1Department of Mechanical Engineering, 2Department of Biomedical Engineering and 3Departments of Biochemistry and Pediatrics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia
Submitted 25 August 2004 ; accepted in final form 9 February 2005
ABSTRACT
Passive mechanical properties of strips of rabbit detrusor smooth muscle were examined and found by cyclic loading in a calcium-free solution to display viscoelastic softening and strain-induced stress softening (strain softening). Strain softening, or the Mullins effect, is a loss of stiffness attributed to the breakage of cross-links, and appeared irreversible in detrusor even after the return of spontaneous rhythmic tone during 120 min of incubation in a calcium-containing solution. However, 3 min of KCl or carbachol (CCh)-induced contraction permitted rapid regeneration of the passive stiffness lost to strain softening, and 3 µM of the RhoA kinase (ROK) inhibitor Y-27632 prevented this regeneration. The degree of ROK-induced passive stiffness was inversely dependent on muscle length over a length range where peak CCh-induced force was length independent. Thus rabbit detrusor displayed variable passive stiffness both strain- and activation-history dependent. In conclusion, activation of ROK by KCl or CCh increased passive stiffness softened by muscle strain and thereby attributed to cross-links that remained stable during tissue incubation in a calcium-free solution. Degradation of this signaling system could potentially contribute to urinary incontinence.
muscle mechanics; preconditioning; Y-27632; RhoA kinase; passive force
There are many candidate structures that may contribute to smooth muscle passive force. Extracellular collagen and elastin play a role, especially at long muscle lengths (43, 47). The intracellular sarcomeric protein titin was recently identified as the major passive force-bearing structure in striated muscles (28, 60), and a related molecule, smitin, was recently identified in smooth muscle (17). Whether this protein plays a role in passive force is unknown. In addition to smitin, other intracellular cytosolic proteins associated with force transmission through dense body/focal contacts (26), as well as actomyosin cross-bridges or ancillary actin and myosin cross-linking proteins, may contribute to passive force in smooth muscle. For example, filamin is a cross-linking protein involved in forming F-actin networks and bundles, and in attaching F-actin to focal contacts (51, 58). Whereas desmin is not required for maintenance of passive force, this intermediate filament appears to play a role in transmission of both active and passive forces (2, 48), and the filamin-actin-desmin domain has been proposed to play a force-bearing regulatory role in smooth muscle (35). Calponin, a proposed thin-filament regulatory protein, can bridge intermediate filaments and actin at dense bodies (22) and cross-link microtubules with actin-based microfilaments (5). Moreover, smooth muscle thin and thick filaments might become cross-linked and the level of cross-linking might be regulated. For example, caldesmon and calponin can bind both actin and myosin, and both proteins have been associated with formation of cross-links between actin and myosin (12, 24, 53, 59). Although the physiological and biomechanical significance of this biochemical finding remains obscure, reductions in cross-bridge activity by caldesmon and calponin may be relieved by elevations in cytosolic calcium and phosphorylation of caldesmon and calponin (see Refs. 25 and 54 for reviews). Also, at levels of cytosolic calcium and myosin light chain (MLC) phosphorylation only marginally above basal levels, actomyosin cross-bridges, which normally cycle rapidly (and therefore are not considered "cross links" between actin and myosin) may enter a "latch" state, where they cycle slowly or not at all, effectively cross linking actomyosin (33, 40). However, high passive stiffness attributed to attached cross-bridges is abolished when smooth muscle tissues are incubated in a Ca2+-free solution (47).
To obtain reliable preloads (i.e., passive force), smooth muscle tissues must be preconditioned by applying cyclic loading (6). Preconditioning is characterized by reductions towards a steady state in stiffness with each subsequent cycle, and has been identified in whole organs (8, 29), isolated tissues (18), and recently, in isolated titin (16). Although the mechanism responsible for preconditioning remains to be determined, two hypotheses have been put forth to explain this phenomenon. One hypothesis is that loss of stiffness is reversible and due to viscoelastic properties of muscle (6). The other is an essentially irreversible loss of stiffness termed strain-induced stress softening, or simply, strain softening, that is due to elastic structural changes involving cross-link breakage (4, 8).
Stress-relaxation and stress-strain hysteresis classically characterize viscoelastic materials, and a reversible loss of stiffness with loading cycles can be attributed solely to the time (and rate)-dependent viscous structures within muscle (6). At the molecular level, reversible mechanical fatigue of passive molecular structures in muscle can impart a complex (i.e., nonlinear) history dependence on the level of passive force produced at any given length. Titin, for example, has been shown to progressively lengthen when stretched, and the highly charged proline-glutamic acid-valine-lysine segment of this molecule has been proposed to play a role in intrachain unfolding during stretch and slow (minutes), diffusion-limited, refolding during release (16).
Strain softening was originally shown to be a property of rubber, and the loss of stiffness after a stretch to a new force is attributed to breakage of cross-links between rubber molecules (23, 30). Cross-link reformation of rubbery materials is very slow, i.e., taking several days (31), and is therefore considered irreversible in the time frame of a biological experiment. In rat left ventricular myocardium and guinea pig small intestine, preconditioning was recently attributed more to strain softening due to alterations in elastic structures than to viscous effects (4, 8). However, whether this preconditioning was reversible was not examined. We provide a complementary hypothesis, tested in the present study, and propose that strain softening in smooth muscle reflects breakage of cross-links that participate in passive resistance to stretch, and most importantly, that can be rapidly reformed on muscle activation.
Although preconditioning is present in the bladder (29), it is unknown whether the loss of stiffness with increasing strains is caused purely by viscoelastic effects or also due to strain softening. The present study is designed to test the hypothesis that detrusor preconditioning involves elements of both viscoelastic softening (reversible) and strain softening (cross-link breakage). Perhaps most importantly, this study also seeks to determine whether cross-links "broken" by cyclic length changes can be reformed during muscle activation. Finally, this study uses a pharmacological approach to assess whether RhoA kinase (ROK) participates in development of the passive stiffness softened by cyclic stretches. ROK plays a role in focal contact and stress fiber formation (44), microtubule assembly (1), and smooth muscle contraction (49). Thus, because ROK regulates structures involved in both passive and active resistance to stretch, this kinase is a likely candidate as a regulator of reversible passive stiffness in detrusor.
METHODS
Tissue preparation.
Tissues were prepared as described previously (36, 45). Whole bladders from adult female New Zealand White rabbits were removed immediately after pentobarbital-induced death. The bladders were washed several times, cleaned of adhering tissue, including fat and serosa, and stored in cold (04°C) physiological salt solution (PSS), composed of (in mM) 140 NaCl, 4.7 KCl, 1.2 MgSO4, 1.6 CaCl2, 1.2 Na2HPO4, 2.0 morpholinopropanesulfonic acid (adjusted to pH 7.4 at either 0° or 37°C, as appropriate), 0.02 Na2 EDTA (to chelate trace heavy metals), and 5.6 dextrose. High-purity (17 M) water was used throughout the experiment. For clarity, in RESULTS, PSS will be referred to as a "Ca2+-containing solution" or as "+ Ca2+", whereas PSS with no CaCl2 and the addition of 1 mM EGTA to chelate Ca2+ as a "Ca2+-free solution" or as "Ca2+ free." Longitudinal detrusor muscle strips free of underlying urothelium were cut from the wall of the bladder above the trigone. Each muscle strip was incubated in aerated PSS at 37°C in a water-jacketed tissue bath (Radnotti Glass Technology, Monrovia, CA) and secured by small muscle clips to a micrometer for manual length adjustments and an computer-controlled electronic lever (model 300H, Aurora Scientific, http://www.aurorascientific.com/index.asp) to record force and to induce time-controlled muscle length changes (ramp stretches and ramp releases).
Latex strips.
To assess the mechanical behavior of natural rubber, small strips, 3 mm x 10 mm, were cut from new latex gloves (Microflex, Reno, NV) and secured by small muscle clips to a micrometer and lever to record force and to induce time-controlled muscle length changes as described for detrusor strips.
Contraction of isolated detrusor strips.
Isometric contraction was measured as described previously (37, 45). Voltage signals were digitized (model PCI-6024E, National Instruments; http://www.ni.com), visualized on a computer screen as force (g), and stored for analyses. All data analyses were performed with the use of multichannel data-integration software (DMC from Aurora Scientific and DASYLab from National Instruments; http://www.dasylab.net/dasylab_english). Each tissue was secured to muscle clips such that its initial (cold) zero preload length was 5 mm and equilibrated for 1 h at 37°C to permit development of spontaneous rhythmic contraction. Tissues were then incubated in a Ca2+-free solution to eliminate spontaneous contractile activity (46), stretched in 0.5-mm step increments and allowed to stress relax with each step increase until a stable preload 0.05 g above zero was established. This was considered slack length (Ls) at 37°C.
Cyclic loading protocol for analyses of strain softening and viscoelastic softening.
Each tissue was incubated in a Ca2+-free solution, stretched using a manual micrometer to 120% Ls, equilibrated in a Ca2+-containing solution until spontaneous rhythmic tone returned (30'), and contracted with 110 mM KCl (substituted isosmotically for NaCl) for 3 min to determine the maximum force (Fig. 1). The average KCl-induced peak stress (force/cross-sectional area) produced at 120% Ls was 1.7 ± 0.1 x 105 N/m2, n = 17, a value comparable to that produced by carotid artery preparations (32). After washout of KCl to permit relaxation, tissues were incubated in a Ca2+-free solution for 10 min to eliminate rhythmic tone and subjected to 14 consecutive triangle ramp-length changes (cyclic loadings, Fig. 1). Some tissues "rested" for 10 min in the Ca2+-free solution to permit restoration of viscous series elastic force (32), and were then subjected to a second, and 10 min later, a third series of 14 consecutive cyclic loadings. For studies shown in Figs. 6 and 7, these three series of cyclic loadings were termed series 1a (S1a), series 2a (S2a), and series 3a (S3a). All loadings were ramp stretches at 1 mm/s. The peak amplitude for cycles 17 was 1 mm, and that for cycles 814 was 2 mm.
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Cyclic loading protocol for the analysis of the dependency on muscle strain history on passive stiffness induced by muscle activation. For studies shown in Fig. 9, an abbreviated strain softening protocol was used consisting of 7 consecutive loading cycles (1 mm/s, 2 mm peak amplitude) to induce both strain softening and viscoelastic softening, followed 10 min later by a single loading cycle of the same rate and amplitude (cycle 8) to induce only viscoelastic softening. The area of the work loops from the first and eighth cycles were normalized to the seventh cycle area to reduce tissue-to-tissue variability, and the eighth cycle area was subtracted from the first to obtain an estimate of passive work produced by strain softening.
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Statistics. Analysis of variance and the Student-Newman-Keuls test, or the t-test, were used where appropriate to determine significance, and the Null hypothesis was rejected at P < 0.05. The population sample size (n value) refers to the number of animals, not the number of tissues.
RESULTS
Strain softening of detrusor strips in a Ca2+-free solution.
Early studies on smooth muscle show that incubation in a Ca2+-free solution abolishes active cross-bridge cycling responsible for the high passive force and stiffness of smooth muscle at rest (13, 47). Therefore, before length changes were imposed to measure passive force in the present study, tissues were incubated in a Ca2+-free solution to eliminate cross-bridge activation. Incubation of rabbit detrusor in a Ca2+-free solution for 10 min abolishes basal rhythmic contraction, reduces basal MLC phosphorylation from 15% to
3% (39) and prevents 20 mM caffeine, 110 mM KCl, and a maximum concentration of the muscarinic receptor agonist bethanechol from causing contraction (15). For these reasons, we conclude that the force responses to loading cycles produced by rabbit detrusor strips incubated for 10 min in a Ca2+-free solution were due to passive structural elements, and not to actively cycling cross-bridges. As expected, all passive force-vs.-length work loops produced in the present study showed a loss of energy. That is, passive force was greater during the ramp loading (Fig. 1A, inset c, up arrow) than during the ramp unloading (Fig. 1A, inset c, down arrow). Peak passive force and muscle stiffness produced during the first loading (Fig. 2, AC, "1") were much greater than those produced during loadings 27 (Fig. 2). The greater work of extension produced during cycle 1 loading compared with cycles 27 was revealed as a larger work loop produced during cycle 1 (Fig. 2C). The fact that the work loop from cycle 2 was less than the work loop from cycle 1 indicates that energy stored in passive structural elements was lost because of the first imposed loading strain. Work loops produced during the sixth and seventh cycles were superimposable (Fig. 2C), indicating no further loss of energy and that viscoelastic steady state was reached. Although the initial stiffness curve produced during the first millimeter of cycle 8 loading was exactly superimposable on the stiffness curve of cycle 7 loading (Fig. 2D), stiffness during the next loading cycle (Fig. 2D, cycle 9) was greatly reduced, mimicking results from cycles 1 and 2 (Fig. 2C). Also, as with cycles 6 and 7, the work loops produced during cycles 13 and 14 were superimposable, indicating that viscoelastic steady state was achieved at these two different muscle lengths within 6 loading cycles. The additional strain imposed in loading cycle 8 (2 mm) compared with loading cycle 7 (1 mm) produced a higher stiffness, despite having achieved viscoelastic steady state by cycle 7, which revealed the presence of additional force-bearing structures at these longer strains. The loss of stiffness (and energy) between cycles 8 and 9 indicated that loading cycle 8, as with loading cycle 1, induced softening of the muscle's passive properties. These data support the existence of strain-dependent cross-links under passive (i.e., Ca2+ free) conditions. The "extra" stiffness during the first loading curves for a new strain (i.e., cycle 1 and the second millimeter of cycle 8) compared with subsequent loading curves was also revealed by a comparison of the shapes of the first and second curves. Cycle 1 and the second millimeter of the cycle 8 loading curves produced a linear fit (Fig. 3, A and C), whereas cycles 2 and 9 loading curves fit more closely to an exponential curve (Fig. 3, B and D).
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To document that the biomechanical behavior of detrusor smooth muscle is similar to that of rubbery materials, latex strips were subjected to cycling loadings and the resulting changes in force were measured. As demonstrated in detrusor, peak stiffness produced during the first loading (Fig. 5, A and B, "1") was much greater than that produced during loadings 27 (Fig. 5A). The greater work of extension produced during cycle 1 loading compared with cycles 27 was revealed as a larger work loop produced during cycle 1, and work loops produced during the sixth and seventh cycles were superimposable (Fig. 5A), indicating no further loss of energy and that viscoelastic steady state was reached. Also, as in detrusor strips, the initial stiffness curve produced during the first millimeter of cycle 8 loading was exactly superimposable on the stiffness curve of cycle 7 loading (Fig. 5B), but stiffness during the next loading cycle (Fig. 5B; cycle 9) was greatly reduced, mimicking results from cycles 1 and 2 (Fig. 5A). Work loops produced during cycles 13 and 14 were also superimposable, indicating that viscoelastic steady state was achieved at these two different muscle lengths within 6 loading cycles. The presence of additional strain-breakable force-bearing structures when straining from 1 to 2 mm (loading cycle 8 compared with loading cycle 7) was evident by the greater loss of stiffness (and energy) between cycles 8 and 9 of S1 compared with the loss of stiffness and energy between cycles 8 and 9 of S2 and S3 (Fig. 5, CE). These data together indicate that rabbit detrusor muscle strips exhibited classic strain softening behavior because the passive properties were dependent on the maximum previous strain.
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Reversibility of strain softening and inhibition of reversal by the ROK blocker Y-27632. Tissues were contracted with KCl as part of the protocol to establish initial conditions of the muscle before the first cyclic loading series. To reproduce this initial condition and attempt to reestablish the "extra" passive stiffness softened by the first cyclic strain, tissues treated as in the protocol shown in Fig. 6A were contracted for 3 min with KCl during the 30-min incubation period in the Ca2+-containing solution (Fig. 7A). This brief but maximum contraction did permit recovery of the passive stiffness "lost" to strain softening, as shown by the increase in S1b cycle 8 (Fig. 7B). In addition, cycle 8 areas from S2b and S3b were not significantly different than cycle 8 areas from S2a and S3a (Fig. 7B). These data suggest that the small but significant differences in cycle 8 areas shown in the previous figure (Fig. 6), were due to the continued small loss of stiffness produced with each cycle 8 strain. Together, these data indicate that strain softening can be reversed by muscle activation with KCl.
Recent studies by our laboratory show that KCl activates ROK in vascular smooth muscle (56). To determine whether the "extra" passive stiffness produced by KCl-induced detrusor muscle activation was due to ROK activity, a selective ROK inhibitor, 3 µM Y-27632 (14, 55), was added 10 min before and during the 3 min KCl-induced contraction that was induced 20 min into the incubation period between S3a and S1b, when tissues were exposed to a Ca2+-containing solution. As it does in the rabbit femoral artery (56), Y-27632 inhibited steady-state but not peak force produced by KCl (Fig. 7A; "+Y" compared with "Control"). When normalized to the initial KCl-induced peak force (Fmax) produced before strain softening, peak and steady-state force values produced by control tissues stimulated with KCl in the protocol shown in Fig. 7A were, respectively, 1.00 ± 0.06 Fmax and 0.69 ± 0.05 Fmax, and those produced by KCl in the presence of Y-27632 were, respectively, 0.91 ± 0.04 Fmax (n = 4, not different than control) and 0.45 ± 0.06 Fmax (n = 4, P < 0.05 compared with control). Inhibition of KCl-induced steady-state force by Y-27632 prevented the restoration of passive stiffness produced by muscle stimulation with KCl (Fig. 7C).
Evidence for activation of ROK by KCl.
To test the hypothesis that ROK was activated by exposure to a depolarizing concentration of KCl, tissues stretched to 120% Ls and strain softened were incubated in a Ca2+-containing solution for 10 min with or without 3 µM Y-27632, contracted for 2 min with 110 mM KCl (substituted isosmotically for NaCl), quick-frozen, and processed for determination of the degree of MYPT1 site-specific phosphorylation (see METHODS). The results were compared with those produced by tissues incubated in a Ca2+-free solution for 10 min (Basal). KCl increased the degree of MYPT1 T853-p and T696-p by 1.5-fold above the control level produced by tissues in a Ca2+-free solution (Fig. 8). The ROK inhibitor Y-27632 abolished the increase produced by KCl and reduced the basal level of MYPT1 T853-p to < 50% that produced in a Ca2+-free solution (Fig. 8).
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In a final experiment, before undergoing cyclic loadings to measure strain softening, tissues precontracted with KCl (to induce strain softenable passive stiffness) were exposed to 100 µM () blebbistatin for 10 min while they were being incubated in the Ca2+-free solution. Blebbistatin inhibits skeletal and nonmuscle myosin II with an IC50 value of 2 µM, and smooth muscle myosin II with an IC50 value of
80 µM, by blocking myosin in an actin-detached state (19, 21, 52). Thus blebbistatin should abolish strain-softenable stiffness if the stiffness retained in the Ca2+-free solution was the direct result of attached, cycling cross-bridges. We found that 100 µM () blebbistatin did not reduce passive work (measured as fold cycle 7) caused by 2 mm cyclic strains (no blebbistatin, 1.5 ± 0.4, + blebbistatin, 2.3 ± 0.8, n = 3).
DISCUSSION
The results from the present study indicate that both viscoelastic softening and strain-induced stress softening (strain softening, Refs. 30 and 31) were identified in strips of rabbit detrusor smooth muscle under passive conditions [i.e., incubated in a Ca2+-free solution to abolish active cross-bridge cycling and the ability to generate force (15, 39)]. Thus detrusor shares at least two distinct mechanical properties with nonliving rubbery materials subjected to cyclic loading. Viscoelastic softening in rubber is readily reversible (see Fig. 5, D and E), and the passive stiffness due to viscoelastic softening in detrusor returned within 10 min, even when tissues remained in a Ca2+-free solution (see Fig. 4, F and I). Strain softening in rubbery materials is caused by strain-induced breakage of cross-links between polymer chains within the rubber matrix (23). At room temperature, the strain-softened stiffness of rubbery materials returns only very slowly (days) and is considered irreversible over the course of an experiment (31). Strain softening of detrusor in a Ca2+-free solution also induced an apparently irreversible loss of stiffness. This was true even when tissues regained spontaneous rhythmic contractile tone by incubation in a Ca2+-containing solution for up to 120 min between cyclic loadings performed in a Ca2+-free solution. However, the most important aspect of this study was the finding that 3 min of a KCl- or CCh-induced contraction permitted rapid regeneration of the passive stiffness lost to strain softening, and that inhibition of ROK prevented this regeneration. These data support the hypothesis that ROK participates in an active process to increase the degree of passive stiffness, and therefore, passive force for a given muscle length. Because incubation of rabbit detrusor strips in a Ca2+-free solution abolishes basal rhythmic contraction and reduces basal MLC phosphorylation from 15% to
3%, and completely prevents KCl, caffeine and the muscarinic receptor agonist, bethanechol, from causing contraction (15, 39), the implication of these findings is that passive force in rabbit detrusor smooth muscle includes a variable component involving cross-link formation that is dependent on the history of ROK activity and that can be sustained in the absence of extracellular Ca2+. Muscle total force is the sum of passive and active forces (see Ref. 32 for review), and passive force is generally assumed to be a constant value at a given muscle length. Thus the identification of variable passive force in detrusor challenges this assumption. Together, these data indicate that, in addition to a length- and time-dependency due to viscoelastic properties of structural proteins, detrusor smooth muscle passive force also displayed dependencies on strain history and ROK activation state history.
In smooth muscle, ROK is activated by stimulation of G protein-coupled receptors (see Ref. 49 for review) and membrane depolarization with KCl (see Ref. 42 for review). One function of ROK in differentiated smooth muscle is to inhibit MLC phosphatase (see Ref. 50 for review), permitting elevations in MLC phosphorylation despite low levels of [Ca2+]i that occur tonically (34). Thus ROK activation plays a major role in permitting sustained contraction (56). The present study identifies an additional action of ROK in differentiated visceral smooth muscle, namely, to increase passive muscle stiffness. Our data support the hypothesis that the stiffness is not directly due to attached cross-bridges, but might be caused by the action of prior strong cross-bridge activation. That is, reformation of cross-links softened by strain required prior muscle activation by KCl or CCh, and these cross-links were sustained when tissues were incubated in a Ca2+-free solution when cross-bridge activation was entirely absent.
On the basis of several lines of evidence, it seems to be a reasonable conclusion that cross-bridges were not "on" and cycling and therefore likely not the mechanical cross-link directly responsible for the ROK-mediated passive stiffness. The ROK-mediated passive stiffness revealed by strain softening was identified when detrusor strips were incubation for at least 10 min in a Ca2+-free solution, and previous studies have shown that such treatment is sufficient to reduce cytosolic free Ca2+, abolish spontaneous rhythmic contraction (39, 46), decrease basal MLC phosphorylation (39), and completely prevent KCl, caffeine, and a muscarinic receptor agonist from causing contraction (15). However, our data do support a requirement for the history dependence of cross-bridge activation in permitting formation of stable cross-links that participate in resistance to passive stretch.
A potential site for generation of variable passive force is formation of a population of cross-linked cross-bridges. Actomyosin cross-bridges can be cross-linked by forming latch-bridges (attached, dephosphorylated, noncycling, or slowly cycling cross-bridges; see Ref. 34 for review) or because they have become tethered by caldesmon or calponin that contain both actin and myosin binding domains (12, 24, 53, 59). Data from the present study indicate that greater passive stiffness was generated during contraction induced by CCh when muscles were at 100% Ls compared with 140% Ls. However, CCh did not produce a greater peak contraction at the shorter length, suggesting that overlap of actin and myosin filaments, and thus the number of attached (cycling) cross-bridges during CCh-induced peak force, was not strain dependent over the range of muscle lengths used in this study (100% Ls to 140% Ls). If it is assumed that the number of cross-bridges remaining in some cross-linked conformation was a representative fraction of the number of cycling cross-bridges produced during the CCh-induced contraction, then the work done by strain softening should have been equivalent at 100% Ls and 140% Ls. Thus our data do not support a role for cross-linked actomyosin cross-bridges as bearing the variable passive stiffness induced by ROK activation.
More possible sites of action for establishment of variable passive force include focal contacts, the cytoskeletal matrix, including such actin and actin-focal contact cross-linking proteins as filamin, microtubules, and the smooth muscle titin-like protein smitin. Gunst and coworkers (see Refs. 7, 9, and 10 for reviews) provide strong evidence for length-dependent variable stiffness produced during activation of airway smooth muscle. The mechanical plasticity model used to describe length adaptation and increased active stiffness at short lengths in airway smooth muscle involves series elastic plasticity and attached cross-bridges that occur only after, but not before, the muscle is activated (11). Our variable passive stiffness model is complementary to, but fundamentally distinct from the mechanical plasticity model (11). We propose that, in detrusor smooth muscle, stimulation of ROK during muscle activation leads to increased passive elastic stiffness that is retained after muscle stimulation has ceased (and is even retained when tissues are exposed to a Ca2+-free solution). Moreover, our model indicates that the "extra" passive stiffness remains while the muscle is relaxed, unless the muscle was subjected to a strain sufficient to break cross-links formed during muscle activation. Our variable passive stiffness model is complementary to the mechanical plasticity model because variable passive stiffness could reside in cross-linking cytoskeletal proteins. For example, phosphorylation of the focal contact protein paxillin has been proposed to play a role in mechanical plasticity seen in airway smooth muscle, and although paxillin phosphorylation produced by contractile agonists is not prevented by the ROK inhibitor Y-27632 (27), other focal contact molecules may be targets for ROK.
In conclusion, we have identified a novel role for ROK in differentiated detrusor smooth muscle. Our data support the hypothesis that activation by KCl or CCh of a ROK-mediated signaling system induces variable passive stiffness likely caused by cross-link formation because the stiffness was softened by increased muscle strain. We propose that this component of passive stiffness was not a direct result of cross-bridge breakage, but may have been caused by prior cross-bridge activation because formation of the strain softenable stiffness was 1) inversely dependent on muscle strain, whereas CCh-induced peak force was strain independent, and 2) was retained when the muscle was incubated in a Ca2+-free solution. Additional data are required to identify the structures responsible for the variable passive stiffness produced by activation of ROK.
GRANTS
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK59620.
FOOTNOTES
Address for reprint requests and other correspondence: P. H. Ratz, Virginia Commonwealth Univ., School of Medicine, Depts. of Biochemistry and Pediatrics, 1101 E. Marshall St., PO Box 980614, Richmond, VA 23298-0614 (e-mail: phratz{at}vcu.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.
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