Protein Synthesis and Amlno Acid
Transport in the Isolated Rabbit
Right Ventricular Papillary Muscle
EFFECT OF ISOMETRIC TENSION DEVELOPMENT
By Myron B. Peterson and Michael Letch
With the Technical Assistance of Alan G. Ferguson
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ABSTRACT
To investigate the effects of isometric tension development on myocardial
protein metabolism, 14C-phenylalanine incorporation into protein was studied
in the isolated rabbit right ventricular papillary muscle. Amino acid
incorporation was linear for 6 hours in resting muscles and was totally inhibited
by ICHM cycloheximide and ICHM puromycin. Phenylalanine incorporation
into total protein was unaltered by 90 minutes of isometric tension development
at peak tension at stimulation rates of 30, 50, or 100/min. Significant increases
were noted in muscles stimulated at 50 and 100/min for 180 minutes
(P < 0.01). Electrical stimulation, in the absence of isometric tension development, was not responsible for this effect. Passive stretch also appeared to stimulate incorporation, although to a lesser degree than did active tension development. The specific activities of the intracellular phenylalanine pools were
identical in control and stimulated muscles. Alpha-aminoisobutyric acid was used
to evaluate the effect of tension development on myocardial amino acid transport.
Enhanced transport was noted in muscles stimulated isometrically at 30, 50, or
100/min at peak tension. The increased transport ratios could not be solely
attributed to active tension development since passive stretch resulted in
comparable changes. This study indicates that both passive stretch and tension
development are important in regulating myocardial protein synthesis.
KEY WORDS
passive stretch
alpha-aminoisobutyric acid
phenylalanine
• The heart will hypertrophy when it is
exposed to a sustained increase in mechanical
work (1-3). This is a positive adaptation which
permits the myocardium to meet increased
work demands, albeit with a probable decrease in efficiency (4, 5). Alterations in
protein and nucleic acid metabolism have
been described in many investigations of
From the Cardiovascular Division, Department of
Medicine, Harvard Medical School, Peter Bent
Brigham Hospital, Boston, Massachusetts 02115.
This investigation was supported by U. S. Public
Health Service Grants 11306, 2R01-HE09714, and
5T1HE5890-02 from the National Heart and Lung
Institute.
Dr. Lesch is an Established Investigator of the
American Heart Association.
Received December 6, 1971. Accepted for publication June 20, 1972.
Circulation Rtnarcb, Vol. XXXI, Stplemitr 1972
mechanochemical coupling
cardiac work
hypertrophied and mechanically stressed myocardial tissue (2, 3). Enhancement of protein
and nucleic acid synthesis, which appears to
be responsible at the molecular level for the
hypertrophic process detectable at the organ
level, has been routinely found in myocardial
tissue undergoing active hypertrophy. The
inability to initiate (6) or to maintain (7)
these particular changes in protein and nucleic
acid metabolism in response to mechanical
stress has been associated with the appearance
of mechanical failure and suggests that these
biochemical alterations are requisite for the
maintenance of cardiac integrity.
Laboratory study of the hypertrophic process in general and of the mechanical
regulation of myocardial protein and nucleic
317
318
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acid metabolism in particular has been hindered by the lack of a naturally occurring
experimental model. Hypertrophy has been
induced in vivo with surgical and pharmacological procedures which increase the work of
the heart by raising systemic blood pressure or
creating a high output state (8-10). Also,
isolated perfused heart preparations have
been subjected to flow or pressure loads in
vitro, and the biochemical responses of the
myocardium to these interventions have been
evaluated (11). Although these techniques
"stress" the heart, an adequate definition of
the mechanical variables involved is not
possible due to the complexity of intact heart
preparations.
In certain studies, isolated perfused guinea
pig hearts have been used to investigate both
the mechanical stimuli initiating myocardial
hypertrophy and the exact nature of the
induced biochemical responses. These investigations showed that (a) augmented volume
or pressure loads enhanced ventricular protein
synthesis (11), (b) synthesis of contractile
proteins, compared to that of myoglobin and
collagen, was preferentially stimulated (12),
and (c) an increase in nuclear ribonucleic
acid polymerase activity preceded the observed alterations in protein synthesis (13).
Coronary flow rates were adequately controlled in these studies, although alterations in
coronary perfusion pressure and transmyocardial wall stress could not be. In contrast, in
isolated rat hearts no difference was observed
in the rate of amino acid incorporation into
the contractile proteins of hearts obtained
from rats subjected to subdiaphragmatic
aortic constriction 24-48 hours before perfusion
(14). However, the hearts from control and
experimental rats were perfused under identical loading conditions in these studies. In a
similar preparation, Florini and Dankberg
(15) did not detect an early stimulation of
contractile protein synthesis. If these experimental preparations all impose a mechanical
stress on the heart, it is clear that the response
of the protein synthetic mechanism is not the
same in all cases. This could be due to species
differences or to disappearance of the bio-
PETERSON, LESCH
chemical effect as soon as the mechanical
stimulus is removed. The latter seems unlikely
because Moroz (16) demonstrated enhanced
cell free-protein synthesis in a system obtained
from rabbit hearts 24-48 hours after creation of
aortic stenosis. Species differences appear not
to be a factor since Posner and Fanburg (8,17)
observed hypertrophy and induced alterations in ribonucleic acid metabolism in rat
hearts following 24 hours of aortic constriction.
In an attempt to more precisely examine the
relationship of mechanical stress to myocardial
protein metabolism, we used the isolated
rabbit right ventricular papillary muscle preparation. Standard myographic techniques can
be applied to this preparation, and easily
defined mechanical loads can be imposed. In
initial studies (18), we described the influence
of passive stretch on amino acid transport in
the isolated papillary muscle. In the present
study, we investigated the effect of repeated
isometric contraction on myocardial protein
synthesis (amino acid incorporation) and
amino acid transport.
Methods
New Zealand white rabbits (2.0-2.5 kg) were
kept and fed as previously described (18). The
rabbits were killed by a sharp blow to the head,
the hearts were rapidly removed, and the right
ventricular papillary muscles were dissected free.
The cross-sectional area of these muscles varied
from 0.2 to 0.7 mm2, with an average of 0.4 mm2.
The base of the muscle was mounted in a springloaded clamp and the tendon attached directly to
a force transducer by a short length of 4.0
surgical silk (19, 20). Force transducers were
mounted vertically above the muscles on supports
which could be accurately adjusted in 0.1-mm
increments. The muscles were electrically stimulated at rates of 30, 50, or 100/min with field
stimuli (4 msec) at voltages 10-20% above
threshold. Each bath contained 4.0 ml of Krebs
bicarbonate buffer oxygenated with 95* O2-5%
CO 2 at a temperature of 34°C (18). Isometric
tension development was continuously recorded
on a Hewlett-Packard oscillographic recorder.
Only rabbit hearts that contained two suitable
papillary muscles were used to evaluate the
response of the protein synthetic mechanism to
mechanical interventions in these studies, since
preliminary experiments with random muscles
yielded inconsistent data because of biological
Circulation Rtsircb, Vol. XXXI, Sepumbtr 1972
319
ISOMETRIC TENSION AND MYOCARDIAL PROTEIN SYNTHESIS
variability (21). The metabolic dependencies of
protein synthesis in the isolated papillary muscle
were determined by incubating the muscles in 5ml micro-Fernbach flasks placed in a Dubnoff
metabolic shaker, as reported earlier (18).
EXPERIMENTAL PROTOCOL
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Each muscle was incubated for 30 minutes
prior to study. Zero-tension control muscles were
maintained at zero tension and all others at low
tensions during this time. The initial buffer was
replaced with buffer containing hexose-free inulin (7.5 mg/ml), the stimulation rate was set (30,
50, or 100/min), and the length-tension curve
was defined. The experiment was initiated by the
addition of the appropriate 14C-labeled amino
acid (alpha-aminoisobutyric acid, leucine, or
phenylalanine). The individual muscles were
removed from the baths, rinsed with 5 ml of
distilled H2O, blotted, and frozen between blocks
of dry ice to terminate the incubation. If
determinations of wet weight were made, the
samples were weighed before freezing. Each
muscle was then Iyophilized to a constant weight,
which was recorded as dry weight.
AMINO ACID INCORPORATION
Either uniformly labeled 14C-Z-phenylalanine or
uniformly labeled 14C-J-leucine (New England
Nuclear Corporation) was added to the medium
to a final concentration of 80 fjM unless otherwise
stated. Total radioactivity in the medium exceeded the total radioactivity incorporated into muscle
protein by a factor of 400 and ranged in concentration from 3 X 10° to 6 X 106 dpm/ml medium.
Nineteen naturally occurring amino acids other
than the tracer were each present in final
concentrations of 100 ^IM unless otherwise
stated.
At the termination of the incubation period,
muscles were treated as described above. After
lyophilization, the dry muscle was homogenized
in 0.4 ml of 0 . 8 5 M NaOH containing unlabeled
phenylalanine or leucine (1 mg/ml), and the
volume of the homogenate was adjusted to 1.0 ml
with 0 . 0 5 M NaOH. Carrier protein solution (0.3
ml) and 50% trichloroacetic acid (TCA) (0.12
ml) were added to 0.3 ml of the muscle
homogenate, and the mixture was allowed to
precipitate at 4°C for 1 hour. The precipitate was
collected by centrifugation and washed according
to the method of Siekevitz (22). The TCA
solutions used in the protein washing procedure
contained unlabeled phenylalanine or leucine (1
mg/ml). Purified protein was solubilized in 0.5
ml of Soluene (Packard) and quantitatively
transferred to a liquid scintillation vial before
adding 10 ml of scintillation fluid (1 liter of
toluene, 10 g of PPO, 0.5 g of dimethyl-POPOP).
Quench standards were prepared with factoryCtrcaUiion Rtsesrcb, Vol. XXXI, Stpltmbtr 1972
calibrated I4C-toluene (New England Nuclear
Corporation), and efficiency was determined by
the Channels ratio method (23).
Appropriate control studies demonstrated the
adequacy of the protein washing procedure.
Labeled amino acids added to a basic homogenate of unlabeled muscle could be quantitatively
recovered in the wash fractions with no activity
remaining in the protein pellet.
Protein isolated from muscles incubated with
14
C-phenylalanine was solubilized and treated
with dinitrofluorobenzene (24). Less than 5% of
the incorporated radioactivity was found in an Nterminal position regardless of incubation time
(1-6 hours). These data indicate that the
nonspecific absorption of tracer was minimal and
that incorporation could be equated with peptide
bond formation.
The protein content of a 0.5-ml sample of the
initial muscle homogenate was determined by the
method of Lowry et al. (25). Dry weight and
protein content of muscles were linearly related
(Fig. 1), and percent dry weight remained
constant. Therefore, since amino acid incorporation varied linearly with muscle dry weight (Fig.
2), identical comparative results were obtained
when incorporation rates were expressed as
dpm/mg protein, dpm/mg wet weight, or
dpm/mg dry weight (Fig. 3).
CARRIER PROTEIN PREPARATION
A carrier protein solution was prepared by
homogenizing rabbit ventricular muscle in four
volumes of 0.9% NaCl containing unlabeled
phenylalanine or leucine (1 mg/ml) and centrifuging the homogenate at 14,000 g for 20
minutes. The supernatant solution was decanted
and centrifuged at 100,000 g for 90 minutes.
too
700
MO
-
•
WO 400
.
300
.
700
100
.
.
.
'
;
.
"
:
'
•
•
•
'
-
i
ICO
i
WO
i
M0
i
400
i
»0
DIY
i
*OO
i
i
i
no
no
wo
i
i
i
tooo noo 1300
W[ IOHI „,
FIGURE 1
Protein content of 46 individual papillary muscles
plotted as a function of muscle dry weight.
320
PETERSON, LESCH
Protein concentration in the supernatant fluid
from the final centrifugation was adjusted to 2
mg/ml, and the solution was stored at 4°C until
it was used.
, . J9.»«IO'i - 0 . 7 . 10"
, . 0 90
- 8 »
\ I"
O
10
5
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DKY WIIOHI ;-(]
FIGURE 2
Total incorporation of uniformly labeled 1*C-lphenylalanine into myocardial protein in 20 individual
muscles as a function of muscle dry weight. Incubation time was 1 hour.
o
O
u
Z
TIME
IHOUIS]
FIGURE 3
Incorporation of uniformly labeled 1*C-\-phenylalanine into total myocardial protein as a function of
time. The broken line represents incubation with all
amino acids except phenylalanine added to a final
concentration of 100/JM each. The solid line represents
incubation with only labeled phenylalanine at a final
concentration of 80 /UH. All values represent means ±
SE obtained from eight to ten individual muscles.
DETERMINATION OF INTRACELLULAR PHENYLALANINE
Multiple muscles from an experimental group
of rabbits were pooled and homogenized in 0.5 ml
of distilled water. Crystalline sulfosalicylic acid
was added to a final concentration of 5 g/100 ml,
and the suspension was centrifuged. The concentration of free amino acids and the radioactivity
of the individual amino acids in the supernatant
fraction were determined with a Technicon amino
acid analyzer (26) equipped with a streamsplitting device.1 The radioactivity in the supernatant fraction was quantitatively recovered
(9935) in the phenylalanine fraction, indicating
that no significant metabolic conversion of the
tracer phenylalanine had occurred.
INTRACELLULAR RADIOACTIVITY
A precise determination of the specific activity
of the intracellular tracer amino acid pool could
not be repeatedly obtained in individual muscles
due to the small amount of tissue available
(average muscle wet weight was 2-5 mg) and to
the use of carrier amino acid in the protein
washing procedure. However, if the ratio of
intracellular fluid space to extracellular fluid
space and the concentration of the tracer amino
acid in the incubation medium remain constant,
the radioactivity soluble in cold washes of TCA
should be a valid index of the specific activity of
the intracellular pool since metabolic interconversion of phenylalanine was negligible in rabbit
papillary muscles. These two conditions were met
in all experiments employing phenylalanine as the
tracer amino acid. More than 95% of the
unincorporated radioactivity was recovered in the
cold TCA washes. These were pooled and
aliquots counted in 20 ml of scintillation fluid
(700 ml of toluene, 300 ml of absolute ethanol, 4
g of PPO, 100 mg of POPOP). Radioactivity
soluble in TCA was expressed as dpm/mg wet
weight of muscle and assumed to reflect
intracellular phenylalanine concentration. This
value was recorded as TCA radioactivity.
MUSCLE PROTEIN FRACTIONATION
A modification of the procedure of Helander
(27) was used to separate the muscle proteins
and to determine the ratio of tracer incorporation
in the "myofibrillar-connective tissue" protein
fraction to that in the "sarcoplasmic" protein
fraction. In this manner, alterations in the
pattern, as opposed to the overall rate, of protein
x
Dr. Ellen Kang, Children's Hospital, Boston, Mass.,
kindly performed the amino acid analyses.
Circulation Riitarcb, Vol. XXXI, September 1972
321
ISOMETRIC TENSION AND MYOCARDIAL PROTEIN SYNTHESIS
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synthesis were studied. Immediately after incubation, the muscles were homogenized in 0.3 ml of
buffer (0.1M KC1, 5 mM histidine, pH 7.4) and
extracted at 2°C for 90 minutes. The homogenate
was centrifuged at 1,000 g for 20 minutes, and
the pellet was washed twice with 0.15 ml of
homogenizing buffer. The washes were combined
with the original supernatant fraction, and 0.3 ml
of carrier protein solution was added. An
appropriate volume of 50% TCA was added to a
final concentration of 104, and the precipitate
containing the sarcoplasmic proteins was collected, washed, and counted as described. The 1,000g pellet, consisting of the myofibrillar-connective
tissue proteins of the muscle, was suspended in
0.3 ml of KC1 buffer. Carrier protein and 50*
TCA were added, and the protein pellet was
washed and counted.
DETERMINATIONS OF ALPHA-AMINOISOBUTYRIC
ACID DISTRIBUTION RATIOS
Muscle wet weight, dry weight, inulin space
(extracellular fluid space), and the ratio of
intracellular 14C-alpha-aminoisobutyric acid (14CAIB) to extracellular 14C-AIB were measured as
previously described (18) with the following
modifications. (1) Muscle weights were determined to the nearest 0.001 mg with a Mettler M-5
microbalance. (2) Unlabeled AIB, in a concentration of 1 rng/ml, was added to all TCA
solutions used for separating the insoluble
proteins from the soluble extract which contained
the 14C-AIB. (3) The base stability of purified
inulin varied, with up to 15% of each preparation
becoming base labile with time. Since the
appearance of base instability was not a direct
function of storage time or conditions, a basic
hydrolysis blank was routinely run on the inulin
preparation used in each experiment, and appropriate corrections were made in the calculations
of inulin space.
not vary significantly over the 6-hour incubation period.
WATER CONTENT AND EXTRACELLULAR FLUID SPACE
The percent dry weight of control muscles,
as determined in this laboratory, was 24*
(18). Regardless of the rate of stimulation, or
the duration of incubation, no significant
variation from this value was noted in any
muscle group in the present study. The
extracellular fluid space, expressed as percent
of wet weight, did not vary from control
values of 25% in any muscle group (18).
BASIC CHARACTERISTICS OF AMINO ACID
INCORPORATION IN THE ISOLATED PAPILLARY
MUSCLE PREPARATION
14
The incorporation of C-phenylalanine into
myocardial protein was linear for 6 hours in
muscles incubated at zero tension in the
Dubnoff apparatus (Fig. 3). Linearity and
rate of incorporation were identical in muscles
incubated with supplemental amino acids
(100 fAM.) in the medium and in control
muscles incubated with only tracer present
(Fig. 3). Incorporation did not change when
the concentration of each supplemental amino
acid in the medium was varied between 0 and
500 /IM while phenylalanine concentration was
constant. Similarly, the ratio of tracer incorporation in myofibrillar-connective tissue protein to that in sarcoplasmic protein was
•---•
JO'
W HIN
.
- - - • 100' 0 IH
STATISTICAL ANALYSIS
Data were analyzed using Student's f-test (28).
I
1
Results
MECHANICAL PROPERTIES OF THE ISOLATED
PAPILLARY MUSCLE
Peak isometric tension development was
stable for up to 6 hours in muscles stimulated
at 50 and 100/min, but decreased by 50% in
those stimulated at 30/min (Fig. 4). Maximum developed tension ranged from 2 to 9
g/mm2 in muscles stimulated at 30 and
50/min and from 2 to 4 g/mm 2 in muscles
stimulated at 100/min. The peak of the active
length-tension curve was obtained at resting
tensions between 0.5 and 1.5 g/mm2 and did
OrcuUiion Rtiurcb, Vol. XXXI, Siputmttr 1972
-
...
i
tIMI
I
HOUtl
FIGURE 4
Active tension development for representative muscle
groups stimulated at 30, 50, or 100/min plotted as a
function of time. AU values represent means ± SE
obtained from six individual muscles.
322
PETERSON, LESCH
also inhibited by puromycin and by cycloheximide (Table 1). Total inhibition of protein
synthesis by 10~RM puromycin further documented the efficiency of the protein washing
procedure. If any coprecipitation or nonspecific binding of the labeled amino acid had
occurred, total inhibition of incorporation
would not have been seen.
constant at 0.65:0.35 regardless of the concentrations of amino acids in the medium.
Leucine incorporation was significantly inhibited by puromycin at a concentration of
lfr^M, although 10~*M was required for
complete inhibition during 1 hour of incubation (Table 1). The incorporation of 14 Cphenylalanine into myocardial protein was
TABLE 1
Effect of Inhibitors of Protein Synthesis on Amino Acid Incorporation into Papillary Muscle Protein
Tracer amino acid
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M
" C-In corporation
(dpm/mg p rot tin)
Inhibitor
C-Leucine
10,500 ± 1,100
8,503 ± 570
4,100 ± 580
10
290 ±
10
10 =t
Control muscles
Puromycin (lO"8*!)
Puromycin (10~*M)
Puromycin (10~<M)
Puromycin (10~ 3 M)
"C-Phenylalanine
41,600 ± 1,700
60
350 ±
80
290 ±
Control muscles
Puromycin (10~'M)
Cycloheximide (10~'M)
Each value represent*) the mean ± SB of six individual determinations. Incubations were
carried out for GO minutes under standard conditions described in Methods.
TABLE 2
Effect of Isometric Tension Development on Amino Acid Incorporation into Papillary Muscle Protein
w C- In corpora tion
(dpm/mg protein)
TCA radioactivity
(dpm/mg wot wt)
Change
(%)
Change
(%T
90-Minule Incubation
30 stimulations/min
C
E
11,200 =t 1,030
13,800 ± 690
+ 23
2,110 ± 90
2,300 ± 60
10,160 ±
12,150 ±
790
730
+ 20
1,730 ± 60
1,940 ± 90
+ 12
9,700 ±
11,900 ±
930
690
+ 23
1,600 ±
1,730 =t
50
60
+ 8
50 stimulations/min
C
E
100 stimulations/min
C
E
180-Min-ule Incubation
30 stimulations/min
C
E
18,900 ± 1,920
25,900 ± 1,220
+ 37
<0.02
2,110 ± 170
2,140 ± 40
+ 1
17,100 =•= 1,300
26,000 =t 1,620
+.52
<0.01
1,780 ± 110
1,970 ± 80
+ 10
20,500 ± 1,560
31,400 ± 1,260
+53
<0.01
1,770 ± 30
1,770 ± 30
+ 0
50 stimulations/min
C
E
100 stimulations/mio
C
E
Each value represents the mean ± SE of six individual determinations. C := control, E = experimental. A P value
is listed only if a significant difference occurred. No stimulation, zero tension served as the control group.
CircuUium RccMrcb, Vol. XXXI, Stpumbtr 1972
323
ISOMETRIC TENSION AND MYOCARDIAL PROTEIN SYNTHESIS
tion of the 14C label in the individual amino
acids of a distilled-water extract of the
muscles was determined. Radioactivity was
quantitatively recovered as phenylalanine in
control muscles and in muscles stimulated at
100/min. The specific activity of the intracellular phenylalanine pool in the control and the
stimulated muscles was calculated with equations similar to those previously reported
(18). Intracellular phenylalanine specific activity was 7.0 X 107 and 7.1 X 107 dpm//n,mole
in the control and the stimulated muscles
(100/min for 3 hours), respectively.
To rule out the possibility that the observed
effects at 3 hours were due solely to the
electrical stimulation of the muscle, phenylalanine incorporation in unstimulated and
stimulated muscles mounted at zero tension
for 3 hours was compared (Table 3). No
significant difference in the rate of phenylalanine incorporation was noted between the
flaccid stimulated muscles and the flaccid unstimulated muscles at any stimulation rate, although incorporation rates were approximately
20? higher in the stimulated muscles.
To determine if passive stretch alone could
explain the increased tracer incorporation
EFFECT OF MECHANICAL INTERVENTIONS
ON AMINO ACID INCORPORATION
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The effect of isometric tension development
on amino acid incorporation is illustrated in
Tables 2 and 3. At 90 minutes, the rate of 14Cphenylalanine incorporation into protein was
not statistically different for muscles stimulated at 30, 50, or 100/min and controls,
although incorporation was increased by
about 2035 in all stimulated groups. Phenylalanine incorporation was enhanced by approximately 50& in the muscles stimulated at 50 or
100/min for 3 hours (P<0.01) and by 37%
( P < 0 . 0 2 ) in those stimulated at 30/min for
the same period of time. Comparable results
were obtained when these experiments were
repeated using an alternative method of
protein isolation (29).
The radioactivity (dpm/mg wet weight) in
the pooled cold TCA washes was not significantly different in any muscle group at 90 or
180 minutes.
To validate the assumption that radioactivity soluble in TCA accurately reflected the
specific activity of the intracellular phenylalanine pool, groups of muscles were incubated
under standard conditions for 3 hours with
14
C-labeled phenylalanine, and the distribu-
TABLE 3
Effect of Electrical- Stimulation and Passive Stretch on Amino Acid Incorporation into Papillary Muscle Protein
"C-Incorpor«tion
(dpm/mf protein)
TCA r»dioactiTity
(dpm/mg wot wt)
Chinge
(%)
Chance
(%)
Stimulation al Zero Tension
30 stimulations/min
C (5)
E (5)
50 stimulations/min
C (10)
E (10)
100 stimulations/min
C (11)
rc ( i i )
18,700 =•= 800
22,500 ± 2,100
+ 20
21,000 ± 1,400
25,000 ± 1,300
19,900 ± 1,400
23,200 ± 1,400
1,640 ±
60
+ 10
1,540 •*
1,770 ±
60
+ 15
+ 17
1,530 ±
1,600 =•=
30
50
+ 4
1,800 ± 160
1,990 ± 80
+ 11
1,660 ="- 50
+ 1
90
Passive Stretch
C
E
(6)
(6)
19,000 ±
25,900 =t
740
740
+ 36
<0.01
Each value represents the mean ± SE; numbers in parentheses represent individual determinations. C = control,
E = experimental. A P value ia Listed only if a significant difference occurred. Passively stretched muscles were
stimulated to define the peak of the active length-tension curve, stimulation was discontinued, and the muscles were
maintained at the resulting resting tension for the entire incubation. Incubations were carried out for 180 minutes
in all cases.
Circulation Resemrcb, Vol. XXXI, Stpltmbtr
1972
324
PETERSON, LESCH
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noted in the muscles stimulated isometrically
at peak tension for 3 hours, unstimulated
muscles which had been passively stretched to
the resting tension corresponding to this peak
tension were compared to resting controls at
zero tension (Table 3). Incorporation was
increased by 36$ (P<0.01) in the stretched
muscles, but radioactivity in the TCA washes
was not different
Finally, muscle protein was fractionated to
determine if any gross change in the labeling
pattern had occurred in stimulated muscles.
Both control muscles incubated for 3 hours at
zero tension and those stimulated isometrically
at 30, 50, or 100/min at peak tension
incorporated 35$ of the tracer amino acid into
sarcoplasmic protein and 65% into myofibrillarconnective tissue protein.
ALPHA-AMINOISOBUTYRIC ACID TRANSPORT STUDIES
As previously reported (18), mechanical
intervention did not alter AIB transport after
1 hour of incubation. The data obtained for
muscle groups stimulated at 30, 50, and
100/min for 3 hours are summarized in Table
4. A significant increase (P<0.01) in the
intracellular-extracellular AIB distribution
ratio was noted in unstimulated muscles passively stretched to resting tensions of 0.5-1.5
g/mm2. This range of resting tensions corresponded to that found in muscles stimulated
at the peak of their active length-tension
curves. Accumulation of AIB in unstimulated
and stimulated muscles (30/min) mounted at
zero tension was the same, but accumulation
was significantly enhanced (P<0.01) in
muscles stimulated at this rate at the peak of
the length-tension curve. Muscles stimulated
at 50/min and 100/min at the peak of the
length-tension curve also demonstrated increased AIB uptake compared to control
muscles.
Discussion
Although responses of myocardial protein
and nucleic acid metabolism to mechanical
stress have been reported in various systems,
the precise mechanical factors responsible for
the biochemical adaptations, the temporal
pattern of the induced alterations, and the
specific protein classes involved are unresolved. We used the isolated rabbit right
ventricular papillary muscle to study these
problems since the biochemical and mechanical stability of the preparation suggests its
suitability for in vitro studies of myocardial
mechanochemical coupling.
In contrast to isolated perfused heart
preparations, mechanical integrity of the
isolated papillary muscle was maintained over
a wide range of stimulation rates for up to 6
hours (Fig. 4). Percent dry weight and
extracellular fluid space (inulin space) remained constant during this period, demonstrating that the muscles did not become
progressively edematous with incubation.
Amino acid incorporation in resting right
ventricular papillary muscles was linear for 6
TABLE 4
Effect of Mechanical and Elecincal Interventions on Alpha-Aminoisobutyric Acid Distribution in
Isolated Papillary Muscles
Muscle group
No stimulation, zero tension (31)
30 stimulations/min, zero tension (21)
30 stimulations/min, peak LT (38)
50 stimulations/min, peak LT (9)
100 stimulations/min, peak LT (9)
No stimulation, passive stretch (30)
IC-EC ratio
2.7
3.1
3.7
4.4
4.6
4.7
± 0.2
± 0.3
=•= 0.3
± 0.4
± 0.3
± O.o
<0.01
<0.01
<0.01
<0.01
Each value represents the mean ± SE; numbers in parentheses represent individual determinations. A P value is listed only if a significant difference occurred. No stimulation, zero tension
served a3 the control group. Incubations were carried out for 180 minutes. LT = length-tension
curve, IC = intracellular, EC = extracellular. Passive stretch was applied from 0.5-1.5 g/mm*.
Circulation Rtiurcb, Vol. XXXI, Sfpumitr 1972
ISOMETRIC TENSION AND MYOCARDIAL PROTEIN SYNTHESIS
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hours in the absence of supplemental amino
acids or exogenous insulin (Fig. 3), whereas
tracer amino acid incorporation into cardiac
protein was linear for only 1-2 hours in the
isolated perfused rat heart (30, 31).
Just as supravalvular aortic constriction in
vivo imposes an undefined mechanical stress
on the heart, precise mechanical loads cannot
be imposed on the isolated perfused heart
since volumes and pressures are not independently regulated. Furthermore, although coronary flow can be adjusted in such preparations, it is impossible to increase pressure
loads without altering coronary perfusion
pressures. The isolated papillary muscle can
be exposed to defined mechanical stresses
independent of the limitations noted with
intact hearts, thus simplifying the task of
identifying mechanical stimuli responsible for
biochemical adaptations. The present study
demonstrated that a statistically significant
increase in 14C-phenylalanine incorporation
into myocardial protein occurred in response
to repetitive isometric tension development
for 3 hours. Electrical stimulation alone was
not responsible for the effect, since enhanced
phenylalanine incorporation was not found in
muscles mounted at zero tension and stimulated at 30, 50, or 100/min (Table 3).
Total tension developed during the incubation period (tension/beat X stimulation rate X
hours) appears to be directly related to
enhanced amino acid incorporation. Muscles
stimulated at 50/min developed twice the
tension per beat (Fig. 4) of those stimulated
at 100/min, yet they showed identical increases in incorporation, whereas muscles
stimulated at 30/min developed less total
tension and incorporated less amino acid
(Table 2). To isometrically stimulate muscles
at peak tension, it is necessary to apply and
maintain, independent of active tension development, a degree of resting tension during the
incubation. Even though passive tension was
capable of inducing alterations in the rate of
incorporation (Table 3), it appears that
enhanced incorporation in muscles stimulated
at 50 and 100/min cannot be solely ascribed to
stretch since the mean percent increase in
Circulation Risircb, Vol. XXXI, SepMmbtr 1972
325
incorporation was 36% in passively stretched
muscles and 53!? in stimulated muscles. These
data are most consistent with the hypothesis
that both stretch and total tension development are operative in stimulating amino acid
incorporation into myocardial protein, but an
unequivocal separation of these two variables
has not been obtained.
Evidence from other laboratories implies
that intracellular free amino acids partially
regulate the rate of protein synthesis in
mammalian cells (32, 33). Increased concentrations of free amino acids have been found
in rapidly growing tissues, including heart and
skeletal muscle, and it has been postulated
that the rate of myocardial protein synthesis is
closely related to the intracellular free amino
acid concentration (10, 34). In view of these
data, the effect of repetitive isometric tension
development on amino acid transport was
investigated with previously reported techniques (18).
Transport of AIB was clearly enhanced in
papillary muscles stimulated at the peak of the
active length-tension curve. However, since
passive stretch alone was capable of inducing
the same quantitative response, it was not
possible to correlate peak isometric tension
development and enhanced AIB transport.
Although passive stretch influenced AIB transport, this need not be true for all amino acids
since different transport mechanisms for various amino acids have been described in
striated muscle (35). Also, it has been
reported that the transport mechanisms for
AIB (A system of transport) and phenylalanine (L system of transport) are intrinsically
different in heart (36) and that they respond
differently to hypoxic insult (37). Our data
are consistent with these reports in that
enhanced phenylalanine incorporation into
protein occurred independent of an effect on
the transport mechanism, although AIB transport was clearly altered by similar mechanical
interventions. It appears that mechanical
factors are capable of altering the transport of
certain amino acids, but it is not clear if these
mechanical factors function as specific mediators of protein synthesis in this case.
326
PETERSON, LESCH
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Circultiion Rtsttrcb, Vol. XXXI, Stpt+mbtr 1972
Protein Synthesis and Amino Acid Transport in the Isolated Rabbit Right Ventricular
Papillary Muscle: EFFECT OF ISOMETRIC TENSION DEVELOPMENT
Myron B. Peterson, Michael Lesch and Alan G. Ferguson
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Circ Res. 1972;31:317-327
doi: 10.1161/01.RES.31.3.317
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