Simultaneously Measured Isometric Tension
and ATP Hydrolysis in Glycerlnated Fibers
from Normal and Hypertrophied Rabbit Heart
By Philip D. Henry, Gail G. Ahumada, William F. Friedman,
and Burton E. Sobel
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ABSTRACT
Adenosinetriphosphatase (ATPase) activity, isometric tension, and sarcomere length of glycerinated fibers from 14 hypertrophied and 18 normal rabbit
hearts were measured simultaneously to characterize the altered performance of
hypertrophied myocardium. ATPase activity was assessed radiochromatographically, and contributions from mitochondria, sarcolemma, and sarcoplasmic
reticulum were excluded. The active length-tension and the active tension-pCa
relationships in fibers from hypertrophied myocardium were normal in sharp
contrast to the depressed active length-tension curves in corresponding intact
papillary muscles. Total ATPase activity associated with contraction at a given
developed tension was decreased significantly in fibers from hypertrophied
hearts ( P < 0.001). ATPase activity was diminished in glycerinated myofibrils,
actomyosin, and homogenates from hypertrophied hearts as well. On the other
hand, the increase in ATPase activity associated with a given increase in
developed tension was virtually identical in fibers from hypertrophied and
normal hearts. Thus, depressed total ATPase activity in glycerinated fibers from
hypertrophied hearts does not appear to impair maintenance of isometric
tension.
KEY WORDS
myocardial mechanics
energy utilization
myofibrils
length-tension relationship
myofibrillar adenosinetriphosphatase
• The decreased performance of the hypertrophied or failing myocardium might be
related to the diminished adenosinetriphosphatase (ATPase) activity of the contractile
proteins (1-6). However, the functional implications of depressed myosin and actomyosin
ATPase activity in hypertrophied and failing
heart muscle have not yet been elucidated.
Accordingly, simultaneous determinations of
sarcomere length, developed tension, and rate
of adenosine triphosphate (ATP) hydrolysis
From the Departments of Medicine and Pediatrics,
University of California, San Diego, School of
Medicine, La Jolla, California 92037.
This investigation was supported in part by U, S.
Public Health Service Program Project Grant HL
12373 and by Research and Career Development
Awards 5-K4-HL-41, 737-04 (Dr. Friedman) and 1K4-HL-50, 179-01 (Dr. Sobel).
Received May 19, 1972. Accepted for publication
September 11, 1972.
740
actomyosin
contractility
were made in glycerinated rabbit myocardial
fibers to define the relationship between the
maintenance of tension and the depression of
contractile protein ATPase activity associated
with hypertrophy.
Methods
Reagents.—Disodium ATP, imidazole, neutralized ediyleneglycol bis(y3-aminoethylether)-N,
N'-tetraacetate
(EGTA), and dithiothreitol
(DTT) were obtained from Sigma Chemical
Corporation. Gamma-labeled AT32P and "C-ATP
were obtained from New England Nuclear
Corporation. Deionized, doubly glass-distilled
water was used for reagent preparation.
Animal Procedures.—Male New Zealand rabbits, weighing 2.5—3.0 kg, were anesthetized with
sodium pentobarbital (25 mg/kg, iv) and
maintained with positive-pressure ventilation.
Right ventricular hypertrophy was produced by
an 85$ constriction of the pulmonary artery with
Teflon clips. (3.4 mm, i.d.). Sham-operated
rabbits served as controls. Rabbits were killed by
Circulation Rtstsrcb, Vol. XXXI, Normier 1972
ISOMETRIC TENSION AND ATP HYDROLYSIS
741
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a blow to the head 1 week after surgery, and thenhearts were removed rapidly, immersed in 0-4 o C
isotonic KG, and trimmed. The free right
ventricular wall and the left ventricle with the
septum were blotted and weighed. Papillary
muscles used for studies of mechanics were
transferred immediately to a myograph containing
oxygenated, buffered Krebs solution without
being immersed in KC1.
Mechanics of Papillary Muscles.—Right ventricular papillary muscles were suspended vertically in a myograph; the tendinous end was fixed
with 4-0 silk and the other end was attached with
a clip to a Statham Gl-1-100 force transducer.
The bath contained modified Krebs-bicarbonate
buffer, pH 7.4 at 30°C, equilibrated with 95% O25% CO2. The solution had the following miUimolar
composition: NaCl 121, KC1 2.4, MgSO4 1.1,
KH2PO4 1.1, CaCl2 2.5, NaHCO a , 25 and glucose
4.5. The muscles were stimulated with platinum
field electrodes at a frequency of 12/min with
square waves 4 msec in duration at a voltage 10%
above threshold. Relatively large muscles from
normal hearts and relatively small muscles from
hypertrophied hearts were selected to match the
muscles for cross-sectional area. Isometric lengthtension curves were obtained by increasing length
in 0.1-mm increments until actively developed
tension was maximum (Lmax). Muscle diameters at
Lmax were used to calculate cross-sectional areas
assuming a cylindrical model. When experiments
were complete, muscles were fixed at Lmax in
10% formalin prior to light microscopy studies.
Glycerination of Fibers and Myofibrils.—\Jnstretched right ventricular papillary muscles were
placed in a solution containing 50% glycerol (by
volume), 20 mM imidazole, 4 mM EGTA, and 1
mM DTT, pH 7.0 at 0°C. After 24 hours at 0°C,
the muscles were transferred to fresh glycerination medium and stored at —20°C for 1 week.
Right ventricular walls were cut into small
fragments, immersed in 30 volumes of glycerination medium, stored for 24 hours at 0°C, and
subsequently stored for 1 week at — 20° C.
Myofibrils were prepared from glycerinated
fragments as described below.
Mechanics of Glycerinated Fibers.—Fibers
were studied in a micromyogTaph consisting of a
250-/i,liter reaction chamber machined from
polycarbonate (Fig. 1). Horizontal stainless steel
wires (diameter 0.2 mm) penetrated the chamber
without touching its wall from each end through
slits (width 0.16 mm) in the chamber walls.
Glycerinated fibers were suspended horizontally
in the chamber by attachment to the wires with
6-0 silk. The free ends of the fibers were trimmed
very closely to the silk ties. One wire was
attached to a micrometer to adjust fiber length.
The other wire was attached to a Sanbom FT-A-1
strain-gauge transducer. The overall compliance
fl»f
!•"—15 mm — ',
4 --
T>
1) Microscope condenser
2) Modified microscope stage
3) Microscope objective
4) Plexiglass muscle bath chamber
5)
6)
7)
8)
9)
Nfi
Muscle suspension wires
Hinge
Lever arm
Micrometers used as slops
Force transducer
FIGURE 1
The micromyogTaph
diagrammatically.
used
to assess mechanics
CircmUion Rtstsrcb, Vol. XXXI, Novtmbtr 1972
in
glycerinated
fibers
is
represented
742
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of the force-transducing system was 0.12 /im/mg
weight, and the natural frequency was 66 Hz.
Transducer signals were amplified with a Brush
carrier amplifier and recorded with a Brush Mark
200 recorder. The myograph was mounted on a
standard Zeiss microscope fitted with Kpl 12.5
eyepieces, Optovar magnifier, UMK 50 Leitz
objective, filar micrometer, and Polaroid camera.
The reaction chamber could be moved within the
visual field of the microscope with an x-y
manipulator.
Relaxation solution had the following millimolar composition: KC1 100, MgCl2 5, Na2ATP 5,
neutralized ethylenediaminetetraacetate (EDTA)
5, sodium azide 5, and imidazole 20, pH 7.0 at
25°C. Contraction solution was identical except
that it contained calcium (Ca)-EGTA buffer
(4 mM EGTA), rather than EDTA. pCa was adjusted by changing the ratio of Ca 2 + to EGTA
(apparent association constant of the Ca-EGTA
complex = 106- % - ' [7]). Unless otherwise specified, pCa was 5.4.
Glycerinated papillary muscles were incubated
in relaxation solution at 0°C for 1 hour before
use. Fiber bundles 3-4 mm in length and 0.15—0.25
mm in diameter were dissected from the central
core of a papillary muscle, selected microscopically on the basis of regular margins, even widths,
and sharp striation patterns, and mounted on the
myograph. Mean sarcomere length was taken as
one-tenth the distance between 11 consecutive
striations. Preloads were varied so that initial
sarcomere length ranged from 1.68 to 2.32 [x,m.
Mean fiber width, from three measurements taken
in the middle third of the fiber, was used to
calculate cross-sectional area assuming a cylindrical model. Microscopically derived cross-sectional
area and the ratio of fiber protein content to fiber
length correlated closely (r = 0.91).
Contractions were elicited by substituting
contraction solution for relaxation solution. The
relationship between pCa and tension was
examined by exposing fibers to contraction
solutions of increasing Ca2+ concentration without relaxing the fiber between changes of
solution.
Protein Determinations.—Protein was measured
by a modified Lowry procedure (8) with the use
of similarly treated bovine serum albumin as a
standard.
AT Pose Activity of Glycerinated Fibers.—
ATPase activity associated with a given contraction was measured using a radiochromatogTaphic assay. The contraction solution, 200
//liters, containing 5 mM ATP was added to the
reaction chamber. At a specific activity of 3 X 10"
counts/min mmole"1 the chamber contained
3 X 108 counts/min (1 yxmole) of gamma-labeled
AT 32 ?. After a 10-minute isometric contraction,
HENRY, AHUMADA, FRIEDMAN, SOBEL
the solution was quantitatively recovered and
mixed. Duplicate 5-yuliter samples were pipetted
onto separate strips of a washed Baker-flex
cellulose-polyethyleneimine
(PEI)
thin-layer
chromatography plate. Kinetic measurements
were obtained by removing 5-/iliter samples from
the bath at timed intervals following brief stirring.
Blanks included both fresh contraction solution
and contraction solution incubated in the bath for
10 minutes without a muscle fiber. After
ascending chromatography in 0.3M LiCl and 0.5N
HCOOH, pH 2.0 at 25°C, ATP and inorganic
phosphate (P|) were visualized with sodium
molybdate and SnCl2 sprays {9). Uniform strips
encompassing ATP and P, were cut from the
plates and placed in vials containing 15 ml of
Bray's solution (10). Radioactivity was counted
in a Packard Tri-Carb scintillation counter
(model 3375). Counting efficiency was 972.
The ATPase activity of each fiber was
calculated using values for the initial quantity of
ATP in the chamber (1 //.mole) and the ratio of
82
P, (counts/min) to AT 82 P + 82Pj (counts/min).
In kinetic experiments, the sequential removal of
5-fjditer samples from the bath was taken into
account in the calculations. In some experiments,
phosphate liberation from ATP was estimated
simultaneously by the radioactive method and by
a modification of a colorimetric procedure, which
required larger amounts of tissue (11). The
ATPase activity of a fiber was expressed with
respect to the protein content of the fiber.
ATPase Activity of Glycerinated MyofibriUar
Preparations and of Fresh Myocardium.—Glycerinated myocardial fragments were minced, passed
through a tissue press (pore size 1 mm), and
homogenized in a Virtis homogenizer with three
15-second bursts at a speed setting of 5 in 25
volumes of a medium containing 0.32M sucrose,
20 mM imidazole, and 1 mM DTT, pH 7.0. The
homogenate was centrifuged at 60 g for 2
minutes. The supernatant fraction was recovered
and centrifuged at 600 g for 20 minutes, and the
pellet was resuspended in homogenizing medium,
washed five times by the same procedure, and
finally resuspended in 2.5 volumes of a solution
containing KC1 100 mM, MgCl2 5 mM, imidazole
20 mM, DTT 1 mM, and sodium azide 5 mM, at
pH 7.0. In experiments with solution containing
low concentrations of Mg2 + , 1 mM neutralized
EDTA was added to the resuspension medium
and exogenous Mg 2 + was omitted.
ATPase activity was determined at 26°C in a
continuously stirred 2-ml reaction medium, pH
7.0, containing KC1 100 mM, MgCl2 5 mM
(except in experiments with no added M g 2 + ) ,
Na2ATP 5 mM, imidazole 20 mM, sodium azide 5
mM, and Ca-EGTA buffer along with approximately 1.0 mg/ml of myofibrillar protein. In some
CircuUtton Retard,
Vol. XXXI. Ntwmifr
1972
ISOMETRIC TENSION AND ATP HYDROLYSIS
CO
O
O
IC
©
O
experiments, azide was omitted. Serial 0.1-ml
samples were added to 0.5 ml of 0.5N perchloric acid. P, was measured colorimetrically
(11). The reaction was linear for at least 10
minutes. ATPase activity in whole homogenates
of fresh myocardium was measured as described
for myofibrils.
ATPase Activity of Actomyosin.—Actomyosin
content of normal and hypertrophied fresh
nonglycerinated myocardium was estimated by
the extraction procedure of Inchiosa (12).
Actomyosin ATPase activity was assayed under
the same conditions used for assay of myofibrillar
ATPase at a pCa of 5.0 with approximately 0.8 mg
protein /ml reaction mixture. The purpose of the
actomyosin studies was primarily to estimate the
yield from normal and hypertrophied myocardium. Depression of ATPase activity in actomyosin with respect to myofibrillar preparations is not
unexpected, because of loss or depolymerization
of actin during extraction procedures.
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d d
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743
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Results
Extent of Ventricular Hypertrophy.—The
operative procedure resulted in gross right
ventricular hypertrophy 1 weelc after surgery
(Table 1). The ratio of extractable actomyosin to total protein was not significantly
different in normal and hypertrophied hearts.
Histologic study of papillary muscles used in
d o
•g
20
°
•
NORMAL ( 0 = 5 1
HYPERTROPHY ( l - B I
ACTIVE TENSION
RESTING TENSION
88
d d
HI HI
a
3 a
8 s
0.8
04
I
10
HI HI
5 i OS
d rn
HI -5
FIGURE 2
o
I
55 m^
CircxUtion Rttetrcb, Vol. XXXI, Navtmktr 1972
Length-tension curves of normal and hypertrophied
right ventricular papillary muscles. Muscles were
studied in modified Krebs-bicarbonate solution, pH
7.4 at 30°C. Cross-sectional areas in normal and
hypertrophied muscles averaged 1.04 ±0.11 mm*
(mean ± SB) and 1.09 ±0.12 mm', respectively.
744
HENRY, AHUMADA, FRIEDMAN, SOBEL
TABLE 2
1
ATPase Activity (nmoles P,/mg protein minr ) of Fresh Homogenate, Glycerinated Myofibrils, and Actomyosin
Whole homogeaate of fresh myocardium (4)
pCa
Normal
Hypertrophy
P
5
9
33 ± 1.9
25 ± 1.6
9
43 ± 2.8
33 ± 1.5
< 0.01
64 ± 3.0
49 ± 2.7
0.05
0.05
Actomjoiln (4)
Glycerinated myoflbriU (9)
7
57 ± 3 . 1
46.1 ± 2.6
< 0.01
5
59 ± 1.6
38 ± 3.0
<0.001
97.4 ± 3.6
76.0 ± 3.0
<().01
Results are means ± BE. Number of prepara (ions tested is triven in parentheses.
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
at any Ca 2+ concentration examined; this
finding is in agreement with that of a previous
report (1). ATPase activity in the presence of
azide was 5-1035 lower than it was in the
absence of azide, indicating only a modest
contribution of mitochondrial ATPase to total
ATPase activity. ATPase assays were performed under conditions in which pCa was
varied through the range used in the studies of
mechanics.
Mechanics of Glycerinated Fibers.—Fibers
glycerinated longer than 2 weeks contracted in
contraction solution but did not relax completely with EDTA. With fibers glycerinated
less than 2 weeks, reproducible contractionrelaxation cycles were obtained (Fig. 3).
The relationship between glycerinated fiber
sarcomere length and tension is shown in
Figure 4. At similar sarcomere lengths, total
tension was comparable in glycerinated fibers
from normal and hypertrophied hearts. This
finding was in sharp contrast to the depressed
length-tension curves observed in intact,
hypertrophied papillary muscles.
experiments on mechanics revealed no appreciable increase in connective tissue in those
isolated from hypertrophied hearts.
Mechanics of Papillary Muscles.—The
length-tension curves of normal and hypertrophied right ventricular papillary muscles
matched for cross-sectional area are shown in
Figure 2. Active tension was clearly depressed
in hypertrophied muscles at all lengths.
Resting tension did not differ significantly
when hypertrophied muscles were compared
with normal muscles.
ATPase Activities of Whole Homogenates
of Nonglycerinated
Fresh
Myocardium,
Glycerinated Myofibrils, and Actomyosin.—
ATPase activities of these preparations are
summarized in Table 2. At high Mg 2+ concentration, the ATPase activity of glycerinated
myofibrils from hypertrophied hearts was significantly depressed at all Ca2 + concentrations
examined. However, in the absence of added
Mg2 + , preparations from hypertrophied hearts
exhibited myofibrillar ATPase activity which
did not differ from that in normal preparations
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TIME ( m i n )
FIGURE 3
Sequential contractions of a glycerinated fiber. Contractions were obtained by alternating
contraction solution (pCa = 5.4) with relaxation solution (pCa'— 9 /
CinmUtion Resurcb, Vol. XXXI, Novnxbar 1972
ISOMETRIC TENSION AND ATP HYDROLYSIS
745
o Normal ( i t ' l l )
• Hjptrlrophj ( a '10)
08
04
1.6
20
I
fl
SARCOMERE LENGTH
2 2
( jirn)
24
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
FIGURE 4
SoTcomere length-tension relationship in glycerinated
fibers. Points on the bottom curve represent the
tension observed at pCa — 9. Points on the top curve
represent the difference bet-ween the tensions at pCa
5.4 and those at pCa ^— 9. Each point on the bottom.
curve and its corresponding point on the top curve
belong to the first contraction cycle of a different
fiber. Since tension at pCa *-~> 9 approximates resting
tension, the top curve corresponds to developed
tension.
100
o Normol ( n = 8 )
• Hypertrophy (n = B)
K
The tension-pCa response curves of eight
normal and eight hypertrophied glycerinated
fibers studied at the same sarcomere lengths
are shown in Figure 5. It is apparent that the
response at optimal sarcomere lengths was
similar in fibers from normal and hypertrophied hearts. The pCa at half-maximum
tension was approximately 6.43 in both
cases.
Relationship between Glycerinated Fiber
Tension and Simultaneous ATP Hydrolysis.—
The ATPase activity of a representative
glycerinated fiber during a 12-minute contraction is shown in Figure 6; ATP hydrolysis was
linearly related to time. ATPase activity of
fibers from normal and hypertrophied hearts
increased linearly with total tension (Fig. 7).
Identity of the count ratios of fresh and
incubated blanks indicated that neither evaporation nor spontaneous hydrolysis in the
bath influenced the results spuriously. In
additional experiments, agreement was close
between results obtained with the radioactive
ATPase assay system and the colorimetric
=
20 -
75
50
pCa 50
=6.43
25
12
TIME
(min)
FIGURE 6
70
6.5
6.0
55
pCo
FIGURE 5
Glycerinated fiber tension. The range of
sarcomere length was 2.11 to 2.18 (im in the
fibers and 2.08 to 2.20 pm in the hypertrophied
Tensions were expressed as a percent of the
at pCa = 5.4, which were 1.3 ± 2.0 g/mm*
1.8) and 1.5 ±1.8 g/mm' (mean 1.7) in the
and hypertrophied fibers, respectively.
Circulation Rtsttrch, Vol. XXXI, Novtmtttr 1972
initial
normal
fibers.
tension
(mean
normal
ATPase activity of a representative glycerinated fiber
during a contraction at pCa = 5.4. In this experiment,
mixing during incubation of the fiber was achieved
by magnetic stirring with a 1.5-tnm 25-gauge needle
tip. Extrapolation to zero time indicated an apparently
elevated initial rate of hydrolysis. The initial deviation from, linearity was less than 5% and was not
taken into account in the calculation of the steadystate rate of hydrolysis. Conditions: pCa — 5.4,
sarcomere length = 2.12 /im, tension — 1.64 g/mm*.
746
HENRY, AHUM ADA,
100
o Normal (n -18)
• Hypertrophy (n= 14)
80
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20
0
0.8
1.6
TOTAL TENSION
2 4
z
(g/mm )
FIGURE 7
Isometric tension and simultaneously measured ATPase
activity in individually studied glycerinated fibers.
Variations in tension were obtained by initiating contractions at different sarcomere lengths. Regression
lines were obtained by the least-squares method; the
slopes are not significantly different.
phosphate procedure (N = 4 ) , excluding significant interference due to radioisotopic exchange. Glycerinated fibers from hypertrophied hearts exhibited lower ATPase activity
at all tensions, but with any given increment
in tension the increase in ATP hydrolysis was
nearly identical, as reflected by the similar
slopes of the regression lines. Thus, although
the total ATPase activity of glycerinated fibers
from hypertrophied muscle was diminished,
there was no depression in the augmentation
of ATP hydrolysis associated with increments
in tension.
Additional experiments were performed to
characterize further the glycerinated fiber
ATPase assay system. To determine whether
significant myokinase activity was present,
glycerinated fibers were incubated in contrac-
FRIEDMAN, SOBEL
tion solution containing 14C-ATP. Samples
were chromatographed on PEI plates with a
LiCl gradient chosen to separate nucleotides
(13). The absence of radioactivity in the
region to which adenosine monophosphate
migrated excluded the presence of appreciable
myokinase activity under the assay conditions.
Sequential contractions of normal and
hypertrophied fibers with and without 5 HIM
azide ( N = 4) demonstrated that azide did not
influence tension development and that the
contribution of mitochondrial ATPase to total
ATPase activity was less than 10? in both
preparations. Furthermore, polarographic experiments with homogenates of glycerinated
fibers and with 8,000-g fractions prepared
from the same homogenates demonstrated no
oxygen consumption following addition of
adenosine diphosphate with 0.01M glutamate
or 0.01M pyruvate and 0.01M malate, providing further evidence that functionally intact
mitochondria were absent from the preparation. Neither ouabain (10-®M) nor detergents
( IO^M neutralized sodium deoxycholate or
0.556 Tween 80) affected tension or ATP
hydrolysis in glycerinated fiber preparations,
excluding a significant contribution of sarcolemmal or sarcoplasmic reticulum ATPase to
total ATPase activity. This finding was corroborated by the fact that the ATPase activity
of a microsomal fraction prepared from glycerinated fibers (14) was less than 2% of the
total fiber ATPase activity.
Discussion
Glycerinated fibers from hypertrophied papillary muscles exhibited normal isometric
tension with respect to length and pCa and
normal augmentation of ATP hydrolysis associated with increased tension. These characteristics of the glycerinated fibers from hypertrophied hearts contrasted sharply with the
depressed active length-tension curves of the
corresponding intact papillary muscles in this
investigation and of the muscles from hypertrophied hearts described previously (3, 15,
16). Cross-sectional area (17) and cellular
composition of papillary muscles (17-19)
Circulation Rtsarcb, Vol. XXXI, Novtmttr 1972
ISOMETRIC TENSION AND ATP HYDROLYSIS
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
might be important determinants of results
based on tension normalized for cross-sectional area in the intact muscle. In this study,
normal and hypertrophied muscles were
matched for cross-sectional area {Fig. 2), and
there was no histologic evidence of fibrosis.
Furthermore, actomyosin concentrations and
ratios of actomyosin to protein were similar in
hypertrophied and normal tissue. Thus, the
decrease in mechanical performance of intact
papillary muscles from hypertrophied hearts
observed in this investigation probably reflects
an intrinsic abnormality of the myocardial
cells. The absolute tension values in this study
were consistent with those in previous reports,
indicating that normal rabbit papillary muscles develop less tension in vitro than do cat
papillary muscles, possibly because of diffusion limitations (20).
Decreased tension has been reported in
glycerinated trabecular fibers from dogs with
chronic right ventricular failure (2), in
disagreement with the findings in the present
study. However, trabecular muscle might
become fibrotic under conditions of chronic
failure, and the ratio of actomyosin to protein
might be decreased (17). The problem of
diffusion variation in studies of mechanics of
intact muscles must be considered. In the
present study, muscles were matched for
cross-sectional area to minimize disparate
effects of diffusion on muscles from hypertrophied and normal hearts.
Our results with glycerinated fibers indicated that the contractile apparatus in hypertrophied muscles was capable of generating
normal tension. Accordingly, the decreased
active tension in intact muscle preparations
might be related to abnormal activation by
Ca 2+ of the contractile proteins. Previous
findings indicated that Ca 2+ sensitivity was
progressively lost during storage in glycerol
(21-24), a change that was noted also in this
study and that could not be retarded by the
addition of a reducing agent (DTT) (25).
However, by glycerinating the muscles for
only 1 week, preparations with largely preserved Ca 2+ sensitivity were obtained. The
similarity of the relationship between pCa and
Circulation Rtsurcb, Vol. XXXI, Hovrmbtr 1972
747
isometric tension in normal and hypertrophied
fibers glycerinated for 1 week implied that
there was no major difference in the factors
conferring Ca 2+ sensitivity to maintenance of
tension by the contractile apparatus. Thus, it
does not appear that the decreased force
development in the intact muscle is related to
a decreased sensitivity of the contractile
system to activating Ca2 + .
ATPase activity was significantly decreased
in homogenate, glycerinated myofibrils, and
actomyosin from hypertrophied hearts in the
present study. In addition, ATPase activity
was decreased in glycerinated fibers from
hypertrophied papillary muscles despite the
fact that isometric tension was normal with
respect to sarcomere length. A five-fold
increase in total tension was associated with a
doubling of ATPase activity in glycerinated
fibers from both normal and hypertrophied
hearts. The uniformity of this relationship
suggests that the efficiency of biochemical
processes contributing to maintenance of
tension is not impaired in the contractile
system from hypertrophied myocardium even
when the active length-tension curve of the
corresponding intact muscle is depressed.
As demonstrated in this investigation, tension, sarcomere length, and ATPase activity
can be measured simultaneously in glycerinated fibers. The present experiments were
designed to characterize isometric contractions. It has been suggested that muscle
shortening is a function of myosin ATPase
activity and that force is a function of
regulatory proteins conferring Ca 2+ sensitivity
to the contractile apparatus (26-28). However,
the mechanisms responsible for maintaining
tension in the presence of ATP might depend
on cycling of the cross bridges as well (29-31).
Thus, shortening and force might not be
regulated independently. Some data supporting this view indicate that unloaded shortening velocity, as well as force, is Ca 2+
dependent (20, 32-34). The present results
indicate that the capacity of hypertrophied
glycerinated fibers to maintain isometric
tension is not impaired by depressed total
ATPase activity. However, the possibility that
748
HENRY, AHUMADA, FRIEDMAN, SOBEL
decreased ATPase activity is related to or
reflected by abnormalities of other mechanical
properties of the glycerinated fiber preparation such as shortening velocity cannot be
excluded. Accordingly, it is important to
explore the relationship between unloaded
shortening velocity and ATP hydrolysis in
glycerinated fibers from heart muscle. Nevertheless, it seems clear that the capacity of
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Acknowledgment'
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We appreciate the help of Harley Sybers, M.D.,
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PHILIP D. HENRY, Gail G. AHUMADA, WILLIAM F. FRIEDMAN and BURTON E. SOBEL
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Circ Res. 1972;31:740-749
doi: 10.1161/01.RES.31.5.740
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