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1819
Cross-Bridge Dynamics in Human
Ventricular Myocardium
Regulation of Contractility in the Failing Heart
Roger J. Hajjar, MD, and Judith K. Gwathmey, VMD, PhD
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Background. To investigate whether altered cross-bridge kinetics contribute to the contractile abnormalities observed in heart failure, we determined the mechanical properties of cardiac muscles from
control and myopathic hearts.
Methods and Results. Muscle fibers from normal (n5=) and dilated cardiomyopathy (n=6) hearts were
obtained and chemically skinned with saponin. The muscles were then maximally activated at saturating
calcium concentrations. Unloaded shortening velocities (V0) were determined in both groups. V0 in control
was 0.72±0.09 L./sec, whereas in myopathic muscles, V. was 0.41±0.06 L,,./sec at 22°C. The muscles
were also sinusoidally oscillated at frquencies ranging between 0.01 and 100 Hz. The dynamic stiffness
of the muscles was calculated from the ratio of force response amplitude to length oscillation amplitude.
At low frequencies (<0.2 Hz) the stiffness was constant but was larger in myopathic muscles. In the range
of 0.2-1 Hz, there was a drop in the magnitude of dynamic stiffness to approximately one quarter of the
low-frequency baseline. This range reflects cross-bridge turnover kinetics. In control muscles, the
frequency of minimum stiffness was 0.78±0.06 Hz, whereas it was 0.42±0.07 Hz in myopathic muscles. At
higher frequencies, the dynamic stiffness increased and reached a plateau at 30 Hz. There were no
differences in the plateau reached between control and myopathic muscles.
Conclusions. Because myopathic hearts have a markedly diminished energy reserve, the slowing of the
cross-bridge cycling rate plays an important adaptational role in the observed contractility changes in
human heart failure. Although the potential to generate maximal Ca2+-activated force is similar in normal
and myopathic hearts, alterations in contractile protein composition could explain the diminished
cross-bridge cycling rate in failing hearts. (Circulation 1992;86:1819-1826)
KEY WORDS * calcium * myofilaments * cardiomyopathy * cross-bridge kinetics
D ilated cardiomyopathy is characterized by impaired contractile function and dilation of one
or both ventricles. The cellular mechanisms
underlying the systolic and diastolic dysfunction in heart
failure are being slowly unraveled.1-6 Recent experiments on isolated human myocardium have revealed
that myopathic hearts have altered metabolic and structural properties.' When compared with normal myocardium, myopathic hearts show differences in rates of
tension development,2 creatine kinase activity,3 sarcolemmal receptors,45 and cAMP content.6'7 These differences have been shown to result in alterations in
From the Department of Medicine (J.K.G.), HarvardThorndike Laboratory of Beth Israel Hospital, Beth Israel Hospital, Boston; Harvard Medical School and Department of Molecular and Cellular Physiology (J.K.G.), Harvard University,
Boston; the Institute for the Study of Treatments for Cardiovascular Diseases (J.K.G.) and Medical Services (R.J.H.), Massachusetts General Hospital, Boston.
Supported in part by a Biomedical Research Grant from Beth
Israel Hospital (J.KG., R.J.H.) and National Institutes of Health
grants HL-36797 and HL-39091, Glaxo Inc., Upjohn Co., and the
Cardiovascular Diseases Institute (J.KG.). J.K.G. is an Established Investigator of the American Heart Association.
Address for correspondence: Judith K. Gwathmey, VMD, PhD,
Cardiovascular Division, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215.
Received March 5, 1992; revision accepted September 2, 1992.
excitation-contraction coupling.2 More recently, we investigated Ca2+ activation in myopathic hearts and
found no differences in the sensitivity of the myofilaments to Ca2+ or in the maximal Ca2+-activated force
between control and myopathic hearts.8,9 Additional
results revealed that the dysfunction underlying diseased myocardium is not due to reduced calcium release
from the sarcoplasmic reticulum.10'11 It has been proposed that subcellular alterations in the number of
cross-bridge interactions may result in myocardial failure.12 Because changes in cross-bridge kinetics and
number of cycling cross-bridges could affect contractile
function, we became interested in examining the regulation of cross-bridge dynamics in heart failure. We
therefore used two direct techniques to measure the
cross-bridge cycling rate: 1) unloaded velocity of shortening and 2) dynamic stiffness calculated from the ratio
of force response to length perturbation amplitude.
Methods
Muscle Preparation
Trabeculae carneae were obtained from the right
ventricles of hearts of patients with end-stage heart
failure undergoing heart transplantation (n=6) as described earlier.2'8-10 All patients from this group were
diagnosed as having idiopathic dilated cardiomyopathy
characterized by an increase in ventricular size and
1820
Circulation Vol 86, No 6 December 1992
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impaired ventricular function without angiographic evidence of significant coronary artery disease (no lesions
resulting in obstruction of >50%), valve disease, insulin-dependent diabetes, congenital malformations,
hemochromatosis, amyloidosis, or infections. The mean
age of the patients was 41.3±2.7 years. None of the
patients had a reported family history of cardiomyopathy, a personal history of alcoholism, or human immunodeficiency virus infection. On endomyocardial biopsy,
there was no evidence of active myocarditis among these
patients. On gross inspection, the hearts were markedly
enlarged, with severe four-chamber dilation and an
average weight of 730.4±24.5 g. Histologically, all patients had generalized cellular hypertrophy and diffuse
interstitial fibrosis measured by light microscopy. None
of the patients were receiving p-blockers or Ca'+ blockers before transplant. The mean ejection fraction measured by two-dimensional echocardiography was
17±1%. Areas containing large amounts of fibrosis were
avoided during muscle selection. Control hearts (n=5)
were obtained from brain-dead organ donors (mean
age, 35.4±2.3 years), without known cardiac disease
who died from trauma. On gross inspection, the control
hearts were normal without areas of scarring or thinning
and with an average weight of 210±23 g. Histologically,
cellular dimensions were within normal limits with no
evidence of hypertrophy or extensive fibrosis. All experimental protocols were reviewed and approved by an
internal review board.
After removal from the chest, the left and right
ventricles were opened surgically and placed into an
oxygenated physiological solution (see composition below). The tissue was then transported to our laboratory,
arriving 20 minutes to 2 hours after removal from the
thoracic cavity for both control and myopathic hearts.
Suitable thin trabeculae carneae were removed and
studied immediately. Muscle dimensions were measured by a light microscope (x 10) equipped with a
calibrated eyepiece. Two measurements of diameters
were assessed at two different muscle orientations perpendicular to each other, and the average of these two
measurements was taken as muscle diameter. Trabeculae diameters were 455±60 gm (n=5) from control
hearts and 416±75 ,um (n=6) from myopathic hearts,
corresponding to cross-sectional areas <0.5 mm2. The
muscle lengths measured at L,,. (defined below) were
5.05±0.34 mm (n=5) from control hearts and 5.33±0.22
mm (n=6) from myopathic hearts. There were no
physiological differences in muscle parameters by Student's t test. The muscles were clamped at one end of a
muscle holder and attached at the other end to a
Servo-controlled electromagnetic lever system (Cambridge 300H, Watertown, Mass.). The signals from the
force and displacement transducers were displayed simultaneously on a Gould diode array recorder. The
muscles were superfused with a bicarbonate-buffered
oxygenated solution with the following composition
(mM): NaCI 120, KCl 5.9, NaHCO3 25, NaH2P04 1.2,
MgCl2 1.2, CaCl2 2.5, dextrose 11.5, bubbled with 95%
0° and 5% CO2, pH 7.4. Muscles were stimulated with
a square wave pulse of 5-msec duration at threshold
voltage at a frequency of 0.33 Hz. The muscle length was
then adjusted to L,.,,, a length at which maximal active
twitch force (total force minus diastolic force) was
achieved.
Skinning Procedure
After setting L,,. in the intact state, the muscles were
chemically skinned by exposure to a solution containing
saponin 250 ,ug/ml, K2ATP 5 mM, MgCl2 7 mM, EGTA
(ethyleneglycol-bis-N,N,N,N'-tetraacetic acid) 5 mM,
KCl 60 mM, MOPS 60 mM, phosphocreatine 12 mM,
creatine phosphokinase 15 units/ml, pH 7.1 at 22°C.
The total salt concentrations necessary for obtaining
the desired pCa (-log,o[Ca2]), pMg (-log1o[Mg2+ ),
pMgATP (-log,o[MgATP]), and pH at a constant ionic
strength were calculated using the program described by
Fabiato.13 The solutions were prepared at a temperature of 22°C with a pMg of 2.5, pMgATP of 2.5, an
EGTA concentration of 10 mM, an ionic strength of
0.16 M, and a pH of 7.1 adjusted using 30 mM TES. The
solutions also contained 12 mM phosphocreatine and 15
units/ml creatine phosphokinase. All stock solutions
were prepared in plasticware: 5 mM K2CaEGTA was
prepared with CaCO3, EGTA, and KOH at 80°C; 5 mM
K2EGTA was prepared with EGTA and KOH. Creatine
phosphate and creatine phosphokinase were added
immediately before the experiment. The relaxation solution had a pCa >8.0, whereas the maximal activation
solution had a pCa of 4.0.
Unloaded Shortening
As described above, the muscles were skinned, maximally activated, and then quickly released from the end
attached to the galvanometer (in less than 1 msec),
causing a rapid fall in tension. The time At was measured from the onset of release to the point at which the
force started to redevelop. The data relating amplitude
AL and duration At of unloaded shortening after various amplitudes of quick releases were fitted with a
regression line using the least-squares method as described by Edman.14
Dynamic Stiffness
Once the skinned muscles achieved maximal force at
a pCa of 4.0, they were subjected to sinusoidal oscillations from the end of the muscle attached to the
galvanometer at 20 discrete frequencies from 0.01 Hz to
100 Hz at a fixed amplitude of 1% L,, peak to peak.
Both AL and AF were measured throughout these
frequencies. Dynamic stiffness of the muscle was defined as AF/AL and then normalized to the value that
would be obtained if each muscle had a length of 5 mm
and a cross-sectional area of 0.5 Mm2m. Dynamic stiffness
was then plotted as a function of frequency of oscillations.1516 Phase shift between force and muscle length
was measured by the time difference between the peaks
of force and muscle length. We generated the dynamic
stiffness spectrum using whole muscle length, which
generally includes damaged end regions.
Statistical Analysis
Results are presented as mean+SEM. Statistical significance in the comparisons made between frequencies
of minimum stiffness, velocities of unloaded shortening,
and stiffness measurements between control and myopathic muscles were determined by the Student's t test.
Results
Skinned Fibers
The activation range for both control and myopathic
skinned muscles was between 10`7 M and 10`4 M, with
Haijar and Gwathmey Cross-Bridge Dynamics in Heart Failure
1821
noted in Figure 2, myopathic muscles had series elasticities that were similar to control muscles (0.04+ 0.01 L..
versus 0.03+0.01 Ln,, respectively).
Dynamic Stiffness
As length was oscillated sinusoidally, the force out-
C.)
ILI.
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8
7
6
5
4
PCa
FIGURE 1. Force-[Ca21] relations of control (n=5) and
myopathic muscles (n=6) derived from curves fitted individually to the data of each experiment. [Ca 2+Jso% was
1.06±0.09 ,uM and 1.02±0.06 gM, and the Hill coefficients
were 2.16±0.10 and 2.24+0.14 for the control and myopathic
muscles, respectively. Computer-generated curves from pooled
Hill parameters are illustrated.8'9
both groups being maximally activated at a pCa <4.5.
The force development saturated at a pCa of 4.5 and
remained maximal at a pCa of 4.0. As shown in Figure
1, there were no differences in Ca2+ sensitivity or
cooperativity (steepness of the force-[Ca21] relation,
reflecting long-range associations among cycling crossbridges) between skinned trabeculae from control and
myopathic hearts. Maximal force measured at a pCa of
4.0 was 0.35±0.06 g/mm2 for control and 0.33±0.08
g/mm2 for myopathic muscles.
Velocity of Unloaded Shortening
Control and myopathic muscles were maximally activated at a pCa of 4.0 and then quickly released to
different lengths as shown in Figure 2. The release
caused a rapid fall in tension to zero. The tension
remained at zero until force redeveloped with an exponential rise and then reached a plateau. The releases
started at the same muscle length, Ll,., and the redevelopment of tension occurred at different lengths,
depending on the amplitude of the release (the smallest
one being 5%). The muscles were then restretched to
L,. There was a linear relation between AL and At, as
shown in Figure 2. The slope of the line has been shown
to provide a measure of the velocity of unloaded
shortening.14 In control muscles, the unloaded shortening velocity V0 was 0.72±0.09 L,/ajsec, whereas in
myopathic muscles, V0 was 0.41±0.06 Lmax/sec
(p<O.OOS). The y-intercept of the fitted line is a measure of the series elastic element of the muscles. As
puts were sinusoidal in both control and myopathic
muscles, as shown in Figure 3. At low frequencies of
oscillations, the amplitude of force oscillation was constant in both control and myopathic muscles. As the
frequency increased, the amplitude of force decreased
and reached a minimum at about 0.78±0.06 Hz (n=5)
for control muscles and 0.42±0.07 Hz (n=6) for myopathic muscles (p<0.01). This frequency has been
termed frequency of minimum stiffness (fm,u). Further
increases in the frequency of oscillation resulted in an
increase in stiffness that reached a plateau >30 Hz.
Representative frequency spectra of the stiffness measurements of normal and myopathic skinned trabeculae
are shown in Figure 4A. Table 1 depicts the stiffness
measurements at low, minimum, and high frequencies
(KOw K~ , and K,igb, respectively). The stiffness measurements were significantly higher in myopathic muscles compared with control muscles at low frequencies
(<0.2 Hz). The values for minimum and high stiffnesses
were not different between control and myopathic muscles. The phase shift between force and muscle length
also showed a strong frequency dependence, as seen in
Figure 4B. The phase shift was close to zero at low
frequencies. Above the frequency of minimum stiffness,
the phase shift became abruptly positive, reaching a
maximal value of -80°. At higher frequencies, the phase
shift gradually decreased toward zero.
The stiffness spectra of skinned trabeculae at a pCa of
8.0 increased slightly with increasing frequency. Myopathic muscles had higher resting stiffness than control
muscles throughout all frequencies.
Force-High-Frequency Stiffness Relation
To examine whether the stiffness at high frequencies
is governed by the active level of force, we related the
stiffness at 100 Hz versus the mean level of force at 100
Hz. We combined the data points for both control and
myopathic muscles because there were no statistical
differences between the coefficients of linear regression.
Figure 5 shows the correlation of the combined data
between force and high-frequency stiffness to be
KYhig=16.31f+0.14, where f is force developed. This
linear relation shows that the high-frequency stiffness is
nearly proportional to mean force, suggesting that the
elasticity measured at high frequencies resides within
actively cycling cross-bridges. From this correlation, the
required size for a sudden shortening step that would
bring force to zero can be calculated as 1/(16.31
n1m-))=0.06 mm. Relative to the average length of our
muscle preparations, =5 mm, this represented a change
of 1.2%.
Discussion
Validity of the Techniques Used to Evaluate
Cross-Bridge Cycling Rate
To avoid the ambiguities of possible effects of excitation-contraction coupling and different intracellular
calcium levels on cross-bridge cycling rate, we maximally
1822
Circulation Vol 86, No 6 December 1992
n-ga
.
1
0.16
Control
a
0.12
A
-I
J
0.0
Myop/him
0S.
0.0
-
0
s0
-
100
150
2 !00
Tim (At)
MYOPATHIC
CONTROL
0.1 f
Lo*-4% L
o0.
LQ
J
-
95% Le
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L.-92% Le
j
Lo
-
90% Le
L -90% Lo
-1-101
I
100...
m
100 ume
FIGURE 2. Representative skinned fiberpreparations from a control and a myopathic muscle are illustrated. Four quick releases
AL and duration At of unloaded shortening after various amplitudes of
least-squares method.
are shown for each muscle. Relations between amplitude
quick releases were fitted with a regression line using the
activated the myofilaments in skinned fiber preparations. Other methods have been developed to obtain
steady activation in intact cardiac muscle including
barium contracture, which has been used by many
investigators to measure cross-bridge cycling rate15-21
and recently in isolated, perfused working hearts.22 We
have also used tetanizations of human myocardium to
obtain steady-state activation.8 However, we elected to
use membrane-free preparations because we were interested in examining only the contractile proteins
without having to address the possibilities that there
may be differential effects of muscle length stretches on
the sarcoplasmic reticulum, the sarcolemma, and receptors resulting in the observed differences.
We used two techniques to investigate the crossbridge cycling rate in control and myopathic hearts. The
traditional method of assessing cross-bridge cycling rate
has been the maximal unloaded shortening velocity
during contraction developed by Edman.'4 This technique has been extensively used in skeletal muscles and
has become widely accepted for use in cardiac muscles.
It measures the velocity at which the cross-bridges cycle
to take up the slack induced by a length change under
zero load.
Several investigators have interpreted the time course
of force transients in response to rapid, small-amplitude
length changes applied to isometrically contracting cardiac muscle in terms of the kinetics of the active inter-
action of myosin with actin.15-2l23,24 More specifically,
the frequency of minimum stiffness reflects the frequency
at which resonance exists between the mechanical cycle
of the muscle during activation and the mechanical
frequency imposed on the muscle. Based on the complex
stiffness spectra of activated cardiac and skeletal muscle,
several investigators have constructed specific mathematical models in which the frequency of minimum stiffness
reflects the mean cycling rate.1516202324 Furthermore, it
has been shown recently that f,in is similar in skinned
myocardium and intact myocardium.25 The view that the
frequency of minimum stiffness is an indicator of intrinsic
contractile rate has been supported by experiments that
demonstrate this frequency to be independent of muscle
length and degree of activation but to be strongly dependent on temperature,15'16 MgATP concentrations, myosin
isozyme composition,16,23 age,'6 and the presence of
cAMP-varying compounds.l920,26
It is important to note that the information provided
by the two techniques are different in terms of steps in
the cross-bridge cycle. Vo is measured under zero load
starting with all cross-bridges in the detached state and
reflecting the speed with which the cross-bridges,
through multiple cycles, produce shortening and is
limited by the rate of cross-bridge detachment.'4 The
frequency of minimum stiffness is derived under isometric conditions and is an index of the mean cross-bridge
cycling rate. Both measurements are thought to be
Hajjar and Gwathmey Cross-Bridge Dynamics in Heart Failure
0.1 Hz
LENGTH
0.5Hz
1823
30 Hz
% Lmax |
I
M/
CONTROL
FIGURE 3. Frequency responses of maximally
activated (pCa 4.0) skinned control and myopathic muscles at 0.1, 0.5, and 30 Hz at 22°C and
Lmax
FORCE
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AM
intimately related to cross-bridge cycling rate. To the
extent that fmin and V0 represent cross-bridge cycling
rate, our findings show that the cycling rate is slowed in
failing hearts.
We found that the series elasticity was higher with
Edman's slack test when compared with the series elasticity obtained with high-frequency stiffness measurements
(3% versus 1%). One explanation is that with the slack
test, chord stiffness is actually measured, and viscous
properties of the damaged ends' series elasticity predominate, whereas the series elasticity observed with the
dynamic stiffness spectra are the instantaneous stiffness of
elements in series and parallel built into the sarcomeres.
Nevertheless, the series elasticity does not affect V0 or
frequency of minimum stiffness measurements.27
Another consideration in our measurements was control of sarcomere length because it can influence calcium sensitivity and velocity of shortening.28-30 Although we did not measure sarcomere length in our
preparations, the muscles were all activated at L.,,. In
preparations such as ours, the central (healthy) portion
of the muscle preparation shortens during a twitch while
the ends of the excised muscles are held constant.
Depending on the amount of damage in the end regions,
shortening in the middle portion can be as much as 15%
of the total muscle length.31 This leads to shortening of
the sarcomeres in the middle portion while stretching
the sarcomeres in the end region. The greater the
amount of damage in the end region, the higher the
expected series elasticity derived from the Edman slack
test. However, in our preparations, series elasticity
measured with the Edman slack test did not exceed 4%
of L,,, and there were no differences between control
and myopathic muscles, which argues against differential amounts of damaged ends accounting for changes in
the unloaded velocity of shortening.
Cross-Bridge Cycling Rate and Contractile Proteins
In human dilated cardiomyopathy, changes in the
functional properties of the myofilaments have been
MYOPATHIC
reported that may explain altered cross-bridge cycling
rate.89,32-35 Shortening velocity and ATPase activity in
cardiac muscle have been shown to correlate with
myosin isoforms V1, V2, and V3.21,36 The ratio of these
isomyosins varies according to the maturational and
pathological state of animals, with V, being associated
with higher ATPase activity and shortening velocity
(hyperthyroid, fetal) and V3 being associated with lower
ATPase activity and shortening velocity (hypothyroid,
hypertrophy, adult).21,36,37,38 In humans, however, the V3
isoform predominates, and in dilated and hypertrophic
cardiomyopathy, no consistent change in isozyme pattern has been found.37,38
A recent report by Margossian et a139 showed a
decrease in the content of myosin light chain kinase 2
(LC2) in ventricles from patients with dilated cardiomyopathy. In cardiac muscle, LC2 modifies the actomyosin reaction. It has been shown that LC2 removal in
skinned myocardial preparations results in 1) an increase in force production at submaximal [Ca21], 2) a
leftward shift of the force-[Ca2 ] relation, 3) no change
in maximal Ca2'-activated force, and 4) a decrease in
unloaded velocity of shortening.40 Although no significant difference in the sensitivity of the myofilaments to
Ca2 was observed in our preparations, we did find,
consistent with a loss of LC2, diminished velocity of
unloaded shortening and cross-bridge cycling rate, an
unchanged maximal Ca 2+-activated force, and increased
resting stiffness in cardiomyopathy. Furthermore, myofibrillar MgATPase activity has been shown to be reduced in myopathic ventricles41-43; more specifically,
our myopathic preparations demonstrated a decrease in
myosin ATPase activity by -25% when compared with
normal preparations (unpublished results).
Thin filament regulation and structure also have been
shown to be altered in human cardiomyopathy.O8933,35,43
Anderson et a143 recently reported that failing human
hearts demonstrated changes in troponin T (TnT) isoforms with an increase in the expression of troponin T2
1824
Circulation Vol 86, No 6 December 1992
A
DYNAMIC STIFFNESS
B
PHASE SHIFT
E
0
CB
0.
m-
to
1000
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Frequency (Hz)
Frequency (Hz)
FIGURE 4. Dynamic stiffness curves (panel A) and phase shift curves (panel B) of skinned fiber preparations of control (open
circles) and myopathic muscle (closed circles) maximally activated at a pCa of 4.0 at 22°C. Thirty frequencies of oscillations were
used for each preparation. Also shown are the stiffness measurements of the same control (open triangles) and myopathic muscle
(closed triangles) at a pCa of 8.0. Deg., degree.
(TnT2), a fetal isoform that in mammalian myocardium
confers a greater sensitivity of the myofilaments to Ca2'
and a decrease in maximum myofibrillar ATPase activity.42-45 It was argued that the increase in TnT2 in failing
human myocardium may play a role in the regulation of
force development and calcium activation.43 However,
TnT2 was only :15% of the TnT population in the
myopathic hearts, making it unlikely that the significant
decrease in myofibrillar ATPase observed in failing
hearts can be entirely accounted for by TnT2. The fact
that in this study we did not find any changes in
myofilament sensitivity to Ca2' also argues against a
major role of TnT2.
failing human myocardium when stimulated at relatively
slow rates and under hypothermic conditions.2,1047 It
would also explain the finding from our studies and
those of other investigators that maximal Ca 2k-activated
force is unchanged in myopathic trabeculae.8'9'1 A
reduction in cross-bridge cycling rate also translates into
an improvement in thermal economy.48 Recently,
Hasenfuss et a112 found that the average force-time
integral, which is thought to reflect cross-bridge attachment time, was increased by 33% in failing hearts; so the
8
Physiological Implications
_0
E
A decreased cross-bridge cycling rate allows longer
interaction periods between actin and myosin, resulting
in greater force development.46 Therefore, in diseased
human myocardium, the slower cross-bridge cycling rate
would seem to compensate for the decrease in myofibrillar protein content. This would explain in part the
finding of similar contractile performance reported in
0~~~~~~~~~~
TABLE 1. Stiffness Measurements at Different Frequencies
of Oscillations
Control muscles
(n=5)
KO. (g/mm)
Myopathic muscles
(n=6)
1.38+0.23
1.87±0.28*
0.34+0.09
Krin (g/mm)
0.41±0.10
5.97+0.21
5.81 ±0.33
Khigh (g/mm)
Values are mean+SEM. K,, stiffness at low frequencies (<0.2
Hz); K,,,, minimum stiffness; Khigh, stiffness at high frequencies
(100 Hz).
*Significant difference (p<0.05) between control and myopathic muscles.
0.0
0.1
0.2
0.'3
0:4
0.5
FOrc Wm 2
FIGURE 5. High-frequency stiffness-force relation in control
and myopathic muscle fibers. Stiffness measurements were
obtained at 100 Hz, pCa of 4.0, and L,,.. Open circles,
control muscles; closed circles, myopathic muscles* Linear
regression line drawn:. Khgh= 16 31f+ 0. 14; f is the developed
force.
Hajjar and Gwathmey Cross-Bridge Dynamics in Heart Failure
slowing of the cross-bridges may be beneficial in terms
of energy economy, especially because myopathic hearts
have a lower energy reserve when compared with
normal hearts.
Prolonged cross-bridge attachment also results in
reduced rates of relaxation. Gwathmey et a12 have
shown that the time course of intracellular [Ca21] and
twitch force were markedly prolonged in human dilated
cardiomyopathy. Even though a slowing of calcium
uptake by the sarcoplasmic reticulum has been put forth
as the main cause of this observation, a slowing of the
cross-bridge detachment rate may also result in prolongation of twitch and [Ca2"Ji transients. The rate-limiting
step in relaxation may not be entirely due to the uptake
of Ca2' by the sarcoplasmic reticulum but instead may
be due to a slower cycling rate of cross-bridges. This
may also explain the impaired relaxation observed in
patients with dilated cardiomyopathy independent of
[Ca2+].
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The slowing of cross-bridge cycling rate does not
seem to be specific only to human heart failure. Many
animal models of hypertrophy have been associated as
well with a decrease in shortening velocity.49-52 Idiopathic dilated cardiomyopathy in humans is a disease of
unknown cause and may be the result of a variety of
insults. The natural history of this condition is not well
defined because the presenting symptom of affected
patients is usually overt heart failure, the end-stage of
the disease. For this reason, it is not clear whether the
progression of the disease includes a stage of compensated myocardial hypertrophy that resembles that of
most animal models. The decrease in cross-bridge cycling rate seems to reflect an adaptation to both hypertrophy and heart failure.
High-Frequency Stiffness and Maximal
Ca2+-Activated Force
Recent experiments in failing hearts have suggested
that a reduced number of available cycling cross-bridges
are responsible for contractile failure in myopathic
hearts.12 Hasenfuss et a112 found that tension-dependent heat, which is thought to reflect the number of
force-producing cross-bridges, was reduced by 61% in
failing hearts. We measured the stiffness at high frequencies of oscillations, which is governed by the tension generated per cross-bridge.15,16,24 In our experiments, there were no differences in high-frequency
stiffness for control and myopathic muscles. This does
not necessarily translate into a similar number of attached cross-bridges, because if the cross-bridges are
stiffer in myopathic muscles, high-frequency stiffness
would be the same even with a reduced number of
cross-bridges in myopathic tissue. The instantaneous
stiffness measured at high frequencies reflects both the
number of attached cross-bridges in the force-producing
and non-force producing state. However, we found the
same maximal Ca2'-activated force in control and myopathic muscles, which argues that indeed the number
of force-producing cross-bridges are probably similar in
control and myopathic myocardium.
Conclusions
In the present study, we have unambiguously shown
that the cross-bridge cycling rate determined by using
two methods is slower in myopathic compared with
1825
control hearts. Furthermore, we showed that there are
no differences in maximal Ca2' activation between
control and myopathic muscles and that the number of
force-producing cross-bridges is similar. These findings
suggest that 1) the potential for force development is
similar in normal and myopathic hearts and 2) there are
changes at the level of the contractile proteins that
result in reduced cross-bridge cycling rate and lowered
energy requirement.
Acknowledgments
Dr. P.D. Allen, Dr. E. Loh, and Dr. F. Schoen are thanked
for procurement of myopathic myocardium. We also greatly
appreciate Dr. Pieter P. deTombe's critical review of the
manuscript and Dr. Michael Berman's helpful discussion. The
National Disease Research Interchange and the New England
Regional Organ Bank are thanked for tissue procurement.
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Circulation. 1992;86:1819-1826
doi: 10.1161/01.CIR.86.6.1819
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