3. groups of rats with different amounts of myocardial
collagen were studied.
METHODS
Experimental procedure. Thirty-three male Wistar rats
were used in the study. Their care and use conformed with
National Institutes of Health guidelines and the protocol was
approved by the University Animal Care and Use Commit-
tee. In the first group, 10 rats (6 wk old) were anesthetized
with pentobarbital sodium (50 mg/kg ip), and renal hyper-
tension was produced by placing a silver clip around the left
renal artery to constrict it to an external diameter of 0.25
mm; the contralateral kidney remained normally perfused.
After a 5-wk follow-up, the rats were treated for 3 wk with
the angiotensin-converting enzyme (ACE) inhibitor ramipril
(20 mg⅐kgϪ1
⅐dayϪ1
in drinking water; RHTR group). In the
second group (GSSG group, n ϭ 14), myocardial collagen
degradation was induced using the method described by
Caulfield and Wolkowicz (11). Briefly, 10 wk-old rats were
anesthetized and received two intravenous infusions over 3 h
(0.11 ml/min), 1 wk apart, of 20 ml of a 2 mM solution of
oxidized glutathione. The animals were killed 3 wk after the
second infusion, when myocardial hydroxyproline is expected
to be at a minimum (11). A third group (control, n ϭ 9)
consisted of unoperated and untreated normotensive rats
that were the same age as the other two groups at the end of
the experiment (i.e., 14 wk old). All rats were housed in a
temperature-controlled room (24°C) with 12-h light:dark cy-
cles, and food and water were supplied ad libitum. At the end
of the experiment, tail cuff systolic arterial pressure (SAP)
was measured in all rats.
Isolated papillary muscle study. The animals were anes-
thetized with pentobarbital sodium (50 mg/kg ip), and the
body weight (BW) was recorded at the time of death. The
chest was opened by median sternotomy, and the heart was
removed and placed in oxygenated Krebs-Henseleit solution
at 28°C. The LV and septal wall were separated from the
right ventricle, and their weights were determined. One
papillary muscle was dissected from the LV, mounted be-
tween two spring clips, and placed vertically in a bathing
chamber. The lower spring clip was attached to a Kyowa
model 120T-20B force transducer by a thin (1/15,000 in.)
steel wire. The upper spring clip was connected by a thin wire
to a rigid lever arm above which was mounted an adjustable
micrometer stop for the adjustment of unstimulated muscle
length. Oxygenated (95% O2-5% CO2) bathing medium con-
sisted of (in mM) 118.5 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16
MgSO4, 1.18 KH2PO4, 5.50 glucose, and 25.88 NaHCO3 dis-
solved in deionized water. The temperature of the bathing
medium was maintained at 28°C.
The muscle preparation was placed between two parallel
platinum electrodes and stimulated at a frequency of 0.2 Hz,
using square-wave pulses of 5-ms duration. Voltage was set
to a value 10% greater than the minimum required to pro-
duce a maximal mechanical response. After 60 min, during
which the preparation stabilized, the muscle was loaded to
contract isometrically and stretched to the peak length of its
tension-length curve (Lmax).
Once a stable Lmax was determined, the muscle was made
to contract isometrically at Lmax and the resultant isometric
contraction parameters were determined, which included
peak developed active tension (AT, g/mm2
), resting tension
(RT, g/mm2
), peak rate of isometric tension development
(ϩdT/dt, g⅐mmϪ2
⅐sϪ1
), peak rate of tension decrease (ϪdT/
dt, g⅐mmϪ2
⅐sϪ1
), time to peak tension (TPT, ms), and time
from peak tension to 50% relaxation (RT1/2, ms). Active and
passive tension-length curves were derived from data ob-
tained at lengths corresponding to 90%, 92%, 94%, 96%, 98%,
and 100% of Lmax. The muscle length was measured with a
Gaertner cathetometer and telescope. At the end of the ex-
periment, the muscle between the spring clips was weighed
and its cross-sectional area (CSA) was calculated, assuming
cylindrical uniformity and a specific gravity of 1.00. All val-
ues of force were normalized for muscle CSA.
Biochemical study. It has been demonstrated that hy-
droxyproline concentration in the LV free wall is similar to
that in the papillary muscle (15). Therefore, we assumed that
the hydroxyproline observed in the apex of the LV is repre-
sentative of that in the entire ventricle, including the papil-
lary muscle. We measured hydroxyproline in tissue obtained
from the LV apex according to the method described by
Switzer (27). Briefly, the tissue was dried for 4 h using a
SpeedVac Concentrator SC 100 attached to a refrigerated
condensation trap TR 100 and vacuum pump VP 100 (Savant
Instruments, Farmingdale, NY). Tissue dry weight was de-
termined, and the samples were hydrolyzed overnight at
110°C with 6 N HCl (1 ml/10 mg dry tissue). An aliquot of 50
l of hydrolysate was transferred to an Eppendorf tube and
dried in the SpeedVac Concentrator. One milliliter of deion-
ized water was added, and the sample was transferred to a
tube. One milliliter of potassium borate buffer (pH 8.7) was
added to maintain stable pH, and the sample was oxidized
with 0.3 ml of chloramine T solution at room temperature for
exactly 20 min. The oxidation was stopped by the addition of
1 ml of 3.6 M sodium thiosulfate with thorough mixing for
10 s. The solution was then saturated with 1.5 g of KCl, and
the tubes were capped and heated in boiling water for 20 min.
After the tubes cooled to room temperature, 2.5 ml of toluene
were added and the tubes were shaken over 5 min. The tubes
were briefly centrifuged at low speed, and 1 ml of toluene
extract was transferred to a 12 ϫ 75 mm test tube. In the
next step, 0.4 ml of Ehrlich’s reagent was added to allow the
color to develop for 30 min. Absorbencies were read at 565 nm
with a double-beam spectrophotometer (A-160 spectropho-
tometer, Shimadzu) against a reagent blank. Deionized wa-
ter and 20 g/ml hydroxyproline were used as blank and
standard, respectively.
Histology and morphometry. Transverse sections of LV
were fixed in 10% buffered Formalin and embedded in par-
affin. Five-micrometer-thick sections were cut from the
blocked tissue and stained with hematoxylin-eosin and with
the collagen-specific stain picrosirius red (Sirius red F3BA in
aqueous saturated picric acid). Myocyte CSA (MA) was de-
termined for at least 100 myocytes per slide stained with
hematoxylin-eosin. The measurements were performed using
a Leica microscope (ϫ40 magnification lens) attached to a
video camera and connected to a personal computer equipped
with image analyzer software (Image-Pro Plus 3.0, Media
Cybernetics, Silver Spring, MD). MA was measured with a
digitizing pad, and the selected cells were transversely cut
with the nucleus clearly identified in the center of the myo-
cyte. Interstitial collagen volume fraction (CVF) was deter-
mined for the entire section of the heart stained with picro-
sirius red using an automated image analyzer (Image-Pro
Plus 3.0, Media Cybernetics). The components of the cardiac
tissue were identified according to their color level: red for
collagen fibers, yellow for myocytes, and white for interstitial
space. The digitized profiles were sent to a computer that
calculated collagen volume fraction as the sum of all connec-
tive tissue areas divided by the sum of all connective tissue
and myocyte areas. On the average, 35 microscopic fields
were analyzed with a ϫ20 lens. Perivascular collagen was
excluded from this analysis.
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4. Statistics. All grouped data were expressed as means Ϯ SD
and compared by one-way ANOVA and post hoc Tukeys test.
Statistical analyses were performed with SigmaStat statisti-
cal software (Jandel Scientific Software, San Rafael, CA).
Differences with P Յ 0.05 were considered significant.
Straight lines were fit to the systolic tension-length relations
using linear regression analysis (22). The resulting slopes
corresponded to AS, and the means among the groups were
compared by ANOVA. Before the diastolic tension-length
relationship was compared for the three groups, the resting
tension at the muscle length corresponding to 90% of Lmax
(L90) was subtracted from all subsequent tension data in each
experiment to have all tension-length curves intercepting the
y-axis origin at L90.
The diastolic tension-length curves for the three groups
were fit to monoexponential relations of the form RT ϭ
A[e
B
(L Ϫ L0)
Ϫ 1], where A and B are fitting parameters and L0
is the muscle length corresponding to zero resting tension.
These nonlinear relations were compared by constructing an
F ratio from the residual sum of squares. This test deter-
mines whether separate fits to three groups are significantly
better than the fit to data pooled from all groups. Accord-
ingly, a significant F ratio indicates that the two sets of data
being compared were significantly different from one an-
other. For all comparisons, statistical significance was taken
to be P Ͻ 0.05/k where k is the number of comparisons (24).
RESULTS
Average group values for BW, LV weight (LVW),
right ventricular weight, papillary muscle CSA, SAP,
and LVW normalized to BW (LVW/BW) are shown in
Table 1. In the RHTR group, treatment with an ACE
inhibitor for 3 wk significantly reduced systolic blood
pressure from an average value of 202 Ϯ 31 mmHg to
111 Ϯ 11 mmHg (P Ͻ 0.001) and regressed LVW to a
value comparable to the control and GSSG groups. MA
was significantly higher in the GSSG group compared
with control and RHTR groups (Fig. 1). CVF and hy-
droxyproline (Fig. 2) were statistically higher in RHTR
than in the other two groups. The difference between
GSSG and control groups reached a level of signifi-
cance of 10% (Fig. 2A), whereas hydroxyproline was
statistically lower in the GSSG compared with the
control group (Fig. 2B).
The isolated papillary muscle functional parameters
RT, Lmax, AT at Lmax, AT at L90, ϩdT/dt, ϪdT/dt, TPT,
TR1/2, and AS are shown in Table 2. RT was signifi-
cantly higher in the RHTR group (0.64 Ϯ 0.08 g/mm2
)
compared with control (0.47 Ϯ 0.14 g/mm2
) and GSSG
(0.35 Ϯ 0.10 g/mm2
) groups. AT at L90 and at Lmax were
not different among the groups.
In all experiments the relation between peak devel-
oped active tension and muscle length was linear, as
evidenced by the coefficient of determination (r2
),
which was typically Ͼ0.94. This finding means that at
least 94% of the sum of squares of deviations of AT
values about their means is attributable to the linear
relation between AT and muscle length (22). The slope
of these linear regressions corresponds to the myocar-
dial AS, which was significantly increased in the GSSG
group compared with the control group (5.86 Ϯ 1.14 vs.
3.96 Ϯ 1.33 g⅐mmϪ2
⅐%Lmax
Ϫ1
; P ϭ 0.008). The differ-
ences between GSSG and RHTR groups and between
control and RHTR groups were not statistically signif-
icant (Fig. 3).
The passive tension-length curve from the RHTR
group was shifted upward from that of the control
group (F ϭ 14.25; P Ͻ 0.01) and that of the GSSG group
(F ϭ 38.8; P Ͻ 0.01), reflecting an increased passive
stiffness. The GSSG curve was shifted downwards
from the control group (F ϭ 9.95; P Ͻ 0.01), indicating
decreased passive stiffness (Fig. 3).
DISCUSSION
In a previous study (21), we showed that renovascu-
lar hypertension induces marked myocardial hypertro-
phy and interstitial fibrosis. Treatment with ramipril
for 3 wk did not reverse perivascular and interstitial
fibrosis but fully treated the arterial hypertension and
promoted regression of myocardial hypertrophy.
Therefore, we used that experimental model to study
myocardial function in papillary muscle from rat heart
with increased collagen concentration without myocar-
dial hypertrophy. In the present study, collagen
Table 1. Group comparisons of morphometric
parameters and tail cuff systolic arterial pressure
in control, GSSG, and RHTR rats
Control GSSG RHTR
BW, g 329Ϯ17 332Ϯ20 346Ϯ30
SAP, mmHg 136Ϯ14 129Ϯ18 111Ϯ11*
LVW, g 0.66Ϯ0.07 0.65Ϯ0.05 0.69Ϯ0.14
LVW/BW, mg/g 2.01Ϯ0.16 1.96Ϯ0.09 1.90Ϯ0.17
RVW, g 0.21Ϯ0.03 0.19Ϯ0.05 0.21Ϯ0.04
CSA, mm2
0.84Ϯ0.18 0.78Ϯ0.17 0.86Ϯ0.22
Data are presented as means Ϯ SD. BW, body weight; SAP,
systolic arterial pressure; LVW/BW, left ventricle weight (LVW) to
BW ratio; RVW, right ventricle weight; CSA, papillary muscle cross-
sectional area. *P Ͻ 0.05 vs. control. See METHODS for description of
the control, oxidized glutathione (GSSG), and ramipril-treated rat
(RHTR) groups.
Fig. 1. Myocyte cross-sectional area in control, oxidized glutathione
(GSSG), and ramipril-treated rat (RHTR) groups. Data are means Ϯ
SD analyzed by one-way ANOVA with Tukey’s posttest procedure.
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5. amount was measured with CVF and hydroxyproline.
It has been shown that total volume fraction is closely
related to hydroxyproline concentration in the LV (28),
and in our study both measurements indicated that the
interstitial collagen was altered in the treated groups
relative to the control rats. However, we observed that
the CVF measurement was associated with a greater
variation than the measurement of hydroxyproline,
and, consequently, the decrease in CVF in the GSSG
group came close but did not reach the level of statis-
tical significance. The variability of CVF might be
caused in part by the measurement method used. In
the present investigation we used a ϫ20 microscope
objective to obtain a large field. This magnification
would detect only large perimysial collagen fibers,
thereby decreasing the sensitivity of the measurement.
Even so, a power analysis indicated that the difference
between the GSSG and control groups would have
reached the level of significance if but a few additional
histological samples were available.
Despite similar LVW, the papillary muscles were
significantly stiffer in the group with greater collagen
concentration. This result is similar to that obtained by
Narayan et al. (23) using hydralazine to prevent myo-
cyte hypertrophy but not abnormal collagen accumula-
tion in SHR. The collagen excess resulted in abnor-
mally elevated passive myocardial stiffness. In
contrast, Schraeger and co-workers (25) concluded that
ACE inhibitor-induced regression of LV hypertrophy in
Fig. 3. Left ventricular papillary muscle active and passive tension-
length curves obtained from control, GSSG, and RHTR groups.
Results are presented as means Ϯ SD. The active stiffness (AS)
obtained from the GSSG group was statistically higher than that
from controls (P ϭ 0.008). Statistically, no differences were observed
between GSSG and RHTR groups (P ϭ 0.493) and between control
and RHTR groups (P ϭ 0.085). AS was analyzed by ANOVA and post
hoc Tukey’s test. The RHTR passive tension-length curve was shifted
upwards compared with either control (F ϭ 14.25; P Ͻ 0.01) or GSSG
(F ϭ 38.8; P Ͻ 0.01) groups. The curve from the GSSG group was
shifted downwards compared with the control group (F ϭ 9.95; P Ͻ
0.01). The passive tension-length curves were fitted to a monoexpo-
nential relation, and comparisons were made by constructing an F
ratio from the residual sum of squares. Statistical significance was
taken to be P Յ 0.05/k where k is the number of comparisons.
Table 2. Papillary muscle isometric contraction data
for the control, GSSG, and RHTR groups
Control GSSG RHTR
RT, g/mm2
0.47Ϯ0.14 0.35Ϯ0.10 0.64Ϯ0.08*†
AT at Lmax, g/mm2
8.05Ϯ1.59 9.44Ϯ1.86 8.52Ϯ1.72
AT at L90, g/mm2
5.64Ϯ1.26 5.95Ϯ1.64 5.23Ϯ1.58
RT1/2, ms 290Ϯ54 272Ϯ23 280Ϯ47
Lmax, mm 6.23Ϯ0.99 6.07Ϯ0.50 6.31Ϯ0.81
ϩdT/dt, g⅐mmϪ2
⅐sϪ1
75.8Ϯ21.1 84.3Ϯ21.0 72.8Ϯ16.5
ϪdT/dt, g⅐mmϪ2
⅐sϪ1
19.0Ϯ5.5 21.8Ϯ4.9 18.8Ϯ4.1
AS, g⅐mmϪ2
⅐%Lmax
Ϫ1
3.96Ϯ1.33 5.86Ϯ1.14* 5.21Ϯ1.18
TPT, ms 201Ϯ16 193Ϯ12 204Ϯ17
Data are reported as means Ϯ SD. RT, resting tension; AT, active
tension; ϩdT/dt, peak rate of isometric tension development; ϪdT/dt,
peak rate of tension decrease; TPT, time to peak tension; Lmax,
muscle length at peak of the tension-length curve; L90, muscle length
at 90% of Lmax; RT1/2, time from peak tension to 50% relaxation; AS,
active stiffness. *P Ͻ 0.05 vs. control; *†P Ͻ 0.05 vs. GSSG.
Fig. 2. Collagen volume fraction (A) and hydroxyproline concentra-
tion (B) in control, GSSG, and RHTR groups. Data are means Ϯ SD
analyzed by one-way ANOVA with Tukey’s posttest procedure.
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6. SHR significantly decreased the passive stiffness of
skinned trabecular muscle despite abnormally ele-
vated hydroxyproline levels. It is not clear to what
extent, if any, the 48-h incubation in the skinning
solution at 0°C influenced their observations. The dis-
crepancies observed between the studies may be
caused by the animal strains as well as by the different
experimental models used to produce hypertrophy and
fibrosis.
In our study, using the model of presumed collage-
nase activation by oxidized glutathione described by
Caulfield and Wolkowicz (11), it was possible to induce,
in vivo, an 11% reduction of myocardial collagen con-
centration measured by hydroxyproline concentration
and a 19% reduction in the interstitial CVF. These
results are less expressive than the 30–35% reduction
in collagen as reported by Caulfield et al. (10). The
authors have shown that the double infusion of GSSG
resulted in no visible myocyte damage at any time as
examined by light microscopy and scanning electron
microscope (SEM). The collagen matrix alteration was
not visible by light microscopy; SEM revealed damage
to the endomysium with loss of the weave that sur-
rounds groups of myocytes and the struts that inter-
connect myocyte to myocyte and myocyte to adjacent
capillaries, with no change in coiled perimysial fibers.
These changes in the fibrillar collagen network re-
sulted in increased ventricular volume and compliance,
suggesting that damage to the intermyocyte struts and
to the weave complex might be more important than
the decrease in myocardial collagen. Other studies
have shown that the double infusion of GSSG in rats
promotes a reduction in CVF, ventricular dilatation,
and a shift to the right of the diastolic pressure-volume
curve of the entire LV (18, 20). However, a similar
effect in the papillary muscle preparation has not been
studied previously. The main advantage of this prepa-
ration is that the muscle force and length are directly
measured and that the mathematical assumptions re-
quired when myocardial mechanical characteristics
are evaluated in the LV chamber are unnecessary.
The study of cardiac function in the whole heart is
based on the pressure-volume and stress-strain rela-
tionships. In that condition, myocardial stiffness is
derived from chamber measurements using mathemat-
ical models and assumptions regarding LV shape. If
the LV is assumed to be a thick-walled sphere, the
stress will be underestimated (30), whereas the as-
sumption of an ellipsoid shape would result in an
overestimated wall stress (7). Therefore, isolated mus-
cle experiments provide descriptions of myocardial be-
havior without the influence of chamber and wall ge-
ometry. In our study, the diastolic tension-length
relations obtained for the three groups were different
from each other, showing that the changes in collagen
content, measured by hydroxyproline and CVF, are
associated with myocardial passive properties. Com-
pared with the control group, the diastolic tension-
length curves were significantly shifted upwards and
to the left in the RHTR group and downwards and to
the right in the GSSG group. Therefore, our results
allow us to conclude that the decreased passive stiff-
ness in the GSSG group strongly correlates with the
fibrillar collagen loss and that increased collagen con-
tent strongly correlates with the elevated passive stiff-
ness observed in the RHTR group. Previous studies
have suggested that collagen cross-linking (9) may
affect myocardial stiffness, regardless of collagen
amount. In addition to the effect of altered collagen
amounts, it is important to be mindful of the effects of
collagen crosslink density, as well as collagen type
(type I or III) and collagen distribution. At present, we
cannot rule out that changes in the collagen character-
istics might also have influenced myocardial stiffness
in the present study. Nevertheless, the results clearly
indicate that alterations in collagen concentration and
papillary muscle function are correlated.
Ventricular elastance and myocardial stiffness are
indexes of contractility of the ventricular chamber and
myocardium, respectively (8, 26). Elastance is the ratio
of the change in peak isovolumetric pressure for a
given change in volume, and stiffness is defined as the
ratio of the change in active force related to change in
muscle length (8). Myocardial contractility is a very
complex property of the heart that is difficult to mea-
sure directly. During the last two decades it has been
proposed that an ideal index of myocardial contractility
must be able to measure the ability of the myocardium
to generate force independently of loading condition.
The slope of the linear pressure-volume relationship in
the isolated canine heart has been shown to be rela-
tively independent of preload and afterload and there-
fore has been used as an index of contractility (26).
Using the slope of active tension-length (active stiff-
ness) as an index of myocardial contractility, we have
shown an enhancement of active stiffness when the
muscle is stretched from 90% to 100% of Lmax in the
GSSG group. The mechanisms underlying the associa-
tion between decreased myocardial collagen and en-
hanced active stiffness are not well established, and
the results presented in this study do not answer all
the questions concerning this matter. When collagen is
reduced, ventricular dilatation occurs (10) and myocyte
hypertrophy takes place in response to alterations in
the loading state of the ventricle (17). Therefore, myo-
cyte hypertrophy might play an important role in the
improvement in contractility observed in the GSSG
group. Another explanation would be related to the
intracellular glutathione metabolisms. The glutathi-
one level in the heart is ϳ1.2 M/g (16), mainly in the
reduced form, GSH, because of the high activity of
GSSG reductase (13). That means that, inside the cell,
most of the infused GSSG was rapidly converted to
GSH. The action of excess GSH or GSSG in the heart is
not completely elucidated. Bauer et al. (3), working on
fiber bundles from papillary muscle of porcine right
ventricle, observed an increased sensitivity of contrac-
tile protein to calcium and, consequently, an increased
force development in the presence of GSH. In our
study, considering that the half-life of the glutathione
is only a few minutes (1), it is doubtful that the double
infusion of oxidized glutathione might increase the
H1538 COLLAGEN AND MYOCARDIAL MECHANICS
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7. glutathione level in the cardiac tissue after 3 wk.
Nevertheless, this is a very complex matter that re-
quires further study. Active tension and active stiff-
ness in papillary muscles from RHTR rats were similar
to those in the control rats, suggesting that regression
of hypertrophy by treatment with an ACE inhibitor is
associated with preserved myocardial contractility.
We conclude that decreased collagen content induced
by GSSG is associated with myocyte hypertrophy, de-
creased passive stiffness, and increased active stiff-
ness. Abnormally high collagen concentration corre-
lates with myocardial diastolic dysfunction and has no
relation with systolic function.
This study was supported by a grant from Fundac¸a˜o de Amparo a`
Pesquisa do Estado de Sa˜o Paulo (FAPESP), Sa˜o Paulo, Brazil, Proc.
No. 92/4528–1.
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