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HIGHLIGHTED TOPICS
Airway Hyperresponsiveness: From Molecules to Bedside
SELECTED CONTRIBUTION
1Departments of Medicine and 2Department of Pathology and Laboratory Medicine, University of British Columbia, V6T 1Z3; and 3The McDonald Research Laboratories, The iCAPTURE Center, St. Pa's Hospital, Providence Health Care, Vancouver, British Columbia, Canada V6Z 1Y6
Submitted 20 December 2002 ; accepted in final form 24 February 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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muscle mechanics; length-tension curve; muscle plasticity
Knowing that ASM in vitro is able to adapt to length changes by restructuring its contractile apparatus (13) and regain its ability to generate maximal force (17, 18), the consequence of prolonged shortening of ASM in vivo, although it has not yet been demonstrated, could be excessive airway narrowing. The importance of the dynamic L-T relationship in ASM is further investigated in the present study under conditions closer to that in vivo, with an emphasis on long-term length adaptation at body temperature and reversibility of the functional change that accompanies length adaptation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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2.2–2.5 mm wide, 2.0–3.5 mm long, 0.1–0.2 mm thick, with two cartilage bits attached at each end. Length of the muscle between the insertion points with the cartilage was measured in the intact rings and was designated as the in situ length (Lin situ). For day 3, day 7, and day 8 preparations, each pair was dissected from adjacent tracheal rings with comparable Lin situ. Dissection was performed with the tissues submerged in physiological saline solution (PSS; composition in mM: 118 NaCl, 5 KCl, 1.2 NaH2PO4, 22.5 NaHCO3, 2 MgSO4, 2 CaCl2, and 11.1 glucose).
The L-T curve of the CTL0 preparations was determined on day 0 immediately after finishing the dissection. The rest of the preparations were placed on sterilized wax. Under a dissecting microscope, CTL preparations (CTL3, CTL7, and CTL8) were set at their Lin situ. PS preparations (PS3, PS7, and PS8) were stimulated for 3–5 s between two platinum electrodes of an alternating-current stimulator (60-Hz frequency); after the single tetanus, the preparations were set at the shortened lengths. On average, PS preparations shortened to 27.6 ± 3.2% (SD) of their respective controls during stimulation under zero-load. PS preparations then relaxed, on their own, toward their initial lengths after removal of stimulation. The final relaxed lengths of the PS preparations were 50–60% of their Lin situ.
Tissue culturing. The preparations were placed in individual wells of a culture plate where they were washed three times with 4°C Connaught Medical Research Labs (CMRL) medium that contained penicillin (10,000 U/ml) and streptomycin (10,000 µg/ml). ASM preparations were then covered with 8 ml of 4°C CMRL medium enriched with 5% FBS, bovine insulin (4 mg/ml), L-glutamine (10 µl/ml), amphotericin B (250 µg/ml). All the supplements were purchased from GIBCO BRL.
Culture plates were then transferred to a controlled atmosphere gas chamber (BellCo Glass). The chamber was immediately filled with a gas mixture of 4.85% CO2, 44.5% O2, and balanced nitrogen, placed on a rocker platform, and agitated at 10 cycles/min at 37°C. The preparations were kept under these conditions for 3, 7, or 8 days.
Mechanical measurements. The preparations were mounted horizontally in a tissue-bath that contained PSS at 37°C and was bubbled with a 95% O2-5% CO2 mixture. A reference length of the muscle preparation (Lref) was determined during the equilibration period by stretching the muscle until passive tension was first detected [1–2% of maximal isometric force (Fmax)]. A high-K+ PSS (80 mM K+) was used to induce smooth muscle contraction in all experimental protocols, except in setting the initial PS lengths for PS preparations, where electrical stimulation was used. The average time to reach the plateau or Fmax was between 450 and 800 s. This time was nearly constant for all individual ASM strips during repetitive contractions.
Determination of L-T relationship and time course of tension recovery. To obtain the L-T curve, the muscle preparations after being equilibrated at Lref (1–1.5 h) were stretched incrementally by 8% of their own Lin situ (referred to as the step size) after each contraction. To allow the passive force to stabilize, the muscle preparations were stretched 60 s before the next contraction. Both passive and maximal active forces were measured during each contraction. The stretching of CTL preparations was carried out incrementally until the active force passed its peak value by at least two further length increments (Fig. 1A). After completion of measurements, the CTL preparations were fixed in 10% formalin at the length that was associated with Fmax generation (Lmax). The same protocol was applied to the PS preparations before they were subjected to additional measurements. In these additional measurements, after passing the Lmax by one increment of step size and 60 s before the next contraction, the PS preparation was stretched to reach either the Lmax of CTL preparation (CTL Lmax) or until the passive force exceeded the linear range of the force transducer (20 mN). The high passive force (>20 mN) associated with a large stretch was observed to cause permanent reduction in isometric force of the muscle, even after the muscle had been released to a shorter length. The large stretch associated with the high force was therefore avoided during the experiment. The PS preparation was then stimulated periodically (i.e., maximal contraction followed by complete relaxation) at the new length (Fig. 1, A and C). The tension recovery was continued until the active force reached a steady state. The rates of active and passive tension recoveries were obtained by fitting the time course of tension change with exponential functions (Fig. 1, C and D).
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Morphometric measurements. These were performed to determine the cross-sectional areas of the muscle preparations. All the preparations were embedded in paraffin and sectioned with H&E staining. Digital images were obtained, and Image Pro-Plus 3.0 software was used to measure the total cross-sectional area as well as smooth muscle area of the preparations. Smooth muscle area values were then used individually to normalize the measured Fmax.
Data analysis. The active and passive forces as functions of muscle length were plotted to obtain the L-T curves for both the CTL and the PS groups. A Weibull 4-parameter equation of peak function (Sigma Plot 5.0) was used to fit the active force data and to measure the Fmax and Lmax. Paired t-tests were used to compare the values between CTL and PS preparations. Smooth muscle area was measured and used to normalize the Fmax values and calculate the maximal stress (in kPa). The values of maximal stress of CTL and PS groups were also compared with the paired t-test. To show how stretchable the preparations were after 3 and 7 (or 8) days of passive shortening, the L/(CTL Lmax) (%) index was used, where L was the final length of the PS muscle being stretched. This index indicates the length of PS preparations as a percentage of CTL Lmax at which the rate and extent of tension recovery were measured. The poststretch maximal active force and the corresponding passive force were compared with the maximal prestretch active force (i.e., Fmax) and the corresponding passive force (i.e., passive force at Lmax) of PS preparations, respectively. A single three-parameter exponential rise-to-maximum function [F = y0 + a (1 - e-bt), where b represents the rate of recovery, and y0 and a are constants] was used because it provided the best fit for the active force recovery. A single three-parameter exponential decay function (F = y0 + ae-bt) was used to fit the passive force recovery (relaxation). With the use of a paired t-test, the active and passive force recovery rates of days 3 and 7 (or 8) PS preparations were compared with those of freshly dissected muscle preparations. To examine the effect of periodic stimulation on passive force recovery, a comparison was also made between the passive force recovery rates obtained in the presence and absence of periodic stimulation. For all statistical tests, P > 0.05 was not considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Tension recovery measurement. The mean and standard error of L/(CTL Lmax) (%) for day 3 (n = 6) and day 7/8 (n = 14) of PS preparations were 99.9 ± 1.2 and 87.2 ± 3.7%, respectively (Table 2). A t-test (P < 0.05) showed a significant difference between the two groups. It was observed that on day 3, after the PS preparations were stretched to the predetermined length (i.e., the CTL Lmax), the active force of the first contraction was very close to the maximal adapted (recovered) active force. This could be due to the closeness of Lmax values of CTL and PS preparations (and hence a small stretch required). As shown in Table 2, on day 3, the poststretch maximal adapted active force reached the same level as the prestretch active force. As in active force, recovery in passive force after 3 days of passive shortening was also complete. Unlike the behavior observed on day 3, it was found that in most cases the PS preparations on day 7/8 were either too stiff to be stretched to their CTL Lmax and that at the stretched length the active force could not recover to the prestretch value (Table 2). The rates of passive force recovery between day 3 and day 7/8 PS preparations (after stretch) were compared. The rates were not different (P = 0.381).
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Force recovery in the presence and absence of stimulation. The initial passive forces at the beginning of the recovery measurement (Fig. 2) were not different between the group subjected to periodic stimulation and the group that was not stimulated (P = 0.25, n = 8). The rate of passive force recovery (b value) was 0.063 ± 0.007 min-1 for the group with stimulation and 0.074 ± 0.014 min-1 for the group without stimulation. These rates were not significantly different. In Fig. 3A, the active force was measured periodically after a stretch of the muscle (with a protocol similar to that described for Fig. 1C). Results from eight experiments with muscle preparations from eight animals were combined. The equation used for curve fitting was the same as that described for Fig. 1C. In Fig. 3B, the muscle was not stimulated for 2 h after the stretch, but at the end of the "resting" period, the muscle was stimulated periodically as that in Fig. 3A, and the recovery of active force was plotted. The starting level of active force for the group shown in Fig. 3B was 93.4 ± 1.4% of the maximum value. In contrast, this average starting value was achieved in the group shown in Fig. 3A by the fourth contraction.
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Freshly dissected vs. chronically adapted ASM. As shown in Table 3, the rate of active force recovery was 0.081 ± 0.008 min-1 for the PS group on day 7/8 during readaptation to longer lengths and 0.176 ± 0.029 min-1 for the freshly dissected smooth muscle preparations during readaptation to longer lengths. The difference in the rates was statistically significant (P < 0.05). The passive force recovery rates were 0.068 ± 0.008 and 0.046 ± 0.006 min-1 for PS7/8 and the freshly prepared smooth muscle preparations, respectively (Table 3). The difference in the rates was not significant (P > 0.05). The passive force at the beginning of the force measurements was 13.42 ± 1.58 mN for day 7/8 and 13.82 ± 1.26 mN for freshly prepared ASM. There was no significant difference between them. In the first poststretch contraction, the active force was only 11.6 ± 6.1% of the prestretch value for the group of day 7/8. However, with the use of the same stretching protocol, the active force was significantly greater (39.2 ± 5.3% of the prestretch value) for the freshly dissected ASM preparations. The ability to generate force in fresh preparations, therefore, was less affected by the length change compared with the chronically shortened preparations.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Possible mechanism producing shifts in the L-T relationship. The plastic behavior of ASM has been attributed to a restructuring of the muscle's contractile apparatus (13) and cytoskeleton (7, 8), and, it is likely, the underlying mechanism for the observed shifts in L-T relationship. The initiator of the plastic restructuring is often a large change in muscle cell length that cannot be accommodated by sliding of the contractile filaments. The length-perturbation-induced loss of contractile force has been shown to be associated with a decrease in the myosin thick-filament density (10). Previous studies (14, 17) suggest that periodic stimulation accelerates the process of force recovery and also that phosphorylation of the myosin regulatory light chain facilitates formation of myosin thick filaments.
Consequence of a left shift in L-T relationship. When an airway narrows due to contraction of the encircling smooth muscle cells, tension generated in the muscle normally decreases, following the L-T curve, as the muscle cell length decreases. Shortening would stop when the force generated by the muscle is counterbalanced by forces that oppose the shortening, and it therefore prevents excessive narrowing of the airway. Shifting the active L-T relationship to the left (toward shorter muscle length) due to adaptation will allow the muscle to generate more force at shorter lengths, resulting in more constriction of the airway. Periodic stretching of the airways due to tidal breathing and deep inspirations is crucial in keeping the airways from excessive narrowing (15); a left shift in the passive L-T relationship will reduce the effectiveness of breathing in stretching the muscle because of the increase in passive muscle stiffness. This, together with the increased active force at short lengths due to adaptation, will likely result in exaggerated airway narrowing that is refractory to the relaxing effect of stretching that accompanies tidal breathing and deep inspirations.
Force recovery in the presence and absence of stimulation. Force recovery after a large change in muscle length is used to indicate the time course and extent of restructuring of plastic adaptation occurring in the muscle (10, 17). Although the rate of force recovery is accelerated by frequent stimulation of the muscle (17), the recovery does occur in the absence of stimulation (Fig. 3). The exact mechanism for the force recovery is not entirely clear; it appears that the underlying mechanism for the force recovery is operative without periodic activation of the muscle, and that the basal activity of the muscle cell is sufficient to sustain the recovery process. The passive force recovery appears to be due to a different mechanism; the rate of recovery was not altered by periodic stimulation of the muscle (Fig. 2). The passive force appears to be borne by a passive parallel viscoelastic element that, when stretched, undergoes a characteristic time-dependent stress relaxation. This parallel element, however, is not a "passive" element in the sense that it can be restructured just like the contractile element, as evidenced by the shifts in both the active and passive L-T relationships (Fig. 1; Table 1).
A significantly higher rate was observed in the active force recovery of freshly dissected ASM tissues (CTL0) compared with that of the PS7/8 preparations (Table 3). It appears that more permanent structural changes occur progressively in ASM during prolonged length adaptation so that readaptation of the shortened muscle at a longer length becomes more and more difficult and often incomplete after 7 days. It is interesting that after an identical stretch applied to the ASM preparation, a smaller decrease in active force was observed in freshly dissected smooth muscles compared with the PS7 preparations. It appears that the ability of the muscle to rapidly adapt to length change is partially impaired in the day 7 preparations. The rate of passive force recovery is, however, not different between cultured (PS7/8) and freshly isolated (CTL0) ASM preparations (Table 3). The difference between the time course and characteristics of active and passive force recovery suggest that these two processes may be governed by different mechanisms.
Time dependent change in L-T relationship. When incubated under the same condition, the PS ASM preparations produced different degrees of shifting in the L-T relationship depending on the incubation time; the longer the preparations were incubated the larger the shifts, even though the preparations were set at the same length during the incubation period (Table 1). For the day 3 preparation, the shift in Lmax was 10%, even though the muscle length was kept at 50–60% of its Lin situ the whole time during incubation. This suggests that either the adaptation to a 40–50% length shortening was not completed after 3 days or the adaptation was not permanent and that some of the adapted changes were partially reversed during the measurement. The shift in Lmax after 7 or 8 days was 38%, close to the 40–50% length change imposed on the muscle during the incubation period. This suggests that, after 7/8 days, adaptation was nearly complete.
Readaptation of shortened muscles to longer lengths also revealed time-dependent change in the L-T relationship in terms of its permanence. After 3 days of adaptation at short lengths, the muscle could be restretched to its CTL Lmax where full recovery of isometric force could be achieved (Table 2). On the other hand, after 7 or 8 days of adaptation, most of the preparations became so stiff that it was not possible to restretch them to their CTL Lmax. Something more permanent appears to have occurred in the process of restructuring during the period of adaptation. It should be pointed out that the irreversibility of the PS7/8 preparations evidenced in short-term (hours) readaptation to longer lengths may not indicate a permanent condition; it is possible that long-term (days) readaptation could reverse the apparent permanent structural change.
These results suggest that if permanent adaptation to short lengths is to be avoided in ASM, prolonged shortening of the muscle either passively or actively due to stimulation by inflammatory mediators should be avoided. Brief episodes of muscle contraction or passive shortening up to 3 days appear to have no permanent effect on the normal structure and function of ASM.
In conclusion, the acute and subacute changes in the L-T curve (up to 3 days of adaptation) are relatively impermanent in that the L-T behavior appears to be plastic over this time period. The finding of a more striking and less reversible change after a prolonged period of length adaptation may be relevant to disease states in which exaggerated airway narrowing is partially or completely irreversible.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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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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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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in A and B), and the CTL length associated with maximal force generation (Lmax) was determined. After the L-T relationship of the PS smooth muscle (
in A and B) and its Lmax were determined, the PS preparation was stretched to a maximally stretchable length (without causing permanent damage to the muscle tissue) and was stimulated isometrically at that length. The active (
) and passive (
) forces were measured. These values were then plotted against time (C and D), and the recovery rates were measured by fitting the data with exponential equations described in the text. Numbers beside the symbols in A and B indicate the time sequence of contractions.
max of the muscle preparations
