustrates a considerable
increase of glycine-2-C1'i incorporation into
the left and right ventricular muscle protein
in animals with aortic stenosis. In aortic stenosis (Group Ic, table 1) as compared to sham
operated animals (Group Ib, table 1), the
increase in incorporation into left ventricular
protein was significant (P < 0.01). Also in
these animals with aortic stenosis (Group Ic,
table 1), incorporation into left and right
ventricular protein was significantly increased
as compared to nonoperated controls (Group
la, table 1, andfig.4); for left ventricle and
right ventricle P < 0.001).
Figure 5 shows that six hours after produc-
tion of aortic stenosis the incorporation of
glycine-2-CJ4 into left and right ventricular
protein was increased to approximately 190%
of that in unoperated controls and remained
at this level during the four-day period of
study. The relative incorporation rose gradually to a constant level, and exceeded that of
unoperated controls by 268% four days after
operation. None of the animals with aortic
stenosis of this group had elevated end diastolic pressure, pleural effusion or ascites during the time of investigation.
ANIMALS WITH REGULAR DIET AND TREATMENT WITH
ACTINOMYCIN D (GROUP I I , TABLE 1)
Actinomycin D had no inhibitory effect on
the incorporation of glycine-2-CI'i into heart
or into skeletal muscle protein in unoperated
(Group Ha, table 1) and sham operated animals (Group lib, table 1) respectively. The
relative incorporation in these two groups of
animals remained almost unchanged. As in
animals without treatment by actinomycin,
sham operations (Group lib, table 1) were
associated with a significant increase (P <
0.005) in incorporation of glycine-2-C1/( into
right ventricular protein (fig. 4). In animals
with aortic stenosis, there was no increase of
300r
O
D
.
6
Operation
18-24
48
Hrs
N=(2)
(5)
post-op.
(5)
Right Ventricle
Skeletal Muscle
OLV/SK
D RV/SK
72
96
(2)
(4)
FIGURE 5
Increase in specific activity and relative incorporation of heart muscle protein 6 to 96 hours
after production of aortic stenosis.
Circulation Research, Vol. XVlll,
May 1966
ZOHLKE, DU MESNIL DE ROCHEMONT, GUDBJARNASON, BING
564
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glycine-2-C14 uptake into heart muscle protein
during the first 24 hours of actinomycin treatment (Group Hci, table 1) as compared to
sham operated animals (Group lib, table 1).
This inhibitory effect was only transient and
as early as two days postoperatively the rate
of incorporation into heart muscle protein of
this group (Group IIc2, table 1) returned to
the level observed in untreated animals with
aortic stenosis (Group Ic, table 1). Despite
further treatment with actinomycin D the
rate of incorporation even increased slightly
within 48 to 96 hours after operation (fig. 6).
Since actinomycin D inhibited glycine-2C1'1 uptake relatively more in skeletal muscle
protein than in heart muscle (fig. 6), the relative activity increased markedly (Groups IIci
and IIc2, table 1). The figures for per cent
activity in animals with aortic stenosis and
actinomycin D (Group IIci, table 1), as compared to sham operated (Group lib, table 1)
and unoperated animals (Group Ila, table 1)
with actinomycin D, were considerably lower
than the corresponding values for animals
without actinomycin treatment (fig. 4). Pleural effusions were found often in animals
treated with actinomycin D, whereas ascites
was not a common finding. End diastolic pressures were elevated in several animals with
aortic stenosis (table 2).
ANIMALS WITH REGULAR DIET AND TREATMENT WITH
PUROMYCIN (GROUP I I I , TABLE 1)
Puromycin inhibited incorporation of glycine-2-CJ;i into heart and skeletal muscle in
animals with aortic stenosis to approximately
the same extent as actinomycin D during 24
hours postoperatively (table 1). The relative
incorporation was almost the same. Again
pleural effusion, ascites and elevated end diastolic pressures were seen occasionally.
ANIMALS WITH PROTEIN-FREE DIET (GROUP IV,
TABLE 1)
In animals on protein-free diet the rates of
incorporation of glycine-2-C"' into the myocardium of unoperated (Group IVa, table 1)
and sham-operated (Group IVb, table 1)
animals were slightly higher than in animals
with regular diet. There was no difference in
the relative incorporation between control and
sham-operated animals. In animals with aortic stenosis (Group IVc, table 1) incorporation of glycine-2-C;/| into left ventricular mus-
120/ig/kg Actinomycin D ip
200
O
O
6
Operation
N=(2)
(4)
Hrs post-op.
(2)
Left Ventricle
Right Ventricle
Skeletal Muscle
O L.V./SK
-a R.V./SK
I
72
96
(3)
(3)
FIGURE 6
Effect of actinomycin D on glycine-2-C'i incorporation into heart and skeletal muscle protein
at various times after production of aortic stenosis in per cent of activity values observed in
untreated animals with aortic stenosis.
Circulation Research, Vol. XVIII, May 1966
565
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
TABLE 2
Signs of Heart Failure in Anitnals Treated with Actinomycin D
Experiment Time postop.
no.
or/ and time of
treatment
Control
1
2
3
4
hours
24
24
48
48
Sham operated
1
2
3
4
24
24
48
48
1
2
3
4
5
6
7
8
9
10
11
12
13
14
6
6
18
18
24
24
48
48
72
72
72
96
96
96
Aortic constriction
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cle protean was again significantly elevated
(P < 0.05| as compared to unoperated animals (Grcjjup IVa, table 1). The glycine-2-C"'
uptake into skeletal muscle protein increased
to 236% of the control values (fig. 4) and
therefore the relative incorporation decreased
(left ventricle 5.3; right ventricle 4.8).
Preliminary results of long term experiments (Group IVd, table 1) in which the
animals were subjected to a change from
regular to protein-free diet during the phase
of chronic cardiac hypertrophy (four months
after production of aortic stenosis) revealed
almost similar incorporation rates of glycine2-C//( into heart muscle protein as in unoperated controls (Group IVa, table 1). Here, too,
the incorporation of glycine-2-CJ4 into skeletal muscle protein was increased to almost
400% of that in unoperated controls. As a result, the relative specific activity of heart
muscle protein decreased markedly (table 1).
In control as well as in sham-operated animals on protein-free diet, end diastolic pressure in the left ventricle remained normal. No
Circulation Research, Vol. XVIII, May 1966
End diastolic
pressure
Pleural
effusion
Ascites
mm Hg
0-1
3-4
14-16
3-5
8
1-2
2-4
5-10
3-4
3
12
27
pleural effusion was found and even slight
ascites was rare. However, in animals with
aortic stenosis including the long-term group,
elevated end diastolic pressure, moderate and
severe pleural effusions and ascites were
found frequently (table 3,fig.2).
Figure 7 summarizes the effects of actinomycin D and of puromycin, as well as the
effect of protein-free diet on animals with
aortic stenosis as compared to untreated
animals with aortic stenosis on regular diet.
The incorporation of glycine-2-C1* was significantly decreased by actinomycin (left ventricle P<0.02; right ventricle P<0.05; skeletal muscle P < 0.005) and puromycin (left
ventricle P<0.02; right ventricle P<0.02,
skeletal muscle P<0.05). In both groups of
animals on protein-free diet and with aortic
stenosis (Groups IVc and IVd, table 1), skeletal muscle protein activity was markedly
increased (P<0.001).
TURNOVER RATE OF GLYCINE-2-C"
The rate of disappearance of radioactivity
from heart and skeletal muscle was deter-
566
ZUHLKE, DU MESNIL DE ROCHEMONT, GUDBJARNASON, BING
TABLE 3
Signs of Heart Failure in Animals on Protein-Free Diet
Time
postop.
Experiment
no.
End distolic
pressure
hours
Control
Sham operated
Aortic constriction
Ascites
mm Hg
1
2
3
4
5
—
1
2
3
4
24
24
48
48
1
2
3
4
5
6
7
8
9
1
2
3
24
48
48
48
72
72
96
96
96
20
4-6
10
6ms
6ms
6ms
6
0
36
—
4-5
3-4
2-3
6
10
6
5
*Animals in this group were kept on protein-free diet for only the last 6 weeks.
o
o
400
zs
Actiivi'
"c
i
t—
*
u
300
100
Right ventricle
D
266
Skeletal muscle
Q
100
75
67.5
66
72
2
25
CD
t/>
O
P
c
<o
o
67.5
56.5
46.5
50
(AN
<5
Q.
Left ventricle
UJ
1-
"o o
>, 1ac- 2 0 0
o
Deci
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Aortic constriction,
long-term*
Pleural
effusion
-50
m
-9.0 -I0.5
-2 5
-325
-34
-28
-325
-43.5
-24
-53.5
N = 7(14)
8
Control
Actinomycin
( 6 - 2 4 hrs:N=7
6-24hrs
2 4 - 9 6 hrs: N=I4)
post-op
Puromycin
2 4 hrs
post-op
Protein free diet
2 4 - 9 6 hrs 6 mo. post-op
post-op
FIGURE 7
Alteration of protein synthesis in heart and skeletal muscle by actinomycin D, puromycin,
and protein-free diet as expressed in per cent activity of values observed in untreated controls
with aortic stenosis and on regular diet.
mined in experiments in which the animals
were maintained on regular and protein-free
diet without operation. The rabbits were
sacrificed 1 to 25 days after injections of
glycine-2-C/4 (fig. 8). In animals of both
groups the incorporation of glycine-2-C'1 into
Circulation Research, Vol. XVIII, May 1966
567
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
2000
r
1500
1-8
Left Ventricle
protein-free diet
•
1000
2
UJ
I-
o
oc
a.
o»
o
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Skeletal muscle
protein- free
diet
I0OJ13
200
5 7
10 13
DAYS
17
21
25
FIGURE 8
Disappearance of radioactivity from heart and skeletal
muscle protein in animals on regular diet and proteinfree diet. Each point represents the mean value of
two experiments.
the left ventricular muscle protein reached a
maximum at about the fifth day, but was
higher in animals on protein-free diet. In
animals with regular diet the disappearance of
radioactivity after the fifth day was very
rapid, while in animals on protein-free diet
the decline was delayed up to the tenth day,
but then decreased at almost the same rate.
In contrast, incorporation of glycine-2-C3<
into skeletal muscle protein was higher in
animals on regular diet (fig. 8). The peak
incorporation occurred between the fifth and
tenth day in both groups and there was almost no decline during the time of study.
Biological half-life of total protein was determined from the semilogarithmic function
of specific activity of protein versus time,
using values obtained between the seventh
and twenty-fifth day (fig. 9).
According to these calculations, the halflife of protein in animals with regular diet was
10.5 ± 1.9 days for the left ventricle, 11.6 ± 1.8
days for the right ventricle; in animals with
protein-free diet the half-life of protein was
Circulation Research, Vol. XV111, May 1966
100
13
5
7
10
13
DAYS
17
25
FIGURE 9
Biological half life of left ventricular protein from
rabbits on regular and protein-free diet. The half life
was determined from the semilogarithmic function of
specific activity of myocardial protein versus time
from values between the 7th to the 25th day after
injection of glycine-2-C1''.
11.4 ± 1.7 days for the left ventricle and 13.8
± 2.6 days for the right ventricle.
Discussion
After the creation of an outflow resistance
the heart passes first through a stage of cardiac hypertrophy, associated with an increased
rate of protein synthesis in heart muscle. In
agreement with Meerson1-2 and Gudbjarnason et al. 3 ' 4 the results described here show
that the specific activity of left and right
ventricular muscle protein increases to 190%
of control values after production of aortic
stenosis. The relative incorporation (the ratio
of left or right ventricular protein activity
to skeletal muscle protein activity) into heart
muscle protein rises 268%. During this period
568
ZUHLKE, DU MESNIL DE ROCHEMONT, GUDBJARNASON, BING
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of induced hypertrophy no signs of heart
failure are observed.
Actinomycin D is known to inhibit DNA dependent BNA-synthesis11"14; this is the mechanism by means of which protein synthesis is
affected. No effect of actinomycin D on the
incorporation of glycine-2-C'* into heart muscle protein is found in animals without aortic
stenosis. Only if protein synthesis is stimulated
by means of aortic stenosis does actinomycin
D inhibit myocardial incorporation of glycine2-C'7' (table 1, fig. 4). This is in agreement
with findings of Schwartz,15 who investigated
the effect of actinomycin D on the incorporation of leucine-C' into protein of intact and
regenerating liver. Incorporation into intact
liver was unaffected but a moderate inhibition (64%) developed in the regenerating liver. Myocardial incorporation of glycine-2-C'
increases in untreated animals with aortic
stenosis to 190% of unoperated controls
(Group Ic, table 1). A lesser incorporation is
noticed during the first 24 hours in these
animals after treatment with actinomycin D
(Group IIci, table 1 and fig. 4); later, from
48 to 96 hours, protein synthesis increases
to more than 200% (Group IIc2, table 1).
Schwartz et al.15 also found only short duration of maximal inhibition of BNA synthesis by
a single dose of 1 mg/kg body weight of
actinomycin D. The escape from inhibition in
overall protein synthesis in the heart muscle
of animals with aortic stenosis during continued treatment with actinomycin D may
be related to observations by Giudice and
Novelli.16 They found that actinomycin D
leads only to a slight inhibitory effect on amino
acid incorporation into regenerating liver protein, but interferes with the synthesis of DNA
polymerase. They conclude that this finding
may be correlated to the relative stability of
messenger BNA. Revel and Hiatt17 observed,
in rats treated with actinomycin D, effects
which they ascribe to the possibility that a
reduction of even 50SS in nuclear messenger
BNA might be unaccompanied by diminution
of nuclear protein synthesis, since the nucleus
contains a much higher stimulatory activity
for incorporation of amino acids into protein
per unit BNA than that found in cytoplasm.18
Since in our experiments no determinations
relating BNA synthesis to protein synthesis
were made, no definite conclusions can be
drawn. It seems likely, however, that the
relatively low doses of actinomycin D were
not sufficient to bind all activated sides on
DNA following the stimulatory effect of the
aortic stenosis in long term experiments. By
this means increase of messenger BNA production could occur gradually with a related
increase in protein synthesis.19 An increase
in relative incorporation on the part of animals
with aortic stenosis, and treated with actino-,
mycin D, is noticed during the whole period of observation. However, during the first
24 hours it is the result of a relatively greater
diminution in protein synthesis in skeletal muscle, while during the later period it results
from both a decrease in skeletal muscle activity and an increase in protein synthesis in
heart muscle.
The finding of pleural effusion together
with elevated end diastolic pressure in 5 out
of 14 animals treated with actinomycin D
(Groups IIci and Co, table 1 and table 2)
illustrates that inhibition of myocardial protein synthesis makes these animals more susceptible to myocardial failure.
Puromycin inhibits protein synthesis mainly by blocking some step in the transfer of
activated amino acid from transfer BNA into
protein or by interfering with the combination of activated amino acid with its specific
transfer BNA molecule.20"27 For 24 hours puromycin decreased the incorporation of glycine2-C14 into heart muscle protein in animals
with aortic stenosis to about the same extent
as actinomycin D did (Group III, table 1
and fig. 7). As with actinomycin D the relative activity was increased. In this group 3
animals developed signs of heart failure, but
the period of observation was shorter than
that with actinomycin D.
With protein-free diet, control (Group IVa,
table 1) and sham operated (Group IVb,
table 1) animals show that the incorporation
of gIycine-2-Ci/( into left and right ventricular
muscle protein is higher than in animals on a
Circulation Research, Vol. XVIII, May 1966
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
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regular diet. It can be assumed from experimental results by Allison28 that on a proteinfree diet the free amino acid pool in tissues is
decreased, affecting the concentration of added
tracer material appearing in the protein synthesized by the tissue cell. Since in our experiments the animals received a constant
dose of glycine-2-CJ/' per body weight, the
relative concentration of the labelled amino
acid becomes greater and therefore the specific activity of protein is higher. In animals
on protein-free diet the specific activity is
increased markedly after production of aortic
stenosis (Group IVc, table 1); however, compared to animals on regular diet (Group
Ic, table 1) the increase in incorporation is
reduced (140% as compared to 190%, fig. 4).
The relative incorporation of glycine-2-C'4
into heart muscle protein in animals with
protein-free diet is decreased markedly in
animals with definite signs of heart failure
(Group IVc and IVd, table 1). In these
animals the low relative incorporation is the
result of a marked increase in the specific activity of skeletal muscle protein, which rose
to approximately 240% (P<0.05) of the values found in unoperated controls (fig. 4).
These results are in agreement with previous
results from this laboratory.3-4
The reason for the elevation in incorporation of glycine-2-CJ/| into skeletal muscle protein in heart failure is not understood. This
increased protein synthesis in skeletal muscle
in animals with heart failure is inhibited by
actinomycin D and puromycin. The results
demonstrate that inhibition of protein synthesis during the first stage of induced cardiac
hypertrophy is associated with the development of heart failure, regardless of whether
the inhibition occurs as the result of actinomycin D, puromycin or of protein-free diet.
In the long term experiments (Group IVd,
table 1) in which animals were placed for
six weeks on a protein-free diet, four months
after production of aortic stenosis, the decrease in relative specific activity of heart
muscle is due mainly to the increase in the
specific activity of skeletal muscle protein;
the specific activity of heart muscle protein
Circulation Research, Vol. XVIII, May 1966
569
is not different from that of unoperated controls. However, in one of these three animals
(Group IVd, table 1) protein-free diet for a
period of six weeks was followed by the development of cardiac failure (fig. 2), although
myocardial protein synthesis appeared to be
unchanged (table 3).
A considerable number of animals with
aortic stenosis, maintained on protein-free diet,
exhibited signs of heart failure (table 3). This
could have been the result of inhibition of
protein synthesis or of altered protein turnover rate. Gudbjarnason et al. 3 ' 4 have already
demonstrated that in animals during the first
stage of cardiac hypertrophy, protein turnover
rate is not increased. Since animals with
aortic stenosis maintained on a protein-free
diet usually fail to survive for periods longer
than four days, protein turnover rate had to
be studied in rabbits maintained on proteinfree diet alone. Therefore the results reported
here permit no conclusions on myocardial
protein turnover rate in animals with aortic
stenosis maintained on a protein-free diet, but
they do furnish information if the myocardial
protein turnover rate is changed by proteinfree diet alone. The interpretation and comparison of the results are complicated further
by evidence that protein depletion may affect protein metabolism in several ways. The
concept of "labile" protein seems to play a
central part in any description of the effects
of protein depletion as outlined by Waterlow
et al.20 He mentioned the possibility that in
protein malnutrition there may be an increase in the reutilization of amino acids liberated by tissue catabolism and a concentration of protein synthesis in the more essential
organs at the expense of the less essential
ones. Allison et al.28f 30 suggest also that the
muscle can contribute amino acids to the overall metabolic pool during periods of protein
deficiency.
Figure 8 shows that during the period of
disappearance of radioactivity from heart
muscle, the specific activity in the myocardium is always higher in animals with proteinfree diet than in animals with regular diet.
In contrast, the specific activity of skeletal
ZUHLKE, DU MESNIL DE ROCHEMONT, GUDBJARNASON, BING
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muscle is lower in animals with protein-free
diet than in those with regular diet. This difference between protein activities of heart
and skeletal muscle in these two groups could
be explained by the hypothesis of Waterlow
and Allison that in the protein depleted animal there occurs a concentration of protein
synthesis in the more vital organs.28"30 The
rate of disappearance of radioactivity from
heart muscle protein is almost identical for
animals on protein-free diet and on regular
diet (fig. 8). There is actually no decrease
in activity of skeletal muscle during the
whole period of observation (25 days, fig. 8).
This was the case in animals maintained on
regular and on protein-free diet. This slow
turnover rate agrees with results by Dreyfus,
Kruh and Schapira.31 They showed that myosin behaves like a nondynamic protein such
as hemoglobin, so far as its turnover rate is
concerned. They found that the radioactivity
of myosin remains constant until the thirtieth
day and then decreases to a lower plateau
in animals fed a diet containing 24% of protein. On the other hand, the water soluble
proteins display an exponential type of decrease in radioactivity.32
The rates of disappearance in our experiments represent only average values, since we
were dealing with a mixture of proteins. Because the disappearance of radioactivity
from heart muscle protein is almost the same
in animals maintained on protein-free or on
regular diet, it is likely that the rate of turnover of myocardial protein is not altered
greatly by protein-free diet. Calculation of
the half life of heart muscle protein, from
values obtained between the seventh to the
twenty-fifth day after injection of gIycine-2C"1, indicates only a slight prolongation of
half life in animals with protein-free diet (fig.
9). This finding is substantiated by the demonstration that the relative specific activity of
left ventricular protein, as calculated from the
seventh day on, decreases at the same rate
in both groups of animals (fig. 10). Using
essential amino acids such as methionine,
Steinbock and Tarver33 found increased half
life in plasma proteins of rats maintained on
10.0
8.0
6.0
' 4.0
Left Ventricle
protein-free diet
2.0
o
o
o
*•"<J
<D
Q.
1.0
0.8
Left Ventricle
regular diet
0.6
m
<D
0.4
ela'
570
cr
n 5
O.I
I
I
10
13
17
21
25
DAYS
FIGURE 10
Decline of relative incorporation by left ventricular
protein in animals maintained on regular and on protein-free diet.
a protein-free diet. However, it is possible that
essential amino acids are more likely to be
reutilized for protein synthesis.29
Summary
The rate of myocardial protein synthesis
was studied in hearts of rabbits with experimentally produced cardiac hypertrophy and
treatment with actinomycin D and puromycin
as well as in animals maintained on a proteinfree diet. Actinomycin D and puromycin inhibited myocardial incorporation of glycine2-CJi only if protein synthesis was stimulated
by means of aortic stenosis. Actinomycin D
had no effect on the normal myocardium.
Animals on protein-free diet also showed a
decreased rate of glycine-2-C1;i uptake into
heart muscle protein after production of aortic stenosis, but an increased incorporation
into skeletal muscle protein. Inhibition of
protein synthesis during the first stage of
cardiac hypertrophy was associated with the
development of heart failure, regardless of
whether the inhibition occurred as a result of
actinomycin D, of puromycin or of proteinfree diet. As compared to animals maintained
Circulation Research, Vol. XVIII, May 1966
CARDIAC HYPERTROPHY, FAILURE AND PROTEIN SYNTHESIS
on a regular diet, myocardial protein turnover
rate in unoperated animals was not altered
significantly by protein-free diet.
control. New Engl. J. Med. 271: 1252 and
1301, 1964.
15.
16.
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Circulation Research, Vol. XVIII, May 1966
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Inhibition of Protein Synthesis in Cardiac Hypertrophy and its Relation to
Myocardial Failure
Volker Zühlke, Wolfgang du Mesnil de Rochemont, Sigmundur Gudbjarnason and
Richard J. Bing
Circ Res. 1966;18:558-572
doi: 10.1161/01.RES.18.5.558
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue,
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Copyright © 1966 American Heart Association, Inc. All rights reserved.
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