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Protein and Nucleic Acid Synthesis
During the Reparative Processes
Following Myocardial Infarction
By Sigmundur Gudbjarnason, Ph.D., Christian De Schryver, M.D.,
Chiyo Chiba, M.D., Jiro Yamanaka, M.D., and Richard J. Bing, M.D.
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• In the past, investigations dealing with
biochemical changes accompanying myocardial infarction have been primarily directed
toward studies concerning the release of enzymes by the heart muscle.1' 2 Studies in dogs
have led to the conclusion that the first reaction in myocardial infarction is necrosis
followed by infiltration with polymorphonuclear leukocytes; finally collagen formation
takes place.8-4 However, detailed investigation of the biochemical nature of the reparative processes of infarction as a function of
time has not yet been performed. The purpose of this report is to study the protein and
nucleic acid synthesis at the subcellular level
during the reparative processes following
myocardial infarction. Of particular interest is
the sequence of events in the reparative process. The question will be examined as to the
portion of the cell in which the reparative
process commences.
Methods
Surgical production of myocardial infarction
was performed on 37 mongrel dogs (10 to 15
kg) under phenobarbital anesthesia (25 mgAg).
An area of the left ventricle was chosen which
was supplied by the various branches of the anterior or posterior descending coronary artery.
An infarcted area, two to three cm in diameter,
was obtained by ligan'on of the branches of the
From the Department of Medicine, Wayne State
University College of Medicine, and Harper Hospital, Detroit, Michigan.
Supported by Grant HE-05043, U.S. Public Health
Service, and by the American Heart Association,
Michigan Heart Association, Life Insurance Medical
Research Fund, Tobacco Industry Research Committee, Burroughs-Wellcome Fund, and John A. Hartford Foundation.
Received for publication March 26, 1964.
320
arteries and accompanying veins with silk thread.
Histopathologic studies were performed on infarcted tissue, borderline or marginal tissue, and
the control sample. Since the results have been
reported previously, no further reference will be
made to the histological pattern of the infarcted
areas.8
Femoral blood pressure and electrocardiogram
were recorded with a Sanborn multichannel recorder before and after the surgical procedure.
Systolic blood pressure fell on the average 40 mm
Hg in 50% of the dogs immediately after the
infarction; in the remaining dogs it did not
change. Although the infarct involved a relatively
small area of the left ventricle, only 70% of the
dogs survived the operation; the remainder died
shortly after occlusion.
The classical signs of acute myocardial subepicardial infarction were present in the electrocardiogram, with an elevation of the ST segment,
inversion of T waves and deep Q and QS waves,
especially in AVL and AVF, confirming the
presence of subepicardial infarction. Arrhythmias
were uncommon in animals which survived. Before sacrifice the electrocardiogram revealed deep
Q and QS waves.
Glycine-2-C^ (specific activity 15.1 me/
mmole) was injected intravenously, 30 /tcAg
body weight. This amino acid was selected because of its incorporation into both protein and
nucleic acids, which allows a simultaneous study
of protein- and nucleic acid synthesis. The animals
were sacrificed at intervals, from four hours to six
weeks, after infarction. Injection of glycine-CJ<
was carried out four hours prior to sacrificing the
animals.
The study was divided into two parts. In the
first, the overall changes in protein and nucleic
acid metabolism were studied. In the second,
the changes in protein and nucleic acid synthesis
of subcellular fragments were observed during
the reparative process. In the first group three
samples were obtained from the left ventricular
muscle: a) the infarcted tissue, b) borderline
tissue (close to the infarcted tissue), and c) normal left ventricular tissue, obtained close to the
left atrium. The latter sample served as control.
The incorporation of glycine-C1* into this healthy
Circulation Rtserrcb,
Vol. XV, Ocioter 1964
PROTEIN SYNTHESIS AND MYOCARDIAL INFARCTION
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tissue did not diifer significantly from the incorporation into left ventricular protein collected
from normal dogs without myocardial infarction.
In addition, a sample of right ventricular muscle
was also collected. All tissues were rapidly removed, washed with cold isotonic NaCl solution,
and frozen in liquid nitrogen. Proteins were isolated according to the method of Schneider et
al.5 RNA was determined with the orcinol reaction, according to Dische.0 The RNA standard was
made of purified yeast RNA, digested in 1 N NaOH
and acidified with perchloric acid. Two standards
of 10 and 20 fig RNA/ml were used with even'
analysis. The final RNA concentration in the standard solution was determined by ultraviolet spectrophotometry. The conversion factor used was
33.16, i.e., OD (optical density) at 260 myx X
33.16 = fig RNA/ml. This factor was calculated
on the basis of a 9.OX phosphorus content for
RNA.7 Since DXA showed positive reaction with the
orcinol method, the optical density due to DNA
was subtracted from total absorption before calculating the concentration of RNA.
DNA was determined with the indole reaction
according to Ceriotti.8 DNA standards were prepared from purified calf thymus DNA * which was
treated the same way as the sample. The final
concentration of the standard solution was determined by ultraviolet spectrophotometry using the
conversion factor 32.94, i.e., OD at 267 m^t, X
32.94 = fig DNA/ml. This factor was calculated
on the basis of a phosphorus content for DNA of
Cell fractionation was carried out by differential
centrifugation. The control specimen and infarcted tissue were homogenized in isotonic
sucrose (0.25 M ) containing 0.003 M CaCl2, and
filtered through cheesecloth. Nuclear fractionation was performed according to a modified
method of Sibatani et al.10 The nuclear fraction
(containing also the bulk of myofibrils) obtained
from the sucrose-CaCb medium was metabolically
intact and incorporated labeled amino acids
(C 4 -protein hydrolysate) into the proteins as
determined with the method of Allfrey et al.11
The nuclear fractionation consisted of: (1) Extraction of nuclear ribosomes (Nj), together with
other proteins of the soluble phase of the nucleus,
with 0.1 M tris HC1 buffer, pH 7.6, containing
0.003 M CaCU. The particles were then removed
from the extract by centrifugation at 100,000 g.
The nuclear ribosomal RNA was hydrolyzed with
1 N NaOH for one hour at 37°C and purified on
a charcoal column.12 (2) Extraction of DNA, RNA,
and nucleoprotein (N n ) with 1 M NaCl for 16
hours at 2°C. The viscous solution was centrifuged
* Mann Research Laboratory.
Circtldtio* Raurcb, Vol. XV, October 1964
321
at 40,000 g and the supernatant was decanted
and acidified with perchloric acid, RNA was hydrolyzed with 1 N NaOH for one hour at 37°C and
the residue subjected to further digestion overnight with 0.1 N NaOH to remove residual RNA.
The DNA was hydrolyzed with 1.6 N PC A at
70°C and purified on charcoal. (3) Phenol fractionation. The 1 M NaCl residue was suspended
in 10 volumes of 0.05 M phosphate buffer at pH
6.85. An equal volume of 90% (w/v) phenol was
added to the suspension and the mixture was
shaken for one hour at 25°C. Centrifugation at
1000 g for 30 minutes resulted in the separation
of an aqueous phase which overlay a considerable interphase layer, a phenol phase, and a sediment at the bottom of the tube. The aqueous
layer was removed and the phenol phase plus
interphase washed once with an equal volume of
phosphate buffer. This phenol treatment separated
the RNA into two phases: an "aqueous phase" RNA
fraction of relatively low activity (P-RNA), and an
"interphase" fraction with very high activity (mRNA) . The p-RNA was precipitated with potassium
acetate (2%) and two volumes of 95% ethanol.
The m-RNA was digested with 1 N NaOH and
purified on charcoal. All nucleic acid fractions
were purified on charcoal, except p-RNA, which
did not need such purification, but was washed
repeatedly with ethanol-ether to remove residual
phenol. The purity of the nucleic acids was tested
in Beckman DK2 spectrophotometer and the
absorption at 260 mfi and 267 mfi was used for
the quantitative estimation of RNA and DNA respectively.
The protein in the interphase was labeled
N l l r and the protein fraction from the sediment
at the bottom of the tube was labeled N IV . The
N t v protein fraction contained the bulk of contractile proteins. In order to identify more clearly
the protein fraction in which the contractile
proteins were located, myosin was isolated from
dog heart muscle and subjected to identical treatment. At the end of the phenol treatment the
myosin was collected as Ntv fraction. Sibatani et
al.,10 working with calf thymus, obtained only a
slight sediment on the bottom of the tube following phenol treatment (N1V fraction). In our case
most of the protein of cardiac muscle was collected as the N IV fraction. It can therefore be
assumed that most of the protein obtained as N IV
fraction consists of contractile protein mixed with
some nuclear protein. Hereafter the NIV fraction
will also be called the "contractile protein"; it is
understood, however, that this fraction also contains some nuclear protein.
The radioactivity of nucleic acids was determined in a liquid scintillation counter using the
dioxane scintillator of Bray.18 The protein fractions were treated with 1.6 N PCA at 70°C to
GUDBJARNASON, De SCHRYVER, CHIBA, YAMANAKA, BING
322
remove possible residual nucleic acids; lipids were
removed with ethanol, ethanol-chloroform, ethanol-ether, and ether. The protein was washed,
dried, and the radioactivity was determined by
suspending the protein powder m a toluene-gel
scintillator.34
100-
•s
Results
5<
H
EFFECT OF MYOCARDIAL INFARCTION UPON OVERALL
SYNTHESIS OF PROTEIN AND NUCLEIC ACIDS
IN INFARCTED TISSUE
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The effect of myocardial infarction upon
the incorporation of glycine-2-C14 into the
protein of infarcted and borderline tissue is
illustrated in figure 1. The incorporation into
the control tissue is considered 100% and the
specific activity of protein obtained from infarcted and borderline zones is related to this
control sample. The infarcted tissue showed a
rapid diminution in incorporation of glycine
into cardiac protein after infarction; the incorporation fell immediately after infarction to
70$ of normal. Two days after infarction the
rate of incorporation rose to normal and
reached a maximum of 207$ of the control on
the fourth day. Then the rate of protein synthesis declined and approached control values
six weeks after infarction. The borderline tissue showed an earlier increase in protein synthesis with maximum incorporation two days
after infarction. The rate of incorporation in
the borderline tissue approached normal in200-
I (Infarct)
50
2 4 6 8 10
21
42
DAYS
FIGURE 1
Effect of myocardial infarction on protein synthesis
of infarcted and borderline tissue. Each point represents mean value of three experiments.
42
2 4 6 8 10
DAYS
FIGURE 2
Specific activity of leukocyte protein compared to
specific activity of protein from infarcted tissue. Incorporation into infarcted tissue is considered as
100%.
corporation sooner than in the infarcted tissue.
Since infarcted tissue shows an early infiltration of leukocytes,8 it was thought possible that these cells may be responsible for
the increased radioactivity observed in protein from infarcted tissue. In order to study
the magnitude of radioactivity transported by
the leukocyte protein into infarcted tissue,
leukocytes were isolated from blood by centrifugation and repeated washing with isotonic NaCl solution and sucrose solution. The
protein obtained from leukocytes was then
treated in the same manner as heart muscle
protein obtained from the infarcted area and
the specific activity was determined. It was
found that the specific activity of leukocyte
protein varied from 24 to ffl% of the activity
of the protein obtained from infarcted muscle
(fig. 2). Since the specific activity of protein
obtained from leukocytes was lower than that
of infarcted tissue, it is considered unlikely
that infiltrating leukocytes were responsible
for the specific activity of the protein obtained from the infarcted tissue.
Concentrations of the nucleic acids (mg/g
tissue) of the damaged tissue changed considerably following infarction (fig. 3). In
general, alterations in the concentration of
RXA followed a course similar to that observed
for the incorporation of glycine-2-C14 into
protein. Thus, immediately following operaCircuUiion Resurcb, Vol. XV, Oaobtr 1964
323
PROTEIN SYNTHESIS AND MYOCARDIAL INFARCTION
TABLE 2
200-
Specific Activity of Nucleic Acids in
Two Days After Infarction
Normal
Sample
Microsomal
Mitochondrial
Nuclear ribosomal
891
981
323
108
1.840
p-RNA
m-RNA
100-
50
2 4 6 6 10
42
21
counts/min/mg
Infarct
4.107
1.518
1.314
360
8.401
300-
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DAYS
FIGURE 3
Effect of myocardial infarction on RNA and DNA
content of infarcted tissue.
1200-
TABLE 1
Specific Activity of Myocardial Protein Fractions in
counts/min/10 mg Two Days After Infarction
Sample
Cytoplasma
n= 7
Microsomes
n= 7
Mitochondria
n= 7
Nuclear ribosomes
n= 7
n= 7
n= 7
N,v
n= 7
Normal
Infarct
951
±60*
1243
±213
911
±111
890
±92
740
±80
606
±47
590
±65
1249
±83
2239
±140
1739
±247
2099
±250
1122
±94
837
±71
464
±92
* ± standard error of the mean.
tion, the RNA content of the infarct decreased,
but rose again above normal four days after
infarction (fig. 3). The concentration of DNA
increased on the second day after infarction
and reached a maximum of 90% above normal
eight days later. The borderline tissue showed
little change in the concentration of nucleic
acids.
EFFECT OF MYOCARDIAL INFARCTION UPON
SUBCELLULAR FRACTIONS OF INFARCTED TISSUE
The specific activities of protein fractions
CircmUtion Rtiurcb,
Vol. XV, Oaobtr
1964
100-7
4hr
4
6
^k _ 10
Doyt offtr infarction
FIGURE 4
Effect of myocardial infarction on incorporation of
glycine-C14 into subceUular protein fractions of infarcted tissue.
from normal and infarcted tissue two days after infarction are shown in table 1. The greatest increase in incorporation was observed in
nuclear ribosomes, cytoplasmic microsomes,
and mitochondrial fractions; there was subnormal incorporation into the NIV fraction
which contains, along with some nuclear proteins, the contractile proteins.
The incorporation of glycine-2-C14 into
nucleic acids is illustrated in table 2, which
shows the specific activities of HNA fractions
two days after infarction and the increased
activity of all fractions in the infarcted tissue.
Of interest is the marked difference in specific activity of "nucleolar" HNA (P-HNA) and
"messenger" RNA (m-RNA).
GUDBJARNASON, De SCHRYVER, CHIBA, YAMANAKA, BING
324
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Figure 4 shows the incorporation into subcellular protein fractions of infarcted tissue
compared to the control sample as a function
of time after infarction. Each point represents
the mean value of three to seven experiments. Incorporation into the nuclear ribosomal fraction precedes all other fractions,
followed by mitochondria and cytoplasmic
microsomes. The NIV fraction, which contains
the bulk of the contractile proteins, shows
an early fall in incorporation which remained
subnormal during the period of observation.
The incorporation of glycine-C14 into nucleic
acids of infarcted tissues as compared to the
control sample is shown in figure 5. The
earliest increase in incorporation was observed in "nucleolar" RNA or p-RNA, followed
by nuclear ribosomal RNA. The activity of the
DNA fraction increased 48 hours after infarction.
Discussion
An attempt is made in this study to follow
the reparative process in cardiac muscle, following myocardial infarction, as connective
tissue replaces the necrotic muscle.
The main steps in the mechanism of protein
synthesis involve the formation of messenger
p-RNA(nucleolar RNA)
400-RNA (nuclear ribosomal RNA)
attachment of HI-RNA to ribosomes and
formation of ribosomal aggregates, transfer of
activated amino acids by transfer RNA to the
m-RNA template, and polymerization of the
amino acids into polypeptide chains. In our
study an effort was made to follow the incorporation of glycine-2-C14 into the purine
ring of m-RNA, DNA, p-RNA (which is a nucleolar RNA), and ribosomal RNA. The incorporation of glycine-C14 into subcellular protein
fractions is also studied.
The results indicate that four hours after
coronary occlusion the incorporation of glycine-C14 into myocardial protein of infarcted
tissue was reduced to 70% of control (fig. 1).
The question may be asked: Could this fall in
incorporation be the effect of the circulatory
arrest on the transport of the labeled compound to the infarcted area or could it be the
result of inactivation of the protein synthesizing mechanisms of the cell? Most probably
both factors contribute to the diminution in
incoqjoration. The circulatory arrest results
in cell destruction and inactivation of the protein synthesizing mechanism. The first samples were obtained four hours after infarction and represent changes during that
period. The acute circulatory arrest after infarction is followed by compensatory vascular
dilatation of collaterals. The collateral blood
supply will provide the infarcted area with
more labeled precursor, but these changes in
local precursor concentration may have an
effect upon overall incorporation. However,
the differences in the rate of incorporation into the various cell components will not be
influenced by an overall increase or decrease
in precursor concentration or specific activity.
RNA,
The changes in concentration of RNA subsequent to infarction follow a course similar to
that observed for the incorporation of glycine-Ci4 into the protein (fig. 3). This indicates an early inactivation of the protein
synthesizing mechanisms of the cell, paralleled by a decrease in the cellular content of
HNA.
1
Incorporation of glydne-C * into intranuclear nucleic
acid fractions of infarcted tissue.
The subsequent increase in cellular HNA
content and incorporation of glycine-C4 into
the protein is in line with the function of RNA
CircnUiioH Rtsurcb,
Vol. XV, October 1964
PROTEIN SYNTHESIS AND MYOCARDIAL INFARCTION
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in protein synthesis and increased quantity of
microsomes in growing cells.15'16 The decrease in RN'A content is not paralleled by a
similar change in the DNA content. The concentration of DNA increases on the second day
after infarction and reaches a maximum of
902 above normal eight days later. These
changes reflect the renewal of cell material in
the injured area. Histologic findings parallel
these changes and show early appearance of
mitotic figures and an increase in number of
fibroblasts, as necrotic muscle is being replaced by connective tissue.3 A rise in overall
incorporation occurring 48 hours after infarction illustrates the rapid recovery of protein
synthesis. The early increase in protein synthesis of the borderline tissue demonstrates
the compensatory reaction of the uninvolved,
neighboring portion of the myocardium. However, the borderline tissue fails to show significant changes in concentration of nucleic
acids. Histologically, this area is characterized
by hypertrophy and an increase in the dimensions of the cells. These observations indicate
that local factors may play an important role
in the development of cardiac hypertrophy.
The studies on the subcellular fractions indicate that the reparative process starts in the
cell nuclei as early as 24 hours after infarction. The nuclear ribosomal fraction shows the
earliest increase in incorporation followed by
that in mitochondria and microsomes (fig. 4).
The increased incorporation into the subcellular protein fractions is a function of the reparative process and formation of scar tissue.
True muscle regeneration does not take place,
as indicated by the decreased incorporation
into the "contractile protein" fraction (Ni V ).
The early renewal of cell material is further
demonstrated by the incorporation of glycineC14 into nucleic acids of the infarcted tissue
(fig. 5). The earliest increase in incorporation
was observed in "nucleolar" M A or p-RNA,
followed by nuclear ribosomal ENA. DNA
shows an increase in incorporation 48 hours
after infarction, which coincides with the
appearance of new fibroblasts.
The metabolic heterogeneity of the isolated
RNA fractions, shown in table 2, reflects disCircuUtion Rtlurch, Vol. XV, Octobtr 1964
325
tinct origins and functions for the different
fractions. It has been demonstrated by Sibatani et al.10 that treatment of nuclei with
aqueous phenol under appropriate conditions
separates RNA into a "low turnover" RNA or
"nucleolar RNA I" released into the aqueous
phase, and a "high turnover" HNA or "nucleolar RNA II" (messenger RNA ) remaining in the
interphase formed between the aqueous layer
and the phenol phase. Our observations are in
agreement with these studies and show a
metabolically relative inert p-RNA or "nucleolar" fraction and a m-RNA fraction with a high
turnover rate. The m-RNA fraction has 18
times greater specific activity than p-RNA.
The role of cytoplasmic microsomes in protein synthesis has been studied extensively in
the past; more recent discoveries suggest1T
that the functional units of protein synthesis
are ribosomal aggregates, "ergosomes," consisting of particles held together by m-RNA.
The accelerated rate of protein synthesis after
infarction is reflected in the increased incorporation into m-RNA. Two days after infarction the incorporation into m-RNA was increased 4.6 times. Similarly, the incorporation
into cytoplasmic microsomal RNA also increased 4.6 times two days after infarction.
The observed similarity in increase in the
metabolic activity of both m-RNA and microsomal RNA can be explained by their functional relationship in the intensified protein
synthesis in the infarcted tissue. The identical
increase in specific activity of m-RNA and
microsomal RNA could, however, also be explained as the result of m-RNA being a precursor to microsomal RNA, although this
hypothesis is not popular at the present time.
The sequence of labehng of the protein
fractions indicates a possible nuclear origin of
cytoplasmic particles.18 The first step in the
reparative process seems therefore to be synthesis of nuclear material, followed by reconstruction of mitochondria and microsomes.
Summary
The effect of myocardial infarction upon
protein and nucleic acid synthesis of the heart
was studied. Myocardial infarction was pro-
GUDBJARNASON, De SCHRYVER, CHIBA, YAMANAKA,
326
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duced in dogs by ligation of branches of the
anterior or posterior descending coronary artery. The rate of incorporation of glycine-2C14 into heart muscle protein was studied
over a period of six weeks.
The infarcted tissue showed a rapid diminution in protein synthesis after infarction; on
the third day the incorporation into the infarcted tissue rose above normal and reached
a maximum on the fourth. The borderline tissue also demonstrated an early increase in
protein synthesis with a maximum incorporation 48 hours after infarction.
The changes in the concentration of KNA
paralleled those of the incorporation of glycine-CJ4. The concentration of DNA increased
on the second day after infarction and
reached a maximum on the tenth day.
Studies on the incorporation of glycine-Ci4
into subcellular fractions showed an early
renewal of cell material in the infarcted area.
Incorporation into nuclear ribosomal fraction
increased first, followed by mitochondria and
microsomes. The incorporation into "contractile proteins" remained subnormal. The earliest increase in nucleic acid synthesis was
observed for "nucleolar RNA" (P-HNA) followed by nuclear ribosomal BNA. DNA showed
an increase in incorporation 48 hours after
the infarction.
The studies demonstrate the rapid reaction
of the heart muscle to injury and the increase
in protein and nucleic acid synthesis of infarcted and borderline tissue. The first step in
these reactions is the synthesis of nuclear material followed by the reconstruction of mitochondria and microsomes.
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Circulation Rttetrd,
Vol. XV, Octoktr 1964
Protein and Nucleic Acid Synthesis During the Reparative Processes Following Myocardial
Infarction
SIGMUNDUR GUDBJARNASON, CHRISTIAN DE SCHRYVER, CHIYO CHIBA, JIRO
YAMANAKA and RICHARD J. BING
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Circ Res. 1964;15:320-326
doi: 10.1161/01.RES.15.4.320
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1964 American Heart Association, Inc. All rights reserved.
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