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J Appl Physiol 90: 741-749, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 2, 741-749, February 2001

HIGHLIGHTED TOPICS
Plasticity in Skeletal, Cardiac, and Smooth Muscle
Selected Contribution: Plasticity of airway smooth muscle stiffness and extensibility: role of length-adaptive mechanisms

Susan J. Gunst and Ming-Fang Wu

Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airway smooth muscle exhibits the property of length adaptation, which enables it to optimize its contractility to the mechanical conditions under which it is activated. Length adaptation has been proposed to result from a dynamic modulation of contractile and cytoskeletal filament organization, in which the cell structure adapts to changes in cell shape at different muscle lengths. Changes in filament organization would be predicted to alter muscle stiffness and extensibility. We analyzed the effects of tracheal muscle length at the time of contractile activation on the stiffness and extensibility of the muscle during subsequent stretch over a constant range of muscle lengths. Muscle strips were significantly stiffer and less extensible after contractile activation at a short length than after activation at a long length, consistent with the prediction of a shorter, thicker array of the cytoskeletal filaments at a short muscle length. Stretch beyond the length of contractile activation resulted in a persistent reduction in stiffness, suggesting a stretch-induced structural rearrangement. Our results support a model in which the filament organization of airway smooth muscle cells is plastic and can be acutely remodeled to adapt to the changes in the external physical environment.

length adaptation; deep inspiration; stretch; tidal breathing; airway responsiveness


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CHANGES IN LUNG VOLUME that occur during breathing are important in regulating airway reactivity. In normal human subjects, deep inspiration after bronchoconstriction causes a reduction in airway resistance (18). Conversely, the prevention of deep inspiration during methacholine challenge of normal subjects results in an increase in airway reactivity that can reach levels comparable with those observed in asthmatic subjects (14, 27). In experimental animals, the increase in airway resistance in response to methacholine is inhibited by tidal breathing (23, 31, 34). The effects of tidal breathing and deep inspiration on airway responsiveness have been attributed to a reduction in muscle force caused by stretch or mechanical oscillation of the airway smooth muscle (6, 8, 23, 25, 31). This is supported by observations that the contractility of isolated bronchial segments and of isolated tracheal strips is reduced by volume or length oscillation (6, 8, 12, 25). When isolated muscle tissues are stimulated at a long muscle length and then shortened, a depression of both force development and shortening velocity is observed at the shorter length (9, 13). These muscle properties may result from a length-adaptive mechanism in smooth muscle that enables the muscle to optimize its contractility to the length at which is activated (10, 11, 13, 21). The effects of deep inspiration and tidal breathing on airway reactivity in vivo may result in part from length-adaptive mechanisms that modulate airway smooth muscle contractility.

We have proposed that this property of length adaptation may result from an ability of the muscle cells to modulate the organization of their contractile apparatus to accommodate to changes in cell shape that occur at different muscle lengths (10, 11, 13). This hypothesis predicts that differences in the organization of contractile filaments after activation of the muscle at different lengths would lead to differences in muscle stiffness. Activation of the muscle at a short length would be predicted to result in reorganization of the contractile apparatus into a shorter, thicker filament array adapted to shorter, thicker muscle cells. In contrast, activation at a long length would result in the organization of the contractile filaments in a longer, thinner array. If contractile activation at different lengths results in such changes in contractile filament organization, the muscle should be stiffer and less extensible after contractile activation at a short length than after activation at a long length.

In the present study, we tested these predictions by analyzing the effects of muscle length at the time of contractile activation on the stiffness and extensibility of the muscle. We also assessed the modulation of muscle stiffness and contractility caused by stretch. Muscle stiffness was evaluated by imposing high-frequency, small-amplitude length oscillations on the muscle that were too small to disrupt cross-bridge attachments. The extensibility of contracted muscles was assessed by measuring changes in length during imposed stretches over a given force range. Our results demonstrate that the muscle is significantly stiffer and less extensible after activation at a short muscle length than after activation at a long length. These results are consistent with a model in which the filament organization of the muscle is actively remodeled to adapt to the length of the smooth muscle cell at the time it is contracted. Structural plasticity of airway smooth muscle cells could result in a decrease in airway distensibility and an increase in airway responsiveness with bronchoconstriction at low lung volumes.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation and experimental apparatus. Mongrel dogs (20-25 kg) were anesthetized with pentobarbital sodium and quickly exsanguinated. A 10- to 15-cm segment of extrathoracic trachea was immediately removed and immersed in physiological saline solution (PSS) of the following composition (in mM): 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose. The solution was aerated with 95% O2-5% CO2 to maintain a pH of 7.4. Trachealis muscle strips (2-3 × 10 mm) were dissected from the trachea after removal of the epithelium and connective tissue layer. Muscle strips were mounted horizontally in a 25-ml rectangular Plexiglas tissue bath containing 37°C PSS that was bubbled with 95% O2-5% CO2 to maintain a pH of 7.4. One end of each strip was fixed tightly to a stationary platinum hook while the other end was attached to an servo-regulated electromagnetic lever (Cambridge Technology, model 300B). The static compliance of the entire system excluding the muscle was negligible with respect to muscle compliance. The resolution of the force signal was 30 mg.

After placement in the tissue bath, muscles were equilibrated for 60-90 min. During this time, they were stimulated by electrical field stimulation at 5- to 10-min intervals using 20-V, 15-pulses/s (pps), 0.5-ms duration square waves by means of rectangular platinum electrodes (55 × 10 × 0.3 mm) connected to a Grass stimulator and a current amplifier. To determine Lo, the length at maximal active force, muscle length was increased progressively after each stimulation until the force of active contraction reached a maximum (Fo). The muscle was then subjected to one of several paradigms of mechanical manipulation.

Measurement of muscle stiffness. Muscle length was oscillated using a 25-µm, 40-Hz sine wave applied to the position input of the Cambridge Technology servo-system as previously described (11). Muscle stiffness was computed from the resulting force perturbation (dF) by taking the ratio of the force perturbation amplitude to the length perturbation amplitude (dL). At a 40-Hz frequency, a 6° phase shift between the length and force signals was present, which was constant throughout the experiment. This had a negligible effect on the measurement of dF. The length oscillation was added to the position command signal; the force oscillation recorded during contraction was a superimposed component of the muscle force signal. These compound signals were separated using a set of digitally controlled band-pass and low-pass filters slaved to the sine-wave generator. The amplitudes of the force and length perturbations (representing dF and dL, respectively) were measured by full-wave rectification and subsequent short-term averaging of the output of the band-pass filters (11). Force, length, and the filtered signals of dF and dL were recorded continuously on a Gould strip-chart recorder and captured simultaneously on a Nicolet digital oscilloscope at an acquisition rate of 5-50 pps. Digital records of force, length, dF, and dL were stored on disk for later computer analysis.

Data analysis. Values of dF were divided by dL throughout each contraction to obtain a continuous measure of muscle stiffness. These measurements were plotted against time and also against each other during each individual contraction. Instantaneous values of stiffness were divided by instantaneous values of force to assess changes in the ratio of stiffness to force during the course of each contraction. In each muscle, all values of both stiffness and force were normalized to the maximal values (So or Fo) obtained during isometric contraction at Lo.

The extensibility of each muscle after contraction at each muscle length was computed by measuring the change in muscle length that occurred during stretch over a range of force common to all stretches (illustrated in Fig. 3).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of contractile activation at different muscle lengths on muscle stiffness and extensibility. We hypothesized that the activation of tracheal smooth muscle at different muscle lengths would result in adaptive changes in the organization of the contractile filaments and that this would result in differences in the stiffness and extensibility of the muscle (see Figs. 1-3). The following protocol was designed to evaluate the effects of activation of the muscle at different muscle lengths on its stiffness and extensibility during the same activation period.


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  		Fig. 1.   Effect of muscle length during contractile activation on force and stiffness. A: protocol. Each muscle was contracted isometrically with 10-5 M ACh at Lo (length at maximal active force), 0.75 Lo, and 0.5 Lo in random sequence. After 5 min, the muscle was rapidly retracted to minimal length (0.2-0.3 Lo) and then slowly stretched back to the length at which it was initially activated. B: force vs. muscle length during stretch after contraction at 0.5 Lo, 0.75 Lo, and Lo. C: stiffness vs. muscle length during stretch after contraction at 0.5 Lo, 0.75 Lo, and Lo. D: stiffness vs. force for stretch after contraction at 0.5 Lo, 0.75 Lo, and Lo. Data shown are normalized values: force, F/Fo; stiffness, S/So; Length L/Lo (where Fo and So are maximal force and stiffness, respectively).



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  		Fig. 2.   Mean values for force (F/Fo; A), stiffness (B), and the ratio of stiffness to force (C) during stretch after isometric contractions at 0.5 Lo, 0.75 Lo, and Lo. Values for force, stiffness, and the ratio of stiffness to force were measured during stretch at a muscle length of 0.5 Lo. Values are means ± SE (n = 4). *Values significantly different from those obtained for 0.5 Lo.



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  		Fig. 3.   Effect of muscle length during isometric contraction on muscle extensibility after isometric contractions at 0.5 Lo, 0.75 Lo, and Lo. A: calculation of extensibility following isometric contraction of muscle strip at different lengths. The extensibility of each muscle following contraction at each muscle length was computed as the change in muscle length that occurs during stretch over a range of force common to all stretches. B: mean values for extensibility during stretch after isometric contractions at 0.5 Lo, 0.75 Lo, and Lo. Values are means ± SE (n = 4). *Values significantly different from those obtained for 0.5 Lo.

Each muscle strip was contracted isometrically at muscle lengths of Lo, 0.75 Lo, and 0.5 Lo in random sequence using 10-5 ACh (Fig. 1A). At each length, the muscle was first contracted isometrically several times. It was then stimulated isometrically for 5-6 min, after which the activated muscle was rapidly shortened to 0.2-0.3 Lo (minimal length) and then slowly (0.3 Lo/min) stretched back to the length at which it was initially contracted. The duration of the period of stretch ranged from 1 to 2.5 min. This protocol was repeated after contraction at each of the three muscle lengths in each muscle strip (stretches 1, 2, and 3). The sequence in which the lengths were studied was randomized among different muscles. Muscle stiffness was assessed during the stretches by superimposing small high-frequency length oscillations, and extensibility was calculated as the ratio of the change in muscle length over the change in force during stretch over a constant range of force (see Fig. 3). Five muscle strips were subjected to this protocol, and analogous results were obtained in all five of them.

Tracheal muscle stiffness was highest and extensibility lowest after the muscle was contracted isometrically at the shortest length (0.5 Lo), whereas muscle stiffness was lowest and extensibility highest after the muscle was contracted isometrically at the longest length (Lo) (Figs. 1, B and C, 2, and 3). The ratio of muscle stiffness to force was highest after contraction at the shortest muscle length (0.5 Lo) and lowest after contraction at the longest muscle length (Lo) (Fig. 1D). Mean values for force, stiffness, and the stiffness-to-force ratio were computed for isometric contractions at each length at a muscle length of 0.5 Lo during stretch. Mean values for extensibility at each muscle length were computed by measuring the changes in muscle length that occurred during the stretches over a common range of force (Fig. 3, A and B). Values of all of these parameters obtained for contractions at different muscle lengths were significantly different (P < 0.05, n = 4) (Figs. 2 and 3).

The differences in muscle extensibility and stiffness induced by isometric contraction at different muscle lengths were fully reversible and reproducible. The force-length curve obtained during stretch after activation of the muscle at any particular muscle length could be reproduced when contraction of the muscle was repeated at that length. The force-length curve obtained after isometric contraction at a particular length also remained the same when the muscle was successively shortened and stretched several times during a contraction (e.g., see Fig. 4, stretches 2 and 3).


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  		Fig. 4.   Effect of stretch beyond the length of contraction on force and stiffness. A: protocol. Each muscle was contracted at Lo for 5 min, after which it was retracted to minimal length (0.2-0.3 Lo) and then stretched back to Lo (stretch 1). ACh was washed from the muscle strip, and the muscle was recontracted at 0.75 Lo, retracted to minimal length, and stretched back to 0.75 Lo (stretch 2). It was again retracted to minimal length and then stretched to Lo (stretch 3). It was again retracted to minimal length and stretched again to Lo (stretch 4). B: relationship between force and muscle length during each of the 4 stretches in the protocol shown in A. C: relationship between stiffness and length during each of the 4 stretches shown in A. Data shown are normalized values: force, F/Fo; stiffness, S/So; length L/Lo.

Stiffness of activated smooth muscle can be reduced by stretch. If the increased muscle stiffness that results from contractile activation at a short length results from the adaptation of contractile filament organization to the shorter cell length, then stretching the muscle beyond the length at which the contraction was initiated might force the reorganization of the contractile filaments, thereby reducing muscle stiffness. We therefore evaluated the effect of stretching muscles beyond the length at which they were isometrically contracted on muscle stiffness and extensibility.

Muscle strips were first contracted isometrically at Lo with ACh. The actively contracted strips were then rapidly shortened to the minimal length and slowly stretched back to Lo (Fig. 4A, stretch 1). The ACh was then washed out of the muscle. A second isometric contraction was stimulated by ACh at 0.75 Lo; the muscle was again shortened to minimal length and slowly stretched back to 0.75 Lo (Fig. 4A, stretch 2). Without the contractile stimulus being washed out, the muscle was again shortened to minimal length and stretched; however, this time the muscle was stretched to Lo (stretch 3). While still contracted, the muscle was again shortened to minimal length and then stretched again to Lo (stretch 4).

As observed during the first protocol (Fig. 1), both force and stiffness were higher during stretch of the muscle when the contraction was initiated at a short length (0.75 Lo, stretch 2) than after the contraction was initiated at the longer length (Lo, stretch 1) (Fig. 4, B and C). The higher level of force and stiffness persisted when stretch of the activated muscle was repeated, this time with the stretch continuing all the way to Lo (stretch 3) (Fig. 4, B and C). However, stiffness began to decrease as the muscle was stretched beyond the length at which the contraction was initiated (see top section of stretch 3 in Fig. 4). When stretch of the activated muscle was repeated again (stretch 4), both muscle force and stiffness were dramatically reduced, approximating the levels of force and stiffness observed previously during stretch 1 after isometric contraction of the muscle at Lo. Thus the lower extensibility and higher stiffness of the muscle after activation at a shorter length (0.75 Lo) was mechanically reversed by stretching the muscle beyond 0.75 Lo, the length at which the isometric contraction was performed. The mechanically induced decrease in muscle stiffness and increase in extensibility (stretch 4) could be completely reversed by washing out the contractile stimulus and recontracting the muscle isometrically at 0.75 Lo (data not shown). The results shown in Fig. 4 were typical of those obtained in five separate muscle strips.

Effect of the duration of isometric contraction on muscle force and stiffness during stretch. We evaluated the effect of the duration of isometric contraction with ACh on muscle force and stiffness during stretch. In these experiments, isometric contractions were performed at the same muscle length, but the duration of each contraction before stretch was varied, lasting 1, 2, 5, or 10 min. After isometric contraction, the muscle was then rapidly shortened to the defined minimum length and then stretched slowly back to Lo. The successive contractions of different duration were performed in random sequence using 10-5 M ACh (Fig. 5A). Muscle force and stiffness during stretch were plotted against muscle length during stretch (Fig. 5, B and C). Both muscle force and stiffness were highest during stretches initiated at early time points during the contraction and lowest during stretches initiated at later time points. However, the ratio of muscle stiffness to force was identical during stretches initiated at different time points during the contraction (Fig. 5D). The constancy of the force-to-stiffness ratio suggests that the time-dependent changes in muscle force and stiffness reflect changes in cross-bridge activation over the time course of the contraction. Active shortening of the muscle may occur during its retraction to minimal length. Because the shortening rate of the muscle is highest early in the stimulation period, somewhat more shortening can occur during retraction of the muscle early in the activation period than when the retraction is imposed later in the activation period. This results in higher levels of both force and stiffness for stretches performed early in the activation period.


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  		Fig. 5.   Effect of duration of contraction on force and stiffness during stretch. A: protocol. Each muscle was contracted isometrically at Lo for 1, 2, 5, or 10 min and rapidly retracted to minimal length and stretched back to Lo. B: relationship between muscle force and muscle length during stretch after 1, 2, 5, or 10 min of contraction. C: relationship between muscle stiffness and length during stretch after 1, 2, 5, or 10 min of isometric contraction. D: relationship between stiffness and force during stretch after 1, 2, 5, or 10 min of isometric contraction. Data shown are normalized values: force, F/Fo; stiffness, S/So; length L/Lo.

We further evaluated the effect of the duration of isometric contraction on muscle stiffness by imposing stretches on the muscles at different time points during isometric contraction without first retracting the muscles to a short length (Fig. 6A). In these experiments, each muscle strip was contracted isometrically at a muscle length of 0.60 Lo and then slowly stretched from 0.60 Lo to Lo either 1 or 10 min after the initiation of contraction. Force and stiffness were measured during the stretch (Fig. 6, B and C). Muscle force during the stretch was similar for contractions of different duration [mean values at a length of 0.7 Lo were 0.62 ± 0.012 Fo at 1 min and 0.60 ± 0.005 Fo at 10 min (n = 5)]; however, muscle stiffness during stretch increased significantly between 1 and 10 min (mean values of stiffness during stretch at a length of 0.7 Lo were 0.74 ± 0.02 So after 1 min of contraction and 0.804 ± 0.01 So after 10 min). The ratio of muscle stiffness relative to force also increased significantly with the duration of contraction (0.63 ± 0.025 So at 1 min vs. 0.73 ± 0.027 So at 10 min during stretch at a force of 0.5 Fo). This reflected a time-dependent increase in muscle stiffness during the contraction despite a relatively constant level force during this time (Fig. 6A). Both muscle force and stiffness could be reduced by imposing a large (10% Lo) oscillation on the muscle for several minutes during the contraction (Fig. 6D). Oscillation of the muscle resulted in significant decreases in the mean values of force and stiffness to 0.524 ± 0.023 Fo and to 0.662 ± 0.019 So, respectively. Values were measured during stretch at a length of 0.7 Lo.


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  		Fig. 6.   Effect of duration of contraction on force and stiffness during stretch beyond the length of isometric contraction. A: protocol. Each muscle strip was contracted isometrically at 0.6 Lo for 1 min and then stretched slowly to Lo (stretch 1). The muscle was allowed to relax, recontracted at 0.6 Lo after 10 min of contraction, and then stretched again to Lo (stretch 2). The muscle was contracted for 8 min at 0.6 Lo, oscillated over 10% of its length (between 0.55 Lo and 0.65 Lo), and then stretched to Lo (stretch 3). B: force vs. length during stretches after 1 min of isometric contraction, 10 min of isometric contraction, and after oscillation. C: stiffness vs. length during stretches after 1 min of isometric contraction, 10 min of isometric contraction, and after oscillation. D: stiffness vs. force during stretches after 1 min of isometric contraction, 10 min of isometric contraction, and after oscillation. Data shown are normalized values: force, F/Fo; stiffness, S/So; length L/Lo.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanisms for the mechanical modulation of airway muscle stiffness. Our results demonstrate that when airway smooth muscle is maintained at a short length during activation it becomes stiffer and less extensible than when contractile activation takes place at a long length (Figs. 1-3). The effects of muscle length on muscle stiffness and extensibility persist for the duration of the period of contractile activation, even during the imposition of subsequent mechanical maneuvers of retraction and stretch. However, the stiffness of the muscle can be decreased by stretching the muscle beyond the length at which it was maintained during contractile activation (Fig. 4). The reduction in stiffness caused by stretch persists during subsequent maneuvers during the same activation period. These observations are consistent with our hypothesis that the contraction of the airway smooth muscle at different lengths results in changes in contractile filament organization that alter its stiffness and extensibility (Fig. 7) (10, 11, 13). The length-adaptive properties of airway smooth muscle may underlie the increase in airway reactivity that is observed in human subjects prohibited from deep inspiration during challenge with bronchoconstrictors, as well as the dilatory effects of deep inspiration on constricted airways in vivo.


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  		Fig. 7.   Effect of filament organization on muscle stiffness during stretch. When the muscle is maintained at a short length during contraction, the organization of the cytoskeletal filaments adapts to the shorter thicker shape of the smooth muscle cell (A, bottom), resulting in more filaments in parallel. Maintenance of the muscle at a longer length during activation leads to adaptation of the filament organization to the longer thinner shape of the smooth muscle cell (A, top). The muscle will therefore be stiffer after contractile activation at a shorter length than after activation at a long length (B).

Muscle stiffness, as measured by the imposition of small high-frequency length perturbations, is commonly used as an index of cross-bridge attachment. The ratio of muscle stiffness to force during active contraction has been interpreted as an index of the proportion of attached cross bridges that contribute to force development (5, 16, 35). In skeletal muscle fibers, maximal active tension is observed when the cross bridges on the thick filaments are fully overlapped by the thin filaments (7). At fiber lengths longer and shorter than the optimum length, isometric force is predicted to decline due to a reduction in the number of cross bridges that interact with actin. This results in a decrease in muscle stiffness that is proportional to the decrease in the number of attached cross bridges (5). In the present study, small high-frequency length oscillations were imposed on the muscle during forced extension (stretch) as a probe for cross-bridge attachment. Both the force and stiffness of the muscle during stretch were higher after the muscle was activated at a short muscle length than after it was activated at a long length (Figs. 1 and 2). In addition, the ratio of stiffness to force was higher after isometric contraction at a short muscle length. The ratio of stiffness to force increases when the proportion of attached cross bridges that contribute to force decreases or when there is an increase in the number of noncontractile elements contributing to muscle stiffness. The latter condition might result from a rearrangement of the organization of the contractile apparatus that results in an increase in the number of contractile units in parallel, requiring the formation of additional noncontractile linkages.

Plasticity of the length-tension and force-velocity properties of airway smooth muscle has been previously reported in a number of studies (11, 13, 17, 21, 29). We have hypothesized that this plasticity results from a length-adaptive property of the smooth muscle cell that enables it to dynamically reorganize its contractile filament network to accommodate to changes in cell shape imposed by its external environment (10, 11, 13). According to our hypothesis, changes in muscle length stimulate active remodeling of the actin cytoskeleton, resulting in the lengthening or shortening of actin filaments and/or the modulation of their sites of attachment to the cell membrane (15, 28). Because the actin cytoskeleton forms the structural lattice on which myosin filaments slide during cross-bridge cycling, changes in its organization would result in a rearrangement of the organization of the contractile apparatus. The actin filament organization present at the time of contractile activation would then persist for the duration of the period of activation. The cytoskeletal lattice would accommodate to contraction of the muscle at a short length by rearranging to form a shorter thicker array of filaments, whereas contraction of the muscle at a long length would result in a longer thinner array of contractile filaments (Fig. 7). Changes in the muscle extensibility and stiffness resulting from contraction at different muscle lengths could therefore reflect differences in the organization of the cytoskeletal lattice in smooth muscle cells. Stretch of the muscle beyond the length at which the contraction is initiated may reduce muscle stiffness and contractility by mechanically inducing a rearrangement of the contractile filament network.

There is molecular evidence in support of such a mechanism in tracheal smooth muscle. The contractile activation of tracheal smooth muscle tissues and cells stimulates actin filament polymerization (15, 32). In addition, the inhibition of actin filament remodeling suppresses the length sensitivity of contractile force (15). The contractile activation of tracheal muscle also stimulates the phosphorylation of talin, paxillin, and focal adhesion kinase (FAK), proteins that are localized to the sites at which actin filaments link to the extracellular matrix via transmembrane integrins (20, 30, 33). In nonmuscle cells, the phosphorylation of these proteins is associated with actin filament remodeling and cytoskeletal reorganization (1, 3, 19, 22). The phosphorylation of both FAK and paxillin is sensitive to mechanical strain, suggesting that an integrin-mediated mechanotransduction process is present in this tissue (30).

We have previously reported that tracheal smooth muscle stiffness (as measured by the imposition of small high-frequency length oscillations) increases disproportionately to force during the plateau phase of an isometric contraction (11). In the present study, we found that activated tracheal muscles are stiffer when they are stretched at a later time point during an isometric contraction than when they are stretched early in the contraction (Fig. 6). The stiffness measured during the imposed stretch increases disproportionately to force as the duration of the contraction is prolonged, suggesting the progressive formation of cellular attachments that do not contribute to active force. These attachments could be non-force-producing cross bridges or other non-force-generating attachments within the cell such as linkages between filaments that "stabilize" the contractile filament array. A process of continuous slow remodeling of the arrangement of the contractile filament network might also result in a slow increase in muscle stiffness relative to force.

We did not observe differences in muscle stiffness relative to muscle force when the muscle was shortened to the defined minimal length at different time points after contraction and then stretched (Fig. 5). Higher levels of force and stiffness during stretch were observed when the muscle was retracted and stretched early in the contraction rather than later. This time-dependent change in force and stiffness probably results from active shortening of the contractile element during the imposed retraction to minimal length. More active shortening would be expected to occur early in the contraction when the shortening velocity is highest, resulting in more cross-bridge attachments and proportionally higher levels of both force and stiffness.

Implications for the regulation of airway tone. Changes in lung volume during breathing modulate airway tone and airway responsiveness in vivo. In normal human subjects subjected to bronchoconstriction, deep inspiration results in a decrease in airway resistance and an increase in expiratory flow (2, 4, 18). Deep inspiration also reduces airway resistance in experimental animals (24, 26). Airway responsiveness is also inhibited by tidal breathing, and the inhibition of airway responsiveness increases with increasing breath volume (23, 31, 34).

Our current observations suggest that length-adaptive properties of airway smooth muscle cells can account for many of the effects of volume maneuvers on airway tone and airway responsiveness that have been observed in vivo. The increase in airway reactivity that occurs with prolonged tidal breathing in the absence of a deep breath (27) may result from the increase in stiffness and contractility that develops in airway smooth muscle that is maintained at a short length. We observed that, when activated airway smooth muscle is stretched beyond the length at which it was activated, a long-lasting and persistent reduction in stiffness and active force results. This property of the muscle could explain the ability of deep breath to reduce airway responsiveness.

The properties of airways in vivo can be further interpreted in terms of the cellular mechanisms that we have proposed. We have hypothesized that the cytoskeletal structure of the smooth muscle cell adjusts to the length to which the cell is stretched. During tidal breathing at functional residual capacity (FRC), stretch of the airway smooth muscle is relatively small (12). Thus, with prolonged tidal breathing at FRC, the structure of the cytoskeletal lattice adapts to a short cell length, resulting in a stiffer, less extensible muscle and a reduced airway compliance, making inflation of the airways more difficult. Deep inspiration would stretch the muscle to a longer length, as demonstrated in Fig. 4, forcing adaptation of the cytoskeletal structure to the longer cell length. This would reduce muscle stiffness and increase its extensibility, thereby increasing airway compliance. However, when tidal breathing was resumed after deep inspiration, the muscle structure would begin to readapt to the shorter cell length, and muscle stiffness would increase. Thus our hypothesis predicts that airway smooth muscle stiffness is an inverse function of end-tidal volume: as end-tidal volume increases, airway smooth muscle stiffness decreases.

During tidal breathing, airway muscle is subjected to cycles of stretch and retraction in which the contractile element shortens and lengthens as a function of the frequency and magnitude of the tidal volume cycle (25). Retraction and stretch of the muscle over a sufficient length range disrupt cross-bridge attachments, thereby reducing active force development. The amount of force reduction expected to result from load or length fluctuations can be predicted as a function of the kinetic constants of cross-bridge attachment and detachment (6). Thus dynamic mechanical oscillation of the muscle can by itself reduce its stiffness, force, and responsiveness (6, 8, 25). However, the effects of load or length oscillation on cross-bridge attachment do not account for the differences in muscle stiffness and extensibility caused by activating the muscle at different lengths. The parallel shifts in the force-reextension curves that we observed following the activation of airway muscle at different lengths under static conditions are consistent with a mechanism in which the dynamic modulation of muscle structure occurs independently of mechanical effects on cross-bridge kinetics. The cytoskeletal structure that is established in response to contractile activation of the muscle at different lengths may form a framework on which cross-bridge interactions are modulated during length or load fluctuations. Thus, in airway smooth muscle, both cross-bridge kinetics and the dynamic modulation of cytoskeletal structure are likely to interact to regulate muscle stiffness and force under dynamic conditions that occur during breathing.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-29289.


    FOOTNOTES

Address for reprint requests and other correspondence: S. J. Gunst, Dept. of Physiology and Biophysics, Indiana Univ. School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120 (E-mail: sgunst{at}iupui.edu).

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.

Received 25 August 2000; accepted in final form 11 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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