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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm; 2Department of Urology, Lund University, Lund, Sweden; 3Laboratory of Physiology and Physiopathology, UMR7079-Centre National de la Recherche Scientifique, Pierre and Marie Curie University; 4Laboratoire de Biologie Moléculaire de la Différentiation, Paris VII University, Paris, France; and 5Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna, Austria
Submitted 12 December 2007 ; accepted in final form 12 June 2008
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ABSTRACT |
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smooth muscle; intermediate filaments; transgenic mice; hypertrophy
In animal models of adaptive growth of small intestine and urinary bladder (13, 15), it has been shown that the increase in smooth muscle mass is to a large extent due to hypertrophy of the smooth muscle cells. This structural change is associated with a synthesis of contractile proteins, with altered myosin isoform expression patterns (23, 30), and with mechanical alterations including a lower active force (5), an increased tissue stiffness (9), and a lower maximal shortening velocity (Vmax; Ref. 30).
Intermediate (10 nm) filaments in smooth muscle are formed by polymerization of the proteins vimentin and desmin and constitute one of the major components of the cytoskeleton (34). In the vascular system, smooth muscles of larger elastic arteries contain mainly vimentin (10), while smooth muscles in some vessels can coexpress vimentin and desmin (7). In visceral smooth muscle such as the bladder, desmin is the sole component of the intermediate filaments (11). In the rat bladder tissue, vimentin is found in nonsmooth muscle cells in the serosa and submucosal layer, as well as in a small number of cells in the interstitium between the muscle bundles (32).
A striking feature of hypertrophic smooth muscle cells, originally observed by Gabella (13), is a relative increase in the number of intermediate filaments. This finding has been confirmed in hypertrophied smooth muscles of the portal vein (6, 22) and hypertrophied urinary bladders of experimental animals and humans (23, 24). The function of the increased number of intermediate filaments in hypertrophying smooth muscle is unknown, but they might have a mechanical function in the hypertrophied cells or possibly be involved in the linking of cellular stretch signals to the nuclear chromatin as proposed for cardiac muscle hypertrophy (8).
In smooth muscle, the intermediate filaments have been proposed to be coupled to the contractile units through the dense bodies (33, 27). We have previously shown that complete absence of intermediate filaments in visceral smooth muscle of a desmin-deficient mouse is associated with a pronounced decrease in active force (31). These results suggest that the desmin intermediate filaments have a role in the intracellular transmission of active force in smooth muscle, possibly by alignment of contractile units and cell-cell or cell-matrix coupling. Earlier studies (18, 19, 25, 35) on striated muscles from desmin knockout mice have shown extensive damage to muscle fibers, with misalignment of sarcomeres and cardiomyocyte degeneration. The disruption of the muscles increased with age and was found to be most pronounced in more active muscles such as the diaphragm and soleus (19). In a previous study (31), we have shown that smooth muscle in urinary bladder and vas deferens of desmin-deficient mice was structurally intact, except for the lack of intermediate filaments. However, as the ultrastructure of smooth muscle is much less organized than striated muscle, misalignment of individual sarcomere equivalents would be virtually impossible to detect. It should be noted that previous studies on smooth muscle from desmin-deficient mice were performed on muscles that were not exposed to pathophysiological conditions in the animal. It is possible that experiments under conditions of increased strain in vivo can reveal further structural and mechanical functions of the intermediate filament system.
In the present study, we have applied a mouse model for urinary bladder hypertrophy in response to partial outflow obstruction. We have used this model in desmin-deficient mice to answer the following questions: 1) Can adaptive growth of the urinary bladder occur in the absence of intermediate filaments, thus excluding that the intermediate filaments have a role in transmitting the mechanical cell-surface strain signal to the nucleus? 2) Are the intermediate filaments required for function and synthesis of the newly formed contractile components in the hypertrophic smooth muscle? 3) Is the increase in intermediate filaments affecting the mechanical properties of hypertrophied smooth muscle?
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METHODS |
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Mechanical Experiments
Isometric force was measured in intact smooth muscle preparations attached with 6-0 silk at one end to a steel rod equipped with a micrometer screw and at the other end to a Grass FT03 force transducer (Grass Medical Instruments, Quincy, MA). The preparations were held in temperature-controlled (37°C) 25-ml open organ baths and gassed with 95% O2-5% CO2. The passive tension was adjusted to 
2 mN, and the preparations were allowed to equilibrate for 45 min in PSS. The muscle preparations were activated with high-K+ by addition of 80 mM KCl and 2.5 mM CaCl2 to the bathing solution. The force was recorded for 5 min. Thereafter, the muscles were allowed to relax in Ca2+-free PSS for 10 min. The high-K+ contractures were repeated until stable force responses were obtained (usually 2–3 times). The length of the muscle strips was then reduced to give zero passive force. The preparations were thereafter adjusted to increasing lengths and activated at each length to give the length-force relationship of the muscle. The peak active force during the high-K+ contracture and the relaxed passive force at the end of the period in Ca2+-free PSS solution were recorded. When the optimal length (L0) for active force was exceeded by 60%, the length of the muscles was adjusted to L0. K+-contractures were then repeated at L0until stable responses were obtained. The rate of relaxation in Ca2+-free solution after the K+ contractures at optimal length was determined as time to relax to half maximal tension (T
). After the series of active contractions at different lengths, the muscles were allowed to relax in Ca2+-free PSS solution containing papaverine (0.1 mM) and EGTA (1 mM). The length-passive force relationship was again recorded by stretching the muscle to the previous lengths used during the first length-force determination. The muscles were allowed to relax 2–5 min after each stretch to reach a force plateau. The preparation length (L0) was measured with a microscope equipped with an ocular scale. At the end of the experiments, the muscle strips were fixed at L0overnight at 22°C in 2.5% glutaraldehyde in phosphate buffer for subsequent microscopy. After fixation, the muscles were stored at 4°C in 125 mM cacodylate buffer at pH 7.4.
Microscopic Analysis Determination of cross-sectional area. Light microscopy was performed on the urinary bladder preparations used in the mechanical experiments to determine the cross-sectional area of the smooth muscle layer. The glutaraldehyde preparations were postfixed in 1% OsO4, dehydrated, and embedded in Epon. Transverse sections with a thickness of 3 µm were cut and stained with Azur III methylene blue. The preparations were cut at two levels, and at each level at least three sections were analyzed. The smooth muscle area was determined by light microscopy using a video system connected to a computer.
Isotonic Quick Release Experiments
Bladder preparations were chemically skinned using Triton X-100 and stored at –15°C in a glycerol containing solution as described by Arner and Hellstrand (4) before being mounted for isotonic quick release experiments to determine the shortening velocity. Thin muscle strips (0.3 x 3 mm) were cut and attached with aluminum foil between an AME 801 force transducer (SensoNor, Horten, Norway) and a lever that could be clamped and released with electromagnetic relays. The afterload on the preparation could be adjusted by varying the load on the lever. The skinned muscle preparations were mounted and stretched to a length where passive tension was just noticeable in a relaxing solution. The force-velocity relation was determined in maximally activated thio-phosphorylated fibers as described previously (2). Maximal thio-phosphorylation was achieved by treating the muscle with Ca2+ containing (pCa 4.5) rigor solution containing calmodulin (0.5 µM) and ATP-
-S (2 mM) for 15 min. The skinned muscles were contracted by introducing MgATP and PCr. At the plateau of contraction, 12–15 releases to different afterloads were performed. Force and length signals were recorded for each release. After each series of releases, the preparations were again thiophosphorylated for about 10 min. Each preparation was subjected to two series of releases. Since the shortening velocity decreases with time after release, the velocity was determined at a fixed point in time (100 ms) as described previously (4). Afterload (P) and velocity (V) were fitted to the Hill (16) equation: V = b(1 – P/P0)/(P/P0 + a/P0), where P0 is isometric force and a and b are constants. Vmax was calculated as bP0/a. The shortening velocity is given as muscle lengths per second.
Quantitative Gel Electrophoresis Frozen detrusor tissue devoid of mucosa was weighed and homogenized in SDS buffer (50 µl/mg tissue wet wt, composition see Solutions). The homogenate was boiled and centrifuged, and the supernatant was loaded on 8% polyacrylamide gels together with standard protein (skeletal actin). Three different amounts of actin and volumes of sample were run in parallel. After electrophoresis, the gels were stained overnight with Coomassie blue, destained, and scanned with a GS-30 densitometer (Hoefer, San Francisco, CA). A linear relationship between the area under the actin peak of the scans and the amount of the actin standard was found. The actin concentration in the sample was calculated and related to the sample weight. The myosin-to-actin ratio was calculated from the area under the peaks of myosin and actin bands. The myosin concentration was calculated from the actin concentration and the actin-to-myosin heavy chain ratio. To determine the relative contents of desmin in Des+/+ urinary bladders using Western blot, the tissue extracts were separated on polyacrylamide gels as described above, transferred to nitrocellulose membranes, and reacted with a desmin antibody and evaluated with ECL. The intensity of the desmin ECL reaction was related to the actin intensity of Coomassie-stained gels run in parallel.
Ultrastructural Analysis Samples were fixed at approximate optimal muscle length in a solution containing 3% paraformaldehyde and 1% glutaraldehyde, postfixed in 2% OsO4, block stained with uranyl acetate, and embedded in Araldite (Taab, Aldermaston, UK). Thin sections were cut on a Reichert ultramicrotome and studied using a Zeiss EM10A microscope.
Solutions Intact muscle preparations were held in a PSS containing the following (in mM): 118 NaCl, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 KH2PO4, 24 NaHCO3, and 5.5 glucose. The Ca2+-free solution was made by omitting CaCl2. High-K+ solution was made by the addition of 80 mM KCl from a 3-M stock. The solutions were gassed with 5% CO2 in O2 to obtain a pH of 7.4 at 37°C.
Experiments on skinned muscle were performed at 22°C in solutions containing the following (in mM): 4 EGTA, 30 TES, 2 Mg2+, 3.2 MgATP, 12 PCr, and 0.5 mg/ml creatine phosphokinase. Free Ca2+ (given in pCa = –log([Ca2+]) units) was adjusted by adding EGTA as K2EGTA or K2CaEGTA. Ionic strength was adjusted with KCl to 150 mM and pH to 6.9 with KOH. The composition of the solutions was calculated as described by Arner (3). Rigor solutions were made by omitting PCr and MgATP.
SDS sample buffer contained 25 mM Tris·HCl (pH 6.8), 2% SDS, 5% mercaptoethanol, and 10% glycerol.
Statistics All values are means ± SE with the number of animals within parenthesis. Statistical comparisons were made using the Student's t-test for unpaired data (two-tailed).
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RESULTS |
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3-fold increase in weight in both groups. Four out of eight obstructed Des–/–bladders showed signs of pathological changes with large hemorrhagic cysts at the bladder base (2 animals) or rupture of the muscular layer from pole to pole with the inner mucosa remaining intact (2 animals). The obstructed Des–/–animals also showed signs of diffuse hemorrhagic areas in the serosa of the bladder to a varying extent. However, no leakage of urine into the peritoneal cavity or macroscopic signs of peritonitis were observed in any of the animals. Such changes were not observed in the unobstructed Des+/+ or Des–/–bladders or in the obstructed Des+/+ bladders. Since the Des–/–and Des+/+ animals were of similar weight and subjected to a standardized degree of obstruction, the growth-inducing challenge was thus similar in the two groups.
Length-Stress Relationships
Figure 1 shows length-stress relationships of urinary bladder preparations from nonobstructed Des+/+ (A) and Des–/–(B) and obstructed Des+/+ (C) and Des–/–(D) mice. The circumference was calculated from the relationship between the preparation length and circumference during the initial dissection (see METHODS). The cross-sectional area of the smooth muscle at optimal length (circumference) for active force (L0) was determined for each preparation, using morphometry on histological sections, and force is given as stress (force/area at L0). The muscle preparations were subjected to two series of length-passive stress determinations giving essentially the same shape of the relationships, the first in Ca2+-free PSS (not shown) and a second in Ca2+-free EGTA/papaverine PSS (Fig. 1) to ensure that the muscle was completely relaxed. The passive stress values appeared lower at all lengths in the nonoperated Des–/–bladders compared with the nonoperated Des+/+ bladders, although the Des–/–muscles still could maintain a substantial passive tension when stretched. In the bladders subjected to urinary outflow obstruction, the passive length-stress relationships of both the Des–/–and Des+/+urinary bladders appeared somewhat steeper. The preparations showed no signs of breaking at maximum stretch (1.6 times L0) or during development of active tension at that degree of stretch. As seen in Table 2, passive stress at optimal length was lower in the Des–/–groups. To further evaluate the passive elastic properties of the different groups, a mono-exponential function T = T* e
(L/L0–1) (12) was fitted to the passive stress (T) relative length (L/L0) relationships; T* is a constant describing passive stress at optimal length (L0) and describes the curvature of the relationship (i.e., the dependence of stiffness of the tissue on stress). The curvature () obtained by fitting the above equation to the mean values of the passive-stress vs length (L/L0) relations of the obstructed bladders is somewhat steeper ( values of nonobstructed group: Des+/+ = 4.09 and Des–/–= 4.02; obstructed group: Des+/+ = 6.65 and Des–/–= 10.05). The active stress was significantly reduced in the Des+/+ obstructed and the Des–/– animals (Fig. 1; Table 2). We observed that the rate of relaxation after a K+ contracture was significantly lower in the obstructed Des–/–urinary bladders (Table 2).
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Force-Velocity Relations Skinned muscle preparations were prepared from urinary bladder tissue from obstructed and nonobstructed Des+/+ and Des–/–mice. By using thiophosphorylated preparations, we obtained force-velocity data for control and obstructed bladders in the maximally activated state. The Vmax of skinned preparations from bladders of Des–/–mice was significantly lower compared with Vmax of Des+/+ mice (Table 2). In both Des+/+ and Des–/–animals, the obstructed bladders had a lower Vmax compared with the nonobstructed bladders.
Tissue Amounts and Concentrations of Actin, Myosin, and Intermediate Filament Proteins The tissue amounts of contractile and cytoskeletal proteins were determined using quantitative polyacrylamide SDS gel electrophoresis. Skeletal actin was used as the standard. Total amounts of actin and myosin were calculated from the concentration of actin and myosin and the total weight of the bladder. When unobstructed Des–/–and Des+/+ bladders were compared, no significant differences were found in the contents of actin and myosin (Table 3). The total actin and myosin contents per bladder increased approximately two- to threefold in the obstructed Des–/–and Des+/+ bladders. The myosin concentration of the bladder and the myosin-to-actin ratio were significantly lowered in the obstructed Des+/+ animals compared with their nonobstructed controls (Table 3).
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Tissue Morphology Figure 2 shows electron microscopy pictures from obstructed urinary bladders from Des+/+ and Des–/– mice. In the former group, intermediate filaments were observed whereas they were lacking in the Des–/–. We have previously shown that intermediate filaments are absent in other smooth muscles from these Des–/–mice (31). Although no morphometry was performed, the impression was, based on low magnification electron micrographs, that there was no pronounced difference in the contours or cross-sectional area of cells in the Des+/+ and Des–/–groups. The cell profiles appeared more rounded in the hypertrophic bladders of both Des+/+ and Des–/–groups.
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DISCUSSION |
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We show that the desmin intermediate filaments are not required for hypertrophic growth of the urinary bladder. Stretch of the smooth muscle cells seems to be the main physical initiator of the growth (14), and the cytoskeleton has been proposed to be a part of the signaling from cell surface to the nucleus, conveying and sensing mechanical strain (cf. 17). Our data thus suggest that the cellular signaling from the extracellular matrix via the cell membrane to the nucleus does not require the intermediate filaments but rather rely on other pathways.
The growth of the bladder after partial urinary obstruction was associated with a significant increase in the total amounts of actin and myosin in the desmin-deficient animals in a similar manner as in the wild-type mice. This indicates that the ability of the smooth muscle cells to generate new contractile proteins was not impaired by the lack of intermediate filaments. Interestingly, the expression of myosin and actin was not increased to a larger extent in the desmin-deficient mice, and the maximal active stress was substantially lower. This would suggest that the content of contractile proteins and their synthesis during hypertrophy are not regulated primarily to achieve a specific active stress level. In the obstructed wild-type mice, the tissue concentration of myosin and the myosin-to-actin ratio decreased with growth, suggesting that the synthesis of contractile proteins is not completely in pace with the increase in smooth muscle content at this point in time. The measurements of active force per cross-sectional area of the intact muscle showed a lower force generation in the obstructed Des+/+ group. This result is similar to the results in hypertrophic rat urinary bladder (5, 23) where a decrease in myosin concentration was correlated with a lower active force.
We have in a previous study (31) shown that the reduction in active force of Des–/–smooth muscle compared with Des+/+ muscle is not due to an alteration in the excitation-contraction coupling or in the content of contractile proteins. In a study by Thornell et al. (35) on Des–/– cardiomyocytes, supercontraction of myofibrils was evident, suggesting sarcomere inhomogeneity. Similar observations were made in skeletal muscle (19), where the muscle and the sarcomeric structures seem to assemble correctly at birth, but as the animal grows older, an increased irregularity of sarcomere structure and muscle degeneration were observed. As we have suggested previously (31, 37), it is possible that the lower active force of smooth muscle from Des–/–mice reflects dysfunctional force transmission, possibly via inhomogeneities in the sarcomere equivalent length distribution or in the coupling between the contractile units. This points to the possibility that desmin may play a vital role in the organisation of dense bodies and in the optimal alignment of the contractile machinery for force generation.
We describe a pronounced increase of desmin in hypertrophic urinary bladder from the wild-type mice, which is consistent with previous observations from hypertrophic urinary bladder and portal vein from the rat and rabbit (6, 22, 23) and ileum from guinea pig (13). Vimentin and desmin have been suggested to coexist in the same smooth muscle cell from the portal vein of the rabbit (7). In the urinary bladder of the rat, vimentin is found in the serosa and in a small population of interstitial cells (32). The Western blot analysis of the mouse urinary bladder revealed low amounts of vimentin, most likely of nonsmooth muscle origin, consistent with the results from the rat bladder. We could not detect any changes in vimentin expression after obstruction in Des+/+ mice or in Des–/–compared with the Des+/+group. In addition, electron micrographs of both nonobstructed and obstructed Des–/–mice failed to reveal any intermediate filaments. These results suggest that desmin and vimentin are independently regulated in the urinary bladder wall, i.e., an increase in desmin expression during hypertrophy does not lead to an increase in vimentin and a complete removal of desmin is not compensated by an increase in vimentin.
Gabella (13) showed that low numbers of contractile filaments. Our results show an interesting difference between obstructed Des+/+ and Des–/–bladders. The Des+/+ bladders that have hypertrophied have a lower active force than their controls, whereas there is no difference between control and obstructed Des–/– bladders. The increase in intermediate filament proteins during hypertrophy in the wild-type mice could be involved in the decrease in active force. A simple explanation would be that the increase in intermediate filament decreases the number of contractile filaments per unit cross sectional area. Intermediate filaments appear to have a role in active force transmission in normal cells, but why their number increases during obstruction is not clear. Intermediate filaments might be required for alignment of sarcomere equivalents or be important for other functions, e.g., maintaining cell structure in the hypertrophic smooth muscle.
Passive tension was lower in the urinary bladder tissue of the Des–/– mice, suggesting that the desmin intermediate filaments contribute to some extent to the passive resistance to stretch in the length range where active force is generated. However, since passive tension of the detrusor muscle could be maintained in the desmin-deficient mice, the intermediate filament system does not appear to be the sole support of passive tension in the urinary bladder muscle. During hypertrophy of both Des+/+ and Des–/– animals, the stiffness of the passive elasticity was increased. This is consistent with previous observations from hypertrophic rat urinary bladders (9). Since passive stiffness was increased also in the Des–/– bladders after obstruction, the increase in stiffness during hypertrophy is not caused by an increase in intermediate filaments.
In our mechanical analysis, we focused on the properties of the isolated bladder wall. The advantage compared, e.g., to a cystometrical analysis is that the influence of sensory and motor nerve activity are eliminated making conclusions regarding the influence on wall mechanics by the intermediate filament system more straight forward. However, the more complicated in vivo functional effects on micturition, including compensatory mechanisms, induced by the absence of detrusor desmin remain to be elucidated.
Urinary outflow obstruction in the rat induces a dramatic hypertrophic response of the urinary bladder (15). The relatively limited weight gain (3-fold) in the obstructed mouse bladder compared with that in the rat (6- to 10-fold) might explain why the cell cross-sectional area did not seem to increase to a larger extent in the obstructed bladders in this study. The cell profiles appeared, however, more rounded in the obstructed bladders although a detailed morphometric analysis was not performed.
Tissue degeneration has been observed in striated muscle from Des–/–mice (18, 25, 35). In a study by Thornell et al. (35), degenerating cardiomyocytes were seen almost immediately after birth, with an accumulation of macrophages, fibrosis, and calcification, preferentially in the interventricular septum and the wall of the right ventricle. It was concluded that the stress related to lengthening of the myocytes might be a factor involved in the formation of the lesions and not the workload per se, as the left ventricle was largely unaffected. We have shown that smooth muscle from vas deferens from Des–/–mice did not have any apparent alterations in ultrastructure, except for the absence of intermediate filaments (31). In the present study, we observed macroscopic lesions, with rupture of the muscle layer, in several of the obstructed urinary bladders from Des–/–mice. This was not observed in the nonobstructed Des–/–or obstructed Des+/+ mice, indicating that the intermediate filament system is important for maintaining tissue integrity in situations of increased strain. In the study by Janmey et al. (17) on vimentin, it was proposed that intermediate filaments are mechanical integrators that prevent cell breakage under higher strains. Under normal conditions, the urinary bladder is not exposed to higher transmural pressure (21). During outflow obstruction, the bladder pressure is increased severalfold and is maintained over longer periods of time (21). The strain and tension on the bladder wall are further increased, due to the law of Laplace, as the bladder radius increases during hypertrophy. In the present study, outflow obstruction could have a severe effect on the smooth muscle tissue, as obstructed bladders of the Des–/–mice showed signs of rupture of the bladder wall. As discussed above, the obstructed detrusor from Des–/–mice was somewhat more compliant and this might in combination with a lower active tension result in an increased strain on noncontractile components with subsequent damage to the bladder wall. The tissue degeneration was not a generalized phenomenon, since no apparent change in the tissue architecture or cellular organisation was noted in the detrusor preparations from the midportion of the bladder used in our mechanical experiments and structural investigation. Rather, the rupture appeared to be focal and primarily localized to the ventral surface of the bladder. This could reflect a generally thinner wall, a predominantly longitudinal muscle bundle orientation (15), or an increased strain in this region. How the absence of desmin intermediate filaments leads to tissue degeneration is, however, unknown at present.
We have previously shown that hypertrophy of the rat urinary bladder is associated with a decreased shortening velocity and altered expression of myosin heavy and light chain isoforms (30). In slow skeletal and cardiac muscle of Des–/–mice, an increased expression of the slower β-heavy chain of myosin has been described previously (1). In the present study, we found that the maximal shortening velocity was decreased in the Des–/–mice compared with the Des+/+ mice and that the velocity was further decreased after obstruction. In addition, the half-time for relaxation was longer in the obstructed Des–/–. The relaxation parameter might reflect slow deactivation kinetics (e.g., Ca2+ removal or cellular deactivation) as well as slow cross-bridge cycling. The maximal shortening velocity was measured under fully activated conditions in the permeabilized preparations and thus suggests a slow cross-bridge cycling as one important change. This can reflect a change in smooth muscle myosin isoform expression as has previously been reported for obstructed rat bladders (30). An interesting possibility is that the expression of nonmuscle myosins is altered in hypertrophy. These myosin isoforms have been shown to be mainly located in the serosa and interstitial cells in rat and mouse bladder (20, 32). However, nonmuscle myosin can form filaments and have mechanical functions in the newborn bladder (20) and in elastic arteries of adult animals (29). If this isoform contributes to the mechanical properties and the slow kinetics remains to be elucidated. From a physiological perspective, it is possible that the changes toward a slower, more economical, contractile phenotype reflect adaptations of the bladder muscle to a situation when the obstruction, and/or the lower active force, necessitates a longer time of active contraction to empty the bladder.
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ACKNOWLEDGMENTS |
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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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