ROK-induced cross-link formation stiffens passive muscle: reversible strain-induced stress softening in rabbit detrusor

John E. Speich,1 Lindsey Borgsmiller,2 Chris Call,1 Ryan Mohr,1 and Paul H. Ratz3

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


NONACTIVATED [i.e., passive (16)] muscle develops force in response to external muscle stretch (resistance to extension) because of viscoelastic elements within muscle cells and the extracellular space (3, 20, 47). In all muscles, passive force restores muscle length upon release, and in smooth and cardiac muscles (3), passive force contributes significantly to the total force produced at the optimum length for muscle contraction, and this is true for tissues as diverse in function as arteries (13) and urinary bladder (57). Although many different proteins may participate in smooth muscle passive force, the precise contribution each plays remains to be determined.

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 (0–4°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{Omega}) 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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Fig. 1. Example of the protocol used to measure strain softening and viscoelasticity in a detrusor muscle strip. A, inset: cycle 1 shows force response (b) to the first loading cycle (a), and that force produced during loading (c, up arrow) was always greater than that produced during unloading (iii, down arrow), reflecting a muscle under passive conditions. B: tissues at 120% slack length (Ls) in a Ca2+-containing solution (+ Ca2+) developed spontaneous rhythmic tone (Rhythm, and inset showing expanded scale). Tissues were contracted for 3' with KCl, washed to permit complete relaxation, and rhythmic tone was eliminated by replacing the Ca2+-containing solution with a Ca2+-free solution (inset, Ca2+ free). Tissues were then subjected to 14 consecutive ramp loading and unloading cycles (A) producing 14 force cycles lasting 42 s (B). Loading and unloading were at 1 mm/s. Cycles 17 were 1-mm stretches, and cycles 814 were 2-mm stretches. Note that loading produced force responses that were considerably weaker than that produced by KCl.

 


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Fig. 6. A: irreversibility of strain softening. Tissues that had undergone 3 series of 14 loading cycles in a Ca2+-free solution (S1a–S3a) were incubated in a Ca2+-containing solution for 30 and 120 min (+ Ca2+ 30' and 120'), which permitted tissues to redevelop spontaneous rhythmic tone as in Fig. 1. Tissues were then incubated a second time in a Ca2+-free solution and subjected again to 3 series of 14 loading cycles (S1b–S3b). Incubation of tissues in a Ca2+-containing solution for 30 (B) and 120 min (C) did not reverse the softening of passive stiffness produced by the S1a cycle 8 strain. Data (normalized to cycle 14, S3a areas) are means ± SE, n = 3. *P < 0.05, compared with respective series (i.e., S1b compared with S1a, etc.).

 


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Fig. 7. Reversibility of strain softening produced by contraction with KCl and inhibition of stiffness restoration by Y-27632. A: tissues that had undergone 3 series of 14 loading cycles in a Ca2+-free solution (S1a–S3a) were incubated in a Ca2+-containing solution for 30 min and contracted for 3 min with KCl in the absence or presence of 3 µM Y-27632 (+ Ca2+, 30' and inset). Tissues were then incubated a second time in a Ca2+-free solution and subjected again to 3 series of 14 loading cycles (S1b–S3b). B: stimulation with KCl increased passive stiffness, reversing the softening produced by the S1a cycle 8 strain. C: Y-27632, inhibited KCl-induced tonic but not early peak contraction (A, inset, and text) and prevented KCl from increasing passive stiffness. Data (normalized to cycle 14, S3a areas) are means ± SE, n = 4. *P < 0.05, compared with respective series (i.e., S1b compared with S1a, etc.).

 
For studies shown in Figs. 6 and 7, tissues were incubated in a Ca2+-containing solution for 1) 30 min or 2) 120 min to permit spontaneous rhythmic contractions to return. Some tissues incubated for 30 min in the Ca2+-containing solution were contracted with 110 mM KCl for 3 min, 25 min into the incubation period. Some of these tissues were treated with 3 µM Y-27632 for 10 min before and during stimulation with KCl. All tissues were again subjected to 3 series of 14 consecutive cyclic loadings, and were termed series 1b (S1b), series 2b (S2b), and series 3b (S3b) to differentiate them from the first 3 series of cyclic loadings (S1a–S3a).

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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Fig. 9. Tissues stretched to 100%, 120%, and 140% Ls were activated at each muscle length with 10 µM CCh for 3 min in the absence (No Drug, B and C) and presence of 3 µM Y-27632, then strain softened in a Ca2+-free solution at 120% Ls (A) to determine whether the degree of "extra" passive stiffness induced by muscle activation displayed a dependency on the history of muscle length (ML). Seven loading cycles to induce strain softening plus viscoelastic softening were followed 10 min later by 1 loading cycle to induce only viscoelastic softening (A, inset). Passive work done by strain softening was calculated as the work produced by loading cycle 1 minus that produced by loading cycle 8, normalized to cycle 7 to reduce tissue-to-tissue variability (B). Peak carbachol (CCh)-induced force was not dependent on muscle length (C). Data are means ± SE, n = 4–5. *P < 0.05.

 
Myosin phosphatase targeting subunit phosphorylation. The degree of myosin phosphatase targeting subunit (MYPT1) phosphorylation was measured as described previously for ERK phosphorylation (38). Detrusor strips were quick-frozen in an acetone-dry ice slurry, thawed, homogenized in 1% SDS, 10% glycerol, 20 mM dithiothreitol, 25 mM Tris·HCl (pH 6.8), 5 mM EGTA, 1 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 20 mg/ml leupeptin, 2 mg/ml aprotinin, and 20 mg/ml (4-amidino-phenyl)-methanesulfonyl fluoride, heated 10 min at 100°C, clarified by centrifugation at 10,000 g for 10 min, and stored at –70°C. Thawed homogenates were assayed for protein concentration and proteins were separated (SDS-PAGE) on 12% polyacrylamide gels (12 mg of protein per well), followed by Western blotting onto Immobilon-P membranes (Millipore; Bedford, MA). Threonine 853 (T853) phosphorylated MYPT1 (MYPT1 T853-p) and threonine 696 (T696) phosphorylated MYPT1 (MYPT1 T696-p) were identified using phosphoselective antibodies (Upstate, Chicago, IL) and detected with the use of a horseradish peroxidase-labeled secondary antibody and enhanced chemiluminescence (ECL) and ECL film (Amersham). Quantification of visualized bands was obtained by digital image analysis software. To compensate for gel-to-gel variabilities in efficiencies of Western blot analysis, antibody labeling, ECL reaction, and film development, band intensities were reported as the degree of change from the Ca2+-free basal value. To double check that protein loading was consistently uniform across all lanes of the gel, membranes were also probed with MYPT1 primary antibody (BD Biosciences, Franklin Lakes, NJ).

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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Fig. 2. Example of a 42-s force tracing (B) produced by a detrusor muscle strip incubated in a Ca2+-free solution resulting from 7 sequential loading cycles (1,2...6,7) of 1 mm amplitude and at 1 mm/s, followed immediately by 7 sequential loading cycles (8,9...13,14) of 2 mm amplitude and at 1 mm/s (A). Examples of force vs. length (work loops) for the cycles 1, 2, 6, and 7 (C) and cycles 79, 13, and 14 (D) show high, linear stiffness produced during the first loading and the second millimeter of the eighth loading, curvilinear stiffness produced by all other loadings, identical stiffness produced by the seventh loading and first millimeter of the eighth loading, and identical work loops produced by the cycles 6 and 7 and cycles 13 and 14.

 


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Fig. 3. Stiffness analyses of data collected during loading for cycle 1 (A), the second millimeter of cycle 8 (B), cycle 2 (C), and cycle 9 (D). Data for cycles 1 and 8 (second millimeter) displayed linear stiffness curves, whereas those for cycles 2 and 9 fit a single exponential with a high correlation coefficient (R2). Raw data and regression lines are representative, R2 values are means ± SE for n = 13.

 
Irreversibility of strain softening. To determine whether strain softening in detrusor is irreversible, as it is in rubbery materials, tissues incubated in a Ca2+-free solution were subjected to 3 series of 14 loading cycles. Tissues "rested" for 10 min between each series to permit reestablishment of viscous stiffness. Peak force responses to each imposed loading cycle was highest in series 1 (S1; Fig. 4A) compared with respective cycles in series 2 (S2; Fig. 4D) and series 3 (S3; Fig. 4G). Likewise, work loop areas for each cycle from S1 (Fig. 4C, areas from cycles 1–7 are not shown) were greater than respective work loop areas for each cycle from S2 (Fig. 4F) and S3 (Fig. 4I). Respective work loop areas for each cycle from S2 and S3 were not significantly different. These data suggest that strain softening was irreversible when tissues remained in a Ca2+-free solution.



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Fig. 4. Representative force tracings from a single detrusor muscle strip incubated in a Ca2+-free solution and subjected to 14 loading cycles 3 times [series 1, S1 (A); series 2, S2 (D); and series 3, S3 (G)]. Tissues remained in a Ca2+-free solution for 10 min between S1 and S2 and between S2 and S3. Representative work loops for cycles 79, and 14 of S1 (B) are compared with those produced by cycles 79 and 14 of S2 (E) and S3 (H). Work loop area average ± SE values for n = 12 tissues are shown for cycles 814 for S1 (C), S2 (F), and S3 (I). The large loss of stored work seen after completion of cycle 8, S1 (double arrow: difference between areas produced by cycles 8, S1 and 8, S2) represented irreversible loss of stiffness due to strain softening. The loss of work from cycle 8, S2 to cycle 14, S2 (and similar changes in S3), and gain of energy from cycle 14, S1 to cycle 8, S2 and from cycle 14, S2 to cycle 8, S3 represented reversible viscoelastic softening ({Psi}P < 0.05 compared with cycle 14 from previous series). Both strain and viscoelastic softening are evident in S1. *P < 0.05, compared with respective cycles in S1. Cycles 814 of S2 were not significantly (NS) different than respective cycles of S3.

 
As expected, softening due to viscoelasticity was reversible, as shown by the redevelopment of stiffness from the low level produced during S1 cycle 14 (Fig. 4C), to the higher level produced during S2 cycle 8 (Fig. 4F), and likewise, the increase from S2 cycle 14 (Fig. 4F) to that produced during S3 cycle 8 (Fig. 4I). Similar results were obtained when examining cycles 1 and 7, but for the sake of clarity, these data (except as raw force tracings in Fig. 4A, D, and G) were not shown. The degree of viscoelastic softening could therefore be estimated as the difference between cycles 8 and 14 in S2 or S3 (Fig. 4F, dotted lines and double arrows). On the basis of these results, we estimated that the degree of strain softening was approximated by the difference between the cycle 8 areas of S1 and S2 (Fig. 4C, double arrows).

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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Fig. 5. Representative force tracings from a single latex strip subjected to 14 loading cycles (A) 3 times (S1–S3), and work loop area average ± SE values for n = 3 latex strips for cycles 814 for S1 (C), S2 performed 20 min after S1 (D), and S3 performed 80 min after S1 (E). As in strips of detrusor, the large loss of stored work after completion of cycle 8, S1 (double arrow: difference between areas produced by cycle 8, S1 and 8, S2) represented irreversible loss of stiffness due to strain softening. Also as in strips of detrusor, the loss of work from cycle 8, S2 to cycle 14, S2 (and similar changes in S3), and gain of energy from cycle 14, S1 to cycle 8, S2 and from cycle 14, S2 to cycle 8, S3 represented reversible viscoelastic softening ({Psi}P < 0.05 compared with cycle 14 from previous series). Both strain and viscoelastic softening are evident in S1. *P < 0.05, compared with respective cycles in S1. Cycles 814 of S2 were not significantly different than respective cycles of S3.

 
These results indicate that the original stiffness "lost" to strain softening in rabbit detrusor strips could not be regained while the tissues were incubated in a Ca2+-free solution. To determine whether reestablishment of the muscle active state would also reestablish passive stiffness "lost" to strain softening, tissues that had undergone 3 series of 14 cyclic loadings (Fig. 6A, S1a–S3a) were incubated in a Ca2+-containing solution for 30 or 120 min, permitting development of spontaneous rhythmic contractions (average force was 3.0 ± 0.3%, n = 6, of the initial KCl-induced peak contraction), and subjected again to 3 series of 14 cyclic loadings in a Ca2+-free solution (Fig. 6A; S1b–S3b). Cycle 8 areas produced during the 6 series were compared. To reduce tissue-to-tissue variability, areas were normalized to area 14 produced during S3a. The cycle 8 area produced during series-1a was more than sevenfold greater than the area produced during cycle 14 of S3a, whereas cycle 8 areas for S2a and S3a were greatly reduced to ~3-fold cycle 14 (Fig. 6B; S1a, S2a, and S3a). After incubation in a Ca2+-containing solution for 30 min (Fig. 6A, "+ Ca2+"), the cycle 8 area for S1b remained at the low level of <3-fold cycle 14, and S1b cycle 8 area was not significantly different than S3a cycle 8 area (Fig. 6A; S1b). Interestingly, cycle 8 areas from all three series produced after incubation in the Ca2+-containing solution were significantly lower than their series-respective cycle 8 areas produced before incubation in the Ca2+-containing solution (Fig. 6). As shown in Fig. 4, the degree of strain softening can be estimated by measuring the difference between cycle 8 areas produced during S1 and S2 (Fig. 6B, double arrows). Thus, strain softening occurred during S1a, but not during S1b (Fig. 6B), indicating that strain softening was irreversible even when tissues were returned to a Ca2+-containing solution and spontaneous tone was reestablished. A longer period (120') of incubation in a Ca2+-containing solution also did not restore passive stiffness to the prestrain softened level (Fig. 6C).

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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Fig. 8. Examples of (A and B) and average values of Western blots of myosin phosphatase targeting subunit 1-threonine 853 (MYPT1-T853) phosphorylation (C) and MYPT1-T696 phosphorylation (D). KCl increased MYPT1 phosphorylation to ~1.5-fold above that produced by tissues incubated in a Ca2+-free solution (basal). Y-27632 (3 µM) reduced the average values to, respectively, <0.5-fold and ~1-fold basal. C and D are average values of n = 3. *P < 0.05, compared with KCl.

 
Effect of muscle length history on passive force stiffening by carbachol-induced muscle activation. If the "extra" passive stiffness induced by muscle activation and softened by muscle strain involved attached, noncycling cross-bridges acting as cross-links even when tissues were incubated in a Ca2+-free solution, then the numbers of cross-links, and thus the amount of passive stiffness induced, should correlate with the amount of force produced at different muscle lengths. To test this hypothesis, muscle strips were contracted at three different muscle lengths (100%, 120%, and 140% Ls), incubated in a Ca2+-free solution, adjusted to 120% Ls and strain softened (Fig. 9A, Protocol). Carbachol (CCh), a muscarinic receptor agonist known to elevate ROK activity in detrusor (15, 41), was used to activate tissues. Tissues were subjected to an abbreviated strain-softening protocol (see METHODS). The degree of work produced by strain softening was found to correlate inversely with the length at which the muscle was activated by CCh, such that muscle activation at 100% Ls produced over fourfold greater strain softened passive work than did activation at 140% Ls (Fig. 9B). Moreover, 3 µM Y-27634 significantly reduced by ~50% the ability of CCh to induce the "extra" passive stiffness (Fig. 9B). However, CCh-induced peak force values produced above basal values were not dependent on muscle length, at least over this range of lengths (Fig. 9C). Because the strength of Fmax produced by a contractile agent is quantitatively related to the numbers of activated cross-bridges, these data show that equal numbers of cross-bridges were activated at 100%, 120%, and 140% Ls, but a higher number of cross-links were formed upon stimulation with CCh at 100% than 140% Ls. Together, these data do not support the hypothesis that attached cross-bridges formed the cross-links causing the stiffness revealed by strain softening of tissues incubated in a Ca2+-free solution.

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.

REFERENCES

1. Amano M, Kaneko T, Maeda A, Nakayama M, Ito M, Yamauchi T, Goto H, Fukata Y, Oshiro N, Shinohara A, Iwamatsu A, and Kaibuchi K. Identification of tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase. J Neurochem 87: 780–790, 2003.[CrossRef][ISI][Medline]

2. Boriek AM, Capetanaki Y, Hwang W, Officer T, Badshah M, Rodarte J, and Tidball JG. Desmin integrates the three-dimensional mechanical properties of muscles. Am J Physiol Cell Physiol 280: C46–C52, 2001.[Abstract/Free Full Text]

3. Brady AJ. Mechanical properties of isolated cardiac myocytes. Physiol Rev 71: 413–428, 1991.[Abstract/Free Full Text]

4. Emery JL, Omens JH, and McCulloch AD. Strain softening in rat left ventricular myocardium. J Biomech Eng 119: 6–12, 1997.[ISI][Medline]

5. Fattoum A, Roustan C, Smyczynski C, Der Terrossian E, and Kassab R. Mapping the microtubule binding regions of calponin. Biochemistry 42: 1274–1282, 2003.[CrossRef][ISI][Medline]

6. Fung YC. Biomechanics. New York: Springer-Verlag, 1993.

7. Gerthoffer WT and Gunst SJ. Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. J Appl Physiol 91: 963–972, 2001.[Abstract/Free Full Text]

8. Gregersen H, Emery JL, and McCulloch AD. History-dependent mechanical behavior of guinea-pig small intestine. Ann Biomed Eng 26: 850–858, 1998.[CrossRef][ISI][Medline]

9. Gunst SJ and Wu MF. Selected contribution: plasticity of airway smooth muscle stiffness and extensibility: role of length-adaptive mechanisms. J Appl Physiol 90: 741–749, 2001.[Abstract/Free Full Text]

10. Gunst SJ, Tang DD, and Opazo Saez A. Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung. Respir Physiol Neurobiol 137: 151–168, 2003.[CrossRef][ISI]

11. Gunst SJ, Meiss RA, Wu MF, and Rowe M. Mechanisms for the mechanical plasticity of tracheal smooth muscle. Am J Physiol Cell Physiol 268: C1267–C1276, 1995.[Abstract/Free Full Text]

12. Haeberle JR. Calponin decreases the rate of cross-bridge cycling and increases maximum force production by smooth muscle myosin in an in vitro motility assay. J Biol Chem 269: 12424–12431, 1994.[Abstract/Free Full Text]

13. Herlihy JT and Murphy RA. Length-tension relationship of smooth muscle of the hog carotid artery. Circ Res 33: 257–283, 1973.

14. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, and Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of Rho-associated kinases. Mol Pharmacol 57: 976–983, 2000.[Abstract/Free Full Text]

15. Jezior JR, Brady JD, Rosenstein DI, McCammon KA, Miner AS, and Ratz PH. Dependency of detrusor contractions on calcium sensitization and calcium entry through LOE-908-sensitive channels. Br J Pharmacol 134: 78–87, 2001.[CrossRef][ISI][Medline]

16. Kellermayer MS, Smith SB, Bustamante C, and Granzier HL. Mechanical fatigue in repetitively stretched single molecules of titin. Biophys J 80: 852–863, 2001.[Abstract/Free Full Text]

17. Kim K and Keller TC III. Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro. J Cell Biol 156: 101–111, 2002.[Abstract/Free Full Text]

18. Kirton RS, Taberner AJ, Young AA, Nielsen PM, and Loiselle DS. Strain softening is not present during axial extensions of rat intact right ventricular trabeculae in the presence or absence of 2,3-butanedione monoxime. Am J Physiol Heart Circ Physiol 286: H708–H715, 2004.[Abstract/Free Full Text]

19. Kovacs M, Wang F, Hu A, Zhang Y, and Sellers JR. Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform. J Biol Chem 278: 38132–38140, 2003.[Abstract/Free Full Text]

20. Labeit S, Kolmerer B, and Linke WA. The giant protein titin. Emerging roles in physiology and pathophysiology. Circ Res 80: 290–294, 1997.[Abstract/Free Full Text]

21. Limouze J, Straight AF, Mitchison T, and Sellers JR. Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil 25: 337–341, 2004.[CrossRef][ISI][Medline]

22. Mabuchi K, Li B, Ip W, and Tao T. Association of calponin with desmin intermediate filaments. J Biol Chem 272: 22662–22666, 1997.[Abstract/Free Full Text]

23. Marckmann G, Verron E, Gornet L, Chagnon G, Charrier P, and Fort P. A theory of network alteration for the Mullins effect. J Mech Phys Solids 50: 2011–2028, 2002.[CrossRef][ISI]

24. Marston S, Pinter K, and Bennett P. Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments. J Muscle Res Cell Motil 13: 206–218, 1992.[ISI][Medline]

25. Marston S, Burton D, Copeland O, Fraser I, Gao Y, Hodgkinson J, Huber P, Levine B, el-Mezgueldi M, and Notarianni G. Structural interactions between actin, tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments. Acta Physiol Scand 164: 401–414, 1998.[CrossRef][ISI][Medline]

26. McGuffee LJ, Mercure J, and Little SA. Three-dimensional structure of dense bodies in rabbit renal artery smooth muscle. Anat Rec 229: 499–504, 1991.[CrossRef][ISI][Medline]

27. Mehta D, Tang DD, Wu MF, Atkinson S, and Gunst SJ. Role of Rho in Ca2+-insensitive contraction and paxillin tyrosine phosphorylation in smooth muscle. Am J Physiol Cell Physiol 279: C308–C318, 2000.[Abstract/Free Full Text]

28. Minajeva A, Kulke M, Fernandez JM, and Linke WA. Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils. Biophys J 80: 1442–1451, 2001.[Abstract/Free Full Text]

29. Minekus J and van Mastrigt R. Length dependence of the contractility of pig detrusor smooth muscle fibres. Urol Res 29: 126–133, 2001.[CrossRef][ISI][Medline]

30. Mullins L. Effect of stretching on the properties of rubber. J Rubber Res 16: 275–289, 1947.

31. Mullins L. Softening of rubber by deformation. Rubber Chem Technol 42: 339–362, 1969.

32. Murphy RA. Mechanics of vascular smooth muscle. In: Handbook of Physiology: The Cardiovascular System. Bethesda, MD: Am. Physiol. Soc., 1980, sect. 2, vol. II, p. 325–351.

33. Murphy RA. Muscle cells of hollow organs. News Physiol Sci 3: 124–128, 1988.[ISI]

34. Murphy RA. What is special about smooth muscle? The significance of covalent crossbridge regulation. FASEB J 8: 311–318, 1994.[Abstract/Free Full Text]

35. Rasmussen H, Takuwa Y, and Park S. Protein kinase C in the regulation of smooth muscle contraction. FASEB J 1: 177–185, 1987.[Abstract/Free Full Text]

36. Ratz PH. High {alpha}1-adrenergic receptor occupancy decreases relaxing potency of nifedipine by increasing myosin light chain phosphorylation. Circ Res 72: 1308–1316, 1993.[Abstract]

37. Ratz PH. Receptor activation induces short-term modulation of arterial contractions: memory in vascular smooth muscle. Am J Physiol Cell Physiol 269: C417–C423, 1995.[Abstract/Free Full Text]

38. Ratz PH. Regulation of ERK phosphorylation in differentiated arterial muscle of the rabbit. Am J Physiol Heart Circ Physiol 281: H114–H123, 2001.[Abstract/Free Full Text]

39. Ratz PH and Miner AS. Length-dependent regulation of basal myosin phosphorylation and force in detrusor smooth muscle. Am J Physiol Regul Integr Comp Physiol 284: R1063–R1070, 2003.[Abstract/Free Full Text]

40. Ratz PH, Hai CM, and Murphy RA. Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle. Am J Physiol Cell Physiol 256: C96–C100, 1989.[Abstract/Free Full Text]

41. Ratz PH, Meehl JT, and Eddinger TJ. RhoA kinase and protein kinase C participate in regulation of rabbit stomach fundus smooth muscle contraction. Br J Pharmacol 137: 983–992, 2002.[CrossRef][ISI][Medline]

42. Ratz PH, Berg KM, Urban NH, and Miner AS. Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus. Am J Physiol Cell Physiology 288: C769–C783, 2005.[ISI]

43. Roach MR and Burton AC. The reason for the shape of the distensibility curves of arteries. Can J Med Sci 35: 681–690, 1957.

44. Sastry SK and Burridge K. Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics. Exp Cell Res 261: 25–36, 2000.[CrossRef][ISI][Medline]

45. Shenfeld OZ, Morgan CW, and Ratz PH. Bethanechol activates a post-receptor negative feedback mechanism in rabbit urinary bladder smooth muscle. J Urol 159: 252–257, 1998.[CrossRef][ISI][Medline]

46. Shenfeld OZ, McCammon KA, Blackmore PF, and Ratz PH. Rapid effects of estrogen and progesterone on tone and spontaneous rhythmic contractions of the rabbit bladder. Urol Res 27: 386–392, 1999.[CrossRef][ISI][Medline]

47. Siegman MJ, Butler TM, Mooers SU, and Davies RE. Calcium-dependent resistance to stretch and stress relaxation in resting smooth muscles. Am J Physiol 231: 1501–1508, 1976.[Abstract/Free Full Text]

48. Sjuve R, Arner A, Li Z, Mies B, Paulin D, Schmittner M, and Small JV. Mechanical alterations in smooth muscle from mice lacking desmin. J Muscle Res Cell Motil 19: 415–429, 1998.[CrossRef][ISI][Medline]

49. Somlyo AP and Somlyo AV. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185, 2000.[Abstract/Free Full Text]

50. Somlyo AP and Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83: 1325–1358, 2003.[Abstract/Free Full Text]

51. Stossel TP, Condeelis J, Cooley L, Hartwig JH, Noegel A, Schleicher M, and Shapiro SS. Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol 2: 138–145, 2001.[CrossRef][ISI][Medline]

52. Straight AF, Cheung A, Limouze J, Chen I, Westwood NJ, Sellers JR, and Mitchison TJ. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299: 1743–1747, 2003.[Abstract/Free Full Text]

53. Sutherland C and Walsh MP. Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin. J Biol Chem 264: 578–583, 1989.[Abstract/Free Full Text]

54. Szymanski PT. Calponin (CaP) as a latch-bridge protein–a new concept in regulation of contractility in smooth muscles. J Muscle Res Cell Motil 25: 7–19, 2004.[CrossRef][ISI][Medline]

55. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–994, 1997.[CrossRef][ISI][Medline]

56. Urban NH, Berg KM, and Ratz PH. K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle. Am J Physiol Cell Physiol 285: C1377–C1385, 2003.[Abstract/Free Full Text]

57. Uvelius B. Isometric and isotonic length-tension relations and variations in longitudinal smooth muscle from rabbit urinary bladder. Acta Physiol Scand 97: 1–12, 1976.[ISI][Medline]

58. Wang K and Singer SJ. Interaction of filamin with F-actin in solution. Proc Natl Acad Sci USA 74: 2021–2025, 1977.[Abstract/Free Full Text]

59. Wang Z, Jiang H, Yang ZQ, and Chacko S. Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity. Proc Natl Acad Sci USA 94: 11899–11904, 1997.[Abstract/Free Full Text]

60. Yamasaki R, Berri M, Wu Y, Trombitas K, McNabb M, Kellermayer MS, Witt C, Labeit D, Labeit S, Greaser M, and Granzier H. Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1. Biophys J 81: 2297–2313, 2001.[Abstract/Free Full Text]





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