Collagen Synthesis in Development and Reversal of

Cardiac Hypertrophy in Spontaneously Hypertensive Rats

SUBHA SEN, PhD, DSc
F. MERLIN BUMPUS, PhD
Cleveland, Ohio
An alteration in the rate of collagen synthesis was observed in sponta-
neously hypertensive rats during evolution of hypertension. An increase
in rate of synthesis of collagen and a parallel increase in collagen content
were observed in 4-8 week and 24 week old hypertensive rats. In the 4
week old rats, blood pressure was normal or nearly normal, whereas in
the 24 week old rats the arterial pressure was significantly elevated. Use
of some antihypertensive drugs, namely, a-methyldopa, converting en-
zyme inhibitor and a combination of reserpine, hydrochlorothiazide and
apresoline, prevented hypertension and the late increase in collagen
synthesis that was observed in the 24 week old hypertensive rats. In these
rats, prevention of hypertension also reversed myocardial hypertrophy
and reduced collagen content of the myocardium. The alteration of
myocardial collagen synthesis in spontaneously hypertensive rats is a
complex process in which at least two phases can be observed. In the
young rats, in which blood pressure is normal, the stimulus to increase
in collagen may be a humoral factor or a hemodynamic alteration such
as hyperkinetic circulation or it may be a genetic factor. In the older hy-
pertensive rats hypertension seemed more important in altering collagen
synthesis because antihypertensive therapy inhibited the rate of collagen
synthesis and protected the heart from excess accumulation of col-
lagen.
Hypertension is an important risk factor for cardiovascular disease in
man, but its relation to the biochemical changes in the cardiovascular
system is not well established. Wolinsky et a1.Q showed that lysosomal
enzyme activity is increased in the aorta of rats with experimentally
induced hypertension. They concluded that lysosomal enzymes may have
a role in the development of the atherosclerotic plaque. Recently, in-
vestigators in several laboratories3-5 reported that vascular collagen
synthesis is greatly increased when arteriosclerosis is induced by
chemical or mechanical damage to the arterial wall. Othersly reported
that the long-term effect of hypertension and arteriosclerosis was similar
to that of vascular fibrosis. Freis et a1.7 reported that dilatation of the
arterioles due to hypertension causes damage to elastic laminae and
stimulates collagen synthesis.
From the Research Division, Cleveland Clinic
Foundation, Cleveland, Ohio. This study was
supported in part by Grants 6835 and 15837 from
the National Heart, Lung, and Blood Institute, Na-
tional Institutes of Health, Bethesda, Maryland.
Manuscript received July 17, 1979; accepted July
20, 1979.
Address for reprints: Subha Sen, PhD, Research
Division, Cleveland Clinic Foundation, 9500 Euclid
Avenue, Cleveland, Ohio 44106.
We have reported an age-dependent increase in collagen content of
the myocardium of spontaneously hypertensive rata. In the 3 and 24 week
old rats, the collagen content was significantly elevated, whereas in lo-15
week old rats the content was the same as in the control rats. The rate
of synthesis of collagen showed parallel changes.8 The stimulus for in-
creased collagen synthesis in the young or old spontaneously hyper-
tensive rat is not known. Because arterial nressure had been normal in
the 4 week old hypertensive rat, pressure is not likely to be a causal factor.
In the older hypertensive rat, however, elevated blood pressure persisting
for a long time may have a role in triggering collagen synthesis. The
954 October 22, 1979 The American Journal of CARDIOLOGY Volume 44
COLLAGEN SYNTHESIS AND CARDIAC HYPERTROPHY-SEN AND BUMPUS
present study was conducted to evaluate the effect of
long-standing hypertension on rate of collagen synthesis
and collagen content in spontaneously hypertensive rats
by preventing the development of hypertension by oral
antihypertensive therapy.
Methods
Study groups: All spontaneously hypertensive rats used
in this study were the Okamoto-Aoki strain obtained from
Taconic Farm (Germantown, New York). The normotensive
controls were age- and sex-matched Kyoto Wistar rats. Both
normotensive and hypertensive rats were kept under the same
conditions, properly housed and fed (Purina@ Rat Chow).
Arterial pressure was measured by using a tail cuff in a method
similar to that described by Friedman and Freed.g In all ex-
periments, rats were killed by decapitation and ventricles were
cleaned as described previously.1
Collagen synthesis study: For the study of collagen syn-
thesis, ventricles were homogenized in cold Krebs bicarbonate
buffer, pH 7.5 (150 mg/ml). Ten PCi of Carbon-14 proline
(New England Nuclear) was added and incubated under a
mixture of 95 percent oxygen and 5 percent carbon dioxide for
3 hours at 37 C with rapid shaking. Reaction was stopped by
adding O.lN HCl:ethanol(1:20) and washing was carried out
three times with same acid ethanol, two times with ethanol
ether (1:3), and finally with ether. Free proline in the com-
bined supernatant was determined (both radiometrically and
calorimetrically) by the method of Troll and Lindsley.
The residue was homogenized in cold HEPES buffer (pH
7.4). The following concentrations were determined by taking
a known aliquot: (1) Hydroxyproline concentration was de-
termined after hydrolysis for 24 hours with 6N HCl; the
method described by Bergman and Loxley12 was used. (2)
Total carbon-14 counts were determined before collagenase
treatment. (3) A third aliquot was treated with chromato-
graphically pure collagenase (Worthington Chemicals) and
incubated for 4 hours at 37C with continuous shaking. At the
end of 4 hours, reaction was stopped by adding 5 percent TCA
= 0.25 percent tannin, and washing was carried out to three
times with 3 to 4 volumes of the same mixture, and centri-
fuged. The radioactivity of the precipitate was determined in
a Packard scintillation counter. The supernate (collagen) was
dialyzed overnight at 4C, evaporated to dryness, and hy-
drolyzed by adding 6N HCl at 110 for 24 hours. The proline
and hydroxyproline in the hydrolysate were separated by
using a Dowex-50 (H+ form) column and radioactivity was
counted. The absolute rate of hydroxylation of proline to
OH-proline was calculated as described by Ehrhart et al.13
Antihypertensive drugs: All antihypertensive drugs were
given in drinking water in the following doses:
A. Converting enzyme inhibitor (CEI or SQ 14,225)-45
mg/liter
+
Hydrochlorothiazide-500 mg/liter
B. Alpha methyldopa-2.5-5 g/liter
C. Reserpine-0.4 mg/liter
+
Hydrochlorothiazide-500 mg/liter
+
Apresoline-60 mg/liter
D. Apresoline--80 mg/liter
These drugs were selected because they effectively lowered
blood pressure and reversed myocardial hypertrophy in
spontaneously hypertensive rata. l4 Ten rats were used in each
group.
Results
Effect of hypertension on collagen synthesis: The
data on blood pressure and rate of collagen synthesis
during evolution of hypertension in spontaneously hy-
pertensive rats and Wistar-Kyoto (control) rats are
shown in Figure 1. In the 4 week old hypertensive rats,
when blood pressure was not elevated (120 f 10 versus
112 f 8 mm Hg), a significant increase in the rate of
collagen synthesis was found (52.5 f 8 versus 17 f 4 ng
collagen/100 mg tissue per 3 hours) (P <O.OOl). At 8
weeks, a similar increase in the rate of collagen synthesis
was observed, except that the blood pressure in the
hypertensive rats was significantly elevated (174 f 6
versus 123 f 3) (P <O.Ol). When the hypertensive rats
were 10 and 15 weeks old, the rate of collagen synthesis
was slightly, but not significantly, elevated (P <O.l)
compared with the rate in age-matched control rats,
despite persistence of hypertension (191 f 4 versus 134
f 3 and 189 f 5 versus 130 f 4, respectively) (P <O.OOl).
In rats 24 weeks of age, the rate of collagen synthesis was
significantly elevated again (23 versus 11 ng/lOO mg per
3 hours) in parallel with sustained hypertension (191 f
6 versus 125 f 3 mm Hg).
Effect of antihypertensive therapy: The effect of
prevention of hypertension by antihypertensive drugs
on heart weight, blood pressure and kidney weight is
shown in Figure 2. Hypertension was prevented by all
drugs used. Alpha-methyldopa, converting enzyme in-
hibitor plus hydrochlorothiazide, and a combination of
reserpine, apresoline and hydrochlorothiazide pre-
vented the development of hypertrophy in all hyper-
tensive rats. The heart weight/body weight ratios were
2.8 f 0.02,2.5 f 0.03 and 2.8 f 0.001 mg/g, respectively,
compared to 3.3 f 0.04 mg/g in untreated control rats.
Apresoline therapy alone did not prevent the develop-
ment of hypertrophy, despite giving excellent blood
70
1
60-
P
2 50-
:
.Z
a 40-
8
c
5 30-
2
2
0

20-
z
IO -
I I
4 8 IO 15 24
AGE (weeks)
W ImW
3K 12otl o 174tb 19l t4* 169t5 191tb
WKy llZt8 rat3 Kut3 130t4 115t3
FIGURE 1. Rate of collagen synthesis during evolution of hypertension
in spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKy)
control rats. BP = blood pressure.
October 22, 1979 The American Journal of CARDIOLOGY Volume 44 955
COLLAGEN syN~~Esl s AND CARDI AC I ~YPEI TRoPH~--SEN AND BUMPUS
BP 195 112 109 130 128 I20mmHg
WATER 74 73.8 73.7 74.0 - 74.0 %
CONTENT
0 SHR-UNlPEATED
a CEI + ESIDRIX
0 RESERPINE HYDROCHLO APPRESOUNE
I a-MElYLDOPA
B APPRESOLINE
Q WKY
FIGURE 2. Prevention of hypertension in spontaneously hypertensive
rats by long-term (6 months) antihypertensive drug therapy. For details
of doses, see text. CEI -!- Esidrix = converting enzyme inhibitor plus
hydrochlorothiazide; Hydrochloro = hydrochlorothiazide. Other ab-
breviations as in Figure 1.
pressure control. The reduction in heart weight was not
a nonspecific effect, because the kidney weight/body
weight ratio was not altered (Fig. 1). Furthermore, the
water content of the myocardium was not different in
the treated groups, indicating that the reduction of
heart weight was not due to change of water content.
Effect of antihypertensive drug therapy on total
myocardial protein is shown in Figure 3. The concen-
tration of protein in the myocardium was not different
in the drug-treated hypertensive rats compared with
that of untreated control animals (35.4 f 2.44 (CEI),
34.01 f 1.42 (alpha methyldopa), and 34.86 f 2.94
(combination) mg/dl, respectively, in the three treat-
ment groups) compared with 33.76 f 1.96 mg/dl in un-
treated hypertensive rata. However, the protein content
was significantly reduced with all three drug regimens
(280.6 f 20,285 f 16.82, and 330 f 22 mg, respectively)
compared with 394 f 20 mg in the untreated group (P
<O.Ol).
The collagen concentration and content (mgfven-
tricle) are shown in Figure 4. As with protein concen-
tration, the myocardial collagen concentration in the
treated hypertensive rats was not changed-4.98 f 0.28
(converting enzyme inhibitor), 4.80 f 13 (alpha meth-
yldopa) and 5.05 f 0.49 (combination) mg/g-compared
with 4.62 f 0.26 mglg in the untreated group (not sig-
nificant). The content of collagen was significantly re-
duced because of drug therapy (Fig. 4, lower panel); 3.89
f 0.25,3.78 f 0.15 and 4.1 f 0.4 mg, respectively, for the
three treatment groups, and 5.37 f 0.36 in untreated
control rats (P <O.OOl in all cases). The rate of myo-
FIGURE 3. Effect of antihypertensive drugs on myocardial protein
concentration and total content after prevention of hypertension in
spontaneously hypertensive rats. p = probability: SQ 14,225 = con-
verting enzyme inhibitor.
cardial collagen synthesis of treated and untreated
hypertensive rats is shown in Figure 5. The rate of col-
lagen synthesis was significantly reduced with three
drug therapy. In untreated hypertensive rats the rate
of collagen synthesis was 775.7 f 145.2 pg/mg protein
per 3 hours. In the three treatment groups the rate of
collagen synthesis was inhibited to 364.7 f 77,479 f
125, and 387 f 47.7 pg/mg protein per 3 hours, respec-
tively (P <O.OOl for all three groups).
Discussion
Factors altering collagen component of myo-
cardial protein after antihypertensive treatment:
This study describes the changes in protein and collagen
contents and the rate of synthesis of newly formed col-
lagen during evolution of hypertension and after pre-
vention of development of hypertension by antihyper-
tensive drugs. During the evolution of hypertension, the
rate of collagen synthesis was significantly increased in
4 week old spontaneously hypertensive rats (when blood
pressure was not yet elevated) and in 24 week old hy-
pertensive rats in parallel with increased heart weight
and collagen c0ntent.s Several questions arise from this
observation: (1) Why was the rate of synthesis increased
at 4 weeks of age? (2) Why was there a second increase
at 24 weeks? (3) Are the triggering stimuli the same in
both cases? (4) Is the second increase due to persistent
hypertension in spontaneously hypertensive rats?
Several investigators3,15J6 have indicated that in
doca-salt hypertensive rats and spontaneously hyper-
tensive rats, blood pressure may have a role in altering
the rate of protein synthesis of the cardiovascular sys-
tem, especially in the aorta and the mesenteric artery.
In this study, to investigate the effect of persistent hy-
pertension, spontaneously hypertensive rats were
treated with antihypertensive drugs from the prehyp-
956 October 22, 1979 The American Journal of CARDIOLOGY Volume 44
COLLAGEN SYNTHESIS AND CARDIAC HYPERTROPHY-SEN AND RUMPUS
5 6.0
=
2 50 .
s 4.0
g p 3.0
t? t
z-
2.0
?J
I .o
1!
:
** D<. ool
950 r
z 850
1 I
2
p 750 -
0
= 650
f
2 550 -
t!
2 450 -
z
k 350 c
250 L
FIGURE 5. Effect of long-term antihypertensive therapy with three
treatment regimens on rate of myocardial collagen synthesis after
prevention of hypertension in spontaneously hypertensive rats. CEI =
converting enzyme inhibitor; p = probability.
FIGURE 4. Effect of antihypertensive drugs on myocardial collagen
concentration (mg/g ventricle) and content (mg/ventricle) after pre-
vention of hypertension in spontaneously hypertensive rats. SQ 14,225
= converting enzyme inhibitor.
ertensive state at 3 weeks to 24 weeks of age. All three
drug regimens not only prevented the development of
hypertension, but also reduced the rate of collagen
synthesis, and actually lowered the collagen content of
the heart. The reduction in collagen is somewhat dif-
ferent from that which we have reported. In our previ-
ous study17 we showed that short-term (3 to 6 weeks)
therapy with alpha methyldopa administered to 8 week
old spontaneously hypertensive rats reversed myocar-
dial hypertrophy. The concentration of collagen in the
heart in these rats was increased from 4.0 f 0.13 to 4.89
f 0.26 mg/g (P <O.Ol), whereas the content remained
the same because of the smaller size of the heart (3.75
mg versus 3.80 mg). This observation indicated that
duration of drug therapy or the age of the hypertensive
rat could be an important factor in altering the collagen
component of myocardial protein.
Role of hypertension: The response of the vascular
system to hypertension appears to be similar to that of
the heart. Hollander et a1.6 reported that thickening of
the aorta in the spontaneously hypertensive rat was
associated with an increased content of collagen and
protein. These changes were detectable as early as 2
weeks after birth. The increased content of collagen
could be prevented by antihypertensive drug therapy.4,s
An increase in collagen content may alter cardiac per-
formance. Averill et al.ls reported that in chronic renal
hypertensive rats, ventricular performance was signif-
icantly reduced, and the ventricles of these rats had
higher collagen content. However, whether cardiac
performance was reduced in the spontaneously hyper-
tensive rat was not established. Hollander et al.j re-
ported that antihypertensive drug treatment in the
hypertensive rat can arrest the progression of hyper-
tensive cardiovascular disease and cause significant
regression. These observations are consistent with the
clinical findings in hypertensive men reported by Freis
et al. 7
In young spontaneously hypertensive rats, as hy-
pertension is developing, a negative correlation between
rate of collagen synthesis and blood pressure was noted
(Fig. 1). This suggested that the increase of rate of col-
lagen synthesis at 4 and 8 weeks was independent of the
rise in blood pressure. In contrast, in older sponta-
neously hypertensive rata, the rate of collagen synthesis
was increased in parallel to increased blood pressure.
This second increase could be due to the long-standing
hypertension. That hypothesis is supported by the
successful prevention of the second rise in collagen
synthesis by long-term antihypertensive therapy (Fig.
5). Thus, an intriguing dissociation in collagen synthesis
was found between younger and,older hypertensive rata;
only the latter was related to hypertension.
Factors altering myocardial protein synthesis in
hypertensive heart disease: The exact stimulus for
the alteration of myocardial protein synthesis in spon-
taneously hypertensive rats is not clear. In the very
young rats, in which blood pressure is normal or nearly
normal, the stimulus may be a humoral factor like
growth hormone, or a hemodynamic abnormality such
as hyperkinetic circulation. In the older rats, however,
long-standing hypertension appeared to play the more
important role. In the latter case, it is likely that stim-
ulation of protein synthesis in hypertensive cardiovas-
cular disease is largely related to structural damage to
the heart and blood vessels caused by long-standing
increased pressure. Other authorsI have reported re-
duction of collagen content in vessels of hypertensive
rats by antihypertensive therapy; our study appears to
be the first one documenting the reversal of the content
October 22, 1979 The American Journal of CARDIOLOGY Volume 44 957
COLLAGEN SYNTHESIS AND CARDIAC HYPERTROPHY-SEN AND BUMPUS
as well as the rate of collagen synthesis in the myocar-
dium of spontaneously hypertensive rats.
of treatment and type of collagen are also important
factors in the reversal of collagen accumulation.
Implications: The -quantity of collagen in the myo-
cardium & believed to be an important factor in hy-
pertension because accumulation of collagen or fibrous
tissue might reduce cardiac performance. Furthermore,
duration of antihypertensive treatment seems to be
important in the reversal of collagen. Further studies
are necessary to determine whether or not age at start
Acknowledgment
We gratefully acknowledge the skilled technical assistance
of Carolee Hollinger and David Young. We also thank Merck
Sharp & Dohme for a generous supply of alpha methyldopa,
Dr. Z. P. Horovitz of Squibb Pharmaceutical Co. for SQ 14,225
and Ciba Pharmaceutical Co, for apresoline and hydrochlo-
rothiazide.
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958 October 22, 1979 The American Journal of CARDIOLOGY Volume 44