Departments of 1Anatomy, 2Medicine, 3Pathology and Laboratory Medicine, and 4Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3; 5Krannert Institute of Cardiology, Indiana University, Indianapolis, Indiana 46220; 6Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3J7; and 7The UBC McDonald Research Laboratories/The iCAPTURE Centre, Saint Paul's Hospital/Providence Health Care, Vancouver, British Columbia, Canada V6Z 1Y6
Submitted 10 March 2003 ; accepted in final form 13 April 2003
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ABSTRACT |
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muscle contraction; myosin filaments; ATPase activity; electron microscopy
The feasibility of the plasticity model can be tested experimentally based on the model predictions: the recruitment of additional contractile units in muscles adapted to longer lengths should result in an increase in thick filament content (or mass) in individual muscle cells, and as a consequence, the metabolic rate should increase with muscle length. In the present study, we tested the plasticity hypothesis through quantitative analysis of mechanical properties and ultrastructure, and measurement of the rate of ATP consumption in airway smooth muscles adapted to different lengths.
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MATERIALS AND METHODS |
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Solutions. Intact muscle experiments were done at 37°C, pH 7.4, in physiological saline equilibrated with 95% O2-5% CO2 and containing (in mM) 118 NaCl, 22.5 NaHCO3, 5 KCl, 1.2 NaH2PO4, 2 MgSO4, 2 CaCl2, and 2 g/l glucose. Permeabilized muscle was studied at room temperature and pH 7.0 in solutions containing (in mM) 5 EGTA, 85 K-acetate, 20 imidazole, 6.1 MgCl2, 5.6 Na2ATP, 1 dithiothritol, 1 NaN3, 5 phosphoenol pyruvate, 0.2 P1-P5-di(adenosine-5') penta-phosphate, 0.6 NADH, 70 U/ml lactate dehydrogenase, and 50 U/ml pyruvate kinase. Activating solution contained sufficient Ca2+ to buffer the free [Ca2+] to 25 µM, whereas relaxing solution had no added Ca2+. A rinse solution containing 0.1 mM EGTA was used to lower intracellular EGTA just before activation.
Muscle force, velocity, and power measurements. For the group of experiments examining changes in mechanical properties associated with a 1.5-fold change in length, canine trachealis preparations were used (to match the ATPase data), whereas in the group examining changes associated with a 2-fold change in length, swine trachealis preparations were used. The reason for using different animal tissues is because this study is a combination of three independent studies carried out in three laboratories (Seow, Ford, and Stephens).
Methods for measuring force-velocity parameters have been described in detail previously by us (15, 19). Briefly, aluminum clips were attached to both ends of a muscle preparation; one end of the muscle was attached to a stationary hook at the bottom of a vertical muscle bath, and the other end was attached to the force/length transducer (lever system) via a low-compliance surgical silk suture about 10 cm in length. Muscle preparations were equilibrated/adapted for about 1 h at 37°C in physiological saline bubbled with a 95% O2-5% CO2 mixture until the isometric tension produced by the muscle reached a maximal, steady state. During the equilibration period, the muscle was stimulated electrically to produce a brief tetanus (12 s) once every 5 min. During the period, a reference length (Lref) for the muscle was determined. The length at which rest tension was just detectable, approximately the in situ length, defines Lref.
After equilibration, isotonic shortening velocity was measured by quick releasing the muscle from the plateau of an isometric contraction to a preset isotonic load. Velocity of shortening was determined by measuring the slope of the shortening trace 100 ms after the quick release when the servocontrolled shortening reached a steady state. Six to ten such releases were performed on a muscle preparation to obtain force-velocity data used in the curve fitting to generate a hyperbolic (Hill's) force-velocity curve (7) of the form: V = b(Fmax - F)/(F + a), where V is shortening velocity, Fmax is the maximal isometric force, F is the isotonic load, and a and b are Hill's constants. Velocity change in a muscle adapted to different lengths was determined by scaling velocity values of the force-velocity curve obtained at Lref by a variable until the best fit was obtained for the force-velocity data obtained at the test length (1.5 or 2 Lref). The same procedure was used in our previous studies (15). Maximal muscle power output was determined from the force-velocity curve as described previously (19). Briefly, the power output (P) of the muscle as a function of load (F) was obtained by multiplying V and F: P = FV = Fb(Fmax - F)/(F + a). The maximal power (Pmax) was calculated by differentiating the force-power function (with respect to F) to obtain the force associated with Pmax,F' = a[(Fmax/a + 1)-1/2 - 1], and substitute F' for F in the force-power function. The number of animals used for the 1.5 Lref group of experiments was 9; for the 2 Lref group, the number was 6.
ATPase measurements. Muscles (canine trachealis) were first adapted to the reference length as described above. A pair of muscle preparations from one trachea was studied in a single experiment; three such experiments were carried out. One member of the pair was adapted to Lref and the other to 1.5 Lref. Muscles were permeabilized by 2-h immersion in a relaxing solution containing 1% (vol/vol) Triton X-100 and studied in an apparatus designed by Güth and Wojciechowski (5). Both muscles were studied at two lengthsfirst, the length at which it was adapted and, then, at the other length. Structural constraints of the apparatus limited muscle length changes to 1.5-fold.
One end of the muscle was tethered to a stationary hook; the other end was
attached to a force transducer. A quartz cuvette containing solution was
slipped over the muscle, which was illuminated with a 340-nm light source. ATP
hydrolysis was measured by the rate of decline in fluorescence of NADH, a
reduced form of nicotinamide adenine dinucleotide used to regenerate ATP in
conjunction with the phosphoenol pyruvate/pyruvate kinase intermediate. Fresh
NADH was injected into the cuvette at 25-s intervals to maintain a high level.
Fluorescence at 450 nm (wavelength) was measured over a 1-mm length of muscle,
and total muscle ATPase rate was estimated by multiplying the rate of
fluorescence decline by the length of the muscle. The fluoresced light passed
through a rectangular aperture in the photomultiplier, 1-mm wide in the
direction parallel to the muscle and 2 mm in the other dimension. Because the
cross-sectional area of the cuvette was at least 20 times larger than that of
the muscle, and because diffusion of ADP and NADH from the 0.2-mm-wide muscle
is very rapid, whereas diffusion over the 5-mm muscle length is
comparatively very slow, it is likely that the optical signal derived almost
entirely form the NADH in the extracellular space and was not influenced by
either the optical properties of the tissue or by diffusion to and from ends
of the muscle.
Electron microscopy. Samples (swine trachealis) were prepared for
electron microscopy as previously described
(6,
12,
16). Briefly, muscle
preparations were fixed for 15 min while they were still attached to the
experimental apparatus. Care was taken not to "physically" disrupt
the muscle during the fixation. The fixing solution contained 1.5%
glutaraldehyde, 1.5% paraformaldehyde, and 2% tannic acid in 0.1 M sodium
cacodylate buffer that was prewarmed to the same temperature as the bathing
solution (37°C). After the initial fixation, the strip was removed from
the apparatus and cut into small blocks, 1 x 0.5 x 0.2 mm in
dimension, and put in the fixing solution for 2 h at 4°C on a shaker. The
blocks were then washed three times in 0.1 M sodium cacodylate (3 x 10
min). In the process of secondary fixation, the blocks were put in 1%
OsO4 and 0.1 M sodium cacodylate buffer for 2 h, followed by three
washes with distilled water (3 x 10 min). The blocks were then further
treated with 1% uranyl acetate for 1 h (en bloc staining), followed by washes
with distilled water. Increasing concentrations of ethanol (50, 70, 80, 90,
and 95%) were used (10 min each) in the process of dehydration. Ethanol (100%)
and propylene oxide were used (three 10-min washes each) for the final process
of dehydration. The blocks were left overnight in the resin (TAAB 812 mix,
medium hardness) and then embedded in molds and placed in an oven at 60°C
for 810 h. The embedded blocks were sectioned on a microtome using a
diamond knife and placed on 400-mesh cooper grids. The section thickness was
100 nm. The sections were then stained with 1% uranyl acetate and
Reynolds lead citrate for 4 and 3 min, respectively. Images of the cross
sections of cells were made with a Phillips 300 electron microscope at various
magnifications.
Morphometric analysis. The myosin thick filament had an amorphous cross-sectional profile with an average diameter of about 15 nm. The actin thin filament had a circular cross section with a diameter of 6 nm. The number of thick filaments in five cells from each muscle preparation was counted manually (n = 4 for the 2 Lref group and n = 5 for the 1.5 Lref group, where n is the number of pairs of muscle strips from one trachea and also equals the number of animals used). Examples of electron micrographs are given in Fig. 1. The filament density was determined by dividing the total filament number in a cross section by the cross-sectional cytoplasmic area, which was the cell's cross-sectional area minus the areas occupied by organelles such as nucleus, mitochondria, sarcoplasmic reticulum, and caveolae. The same method of quantifying myosin filament density was used in our previous studies (6, 12, 16). Morphometric measurements were carried out "blind": the tissue samples were relabeled by a person not involved in the experiments, and the true identity of the samples was revealed only after all samples were analyzed. Identification of the filaments was done manually, and quantification was done with the assistance of the software Image Pro Plus.
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RESULTS |
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Length dependence of muscle metabolic rate. ATPase activities were measured fluorimetrically in canine trachealis muscles permeabilized by partial dissolution of the plasma membranes and stimulated to contract by the addition of Ca2+. Two lengths were studied, Lref and 1.5 Lref. At these two lengths, force was not significantly different and ATPase activity was 34.6% ± 3.4 (SE) greater for the 1.5-times longer muscle (Fig. 2 and Table 1). The ATPase activity was normalized by isometric force to reduce interpreparation difference in the rates. Without normalization, the ATPase activity increase was 32 ± 8% for a 50% increase in muscle length. This is not statistically different from the normalized value; the SE, however, is larger without normalization. The finding that interpreparation variation in ATPase rate could be reduced when the developed force was taken into account is consistent with the finding in skinned gizzard muscle that ATPase activity was directly proportional to the isometric force that a muscle has developed (10).
Length dependence of myosin thick filament density. Two groups of
experiments were carried out to assess changes in myosin filament density at
different muscle lengths: 1.5 Lref vs.
Lref and 2.0 Lref vs.
Lref. Two adjacent trachealis strips with the same initial
length were dissected from each animal; one was set at
Lref and the other at a test length (1.5 or 2.0
Lref). After at least 1 h of adaptation at a set length,
the muscle was stimulated to produce a 120-s contraction by the addition
of 0.1 mM acetylcholine and fixed for electron microscopy in the contracted
state. Acetylcholine was used for the final stimulation to ensure continued
activation during fixation. Cell cross-sectional areas measured in the
electron micrographs were decreased by approximately half in muscles fixed at
2 Lref (Fig.
3 and Table 1)
compared with that at Lref, indicating that cell volume
was conserved. The myosin filament density increased by 35.6% ± 10.6
(SE) and 76.0% ± 9.8 with 50 and 100% increase in muscle length,
respectively. These values were obtained from density ratios of paired muscle
preparations from the same trachea. The pooled absolute values of filament
density are listed in Table 1.
The filament density found in this study was much higher than that found in
our previous studies (6,
12,
16); this is because the
density values listed in our previous publications were those determined in
the relaxed muscles, whereas the density reported here was determined in the
activated muscles.
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DISCUSSION |
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If the thick filament length is much less than the length of a cell, and also if they are randomly distributed within the cell regardless of the cell length, the thick filament density in a cell cross section should remain constant at different cell lengths, provided the filament content (or mass) is length independent (i.e., no filament polymerization or depolymerization with length change). This is analogous to a bag of black and white marbles; as long as the balls are evenly mixed, the number of black marbles per unit area (density) found in any imaginary planes cutting across the bag will be the same whether the bag is stretched or squashed. The present finding that the thick filament density increased in muscles adapted at longer length suggests filamentogenesis. Furthermore, the filament density in a cross section should reflect filament content of a cell, provided that the cell volume is conserved. [Using the same analogy as above, the number of black marbles found per unit area in a cross section is directly correlated to the total number (or content) of black marbles in the bag]. The inverse relationship between cell cross-sectional area and cell length (Fig. 3 and Table 1) indicates that the cell volume was indeed the same within the lengths studied. The 35.6% ± 10.6 and the 76% ± 9.8 increase in thick filament density found at 50 and 100% length increase, therefore, closely fits the model prediction (Fig. 4).
The present study thus provides the first ever evidence that myosin filament content in airway smooth muscle is regulated by muscle length. How is it regulated is unknown. It could, however, be speculated that cytoskeletal deformation could lead to activation of integrin-linked kinase that, in turn, promotes myosin light chain phosphorylation (2) and thick filament formation (16). The observation that there is a large increase in the thick filament density (in a matter of seconds) when airway smooth muscle is activated (6) suggests that there is a large pool of monomeric myosins in the trachealis cells.
There are alternative explanations of our data. The increase in mechanical power output at longer lengths could be a result of increased energetic efficiency in the muscle, although this is unlikely because the rate of ATP consumption found in the present study was similarly dependent on the muscle length (Fig. 2). In fact, the results suggest that the simplest explanation is that the efficiency of the muscle is unchanged at different lengths. The increased ATPase activity at longer muscle length could also be due to activation of cross bridges previously not activated at short lengths. Measurements of thick filament density, however, suggest that there is a net increase in thick filament number in the cell as the cell length increases (Figs. 2 and 3). A simpler explanation, therefore, is that the increase in ATPase rate is due to a matching increase in the thick filament content. Furthermore, the findings that shortening velocity and ATPase activity increase in the same proportion, together with the finding that the force-velocity curves obtained at different lengths can be superimposed by velocity scaling, imply that cross-bridge function (in terms of cycling rate and force per bridge) is the same at the two lengths. The absence of a change in cross-bridge function would require that the individual filaments slide at the same rate in long and short muscles, so that the amount of increase in velocity and power would require the same amount of increase in filaments in series when length was increased. Together, the results are consistent with the interpretation that the increase in the cell content of myosin filaments is the underlying cause for the increases in the other three measures of muscle activity (velocity, power, and ATPase activity).
The model shown in Fig. 4 assumes that the length of thick filaments is the same at different cell lengths, and the increase in thick filament content at longer muscle length is due to more thick filaments added in series and not due to the existing thick filaments getting longer, to account for the present finding that isometric force is length independent. Another model that predicts the same amount of increase in filament content and length independence of isometric force allows the thick filament length to increase in inverse proportion to the number of thick filaments in parallel (to keep isometric force constant). This model also has to allow the number of thick filaments in series to vary with muscle length to account for the length-dependent shortening velocity. Results from the present study do not allow us to favor one model over the other. The model presented in Fig. 4, however, is simpler in the sense that it does not rely on a potentially complicated mechanism that adjusts the thick filament length and the number of filaments in parallel precisely according to the cell length.
Although there is always a danger in extrapolating interpretation on one smooth muscle type to another, it could, however, be speculated that the very long functional length range (up to 10-fold change in length) of rabbit urinary bladder muscle (18) and pedal retractor muscle of Mytilus edulis (blue mussel) (8) could be due to a similar plastic mechanism described above.
The length dependence of smooth muscle force (20), intracellular Ca2+ concentration, and myosin light chain phosphorylation (17), ATPase activity (1), and rate of oxygen consumption (14) have been documented. The present results, however, cannot be compared with those from the early studies because the length adaptation protocol (cyclic activation of the muscle at a constant length) used in our experiments has largely eliminated the dependence of isometric force on muscle length and altered the ultrastructure of the cell.
The need for plastic adaptation is quite obvious in smooth muscles lining the wall of hollow organs that undergo large volume changes. It is not clear why such plasticity exists in airway smooth muscle. It is clear, however, that there are a variety of in vivo conditions under which airway smooth muscle could be adapted to pathologically short lengths, especially when inflammation of the airways is involved: frequent and prolonged stimulation of the muscle, adventitial edema of the airway wall that decreases resting muscle length, reduced elastic tethering of lung parenchyma on the airway wall (due to chronic inflammation) that diminishes resting muscle length, to name just a few (11). Elucidating the mechanism of airway smooth muscle plasticity, therefore, will, in addition to offering insights into the basic mechanism of contraction, shed light on the pathophysiology of exaggerated airway narrowing seen in diseases such as asthma and emphysema.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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