Mechanical strain memory in airway smooth muscle

Wah-Lun Chan, Jeanette Silberstein, and Chi-Ming Hai

Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effect of a single rapid stretch on poststretch force and myosin phosphorylation in bovine tracheal smooth muscle. When unstimulated muscle strips were stretched from suboptimal length to optimal length (Lo), poststretch steady-state force was not significantly different from that of unstretched control at Lo. However, when carbachol-activated muscle strips were stretched from suboptimal length to Lo, poststretch force and myosin phosphorylation were lower than control and significantly correlated with initial length. When poststretch muscle strips were allowed to relax for 1 h and then activated by K+ depolarization, the developed force remained significantly correlated with initial length. When the same strain was applied in 23 increments to minimize peak stress, poststretch force and myosin phosphorylation increased significantly, approaching the levels expected at Lo. Furthermore, poststretch force development increased after each cycle of contraction and relaxation, approaching the control level after four cycles. These results suggest that activated airway smooth muscle cells can retain relatively precise memory of past strain when they are stretched rapidly with high stress.

deep inspiration; muscle length; myosin phosphorylation; stretch


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY SMOOTH MUSCLE CELLS, in vivo, undergo cycles of stretching and shortening during inspiration and expiration. Recent studies have indicated the importance of periodic deep inspirations in maintaining brochodilation in normal subjects (22). Furthermore, prolonged withholding of deep inspiration induces asthmalike bronchoconstriction in normal subjects (29). These findings suggest that mechanical strain has a complex modulatory effect on airway smooth muscle contractility, but the underlying mechanism is unknown. Gunst (9) and Fredberg et al. (7) found that sinusoidal force oscillation of ACh-activated airway smooth muscle would lead to muscle lengthening if the amplitude of force oscillation was above a threshold that was equivalent to the force fluctuation during deep inspiration. Therefore, Fredberg and co-workers (5, 6) have proposed the conditional stability hypothesis which postulates that periodic mechanical stretching places actomyosin cross bridges in a mechanically perturbed state that differs from static equilibrium.

Phosphorylation of the 20,000-Da myosin light chain is the central regulatory mechanism of smooth muscle contraction (17). Mehta et al. (21) and Yoo et al. (32) found that carbachol-activated myosin phosphorylation was linearly dependent on muscle length in bovine and canine tracheal smooth muscle. These results indicate that airway smooth muscle activation is length dependent. However, length-dependent activation alone cannot explain the findings of Fredberg et al. (7) because their data suggest two distinct length-force relations during isometric contraction and length oscillations.

The sinusoidal length oscillation experiments as described by Fredberg et al. (7) are complex because both muscle shortening and stretching occur in sequence in these experiments. Gunst et al. (11) have found that, when a tonically activated smooth muscle is allowed to shorten to a new length and redevelop force, the stiffness-force relation was steeper than that during isometric contraction at the same length. Therefore, Gunst et al. (11) hypothesized that contractile and cytoskeletal structures in an activated smooth muscle cell are relatively fixed so that force development after a sudden length change is suboptimal for the new length. However, contractile and cytoskeletal structures in a resting smooth muscle cell are plastic and can reorganize to optimize force production at any muscle length. In a more recent study, Mehta et al. (21) found that rapid release of ACh-activated canine tracheal smooth muscle to a shorter length resulted in a lower force than that during isometric contraction at the same final length. However, the levels of myosin light-chain phosphorylation in smooth muscles with or without prior shortening were not significantly different. Shortening-induced force attenuation can potentially explain the findings of Fredberg et al. (7) because muscle shortening is one phase of strain oscillation.

Muscle stretching is the other phase of strain oscillation. The hypotheses proposed by Gunst et al. (11) and Fredberg et al. (6) would both predict that poststretch force should be lower than isometric force at the same final length. Mehta et al. (21) have stretched canine tracheal smooth muscle from 0.7 to 1.0 optimal length (Lo) at 1 min after activation by ACh. Contrary to the predictions of these two hypotheses, Mehta et al. (21) did not observe significant attenuation of force relative to muscle strips held isometrically at Lo. However, smooth muscle activation as measured by cross-bridge cycling rate and myosin phosphorylation is known to be time dependent and typically does not reach steady state at 1 min after activation. We hypothesize that the effect of applying mechanical stretch to a tonically activated smooth muscle should induce significant force attenuation. In this study, we tested this hypothesis by investigating the effect of applying a single stretch (from suboptimal length to optimal length) to tonically activated bovine tracheal smooth muscle on poststretch force and myosin light-chain phosphorylation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Bovine tracheas were collected from a slaughterhouse and transported to the laboratory in cold (4°C) physiological salt solution (PSS) containing (in mM) 140.1 NaCl, 4.7 KCl, 1.2 Na2HPO4, 2.0 MOPS (pH 7.4), 0.02 Na2EDTA, 1.2 MgSO4, 1.6 CaCl2, and 5.6 D-glucose. A segment of trachea consisting of multiple rings of cartilage was used for each experiment. The smooth muscle layer, together with the adventitia and mucosa, was removed by longitudinal cuts at the cartilage. The dissected piece was placed in a petri dish containing cold PSS. The mucosal and adventitial layers were then carefully removed using microdissecting scissors and fine forceps. Smooth muscle strips were prepared by making cuts along the circumferential direction. One end of the smooth muscle strip was held by a stainless steel clamp connected to a force transducer (Grass FT 03). The other end of the smooth muscle strip was held by a stainless steel clamp secured on a glass rod that was mounted on a length manipulator.

Muscle strips were first stretched to 12.5 g and then allowed to equilibrate for 1 h in PSS bubbled with air at 37°C. After the first hour of equilibration, muscle strips were activated briefly (3 min) by K-PSS, a solution similar to PSS in composition except 104.95 mM NaCl was substituted by KCl. Responsive muscle strips were then allowed to relax in PSS and equilibrate for another hour in PSS. During this second hour of equilibration, muscle strips were restretched to 12.5 g every 15 min. At the end of the second hour of equilibration, muscle strips were quickly released to a passive force of 2.5 g that was found to be associated with Lo for contraction. Muscle strips were then activated by K-PSS at Lo for 10 min, and the force (Fo) developed in this contraction was used to normalize force development in subsequent contractions. After this contraction, muscle strips were allowed to relax in PSS for 1 h before further experimentation. Muscle strips were ~15 mm in length, 5 mm in width, and 0.5 mm in thickness at Lo. We (16) have previously found that the passive force-to-active force ratio was ~0.1 in bovine tracheal smooth muscle activated by K+ depolarization. Preliminary experiments indicated that a passive force of 2.5 g was typical for optimal force development in the smooth muscle preparation used in this study. The Fo induced by K+ depolarization in the first control contraction was typically 20-30 g. The rationale for setting Lo by standardizing the passive force was to match the number and extent of stretch/release cycles in each muscle strip. Length-force relations obtained using this approach (32) have been found to be comparable to published results in the literature (12).

Single and multiple stretch experiments. After equilibration at Lo, muscle strips were released to the following suboptimal lengths: 0.2, 0.3, 0.45, 0.6, and 0.8Lo by adjusting the length manipulator (0.1-mm resolution). The extent of manipulator adjustment was calculated from the measured muscle length (in mm) at Lo and the assigned fraction of Lo. For example, if a muscle strip has a length of 18 mm at Lo, then to release this muscle strip from Lo to 0.6 Lo requires a manipulator adjustment of 0.4 × 18 mm, or 7.2 mm. For control, one muscle strip is always held isometrically at Lo throughout the experiment. After the release, muscle strips were activated by K-PSS for 15 min to induce shortening to the assigned lengths. The rationale was that when a muscle strip has shortened to the limit set by the length manipulator, the muscle strip then contracts isometrically and produces force. Muscle strips held at different suboptimal lengths were then allowed to relax in PSS for 1 h before activation by carbachol. These muscle strips were activated by 1 µM carbachol until steady-state force was reached (30 min) and then stretched rapidly (within 1 s) to Lo. Therefore, the poststretch final lengths of all muscle strips were identical at Lo. Muscle strips were then quickly frozen with an acetone-dry ice slurry (-78°C) at 1, 3, 5, and 10 min after the application of stretch depending on the experiment. For measuring prestretch force and myosin phosphorylation, some muscle strips were frozen at initial length immediately before the application of stretch. Data from these muscle strips were plotted at time 0 in some figures. Active force at each muscle length was calculated by subtracting passive force from total force. Passive force at Lo was determined at the beginning of each experiment when each muscle strip was quickly released to Lo. Passive force at suboptimal length was the lowest force reached after a rapid release from Lo to suboptimal length. The force shown in all figures represents total force minus passive force.

To determine whether carbachol-induced force changed with time and repeated contractions, some muscle strips were set at different initial lengths and then activated five times in the following contraction-relaxation cycles: 1 µM carbachol for 10 min, followed by 1-h relaxation in PSS. To determine whether poststretch force correlated with stretch size, some muscle strips were activated by 1 µM carbachol at an initial length of 0.4Lo for 30 min and then stretched to different final lengths ranging from 0.4Lo (unstretched control) to 1.0Lo. In multiple stretch experiments, carbachol-activated muscle strips were stretched from 0.3 to 1.0Lo in 23 equal increments. The average size of each length increment is ~0.03Lo. These experiments were designed to minimize peak stress during the application of rapid stretch.

Poststretch force recovery. In these experiments, carbachol-activated muscle strips were stretched rapidly from suboptimal length to Lo, remained activated for 30 min, and then were allowed to relax in PSS for 1 h. These muscle strips at the same final length (Lo) were then activated by K-PSS for 10 min and then allowed to relax in PSS for 1 h. This cycle of activation and relaxation was repeated up to four times.

Measurement of myosin light-chain phosphorylation. The frozen muscle strips in acetone/dry ice slurry were allowed to thaw slowly back to room temperature, resulting in the dehydration of the muscle strip. Acetone-dried tissues were homogenized in an aqueous solution containing 1% SDS, 10% glycerol, and 20 mM dithiothreitol on ice. The homogenate was then analyzed by two-dimensional PAGE, as described previously (16). We have found that acetone/dry ice slurry was as effective as 10% TCA/90% acetone/dry ice slurry in preserving myosin phosphorylation in muscle samples (16). Tissue homogenate was first analyzed by isoelectric focusing (Pharmalyte 4-6.5, Pharmacia) in the presence of 8 M urea to separate phosphorylated and unphosphorylated myosin light chains from each other. Sodium thioglycolate (5 mM) was included in the cathodal solution to minimize protein oxidation. After isoelectric focusing, the tube gel was transferred to a slab gel for SDS-PAGE to separate myosin light chains from other proteins by molecular weight. At the end of electrophoresis, the slab gel was stained by Coomassie blue and scanned in a densitometer equipped with an integrator (Helena). Unphosphorylated and phosphorylated myosin light chains appeared as two spots of different isoelectric pH but similar molecular weight. Myosin light-chain phosphorylation in mole phosphate per mole of light chain (mol Pi/mol LC) was calculated from the ratio of the amount of phosphorylated myosin light chain to the total amount of myosin light chains (sum of unphosphorylated and phosphorylated myosin light chains).

Statistics. Data are presented as means ± SE; n represents the number of tracheal rings. Student's t-test was used for the comparison of two means; P < 0.05 was considered significant. Correlation between two variables such as myosin phosphorylation and muscle length was analyzed by Pearson's correlation analysis; P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of a single stretch on force in unstimulated and carbachol-activated smooth muscle. As shown in Fig. 1A, when unstimulated muscle strips were stretched from 0.8 to 1.0Lo, force increased from 0.04 ± 0.04 Fo to a peak of 0.48 ± 0.20Fo, but then decreased with time to 0.18 ± 0.06Fo at 10 min after stretch. When muscle strips were stretched from 0.3 to 1.0Lo, force increased from 0.010 ± 0.002Fo to 1.17 ± 0.25Fo, but then decreased with time to 0.19 ± 0.08Fo at 10 min after stretch. For comparison, force in control muscle strips held isometrically at Lo was 0.10 ± 0.06Fo at time 0, and 0.10 ± 0.07Fo at 10 min. Therefore, poststretch force was higher than control at earlier times but became not significantly different from control in steady state (10 min).


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Fig. 1.   Effect of rapid stretch from 0.3 to 1.0 optimal length (Lo) (triangle ) or from 0.8 to 1.0Lo (open circle ) on force (Fo) in unstimulated (A) and carbachol-activated muscle strips (B). Control data () represent force in muscle strips held isometrically at Lo. A rapid stretch was applied at time 0. The lower force value at time 0 represents the force immediately before the application of stretch, whereas the higher value at time 0 represents poststretch peak force at ~1 s after stretch. Symbols and vertical bars represent means ± SE (n = 3-6). * Significant differences from control (P < 0.05).

As shown in Fig. 1B, when 1 µM carbachol-activated muscle strips were stretched from 0.8 to 1.0Lo, force increased from 0.84 ± 0.01Fo to a peak of 1.72 ± 0.28Fo but then decreased with time to 0.60 ± 0.08Fo at 10 min after stretch. When muscle strips were stretched from 0.3 to 1.0Lo, force increased from 0.096 ± 0.011Fo to 2.05 ± 0.10Fo but then decreased with time to 0.33 ± 0.05Fo at 10 min after stretch. For comparison, force in control muscle strips held isometrically at Lo was 1.15 ± 0.06Fo at time 0 and 1.17 ± 0.09Fo at 10 min. Therefore, poststretch peak force was higher than control, but poststretch steady-state force (at 10 min) was significantly lower than control. Active forces induced by 1 µM carbachol in control muscle strips were typically similar to Fo. Most of the small differences from Fo shown in Fig. 1B and other figures were not statistically significant.

When additional experiments were performed using various initial lengths, we found that poststretch steady-state force correlated significantly with initial length in carbachol-activated but not in unstimulated smooth muscle (Fig. 2).


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Fig. 2.   Dependence of poststretch steady-state force on initial muscle length in unstimulated () and 1 µM carbachol-activated (open circle ) muscle strips. Symbols and vertical bars represent means ± SE (n = 3). Solid and dashed lines represent linear and nonlinear regression fits to data, respectively. P < 0.05 indicates significant correlation, whereas ns indicates insignificant correlation between poststretch force and initial muscle length.

Effect of a single stretch on myosin phosphorylation in carbachol-activated smooth muscle. As shown in Fig. 3A, when 1 µM carbachol-activated muscle strips were stretched from 0.45 to 1.0Lo, myosin phosphorylation did not change significantly with time. Myosin phosphorylation was 0.32 ± 0.05 mol Pi/mol LC before stretch and was 0.27 ± 0.05 mol Pi/mol LC at 10 min after stretch. When muscle strips were stretched from 0.3 to 1.0Lo, myosin phosphorylation also did not change significantly with time. Myosin phosphorylation was 0.13 ± 0.02 mol Pi/mol LC before stretch and was 0.12 ± 0.02 mol Pi/mol LC at 10 min after stretch. For comparison, myosin phosphorylation in control muscle strips held isometrically at Lo was 0.48 ± 0.05 mol Pi/mol LC at time 0 and 0.55 ± 0.04 mol Pi/mol LC at 10 min. Therefore, poststretch myosin phosphorylation remained significantly lower than control at almost all measured time points. Because final lengths in all experimental groups were identical at Lo, these results indicate that poststretch myosin phosphorylation in carbachol-activated muscle strips is dependent on strain history. When additional experiments were performed using various initial lengths, we found that poststretch steady-state myosin phosphorylation correlated significantly with initial length in carbachol-activated smooth muscle (Fig. 3B).


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Fig. 3.   A: effect of rapid stretch from 0.3 to 1.0Lo (triangle ) or from 0.45 to 1.0Lo (open circle ) on myosin phosphorylation in carbachol-activated muscle strips. Control data () represent myosin phosphorylation in muscle strips held isometrically at Lo. Muscle strips were activated by 1 µM carbachol for 30 min before the application of stretch. Data points at time 0 represent myosin phosphorylation levels immediately before the application of stretch. * Significant differences from control (P < 0.05). B: dependence of poststretch steady-state myosin phosphorylation on initial muscle length in carbachol-activated muscle strips. In these experiments, myosin phosphorylation was measured at 10 min after the application of stretch. Symbols and vertical bars represent means ± SE (n = 5-7). Solid line in B represents linear regression fit to data, and P < 0.05 indicates significant correlation between poststretch myosin phosphorylation and initial muscle length.

In this study, basal myosin phosphorylation in unstimulated tissues was 0.17 ± 0.02 mol Pi/mol LC at Lo. As shown in Fig. 3B, myosin phosphorylation in carbachol-activated tissues held at Lo was significantly higher than basal myosin phosphorylation at Lo. However, poststretch carbachol-induced myosin phosphorylation levels at very short initial lengths were not significantly different from the basal myosin phosphorylation at Lo (Fig. 3B). Yoo et al. (32) have found that both basal and carbachol-induced myosin phosphorylation decreased to very low levels at very short muscle lengths. Suprabasal myosin phosphorylation was statistically significant at Lo but became insignificant at 0.1Lo (32). Therefore, the very low level of myosin phosphorylation at very short initial lengths observed in this study were consistent with the findings of Yoo et al. (32).

K+ depolarization-induced force development in previously stretched smooth muscle. In these experiments, poststretch unstimulated or carbachol-activated muscle strips were allowed to relax in PSS for 1 h and then activated by K+ depolarization. As shown in Fig. 4A, muscle strips previously stretched from 0.8 to 1.0Lo in the unstimulated state (PSS) developed an average force of 1.09 ± 0.02Fo in response to 10 min of K+ depolarization. Muscle strips previously stretched from 0.3 to 1.0Lo developed an average force of 0.73 ± 0.15Fo in response to 10 min of K+ depolarization. For comparison, muscle strips held isometrically at Lo developed an average force of 1.21 ± 0.15Fo. These values of average force were not significantly different.


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Fig. 4.   K+ depolarization-induced contractions in muscle strips previously stretched from 0.3 to 1.0Lo (triangle ) or from 0.8 to 1.0Lo (open circle ) in unstimulated state (PSS) (A) and activated state (1 µM carbachol) (B). Muscle strips were previously stretched from suboptimal length to optimal length (Lo), were allowed to relax in PSS for 1 h, and were then activated by K+ depolarization. Symbols and vertical bars represent means ± SE (n = 3). * Significant differences from control (P < 0.05).

As shown in Fig. 4B, muscle strips previously stretched from 0.8 to 1.0Lo in the carbachol-activated state developed an average force of 0.84 ± 0.08Fo in response to 10 min of K+ depolarization. Muscle strips previously stretched from 0.3 to 1.0Lo developed an average force of 0.26 ± 0.05Fo in response to 10 min of K+ depolarization. For comparison, control muscle strips held isometrically at Lo developed an average force of 0.91 ± 0.05Fo. The average force developed by the "0.8 to 1.0Lo" group was not significantly different from control. However, the average force developed by the "0.3 to 1.0Lo" group was significantly lower than control.

When additional experiments were performed using various initial lengths, we found that poststretch force development induced by K+ depolarization correlated significantly with initial length (Fig. 5). In muscle strips previously stretched in the relaxed state (Fig. 5, solid circles, PSS), the maximum force deficit between stretched and unstretched muscle strips (1.0Lo) was 31%. In contrast, in muscle strips previously stretched in the activated state (Fig. 5, open circles, 1 µM carbachol), the maximum force deficit between stretched and unstretched muscle strips (1.0Lo) was 71%.


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Fig. 5.   Dependence of K+ depolarization-induced steady-state force on initial muscle length in muscle strips previously stretched in unstimulated state (PSS) and activated state (1 µM carbachol). In these experiments, muscle strips were previously stretched from different initial muscle lengths to optimal length (Lo), were allowed to relax in PSS for 1 h, and were then activated by K+ depolarization for 10 min. Symbols and vertical bars represent means ± SE (n = 3). Lines represent nonlinear regression fits to data. P < 0.05 indicates significant correlation between force and initial muscle length.

Length dependence of carbachol-induced force in repeated contractions. To determine whether carbachol-induced force at a given initial length changed with time and repeated contractions, multiple muscle strips were set at different initial lengths and then activated five times in the following contraction-relaxation cycles: 1 µM carbachol for 10 min, followed by 1-h relaxation in PSS. As shown in Fig. 6A, carbachol-induced forces remained length dependent throughout the five cycles of contractions in 5 h. As shown in Fig. 6B, when the forces developed at 0.4 and 0.6Lo were normalized by the force developed at Lo for each cycle of contraction, the fractional force did not change significantly throughout the five consecutive contractions. The results shown in Fig. 6 indicate that the length dependence of carbachol-induced force did not change significantly with time and repeated contractions. Furthermore, comparison between Figs. 2 and 6 indicate that forces developed by poststretched and unstretched tissues at the same initial length were similar. Therefore, these results support the conclusion that poststretch force correlated with initial length.


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Fig. 6.   Length dependence of carbachol-induced force in repeated contractions. In these experiments, multiple muscle strips were set at 0.4Lo (triangle ), 0.6Lo (open circle ), and 1.0Lo () and then activated 5 times in the following contraction-relaxation cycles: 1 µM carbachol for 10 min followed by 1-h relaxation in PSS. In A, carbachol-induced force is expressed as fraction of Fo. In B, carbachol-induced force is expressed as a fraction of the force developed at 1.0Lo. Symbols and vertical bars represent means ± SE (n = 5). * Significant differences from the value at 1.0Lo (P < 0.05).

Stretching from the same initial length to different final lengths. In the experiments shown in Figs. 1 and 2, muscle strips having the shortest initial length also experienced the largest stretch size when stretched to the same final length (Lo). Therefore, an alternative hypothesis was that the lower poststretch force at shorter initial length could be due to force deficit induced by a larger stretch step. This "force deficit" hypothesis could be tested by stretching carbachol-activated muscle strips from the same initial length to different final lengths. Because stretch step is proportional to final length in these experiments, the force deficit hypothesis predicts that poststretch force should decrease with final muscle length. In contrast, the mechanical memory hypothesis predicts that poststretch force should be independent of final length. Muscle strips were activated by 1 µM carbachol at an initial length of 0.4Lo, for 30 min, and then stretched to different final lengths ranging from 0.4Lo (unstretched control) to 1.0Lo. As shown in Fig. 7A, poststretch force increased with final length. However, after the stretched muscle strips were allowed to relax in PSS, K+ depolarization-induced force was independent of final length (Fig. 7B). The data shown in Fig. 7, A and B, were inconsistent with the force deficit hypothesis which predicted that poststretch force should decrease with final length. The data shown in Fig. 7A were also inconsistent with the mechanical memory hypothesis, whereas the data shown in Fig. 7B were consistent with the mechanical memory hypothesis which predicted that poststretch force should be independent of final length. Therefore, the mechanical memory hypothesis appears to be valid for poststretch steady state, but the cellular mechanisms leading to the steady state may be a time-dependent process. It is noteworthy that the force-deficit hypothesis does not take into account the potential independent effects of final muscle length per se on force. Even if stretch causes a force deficit that is proportional to the amount of stretch, the deficit may not be sufficient to result in a decrease in force with stretch to longer lengths. It is unlikely that a single mechanism determines the length dependence of muscle force, and contributions of other mechanisms may account for the inconclusiveness of the data.


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Fig. 7.   A: dependence of poststretch force on final muscle length when carbachol-activated muscle strips were stretched from the same initial length (0.4Lo) to different final length. B: dependence of K+ depolarization-induced force on final length in muscle strips previously stretched from 0.4Lo to different final lengths. Symbols and vertical bars represent means ± SE (n = 4). Solid and dashed lines represent linear fits to data, respectively. P < 0.05 indicates significant correlation, whereas ns indicates insignificant correlation between force and final muscle length.

Effect of multiple stretches of small increments on force and myosin phosphorylation in carbachol-activated smooth muscle. In these experiments, 1 µM carbachol-activated muscle strips were stretched from an initial length of 0.3Lo to a final length of 1.0Lo either in a single stretch or in 23 stretches of equal increments. The average size of each increment was therefore 0.03Lo in the multiple stretch experiments. As shown in Fig. 8A, peak force during a single stretch (Fig. 8A, open triangles) was higher than isometric force generated by carbachol-activated muscle strips held at Lo (Fig. 8A, solid circles). In contrast, peak forces during multiple stretches (Fig. 8A, open circles) were always lower than isometric force generated by carbachol-activated muscle strips held at Lo (Fig. 8A, solid circles). As shown in Fig. 8B, poststretch force in the "single stretch" group (0.27 ± 0.05Fo) was significantly lower than that in the "multiple stretch" group (0.67 ± 0.04Fo; Fig. 8B, open bars). However, these values remained significantly lower than the force developed by unstretched muscle strips (1.03 ± 0.07Fo). As shown in Fig. 8B (solid bars), myosin phosphorylation in the multiple stretch group (0.32 ± 0.03 mol Pi/mol LC) was significantly higher than that in the single stretch group (0.12 ± 0.02 mol Pi/mol LC). However, these values remained significantly lower than the myosin phosphorylation developed by unstretched muscle strips (0.55 ± 0.04 mol Pi/mol LC).


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Fig. 8.   A: effect of stretching a muscle strip from 0.3 to 1.0Lo on force when the stretch was applied in a single step (triangle ) or 23 steps (open circle ). Control data () represent force in unstretched muscle strips held isometrically at Lo. B: poststretch steady-state force (open bars) and myosin phosphorylation (solid bars) in muscle strips that were held isometrically at Lo (control), stretched from 0.3 to 1.0Lo in a single step (single stretch) or 23 steps (multiple stretch). Bars represent means ± SE (n = 6 or 7). * Significant difference from control. # Significant difference between the single stretch and multiple stretch groups of data. C: poststretch force development induced by K+ depolarization. Muscle strips from the experiments shown in B were allowed to relax for 1 h in PSS and were then activated by K+ depolarization. Symbols and vertical bars represent means ± SE (n = 3). * Significant differences from control (P < 0.05).

Muscle strips from the single and multiple stretch groups of muscle strips were allowed to relax in PSS for 1 h and then activated by K+ depolarization. As shown in Fig. 8C, K+ depolarization-induced force in the single stretch group was 0.26 ± 0.05Fo, which was significantly lower than that in the multiple stretch group (0.75 ± 0.02Fo). However, these values remained significantly lower than control (0.91 ± 0.05Fo).

Poststretch force redevelopment. In these experiments, 1 µM carbachol-activated muscle strips were stretched rapidly from 0.2Lo to Lo, were allowed to relax in PSS for 1 h, and were then activated by K+ depolarization (contraction 1). After 10 min of contraction, these muscle strips were allowed to relax in PSS for 1 h and were then activated again by K+ depolarization (contraction 2). These cycles were then repeated for two more times to measure poststretch force development. A control muscle strip was held isometrically at Lo and activated by 1 µM carbachol and K+ depolarization at the same time as the stretched muscle strips. As shown in Fig. 9, average force developed by stretched muscle strips was only 45 ± 2% of control (developed by unstretched muscle strips) in the first poststretch contraction. However, average force developed by the stretched muscle strips increased to 85 ± 4% of control by the fourth contraction.


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Fig. 9.   Poststretch force redevelopment induced by K+ depolarization in previously stretched muscle strips. Two muscle strips were activated by 1 µM carbachol for 30 min at 0.2Lo and then rapidly stretched to Lo. Poststretch muscle strips were held isometrically at Lo, activated for another 10 min by 1 µM carbachol, then allowed to relax in PSS for 1 h. These muscle strips were then activated by K+ depolarization for 10 min followed by 1-h relaxation in PSS. This cycle of activation and relaxation was repeated 4 times. A control muscle strip held isometrically at Lo was activated by K+ depolarization together with the stretched muscle strips. Forces developed by stretched muscle strips were expressed as a fraction of the force developed by the unstretched muscle strip at the matched cycle of contraction. Bars represent mean and range of the forces developed by the 2 muscle strips.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airway smooth muscle cells, in vivo, function in a chemically and mechanically active environment because mechanical stretches and releases are normal phases of lung ventilation. In this study, we found that, when tonically activated airway smooth muscle was stretched rapidly with high stress, they exhibited memory-like behavior. Specifically, when mechanical stretch was applied to an activated muscle, poststretch force and myosin phosphorylation remained correlated with initial length but not final length (Figs. 1-3). Subsequent force development induced by K+ depolarization also remained correlated with initial length but not final length (Fig. 4). Therefore, mechanical strain memory could be observed in both receptor-activated and K+-depolarized muscle strips, suggesting that mechanical strain memory was stored in contractile and cytoskeletal structures. These results are consistent with the hypothesis proposed by Gunst et al. (11) that contractile and cytoskeletal structures are relatively fixed in activated airway smooth muscle. Therefore, when a mechanical stretch is applied rapidly, contractile unit length becomes suboptimal for the new length. The novel finding of this study is that poststretch force development remains correlated with initial length.

Mehta et al. (21) have studied the effect of rapid stretching from 0.7 to 1.0Lo on ACh-activated canine tracheal smooth muscle. They found that poststretch myosin phosphorylation and force both redeveloped to the levels expected at Lo. This finding was different from the results from this study. We found that the force developed by a carbachol-activated muscle strip after being stretched from 0.7 to 1.0Lo was only 74% of the level expected at Lo (Fig. 5). It is possible that the relatively small force deficit (26%) may not be resolved in their study. Alternatively, the effect of stretch may be dependent on the time of application relative to the time course of contraction. Mehta et al. (21) applied mechanical stretch at 1 min after the beginning of a contraction. In contrast, we applied mechanical stretch at 30 min after the beginning of a contraction in this study. It is known that smooth muscle activation as measured by cross-bridge cycling rate and myosin phosphorylation is time dependent (15). Furthermore, Gunst et al. (11) have observed time-dependent changes in the force-stiffness relation in ACh-activated canine tracheal smooth muscle and suggested time-dependent cytoskeletal reorganization during airway smooth muscle contraction. These results suggest that mechanical strain memory may be related to the time-dependent cytoskeletal reorganization during smooth muscle activation. Stretch rate has been found to be an important determinant of poststretch force and myosin phosphorylation in this study. Mehta et al. (21) applied a stretch over a period of 15 s instead of the 1 s used in this study. Therefore, the difference in stretch rate could also account for the difference in results found in this study and that by Mehta et al. (21).

Fredberg et al. (6) have hypothesized that ACh-activated airway smooth muscle may exhibit different muscle lengths during static equilibrium and during force oscillations. Similarly, we found that a carbachol-activated airway smooth muscle at the same length (Lo) could develop different forces depending on its past strain history (Figs. 2 and 5). Gunst and co-workers (10, 13) and Sasaki and Hoppin (28) have previously observed that activated smooth muscle exhibited significant hysteresis during cyclical length changes. These results together suggest that multiple length-force relations may exist in airway smooth muscle depending on strain history. The new finding of this study is that a single rapid stretch applied to a tonically activated airway smooth muscle is sufficient to shift the apparent length-force relation toward longer lengths.

Pratusevich et al. (27) measured shortening velocity, compliance, and isometric force development in electrically stimulated airway smooth muscle at initial length, 75% initial length, and 150% initial length. They found that shortening velocity and compliance exhibited strong dependencies on muscle length. In contrast, isometric force changed only initially when muscle length was changed, but eventually redeveloped to similar levels independent of muscle length. Based on these observations, Pratusevich et al. (27) proposed the plasticity hypothesis that the number of contractile units in series changes with muscle length to maintain a relatively constant force independent of muscle length. In their study, muscle stretches and releases were applied sequentially on the same muscle strip to change muscle length. Therefore, strain history was a significant factor in their study, and the observed length independence of force could reflect strain history-dependent shifting of length-force relations.

Myosin phosphorylation is an important determinant of cross-bridge cycling rate and therefore shortening velocity in smooth muscle (15). Mehta et al. (21) and Yoo et al. (32) have found that muscarinic receptor-mediated myosin phosphorylation increased linearly with muscle length. Therefore, the length dependence of shortening velocity observed in some studies could be explained by the length dependence of myosin phosphorylation. Gunst et al. (11) found that both stiffness and force increased during force development in canine tracheal smooth muscle. Gunst et al. (11) also measured stiffness and force at different muscle lengths ranging from 0.3 to 1.0Lo. They found that both stiffness and force increased with muscle length, although the stiffness-force relation was not linear. These results demonstrated the dependence of stiffness on muscle length and suggested that a major portion of stiffness was contributed by attached cross bridges. However, the nonlinearity of stiffness-force relation suggests that a significant portion of smooth muscle stiffness could be contributed by the cytoskeleton.

The observed dependence of myosin phosphorylation on initial length was surprising. The underlying mechanism is unknown. The sensitivity of myosin phosphorylation to actin filament disruption by cytochalasin D suggests that cytoskeletal structure is an important determinant of the ratio of myosin light-chain kinase to phosphatase in smooth muscle cells (30). If a rapid stretch alters the cytoskeletal structure in poststretch smooth muscle cells, then myosin light-chain kinase-to-phosphatase ratio may also be altered in poststretch smooth muscle cells. Clearly, this hypothesized connection between cytoskeletal structure and myosin light-chain kinase-to-phosphatase ratio remains to be elucidated at the molecular level.

This study was focused on muscle lengths below or at Lo because most muscle cells appear to function on the ascending limb of the length-force relation in vivo. Hai (14) has previously measured K+ depolarization-induced myosin phosphorylation at slack length, Lo, and 1.5Lo in swine carotid media. Whereas myosin phosphorylation was lower at slack length than at Lo, myosin phosphorylation at 1.5Lo was not significantly different from that at Lo. These results indicate that the length dependence of myosin phosphorylation becomes nonlinear at muscle lengths longer than Lo. Therefore, it is difficult to extrapolate from the findings from this study to muscle lengths longer than Lo.

Applying mechanical stretch to an activated skeletal muscle is known to cause cell injury and force deficit. Lynch and Faulkner (19) have studied the effect of applying a single stretch to single muscle fibers of maximally activated extensor digitorum longus muscle of the rat. They found that stretch-induced force deficit was dependent on strain amplitude but independent of the velocity of stretch. In contrast, we found that the same strain (from 0.3 to 1.0Lo) applied in a single and 23 steps resulted in significantly different poststretch force and myosin phosphorylation (Fig. 8). After stretch-induced injury in skeletal muscle, a relatively long time (days) is typically required for force recovery (18). In contrast, we observed substantial force recovery after only four cycles of contractions and relaxation within 5 h after a rapid stretch (Fig. 9). Smooth and skeletal muscle cells are also different in the organization and stability of their contractile and cytoskeletal filaments. Whereas actin filaments are stable in skeletal muscle cells, actin filaments are dynamic in smooth muscle cells (24, 30, 31). Whereas actin and myosin filaments are arranged along the longitudinal axis of a skeletal muscle cell, actin and myosin filaments are arranged in a three-dimensional meshwork in a smooth muscle cell (3). The three-dimensional meshwork of contractile and cytoskeletal filaments in smooth muscle is expected to provide greater flexibility in cytoskeletal reorganization in response to mechanical strain. Finally, mechanical strain oscillation is a physiological process in airway smooth muscle but not in skeletal muscle. Therefore, we think that paradigms derived from skeletal muscle studies may not be directly applicable to smooth muscle. Mechanical strain memory may be a physiological property that is specific to smooth muscle.

Recent studies suggest that dense plaques and actin filaments are dynamic and can assemble and disassemble in response to muscle length (11, 20, 30). Gunst and co-workers (10, 11, 13) have studied shortening-induced inactivation of force in airway smooth muscle. They proposed that actin filaments deriving from contractile units could anchor to different dense plaques in response to mechanical strain. In the study by Gunst et al. (11), the applied external stress was lower than the isometric stress developed by smooth muscle to induce muscle shortening. In this study, the applied external stress was higher than the isometric stress developed by the smooth muscle to induce muscle stretching. The membrane cytoskeleton is positioned at the interface between intracellular actin filaments and extracellular matrix. It is conceivable that the difference between external and internal stresses may deform the cytoskeleton differently and thereby initiate different types of cytoskeletal reorganization.

A common conclusion reached by Gunst et al. (11), Pratusevich et al. (27), and this study is that length-force relation could change in response to mechanical strain/stress in airway smooth muscle. However, the proposed mechanisms in these studies are different. Repositioning of actin filament-anchorage to dense plaques and dynamic actin filament remodeling have been proposed by Gunst et al. (11). Regulating the number of contractile units in series has been proposed by Pratusevich et al. (27). The hypothesis proposed by Gunst et al. (11) can potentially explain the results of this study. The hypothesis proposed by Pratusevich et al. (27) predicted that the number of contractile units in series should change in adaptation to different muscle lengths, resulting in a relatively constant contractile force independent of muscle length. Therefore, this hypothesis proposed by Pratusevich et al. (27) does not appear to predict the different forces as a function of initial length observed in this study.

The general hypothesis proposed by Gunst and co-workers (11, 20) suggests that the formation of dense plaques and/or dense bodies may be more dynamic than previously recognized, and contractile activation may trigger their assembly. It is noteworthy that alpha -actinin cross-links actin filaments, binds to integrin (25), and has been found in both dense plaques and cytosolic dense bodies (4, 23, 26). Dynamic assembly and disassembly of focal adhesions in nonmuscle cells has been well documented (1, 2). Dynamic assembly of dense plaques and bodies, perhaps involving actin cross-linking proteins, may be an important property of smooth muscle cells that allows the cells to adapt to a wide range of organ dimensions.

The experimental application of a rapid and sustained strain in this study is different from deep inspiration, in vivo, because the external load on airway smooth muscle is allowed to return to baseline after deep inspiration. However, it may be interesting to consider the implications of the findings from this study in relation to deep inspiration. Results from this study indicated that poststretch force was lower than what was expected at the final muscle length. Therefore, these results would predict that poststretch muscle length should be longer than what was expected at the same external load without prior stretch. Based on this analysis, we think that results from this study are basically consistent with the phenomenon of deep inspiration-induced bronchodilation and suggest that cytoskeletal reorganization could be an important mechanism. In future studies, it would be interesting to simulate the maneuver of deep inspiration in airway smooth muscle in vitro.


    ACKNOWLEDGEMENTS

Bovine tracheas were generously donated by Baker's Farm (Swansea, MA).


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-52714.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C.-M. Hai, Div. of Biology and Medicine, Brown University, Box G-B3, Providence, RI 02912 (E-mail: Chi-Ming_Hai{at}brown.edu).

Received 28 June 1999; accepted in final form 18 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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