J Appl Physiol
90: 1811–1816, 2001.
Myosin thick filament lability induced by mechanical
strain in airway smooth muscle
KUO-HSING KUO,1 LU WANG,2 PETER D. PARÉ,2
LINCOLN E. FORD,3 AND CHUN Y. SEOW1,4,5
Departments of 1Anatomy and 4Pharmacology and Therapeutics, University of British Columbia,
Vancouver V6T 1Z3; 2Pulmonary Research Laboratory, St. Paul’s Hospital, University of British
Columbia, Vancouver V6Z 1Y6; 3Krannert Institute of Cardiology, Indiana University,
Indianapolis, Indiana 46220; and 5Department of Pathology and Laboratory Medicine, St. Paul’s
Hospital, University of British Columbia, Vancouver, British Columbia, Canada V6Z 1Y6
Received 30 October 2000; accepted in final form 20 December 2000
FUNCTIONAL STUDIES INDICATE that adaptation of airway
smooth muscle to a large length change occurs in two
steps. First, there is a decrease in the ability of the
muscle to generate force immediately after length
change; this is followed by a period of force recovery,
when the muscle is allowed to adapt at a fixed length
without further length perturbation (8, 15). Structural
studies of skeletal muscle adaptation to chronic stimulation at short lengths (6) have also revealed two steps in
the process of muscle remodeling. The first phase of
skeletal muscle remodeling is associated with disorganization of the sarcomeric structure, which appears during
the initial hours of stimulation. This is followed by a
period of recovery, during which the muscle regains its
normal sarcomere appearance. Remodeling of the contractile apparatus in a chronically shortened skeletal
muscle is accomplished by deletion of sarcomeres in series so that optimal filament overlap is reestablished (1,
6). The time required for structural remodeling in skeletal muscle (from hours to days) is much longer than that
required for adaptation of airway smooth muscle to
length change, which takes only minutes (1, 6, 8, 15). The
fundamental mechanism, however, could be the same.
Disorganization of the contractile apparatus in airway
smooth muscle after a large length change could be the
first step in the remodeling process that leads to the
reorganization of the contractile apparatus (which could
include deletion or addition of contractile units in series)
that, in turn, would lead to a full adaptation of the muscle
to a new length with optimal overlap of the contractile
filaments. Characterization of structural changes that
accompanies functional changes during the process of
smooth muscle adaptation has never been done and is the
focus of this study.
A stepwise change in length is not the only way to
initiate the process of adaptation in smooth muscle.
We have found that, by applying a brief period of
length oscillation to a relaxed muscle, one can
achieve the same purpose (15). The advantage of
using length oscillation instead of a stepwise length
change is that, after oscillation, the muscle returns
to its original length and that allows measurements
of functional and structural changes associated with
the reorganization of the contractile apparatus during the adaptation period to be made at the same
muscle length. In the present study, we used electron
microscopy to examine the myosin thick-filament
density in cross sections of airway smooth muscle
fixed at various stages during the adaptation process
initiated by length oscillation.
Address for reprint requests and other correspondence: C. Y. Seow,
Dept. of Pharmacology and Therapeutics, Univ. of British Columbia,
2176 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
swine trachea; mechanics; length perturbation; plasticity
http://www.jap.org
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
1811
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Kuo, Kuo-Hsing, Lu Wang, Peter D. Paré, Lincoln E.
Ford, and Chun Y. Seow. Myosin thick filament lability
induced by mechanical strain in airway smooth muscle. J
Appl Physiol 90: 1811–1816, 2001.—Airway smooth muscle adapts to different lengths with functional changes that
suggest plastic alterations in the filament lattice. To look
for structural changes that might be associated with this
plasticity, we studied the relationship between isometric
force generation and myosin thick filament density in cell
cross sections, measured by electron microscope, after
length oscillations applied to the relaxed porcine trachealis muscle. Muscles were stimulated regularly for 12 s
every 5 min. Between two stimulations, the muscles were
submitted to repeated passive ⫾30% length changes. This
caused tetanic force and thick-filament density to fall by 21
and 27%, respectively. However, in subsequent tetani,
both force and filament density recovered to preoscillation
levels. These findings indicate that thick filaments in airway smooth muscle are labile, depolymerization of the
myosin filaments can be induced by mechanical strain, and
repolymerization of the thick filaments underlies force
recovery after the oscillation. This thick-filament lability
would greatly facilitate plastic changes of lattice length
and explain why airway smooth muscle is able to function
over a large length range.
1812
STRUCTURAL LABILITY AND SMOOTH MUSCLE PLASTICITY
METHODS
Fig. 1. Experimental protocol. Force records were made
during 7 tetani produced by 12-s electrical field stimulation at 5-min intervals. Averaged records from 4 experiments are plotted. Muscles were subjected to length
oscillations between the first 2 contractions, and 5 muscle bundles from each of the 4 tracheae were fixed for
electron microscopy at the 5 times indicated by the
numbered arrows.
RESULTS
To examine the effect of length oscillation on myosin filament structural lability, muscle strips were
fixed at different time points before and after a brief
period of sinusoidal length oscillation and during the
time course of force recovery, as illustrated in Fig. 1.
Figure 2 shows examples of electron micrographs of
cross sections of relaxed airway smooth muscle cells
fixed at time 1 (preoscillation) and time 2 (postoscillation), as shown in Fig. 1. The thick filament density
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Muscle preparation. Swine tracheal smooth muscle was
used for the experiments. The tracheae were obtained from a
local abattoir. The tracheas were kept in ice-cold physiological saline immediately after their removal from the animals.
A bundle of muscle cells (⬃6 ⫻ 1 ⫻ 0.1 mm in dimension) was
dissected from a trachea and attached to clips made of aluminum foil. The clipped muscle strip was then mounted in a
muscle bath; one end of the strip was attached to the force
transducer and the other end to the servomotor. The muscle
bath contained physiological saline solution (pH 7.4 at 37°C),
bubbled with a gas mixture (5% CO2-95% O2), and contained
(in mM) 118 NaCl, 5 KCl, 1.2 NaH2PO4, 22.5 NaHCO3, 2
MgSO4, and 2 CaCl2 and 2 g/l dextrose. The muscle preparation was equilibrated in the bath for ⬃1 h to obtain a
maximal, stable isometric force. During the equilibration
period, the muscle was stimulated electrically once every 5
min. Duration of the supramaximal electrical field stimulation was 12 s; stimulator frequency was 60 Hz.
Apparatus. Muscle force was measured with a photoelectric type of force transducer with a resonant frequency of 1
kHz and a signal-to-noise ratio of ⬎50. The length oscillation
was applied through a servomotor. Displacement of the servomotor coil was driven by a signal generated from a function
generator that controlled both the amplitude and frequency
of the oscillation. The highest frequency that the motor could
follow was ⬃1 kHz.
Experimental procedure. Four tracheae were used for
the experiments. Five strips of muscle were isolated from
each trachea; these strips were fixed at five different time
points during the process of mechanical perturbation and
adaptation, as indicated in Fig. 1. Data at each time point
represent results averaged for four strips from four different animals. A reference length (Lref) was obtained for the
muscle preparation during the period of equilibration. Lref
was associated with a resting tension that was 1–2% of
maximal isometric tension. The amplitude of the sinusoidal length oscillation used was 60% of Lref (peak to peak).
The oscillation therefore caused a 30% Lref stretch of the
muscle. The maximal passive force measured during oscillation was about one-quarter of maximal isometric force.
The frequency of the oscillation was 0.5 Hz. This frequency
is close to the respiration rate of pigs.
Electron microscopy. At each time point in Fig. 1, before
and after oscillation and during the process of force recovery,
the muscle was fixed while in the relaxed state and still
attached to the apparatus for 15 min. The replacement of
physiological saline with fixing solution occurred in ⬍1 s. The
fixing solution was prewarmed to 37°C, the same temperature as the bathing physiological saline. The fixing solution
contained 1.5% glutaraldehyde and 1.5% paraformaldehyde
in 0.1 M sodium cacodylate buffer. After the initial fixing (15
min), the sample was removed from the apparatus, cut into
small blocks (⬃1 ⫻ 0.5 ⫻ 0.1 mm in dimension), and placed
in the same fixing solution for 2 h at 4°C on a shaker. This
was followed by three 10-min washes in 0.1 M sodium cacodylate buffer. The tissue was then fixed with 1% OsO4 in 0.1
M sodium cacodylate buffer for 2 h, followed by three 10-min
washes with distilled water. This was followed by en bloc
staining with 1% uranyl acetate for 1 h and three 10-min
washes in distilled water. The tissue was then dehydrated
through a graded series of ethanols of 50, 70, 80, 90, and 95%
for 10 min each, followed by 100% ethanol and propylene
oxide (three 10-min washes for each). The specimen was then
ready for embedding in resin (TAAB 812 mixing, medium
hardness). The specimen was allowed to “soak” in the resin
overnight before it was embedded in molds and placed in an
oven at 60°C for 8 h. The blocks were sectioned on microtome
using a diamond knife. The thickness of the sections was ⬃90
nm. The sections were then stained in 1% uranyl acetate for
4 min followed by a 3-min staining in Reynolds lead citrate.
A Phillips 300 electron microscope was used for obtaining the
images of the cross sections of the muscle cells.
Analysis. Sampling and analysis were carried out in a
“blinded” manner. Sample codes were revealed only after the
analysis for each group was complete. Fifteen cross sections
from each muscle strip were chosen. Five of the cross sections
were selected to contain nuclei, five to contain central clusters of mitochondria, and five to contain scattered mitochondria and no nucleus. This sampling choice ensured that the
central, near-central, and end segments of muscle cells were
all included in the examination. No statistically significant
difference was found in different segments in terms of thick
filament density. Therefore, results were lumped together in
the final analysis. A total of 300 (4 ⫻ 5 ⫻ 15) pictures were
analyzed. Each picture contained one whole cell cross section.
Myosin thick filaments (with a diameter of 15–20 nm) were
identified by eye and counted for the whole cell cross section.
The density was obtained by dividing the number of thick
filaments by the total area of the cell cross section minus the
areas occupied by nuclei, mitochondria, and other cellular
organelles. The measurement of area was aided by the use of
a morphometric digital device made by Carl Zeiss of Germany.
STRUCTURAL LABILITY AND SMOOTH MUSCLE PLASTICITY
1813
is markedly reduced after mechanical perturbation.
There is no obvious change in the density of actin
thin filaments, although accurate measurement of
thin-filament density is not possible with the resolution of our micrographs.
To examine the ultrastructural change that accompanies force change due to length oscillation, the averaged (n ⫽ 4) thick filament densities for the samples
fixed before and after length oscillation (times 1 and 2;
Fig. 1) and during postoscillation force recovery (times
3–5; Fig. 1) are plotted in Fig. 3 with the measurements of isometric force. A close agreement between
density and force is observed. Absolute densities of
myosin thick filament (in units of filament number per
␮m2) for the five conditions are listed in Table 1. The
cross-sectional areas of the muscle cells fixed under the
five conditions are also listed in Table 1. One-way
ANOVA shows that there is no significant difference
among the areas (P ⫽ 0.26).
The time course of isometric force development reflects, to a certain extent, the kinetic properties of the
crossbridges and arrangement of contractile units. Figure 4 shows the averaged isometric force (n ⫽ 4) in the
rising phase of force development elicited by electrical
field stimulation. Isometric force data were obtained
from averaging digitized isometric force traces before
length oscillation (corresponding to the contraction just
before time 1 in Fig. 1); thick filament density data
were obtained from averaging force traces after length
oscillation (corresponding to the contraction just after
time 2 in Fig. 1). The force traces are normalized to
their respective maximal values. The onset of isometric
force development is delayed by 0.20 ⫾ 0.01 s in the
Fig. 3. Time course of changes in tetanic force (F) and myosin
thick-filament density (E) associated with length oscillation. Reference values for force and filament density are values obtained before
oscillation (time 1 in Fig. 1), and time 0 is the time of the onset of the
first tetanus after oscillation. Force recovery is fitted with a monoexponential function having a rate constant of 0.234 s⫺1. Force and
filament density points are the means of 4 values for 4 separate
muscle bundles obtained from each of the tracheae. Error bars
indicate SE.
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Fig. 2. Electron micrographs of cross sections of airway smooth muscle. A: preparation fixed before a length
oscillation (time 1 in Fig. 1); inset shows the area in rectangle at higher magnification. Arrows indicate myosin
thick filament. A thick filament surrounded by thin filaments can be observed. B: preparation fixed immediately
after a length oscillation (time 2 in Fig. 1). Calibration bars indicate a distance of 1 ␮m.
1814
STRUCTURAL LABILITY AND SMOOTH MUSCLE PLASTICITY
Table 1. Myosin filament densities and cell cross-sectional areas
2
Filament density, filament number/␮m
Cross-sectional area, ␮m2
Time 1
Time 2
Time 3
Time 4
Time 5
46.8 ⫾ 1.6
7.4 ⫾ 0.3
33.8 ⫾ 1.6
7.2 ⫾ 0.2
38.7 ⫾ 1.7
7.6 ⫾ 0.3
45.0 ⫾ 2.4
6.9 ⫾ 0.2
48.1 ⫾ 2.2
7.0 ⫾ 0.3
Values are means ⫾ SE. Tabulated values are averages from 4 muscle preparations from 4 tracheae; for each muscle preparation, 15
morphometric measurements (from 15 micrographs) were made. The 5 time points (or conditions) at which the muscles were fixed for
morphometric examination are indicated in Fig. 1. The variation in filament density is significant (P ⬍ 0.05, one-way ANOVA); variation in
cell cross-sectional area is not significant (P ⬎ 0.05, one-way ANOVA).
DISCUSSION
Myosin lability. Lability of myosin thick filament in
smooth muscle has been a contentious issue ever since
the structure of the tissue was first investigated. It was
postulated by Shoenberg (12) and Rice et al. (9) that
myosin thick filaments of smooth muscle dissolved
during relaxation and formed on activation. Their hypothesis was based on the observation that thick filaments were more abundant when the muscle was fixed
for electron microscopy either at low pH (7) or in
high-divalent cation concentration (12), conditions that
were associated with muscle activation. In addition,
thick filaments were often difficult to find when the
muscles were fixed at rest (13). However, later studies
clearly demonstrated the existence of thick filaments
in smooth muscle in the relaxed, nonphosphorylated
state [see review by Somlyo and Somlyo (14)].
More recently, structural studies on rat anococcygeus smooth muscle have provided evidence that myosin filaments may increase in length and number during activation (3, 4, 16, 17). In these studies, it was
found that myosin lability is present only in anococcygeus smooth muscle and not in guinea pig taenia coli.
This tissue specificity may provide an explanation for
the contradictory results in the early studies. On the
basis of more recent structural studies, it can be concluded that myosin filaments are present in relaxed
smooth muscle; however, in some smooth muscle (e.g.,
rat anococcygeus), the length and/or number of thick
filaments may increase on stimulation of the muscle,
and in others (e.g., taenia coli) the thick filaments may
be stable.
Effect of mechanical perturbation on ultrastructure.
Results of the present study show that mechanical
perturbation is another factor that could affect the
appearance of myosin thick filament in smooth muscle.
If a smooth muscle sample is fixed immediately after
dissection, mechanical manipulation associated with
the dissection may cause a drastic decrease in the
number of thick filaments or even a total disappearance of the filaments. The length oscillation applied to
the muscle tissue in the present study is comparable to
that applied in vivo by a deep inspiration and is relatively small compared with the stretches a tissue might
be subjected to during the process of dissection. The
present results may explain the discrepancies found in
some of the early studies in which physical and physiological states of the tissues were not taken into consideration during tissue fixation.
Thick filament density in a cell cross section is proportional to the average length and number of thick
filaments. The change in the density observed in the
present study, therefore, can be interpreted as a
change in thick-filament length or number or both. A
contractile unit in smooth muscle consists of a thick
filament plus the adjacent thin filaments. The change
in the thick-filament length or number has a direct
consequence in terms of the muscle’s mechanical performance.
Functional evidence suggests that airway smooth
muscle is able to undergo plastic rearrangement of its
contractile apparatus to achieve optimal filament overlap over a large length range (5, 8). It was speculated
that myosin lability might facilitate this plastic restructuring (2). Our recent finding of functional evidence suggesting thick-filament lengthening during
isometric contraction also points to the possibility of
myosin lability in trachealis (10). The present finding
of an inducible and reversible myosin thick-filament
Fig. 4. Time course of isometric force development before (F) and
immediately after (E) length oscillation. Plots are signal averages of
4 traces, one from each trachea, normalized to tetanic force at the
end of stimulation.
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postoscillation contraction, compared with that in the
preoscillation contraction. The maximal rate of force
development occurs early in contraction. For the
preoscillation contraction (n ⫽ 4), maximal rate of force
development is equal to 0.265 ⫾ 0.031 (Po/s); and for
the postoscillation contraction (n ⫽ 4), maximal rate of
force development is equal to 0.324 ⫾ 0.043 (Po/s),
where Po is the maximal isometric force for each of the
contractions. The maximal rates of force development
for the pre- and postoscillation contractions are significantly different (P ⬍ 0.05).
STRUCTURAL LABILITY AND SMOOTH MUSCLE PLASTICITY
subjected to a period of length oscillation in the
relaxed state.
The suggestion that mechanical oscillation results
in the formation of more contractile units in series
spanning the cell length can be confirmed only by
examining longitudinal sections of the muscle for thickfilament length and number. Unfortunately, it is technically difficult to obtain reliable estimates of thickfilament length in a longitudinal section because the
filaments are usually not exactly parallel to the cell’s
longitudinal axis. With our technique used in the
present study, it is also difficult to accurately identify
the beginning and end of a thick filament, especially
when there appears to be two filaments lined up end to
end.
It should be pointed out that, despite a large force
decrease caused by the applied length oscillation, the
change in the time course of force development is
relatively small. This implies that the kinetics of contraction have not been altered drastically by length
oscillation.
In conclusion, the ability of a moderate mechanical
perturbation to reversibly alter the ultrastructural appearance of myosin thick filaments in airway smooth
muscle suggests a possible mechanism by which plastic
rearrangement of the contractile apparatus of the muscle can be achieved. Partial depolymerization of the
myosin filaments is initiated by a mechanical strain
applied to the cell. Myosin lability facilitates the process of reorganizing the filaments; redistribution of the
thick filaments through depolymerization/repolymerization ensures optimal interaction between thick and
thin filaments and generation of maximal force over a
large length range.
Special thanks to Pitt Meadows Meats (Pitt Meadows, BC, Canada) for their kind support of this research project by supplying fresh
swine tracheae.
This project is supported by operating grants from the Canadian
Institutes of Health Research to C. Y. Seow (MT-13271) and P. D.
Paré (MT-4725). L. Wang is the recipient of a Medical Research
Council/British Columbia Lung fellowship.
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1815
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