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787
Inhibition of Protein Degradation in Mouse Hearts
by Agents That Cause Lysosomal Dysfunction
KERN WILDENTHAL, JACQULINE R. WAKELAND, PHYLLIS C. MORTON, AND
EDMOND E. GRIFFIN
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SUMMARY Although the heart contains lysosomes, it has been uncertain whether these organelles and their
proteolytic enzymes can play an important role in cardiac protein degradation. Recent studies have demonstrated
that fetal mouse hearts in organ culture sustain selective derangements in lysosomal structure and function during
exposure to chloroquine or nonmetabolizable sugars. Accordingly, we tested the effects of these agents on cardiac
proteolysis under controlled conditions in vitro using two techniques (measurement of loss of radioactivity from
trichloroacetic add-precipitable protein after prelabeling with tritiated phenylalanine and measurement of loss of
cold phenylalanine after blockade of protein synthesis with cycloheximide). Chloroquine (0.1 D M ) reduced the
average rate of protein breakdown in hearts of matched littermates from 45%/24 hours to 32%/24 hours (P < 0.01)
and decreased the release of cold phenylalanine by 31 ± 5%(0.108 vs. 0.075 nmol/mg per hour, P < 0.01).
Exposure to 100 DIM sucrose for 24-48 hours reduced the rate of breakdown from 44%/24 hours to 33%/24 hours
(P < 0.01) and decreased the release of cold phenylalanine by 35 ± 9% (0.092 vs. 0.060 nmol/mg per hour, P <
0.01). The results suggest that interference with lysosomal function in cultured fetal mouse hearts causes a significant
reduction in the cardiac capacity to degrade proteins.
ALTHOUGH the myocardium is known to contain lysosomes, it has been uncertain whether these organelles and
their proteolytic enzymes play an important role in regulating cardiac protein degradation.1 One approach which
might help to resolve this issue would be to determine
whether experimental interference with normal lysosomal
properties alters the rate of proteolysis.
One problem in adopting such an approach is to select
a suitable experimental model. Agents that cause selective
lysosomal derangements, if given in vivo, might cause
systemic noncardiac effects which could confuse the interpretation of their specific myocardial actions. Therefore,
we chose to use isolated hearts in vitro. Because the
effects on lysosomal function might be slow to develop,
we chose to use an in vitro model which can function
stably for many hours to days, i.e., the fetal mouse heart
in organ culture.2
A second problem in approaching this question is to
choose appropriate agents to interfere selectively with
lysosomal function while causing minimal primary abnormalities in other cellular systems. This problem is made
more difficult by the fact that many of the agents which
might otherwise be suitable, such as some specific inhibitors of individual lysosomal proteolytic enzymes, cannot
readily cross cell membranes and gain access to the
lysosomes. Recently, however, two types of agents—nonmetabolizable sugars and chloroquine —have been identiFrom the Pauline and Adolph Weinberger Laboratory for Cardiopulmonary Research, Departments of Physiology and Internal Medicine,
University of Texas Health Science Center at Dallas, Dallas, Texas.
Supported by the Moss Heart Fund and the National Heart, Lung, and
Blood Institute (HL-14706).
Address for reprints: Dr. Kern Wildenthal, University of Texas, Health
Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas
75235.
Received September 26, 1977; accepted for publication February 8,
1978.
fied which become concentrated in lysosomes of living
cells and which produce severe lysosomal dysfunction
without causing major abnormalities in other organelles.3"13
Like many other tissues, fetal mouse hearts in organ
culture react to these agents by developing striking, selective lysosomal abnormalities.14'l5 Exposure to chloroquine
causes the rapid development of bizarre lysosomes with
myelin figures and inclusion bodies:14 at the same time,
the lysosomal pH becomes alkaline (higher than optimal
for most lysosomal hydrolases), and the drug produces a
direc.t biochemical inhibition of at least one lysosomal
proteinase, cathepsin B,.11 Nonmetabolizable sugars such
as sucrose are conventionally viewed as being confined to
the extracellular space. This is true only in the short term,
however, and over a period of many hours the sugars are
slowly taken into cells by endocytosis and progressively
stored in lysosomes.3"5 During exposure to sucrose for 2448 hours, lysosomes of fetal mouse hearts in organ culture
sequester large amounts of the sugar and swell into giant
vacuoles; these vacuoles seem unable to maintain the acid
pH required for effective activity of most of the lysosomal
enzymes, and many inclusion particles are accumulated.15
Accordingly, since chloroquine and sucrose provide means
to produce selective cardiac lysosomal derangements under precisely controlled conditions in vitro, we undertook
studies to test their simultaneous effects on cardiac protein
degradation.
Methods
Culture Conditions
As described in detail previously,2 hearts were removed
aseptically from 18- to 20-day fetal mice (Charles River,
albino outbred), washed gently, and placed on stainless
steel grids at a liquid-oxygen interface in shallow culture
dishes. Hearts of this age weigh 2-5 mg and measure 0.5-
788
CIRCULATION RESEARCH
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2 mm in their major diameters. The hearts beat rhythmically and maintain stable metabolic characteristics for
several days under appropriate culture conditions .2t l6
Control hearts were maintained in 50% Earle's salt
solution (Grand Island Biological Co.) plus 50% fetal calf
serum (Colorado Serum Co.) supplemented with insulin,
50 /J.g/ml (Schwarz/Mann). Experimental hearts from
matched littermates were maintained in identical media to
which had been added 100 mM sucrose, 100 mM urea, or
0.1 mM chloroquine. Previous studies had established that
these concentrations induce major lysosomal alterations
without altering the contractile ability of the hearts or
producing major abnormalities in other organelles.14'15
In some experiments, cycloheximide (Sigma) was added
to control and experimental media at a concentration of
2.5 /ng/ml, a level that, in this system, inhibits incorporation of radioactive amino acid into protein by >90%. 17
For studies requiring the use of radioactive amino acids,
cultures were exposed to 10 /iCi/ml of L-[side chain-2,33
H]phenylalanine (50 Ci/mmol; Schwarz/Mann) plus nonradioactive carrier, 16 /ng/ml. For studies of protein
degradation, the hearts were transferred to medium lacking radioactive amino acids and containing excessive concentrations (500 /ng/ml) of unlabeled phenylalanine to
minimize reutilization of previously incorporated label.
Biochemical Analyses
Analyses were performed on the heart tissue and on the
culture medium after cultivation under various conditions
for 1-2 days. Total protein content of each heart was
determined by the method of Lowry et al.18 after homogenization in a glass Dounce homogenizer.
Measurements of changes in protein degradation were
made by two techniques. In the first procedure, hearts
were allowed to stabilize in culture for 18-24 hours and
then exposed to [3H]phenylalanine for 4 hours. As described in detail previously,19 the hearts were then washed
at hourly intervals with nonradioactive medium containing
excess unlabeled phenylalanine. After three washes, onethird of each litter was harvested for "zero-time" values,
one-third was maintained for an additional 24 hours in
control medium containing excess nonradioactive phenylalanine to minimize reutilization of label, and one-third
VOL. 42, No. 6, JUNE
1978
was maintained for an identical period in the same medium plus the experimental agent. In some experiments,
the final media (both control and experimental) contained
cycloheximide, 2.5 /xg/ml, to block protein synthesis and
reutilization of label.
For experiments involving chloroquine, initial stabilization for all hearts was in control medium, and experimental hearts were exposed to the drug only during the 24hour degradation period. To test the effects of nonmetabolizable sugars, one group of experiments was performed
in which sucrose was present only during the degradation
period (i.e., its effects on proteolysis reflected changes
during the first day of exposure). In addition, a second
group of experiments was performed in which hearts were
exposed to sucrose during the 24-hour stabilization, pulsation, and wash period, as well as during the degradation
period (i.e., the observed changes in proteolysis reflected
differences present during the period spanning the second
day of exposure to the agent); for these experiments, two
groups of hearts (those exposed to control medium and
those exposed to sucrose) were harvested for "zero-time"
values, to make allowance for any differences that might
be produced in incorporation of label by the prior presence of sucrose. To test for any effects that might be due
to the high molarity of the sucrose rather than to its
specific effects on lysosomes, similar experiments were
made with 100 mM urea, an agent which does not alter
lysosomal structure or function significantly.15
Rates of protein degradation for each group of hearts
were calculated from measurements of the amount of
radioactivity remaining in trichloroacetic acid (TCA)-precipitable protein after the 24-hour degradation period, as
compared to the amount in "zero-time" hearts.19 Each
heart was homogenized in ice-cold 20 mM NaCl-2 mM
phosphate buffer (pH 7.4) in a tightly fitting Dounce
homogenizer. Protein was precipitated from a sample of
each homogenate with cold 10% TCA, collected on a
glass filter (Whatman, GF/C) in a funnel precipitation
apparatus (ICN Corp.), washed twice with cold TCA, and
then solubilized with NCS solubilizer (Amersham/Searle)
in a liquid scintillation counting vial. The radioactivity of
each sample was measured in a Packard 2425 tricarb
scintillation counter in toluene-2,5-diphenyloxazole-l ,4bis[2-(5-phenyloxazolyl)]benzene.
TABLE 1 Effects of Chloroquine (0.1 mu) and Sucrose (100 mu) on Protein Degradation
as Measured by Release ofTritiated Phenylalanine from TCA-Precipitable Material in Fetal
Mouse Hearts
Rate of protein degradation (%/24 hours)
Intervention
Control
Experimental
Differences
Chloroquine
(0- to 24-hr exposure)
45 ± 3.2
32 ± 4.8
13 ± 3.7
<0.01
Sucrose
(0- to 24-hr exposure)
46 ± 1.0
40 i 3.9
6± 2.7
<0.10
Sucrose
(24- to 48-hr exposure)
44 ± 2.5
33 ± 3.0
11 ± 2.8
<0.01
P
Each value represents the mean ± SEM of 30 hearts from six litters. Differences are calculated from the six sets of
matched littermates in each experiment.
CARDIAC LYSOSOMES AND PROTEIN DEGRADATION/Wildenthal et al.
In other experiments, changes in net amino acid balance
in the hearts were determined, as described previously,19
by assaying the medium with a Durrum analyzer after 1 or
2 days' cultivation for the net gain or loss of 12 individual
amino acids. Because phenylalanine is not synthesized or
degraded in heart tissue and is involved only in protein
synthesis and breakdown,20 the rate of uptake or release
of total phenylalanine was used as an index of net protein
balance (synthesis and degradation) as well as of phenylalanine balance per se. When protein synthesis was
blocked with cycloheximide, as described above, measurements of phenylalanine release provided an index of the
rate of protein degradation alone.21
Statistical Analyses
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Although results from different litters maintained in
organ culture often vary considerably, results from individual hearts within a single litter are highly reproducible
(SD < 10%). Accordingly, results from hearts exposed to
the experimental agents were compared to matched littermate controls using Student's /-test for paired data. Results are given as the mean ± 1 standard error of the mean
for each condition, with n as the number of litters tested.
Results
As reported in previous studies,17-l9'21 fetal mouse
hearts in organ culture degraded protein at a fairly rapid
rate. Thus, as estimated from the loss of radioactive
phenylalanine from TCA-precipitable material, 45 ±
3.2% of prelabeled protein was degraded in 24 hours in
control hearts (Table 1). In contrast, only 32 ± 4.8% of
prelabeled protein was degraded during the 24-hour period that hearts of matched littermates were exposed to
chloroquine. The difference, which represented a relative
retardation in the rate of proteolysis by a factor of almost
one-third, was statistically significant (P < 0.01). Experiments in which cycloheximide was present during the
degradation period to prevent artifactual differences due
to altered reincorporation of label yielded similar results:
control hearts degraded protein at a rate of 38 ± 6.5% in
100
I
_
80
60
40
3
o
o
<
> Control
> + Chloroquine (0.1 n t l )
20
0
0-time
24 hours
Cycloheximide Absent
0-Hme
24 hours
Cycloheximide Present
FIGURE 1 Release of tritiated phenylalanine from TCA-precipitable protein in hearts cultured in the presence or absence of
chloroquine and ofcycloheximide. Each point represents the mean
±1 SEM of 30 hearts from six litters. * = P < 0.01 compared to
litter-matched hearts cultured in media lacking chloroquine.
789
100
7
il
m^
o
o
<
o:
80
60
40
20
•
• Control
o-—-o + Sucrose (lOOmM tor 2doys)
0-time
24 hours
Cycloheximide Absent
0-time
24 hours
Cycloheximide Present
FIGURE 2 Release of tritiated phenylalanine from TCA-precipitable protein in hearts cultured in the presence or absence of
sucrose. Each point represents the ±1 SEM of 30 hearts from six
litters. * = P < 0.01 compared to litter-matched controls cultured
in media lacking sucrose.
the presence of cycloheximide vs. 22 ± 8.2% when
chloroquine was added (Fig. 1, P < 0.01).
When sucrose was added to the medium during the
degradation period, the rate of protein breakdown was 40
± 3.9% vs. 46 ± 1.0% in matched controls (Table 1).
This fairly small difference during the first 24 hours of
exposure to the sugar was of only borderline significance
statistically (0.05 < P < 0.10). In experiments in which
pre-exposure to sucrose for 24 hours had occurred before
the hearts were pulsed and degradation rates were measured, the effects of the sugar were much more pronounced. Thus, hearts degraded protein at a rate of 33 ±
3.0%/24 hours during the second day of exposure to
sucrose vs. 44 ± 2.5% in littermatched controls tested
over the same period (Table 1, P < 0.01). In the presence
of cycloheximide, the corresponding figures during the
second day of sucrose exposure were 26 ± 4.0%/24 hours
vs. 39 ± 3.1 %/24 hours in matched controls (Fig. 2, P <
0.01). Under identical circumstances, exposure to urea
for 2 days caused no changes in the rate of protein
degradation (43 ± 4.0%/24 hours in controls vs. 44 ±
4.7%/24hours).
Measurements of the release into the medium of cold
phenylalanine during exposure to cycloheximide provided
confirmation of the inhibitory effects of chloroquine and
sucrose on proteolysis. As shown in Figure 3, hearts
exposed to chloroquine during the first day of culture
released phenylalanine at a rate of 0.075 ± 0.0066 mmol/
mg heart weight per hour, as compared to 0.108 ± 0.0057
in litter-matched controls. This 31% decrease was statistically significant (P < 0.01). Sucrose reduced phenylalanine release by 23% during the first 24 hours of cultivation in the presence of cycloheximide (0.083 ± 0.0078
nmol/mg per hour vs. 0.106 ± .0145, P < 0.01). The
sugar's effect was much more pronounced during the
second 24 hours of exposure, when the rate of phenylalanine release was retarded by 35% (0.060 ± 0.0113 vs.
0.092 ± 0.0132, P < 0.01).
When added to media lacking cycloheximide, so that
both synthesis and degradation were active, chloroquine
and sucrose tended to improve the net balance (i.e.,
CIRCULATION RESEARCH
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i.
g
c-OZOh
Cofltrot
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«*
0-24 hours
0-24 hnurs
2 4 - 4 8 hours
FIGURE 3 Rate of net release of total phenylalanine in hearts
cultured in the presence or absence of chloroquine or sucrose,
while protein synthesis was blocked by cycloheximide. Each bar
represents the mean ±1 SEM of 24 matched hearts from six litters.
* = P < 0.01 compared to litter-matched controls.
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reduce the release or increase the uptake) of all the amino
acids measured (Tables 2 and 3).
Discussion
The precise cellular mechanisms by which proteins are
broken down to their constituent amino acids are poorly
understood. In many tissues, lysosomes appear to play a
major role in the process.22'23 In heart and skeletal
muscle, because relatively few lysosomal structures can be
identified microscopically, because autophagic vacuoles
containing myofibrillar elements are seldom if ever encountered, and because nonlysosomal neutral and alkaline
proteinases are abundant, considerable scepticism has
remained about whether lysosomes and lysosomal enzymes are of major importance in the degradation of
proteins in muscular tissues (see refs. 1, 23, and 24 for
reviews).
Direct demonstrations of the quantitative importance of
specific lysosomal proteinases have been accomplished for
liver, using the specific proteinase inhibitor pepstatin
encapsulated in liposomes to facilitate entry into the
lysosomal compartment.25 Similar studies have been diffi-
VOL. 42, No. 6, JUNE
cult in muscle tissues because myocytes do not readily
incorporate liposomes or their contents. Furthermore,
some other proteinase inhibitors, such as TLCK or leupeptin, which can penetrate cells more easily than pepstatin, may block nonlysosomal as well as lysosomal proteinases and also may produce nonspecific toxic effects which
theoretically might alter proteolysis secondarily, independent of their actions on lysosomal enzymes (unpublished
observations). Accordingly, in the present experiments we
sought to test the effects of agents which, in a variety of
tissues including fetal mouse hearts in organ culture,
readily gain access to the lysosomal compartment and
produce their primary effects on lysosomes with little or
no actions in other organelles.3"15
Previous studies on isolated fibroblasts have shown that
chloroquine inhibits the lysosomal breakdown of endogenous proteins10' " as well as of certain exogenous proteins
that are taken up by endocytosis.12-l3 Similarly, our experiments revealed that the breakdown of endogenous cardiac proteins was inhibited by 30% or more during a 24hour exposure to chloroquine.
Exposure to sucrose causes lysosomal derangements in
many tissues, including heart.3""6-l5 In fetal mouse hearts,
the abnormalities can be observed to appear gradually
over a period of 1-3 days.15 In keeping with the rather
slow and progressive nature of the development of sucrose-induced lysosomal derangements, changes in protein
inhibition also were slow to develop: proteolysis was
decreased by only 10-20% during the first day's treatment
but by approximately 30% during the second day. It
should be pointed out that the degree of inhibition observed, if applicable to both contractile and noncontractile
proteins and if sustained in the absence of any change in
synthesis, would result in a net accumulation of total
protein mass of 10-15% per day over control. At this
rate, total cardiac size would tend to double within a
week. In other words, this degree of change is very
significant, not only statistically but also functionally,
when viewed in the context of the development of cardiac
hypertrophy.
2 Effect of Chloroquine (0.1 m\i) on Net Amino Acid Balance in Fetal Mouse
Hearts Maintained in Organ Culture
TABLE
A. Control medium
Lys
His
Arg
Pro
Gly
Ala
Val
Met
lie
Leu
Tyr
Phe
+ 0.140 dt
+0.038 dt
+ 0.068 di
+ 0.020 dt
+0.401 dt
+0.713 dt
-0.047 dt
+0.033 dt
-0.034 dt
-0.035 dt
+0.052 dt
+ 0.062 dt
0.0080
0.0028
0.0045
0.0064
0.0181
0.0389
0.0102
0.0054
0.0032
0.0084
0.0076
0.0068
1978
B. + Chloroquine
C. Difference (B - A)
+0.055 ± 0.0050
+ 0.012 ±0.0035
+0.024 ± 0.0037
-0.035 ± 0.0090
+ 0.255 ±0.0175
+ 0.351 ± 0.0224
-0.088 ± 0.0134
+0.003 ± 0.0039
-0.060 ± 0.0045
-0.089 ± 0.0092
+0.013 + 0.0047
+0.012 ± 0.0073
-0.095 ± 0.0077*
-0.026 ± 0.0047*
-0.044 ± 0.0025*
-0.055 ± 0.0108*
-0.146 ±0.0178*
-0.362 ± 0.0374*
-0.041 ± 0.0097*
-0.030 ± 0.0035*
-0.026 ± 0.0032*
-0.054 ± 0.0056*
-0.039 ± 0.0063*
-0.050 ± 0.0066*
Results in columns A and B represent the net release (positive numbers) or uptake (negative numbers) of each
amino acid (in nmol/mg heart weight per hour) from the heart into the medium during the first day of cultivation;
each value represents the mean ± 1 SEM for 24 hearts from eight litters. Column C gives the mean difference ± 1 SEM
between the eight matched litters; negative numbers in column C indicate either increased net uptake or reduced net
release.
• P < 0.05 by Student's paired West.
CARDIAC LYSOSOMES AND PROTEIN DEGRADATION/W/WemW et al.
TABLE 3 Effect of Sucrose (100
Maintained in Organ Culture
Lys
His
Arg
Pro
Gly
Ala
Val
Met
He
Leu
Tyr
Phe
791
on Net Amino Acid Balance in Fetal Mouse Hearts
A. Control medium
B. + Chloroquine
+0.187 ± 0.0194
+ 0.022 ±0.0153
+ 0.048 ± 0.0107
-0.008 ± 0.0164
+ 0.428 ± 0.0610
+ 0.663 ± 0.0677
-0.060 ± 0.0297
+ 0.031 ±0.0120
-0.058 ±0.0122
-0.047 ± 0.0278
+ 0.065 ± 0.0169
+0.074 ± 0.0105
+ 0.088 ± 0.0423
-0.021 ±0.0251
+ 0.028 ± 0.0066
-0.033 ± 0.0401
+ 0.208 ± 0.1195
+ 0.252 ± 0.1697
-0.111 ± 0.0546
+ 0.012 ± 0.0080
-0.066 ± 0.0209
-0.169 ± 0.0994
+ 0.028 ±0.0210
+0.025 ± 0.0177
C. Difference (B - A)
-0.100 ±0.0275*
-0.043 ± 0.0143*
-0.020 ± 0.0116
-0.025 ± 0.0239
-0.220 ± 0.0748*
-0.411 ± 0.1259*
-0.052 ± 0.0308
-0.019 ± 0.0052*
-0.008 ± 0.0099
-0.122 ± 0.0921
-0.037 ± 0.0094*
-0.048 ± 0.0104*
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Results in columns A and B represent the net release (positive numbers) or uptake (negative numbers) of each
amino acid (in nmol/mg heart weight per hour) from the heart into the medium during the second day of cultivation
(i.e., after pretreatment with sucrose or control medium for 24 hours); each value represents the mean ± 1 SEM for 24
hearts from eight litters. Column C gives the mean difference ± 1 SEM between the eight matched litters; negative
numbers in column C indicate either increased net uptake or reduced net release.
• P < 0.05 by Student's paired r-test.
With any pharmacological agent that is used to produce
selective effects on a single type of organelle or on a single
aspect of cellular function, it is probable that other
organelles and other functional characteristics will also be
altered to some extent. Indeed, this is intrinsically inevitable if the primary disturbance is on an important aspect
of cell function, since the complex integration of all the
cell's processes will secondarily become upset when a vital
link is perturbed. Thus, in the present studies, as in all
others in which efforts are made to produce selective
perturbations of particular aspects of cell function, one
must be concerned whether the final observations represent predominantly the results of the primary perturbation
in question or whether secondary, nonspecific toxic effects
might not be the cause. Specifically, do the major lysosomal abnormalities known to be produced by sucrose and
chloroquine in fetal mouse hearts'4115 account for the
subsequent inhibition of proteolytic capacity observed in
the present experiments, or is some other factor more
important?
Ultimately, of course, this question can never be answered with certainty. Nevertheless, in the case of sucrose,
there is no evidence of apparent abnormalities in any
other organelles,l5 cardiac beating (which is an exquisitely
sensitive indicator of even mild toxicity or damage in
hearts in organ culture) is not depressed over the interval
of time observed,15 and there is a close concurrence of the
time-course of the observed effects on lysosomes and on
proteolysis. These facts lead one to conclude with some
confidence that sucrose-induced inhibition of protein
breakdown probably is caused by specific lysosomal dysfunction rather than by nonspecific toxicity. In the case of
chloroquine, evidence of some mild, nonspecific toxicity
is a bit more disturbing. Although beating remains strong
over the first day, exposure to chloroquine leads ultimately to contractile depression (unpublished results).
Furthermore, some mitochondrial abnormalities develop
after 1-2 days,1'1 which might lead to inadequate oxidative
metabolism. Because interference with normal oxidative
metabolism can itself produce inhibition of cardiac prote-
olysis,2126 one must consider the possibility that the
observed results might result primarily from mitochondrial
damage and inadequate oxidative energy production
rather than from lysosomal dysfunction. If toxic damage
were severe enough to inhibit oxidative metabolism sufficiently to depress proteolysis, however, alanine production and release from the heart would be expected to
increase markedly.27 Since no such increase occurred
(Table 2), it seems unlikely that the observed inhibition of
proteolysis can be explained by nonspecific cellular toxicity and inadequate oxidative energy production.
In summary, considering all the experiments involved
both sucrose and chloroquine, we believe that the most
likely explanation of the results is that the observed
reductions in protein breakdown are caused primarily by
lysosomal derangements. Thus it appears that, in this
experimental model, cardiac lysosomes can play a major
role in the degradation of myocardial protein and that
interference with lysosomal function retards proteolysis.
Acknowledgments
We are grateful to M. Kennedy and S.C. Jasinski for valuable assistance.
References
1. Wildenthal K: Lysosomes and lysosomal enzymes in the heart. In
Lysosomes in Biology and Pathology, vol 4, edited by JT Dingle, RT
Dean. Amsterdam, North Holland Publishing Co., 1975, pp 167-190
2. Wildenthal K: Long-term maintenance of spontaneously beating
mouse hearts in organ culture. J Appl Physiol 30: 153-157, 1971
3. Wattiaux R, Wattiaux-deDoninck S, Rutgeerts M-J, Tulkens P:
Influence of the injection of a sucrose solution on the properties of
rat-liver lysosomes. Nature 203: 757-758, 1964
4. Trump BP, Janigan DT: The pathogenesis of cytologic vacuolization
in sucrose nephrosis; an electron microscopic and histochemical study.
Lab Invest 11: 395-411, 1962
5. Glauert AM, Fell HB, Dingle JT: Endocytosis of sugars in embryonic
skeletal tissues in organ culture. II. Effect of sucrose on cellular fine
structure. J Cell Sci 4: 105-131, 1969
6. Dingle JT, Fell HB, Glauert AM: Endocytosis of sugars in embryonic
skeletal tissues in organ culture. IV. Lysosomal and other biochemical
effects; general discussion. J Cell Sci 4: 139-153, 1969
7. Abraham R, Hendy R, Grasso P: Formation of myeloid bodies in rat
liver lysosomes after chloroquine administration. Exp Mol Pathol 9:
212-229, 1968
792
CIRCULATION RESEARCH
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8. Hendy RJ, Abraham R, Grasso P: The effect of chloroquine on rat
heart lysosomes. J Ultrastruct Res 29: 485-495, 1969
9. Fedorko ME, Hirsch JG, Cohn ZA: Autophagic vacuoles produced in
vitro. I. Studies on cultured macrophages exposed to chloroquine. J
Cell Biol 38: 377-391, 1968
10. Lie SO, Schofield B: Inactivation of lysosomal function in normal
cultured human fibroblasts by chloroquine. Biochem Pharmacol 22:
3109-3114, 1973
11. Wibo M, Poole B: Protein degradation in cultured cells. II. The
uptake of chloroquine by rat fibroblasts and the inhibition of cellular
protein degradation and cathepsin B,. J Cell Biol 63: 430-440, 1974
12. Goldstein JL, Brunschede GY, Brown MS: Inhibition of the proteolytic degradation of low density lipoprotein in human fibroblasts by
chloroquine, concanavalin A, and triton WR 1339. J Biol Chem 250:
7854-7862, 1975
13. Goldstein JL, Brown MS, Anderson RGW: The LDL pathway in
human fibroblasts: Biochemical and ultrastructural correlations. In
International Cell Biology 1976-77, edited by BR Brinkley, KR
Porter. New York, Rockefeller University Press, 1977, pp 639-648
14. Ridout RM, Decker RS, Wildenthal K: Chloroquine-induced lysosomal abnormalities in cultured foetal mouse hearts. J Mol Cell Cardiol
10: 175-183, 1978
15. Wildenthal K, Dees JH, Buja LM: Cardiac lysosomal derangements
after long-term exposure to nonmetabolizable sugars. Circ Res 40:
26-35, 1977
16. Wildenthal K: Studies of foetal mouse hearts in organ culture:
Metabolic requirements for prolonged function in vitro and the
influence of cardiac maturation on substrate utilization. J Mol Cell
Cardiol 5: 87-99, 1973
17. Wildenthal K, Griffin EE: Reduction by cycloheximide of lysosomal
18.
19.
20
21.
22.
23.
24.
25.
26.
27.
VOL. 42, NO. 6, JUNE
1978
proteolytic enzyme activity and rate of protein degradation in organcultured hearts. Biochim Biophys Acta 444: 519-524, 1976
Lowry OH, Rosebrough NJ, Fair, AL, Randall RJ: Protein measurement with Folin phenol reagent. J Biol Chem 193: 265-275, 1951
Wildenthal K, Griffin EE, Ingwall JS: Hormonal control of cardiac
protein and amino acid balance. Circ Res 38 (suppl I): 138-144, 1976
Morgan HE, Earl DCN, Broadus A, Wolpert EB, Giger KE, Jefferson LS: Regulation of protein synthesis in heart muscle. I. Effect of
amino acid levels on protein synthesis. J Biol Chem 246: 2152-2161,
1971
Wildenthal K, Griffin EE: Regulation of cardiac protein degradation:
Use of fetal mouse hearts in organ culture as an experimental model.
In Proceedings of the Third US-USSR Symposium on Myocardial
Metabolism, edited by HE Morgan. Bethesda, Md., DHEW Publications, pp 271-279
Dean RT, Barrett AJ: Lysosomes. Essays Biol 12: 1-40, 1976
Goldberg AL, St. John AC: IntraceUular protein degradation in
mammalian and bacterial cells. Part 2. Ann Rev Biochem 45: 747803,1976
Bird JWC: Skeletal muscle lysosomes. In Lysosomes in Biology and
Pathology, vol 4, edited by JT Dingle, RT Dean. Amsterdam, North
Holland, 1975, pp 75-109
Dean RT: Direct evidence of the importance of lysosomes in degradation of imracellular proteins. Nature 257: 414-416, 1975
Kao R, Rannels DE, Morgan HE: Effects of anoxia and severe
ischemia on the turnover of myocardial proteins. Acta Med Scand 587
(suppl): 117-120, 1976
Taegtmeyer H, Peterson MG, Ragavan W , Ferguson AG, Lesch M:
De novo alanine synthesis in isolated oxygen-deprived rabbit myocardium. J Biol Chem 252: 5010-5018, 1977
Velocity Distribution and Intimal Proliferation in
Autologous Vein Grafts in Dogs
STANLEY E. RITTGERS, PANAYOTIS E. KARAYANNACOS, JULIA F. G U Y ,
ROBERT M. NEREM, GEORGE M. SHAW, JEPTHA R. HOSTETLER, AND JOHN S. VASKO
SUMMARY Autologous femoral veins grafted between the external iliac arteries in 18 dogs provided a model for
studying the hemodynamics and histopathology of vein graft bypasses. The angle of proximal anastomosis was varied
by groups (<90°, 90°, >90°) to produce a wide range of flow conditions within the grafts. Four months after
implantation, point velocity measurements of blood flow and histological examination of the superior and inferior
walls were made at proximal, middle, and distal locations in each graft. Hot-film velocity measurements revealed
outwardly skewed velocity profiles in the proximal locations in all grafts, and flow visualization models showed
secondary fluid motions and separation zones at those regions. Velocity profiles in the middle and distal regions of
the grafts were more symmetrical and showed no flow separation. Histological examination of wall sections along the
graft length showed that intimal proliferation occurred focally and ranged from 1 to 100 fim in thickness. No
signficant correlation between graft angle and degree of intimal proliferation was found. However, there was a weak
inverse correlation between the apparent fluid shear rate and the corresponding degree of intimal proliferation, with
the greatest proliferation occurring in the regions experiencing the lowest shearing forces. Regions of low shear rate
should be avoided by maintaining adequate flow rates through the grafts and thus minimising losses of patency due to
both thrombus formation and intunal proliferation.
HUMAN AORTOCORONARY saphenous vein bypass
procedures are widely employed in the treatment of severe
coronary artery disease. However, loss of graft patency
has led to concern about the overall benefit of this
procedure. Bypass failure rates of up to 23% during the
first yearlw| have been attributed primarily to early occlusion (up to 1 month) by thrombosis or late occlusion (after
1 month) by intimal or subendothelial proliferation. Although its exact etiology is unknown, many investigators
believe intimal proliferation to be a series of repairative
From the Department of Aeronautical and Astronautical Engineering
(S.E.R., R.M.N.), the Department of Surgery, Division of Thoracic
Surgery (P.E.K., J.S.V.), and the Department of Anatomy (J.F.G.,
G.M.S., J.R.H.), The Ohio State University, Columbus, Ohio.
This work was supported in part by Grant HL-17337 from the National
Institutes of Health.
Address for reprints: Stanley E. Rittgers, M.Sc, Department of Medi-
cine, Division of Cardiology, The Ohio State University, Columbus, Ohio
43212.
This investigation is part of a dissertation submitted by S.E. Rittgers in
partial fulfillment of the requirements for the degree of Doctor of
Philosophy, The Ohio State University.
Received August 11, 1977; accepted for publication November 30,
1977.
Inhibition of protein degradation in mouse hearts by agents that cause lysosomal
dysfunction.
K Wildenthal, J R Wakeland, P C Morton and E E Griffin
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Circ Res. 1978;42:787-792
doi: 10.1161/01.RES.42.6.787
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