Cytoskeletal and Cytocontractile Protein Composition of Smooth Muscle Cells in Developing and Obstructed Rabbit Bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, α-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM α-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibers

Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des-/-) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des-/- muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des-/- and Des+/+. In width measurements of single fibers and bundles, Des-/- soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des-/- and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des-/- soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.     

Desmin regulates airway smooth muscle hypertrophy through early growth-responsive protein-1 and microRNA-26a

Bronchial biopsies of asthmatic patients show a negative correlation desmin expression in airway smooth muscle cell (ASMC) and airway hyperresponsiveness. We previously showed that desmin is an intracellular load-bearing protein, which influences airway compliance, lung recoil, and airway contractile responsiveness (Shardonofsky, F. R., Capetanaki, Y., and Boriek, A. M. (2006) Am. J. Physiol. Lung Cell. Mol. Physiol. 290, L890-L896). These results suggest that desmin may play an important role in ASMC homeostasis. Here, we report that ASMCs of desmin null mice (ASMCs(Des-/-)) show hypertrophy and up-regulation microRNA-26a (miR-26a). Knockdown of miR-26a in ASMCs(Des-/-) inhibits hypertrophy, whereas enforced expression of miR-26a in ASMCs(Des+/+) induces hypertrophy. We identify that Egr1 (early growth responsive protein-1) activates miR-26a promoter via enhanced phosphorylation of Erk1/2 in ASMCs(Des-/-). We show glycogen synthase kinase-3β (GSK-3β) as a target gene of miR-26a. Moreover, induction of ASMCs(Des-/-) hypertrophy by the Erk-1/2/Egr-1/miR-26a/GSK-3β pathway is consistent in human recombinant ASMCs, which stably suppresses 90% endogenous desmin expression. Overall, our data demonstrate a novel role for desmin as an anti-hypertrophic protein necessary for ASMC homeostasis and identifies desmin as a novel regulator of microRNA.     

Deletion of one SERCA2 allele confers protection against bladder wall hypertrophy in a murine model of partial bladder outlet obstruction

The sarco(endo)plasmic reticulum Ca(2+)-ATPase2 (SERCA2) is downregulated in cardiac hypertrophy with decompensation. We sought to determine whether mice heterozygous for the SERCA2 allele would develop greater bladder hypertrophy and decompensation than their wild-type littermates following partial bladder outlet obstruction (pBOO). We found that following 4 wk of surgically created pBOO, SERCA2 heterozygous murine bladders showed significantly less hypertrophy, improved in vitro cystometry performance, diminished expression of the slow myosin isoform A analyzed by RT-PCR, a significant drop in nuclear translocation of nuclear factor of activated T cells by EMSA, and decreased cell proliferation within the smooth muscle layer following 5-bromo-2'-deoxyuridine labeling compared with their wild-type littermates. Thus, in contrast to cardiac muscle, deletion of a SERCA2 allele confers protection against bladder hypertrophy in a murine model of pBOO. Compensatory mechanisms in heterozygous mice seem to be related to the calcineurin pathway. Further studies are underway to better define the molecular basis of this observation, which has potential clinical applications.     

Altered distribution of interstitial cells and innervation in the rat urinary bladder following spinal cord injury

Changes in the distribution of interstitial cells (IC) are reportedly associated with dysfunctional bladder. This study investigated whether spinal cord injury (SCI) resulted in changes to IC subpopulations (vimentin-positive with the ultrastructural profile of IC), smooth muscle and nerves within the bladder wall and correlated cellular remodelling with functional properties. Bladders from SCI (T8/9 transection) and sham-operated rats 5 weeks post-injury were used for ex vivo pressure-volume experiments or processed for morphological analysis with transmission electron microscopy (TEM) and light/confocal microscopy. Pressure-volume relationships revealed low-pressure, hypercompliance in SCI bladders indicative of decompensation. Extensive networks of vimentin-positive IC were typical in sham lamina propria and detrusor but were markedly reduced post-SCI; semi-quantitative analysis showed significant reduction. Nerves labelled with anti-neurofilament and anti-vAChT were notably decreased post-SCI. TEM revealed lamina propria IC and detrusor IC which formed close synaptic-like contacts with vesicle-containing nerve varicosities in shams. Lamina propria and detrusor IC were ultrastructurally damaged post-SCI with retracted/lost cell processes and were adjacent to areas of cellular debris and neuronal degradation. Smooth muscle hypertrophy was common to SCI tissues. In conclusion, IC populations in bladder wall were decreased 5 weeks post-SCI, accompanied with reduced innervation, smooth muscle hypertrophy and increased compliance. These novel findings indicate that bladder wall remodelling post-SCI affects the integrity of interactions between smooth muscle, nerves and IC, with compromised IC populations. Correlation between IC reduction and a hypercompliant phenotype suggests that disruption to bladder IC contribute to pathophysiological processes underpinning the dysfunctional SCI bladder.     

Transforming growth factor β1 involvement in the conversion of fibroblasts to smooth muscle cells in the rabbit bladder serosa

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors – transforming growth factor β1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte–monocyte colony-stimulating factor – were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (α-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast–smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These ‘ectopic’ muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor β1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells. © 1998 Chapman & Hall     

Adjustable passive stiffness in mouse bladder: regulated by Rho kinase and elevated following partial bladder outlet obstruction

Detrusor smooth muscle (DSM) contributes to bladder wall tension during filling, and bladder wall deformation affects the signaling system that leads to urgency. The length-passive tension (L-T(p)) relationship in rabbit DSM can adapt with length changes over time and exhibits adjustable passive stiffness (APS) characterized by a L-T(p) curve that is a function of both activation and strain history. Muscle activation with KCl, carbachol (CCh), or prostaglandin E(2) at short muscle lengths can increase APS that is revealed by elevated pseudo-steady-state T(p) at longer lengths compared with prior T(p) measurements at those lengths, and APS generation is inhibited by the Rho Kinase (ROCK) inhibitor H-1152. In the current study, mouse bladder strips exhibited both KCl- and CCh-induced APS. Whole mouse bladders demonstrated APS which was measured as an increase in pressure during passive filling in calcium-free solution following CCh precontraction compared with pressure during filling without precontraction. In addition, CCh-induced APS in whole mouse bladder was inhibited by H-1152, indicating that ROCK activity may regulate bladder compliance during filling. Furthermore, APS in whole mouse bladder was elevated 2 wk after partial bladder outlet obstruction, suggesting that APS may be relevant in diseases affecting bladder mechanics. The presence of APS in mouse bladder will permit future studies of APS regulatory pathways and potential alterations of APS in disease models using knockout transgenetic mice.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

Myocardin and microRNA‐1 modulate bladder activity through connexin 43 expression during post‐natal development

Overactive bladder (OAB) is a pervasive clinical problem involving alterations in both neurogenic and myogenic activity. While there has been some progress in understanding neurogenic inputs to OAB, the mechanisms controlling myogenic bladder activity are unclear. We report the involvement of myocardin (MYOCD) and microRNA‐1 (miR‐1) in the regulation of connexin 43 (GJA1), a major gap junction in bladder smooth muscle, and the collective role of these molecules during post‐natal bladder development. Wild‐type (WT) mouse bladders showed normal development from early post‐natal to adult including increases in bladder capacity and maintenance of normal sensitivity to cholinergic agents concurrent with down‐regulation of MYOCD and several smooth muscle cell (SMC) contractile genes. Myocardin heterozygous‐knockout mice exhibited reduced expression of Myocd mRNA and several SMC contractile genes concurrent with bladder SMC hypersensitivity that was mediated by gap junctions. In both cultured rat bladder SMC and in vivo bladders, MYOCD down‐regulated GJA1 expression through miR‐1 up‐regulation. Interestingly, adult myocardin heterozygous‐knockout mice showed normal increases in bladder and body weight but lower bladder capacity compared to WT mice. These results suggest that MYOCD down‐regulates GJA1 expression via miR‐1 up‐regulation, thereby contributing to maintenance of normal sensitivity and development of bladder capacity. J. Cell. Physiol. 228: 1819–1826, 2013. © 2013 Wiley Periodicals, Inc.     

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death

Partial bladder outlet obstruction of the rabbit bladder results in a rapid increase in mass characterized by remodeling of the bladder wall.In this study we investigated the effect of partial outlet obstruction on microvessel density and distribution in the bladder wall immunohistochemically using CD31 as a marker for vascular endothelium, and on blood flow using a fluorescent microsphere technique. Transverse sections of bladder wall were examined after 0 (unobstructed), 1, 3, 5, 7, and 14 days of obstruction. The microvasculature of obstructed rabbit bladder mucosa and detrusor smooth muscle apparently increased relative to augmentation of these compartments, while new vessels appeared in the thickening serosa. These vascular changes correlated with results showing that, at 1 week after obstruction, blood flow (ml/min/g tissue) to the mucosa and detrusor was unchanged.Thickening of the serosa, apparent after 1 day of obstruction, began before its vascularization. Then, 1 week post-obstruction, there was significant microvessel formation in the transition region between the detrusor smooth muscle and the increasing serosa; after 2 weeks, the entire serosa was vascularized. The vascularization of the muscle-serosal transition region and then the remaining serosa apparently precedes fibroblast differentiation, providing blood supply and thus metabolic support for this process.All obstructed rabbit bladders in this study were in a state of compensated function based on their weights. Our working hypothesis is that blood flow per unit tissue mass is normal in compensated obstructed bladders, thus allowing for normal contractile function and cellular metabolism. The results of this study indicate the presence of an augmented microvasculature in compensated obstructed rabbit bladders that provides adequate blood perfusion for normal function.     

Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology.     

Increased expression of non-muscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder

Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

Strain history and TGF-β1 induce urinary bladder wall smooth muscle remodeling and elastogenesis

Mechanical cues that trigger pathological remodeling in smooth muscle tissues remain largely unknown and are thought to be pivotal triggers for strain-induced remodeling. Thus, an understanding of the effects mechanical stimulation is important to elucidate underlying mechanisms of disease states and in the development of methods for smooth muscle tissue regeneration. For example, the urinary bladder wall (UBW) adaptation to spinal cord injury (SCI) includes extensive hypertrophy as well as increased collagen and elastin, all of which profoundly alter its mechanical response. In addition, the pro-fibrotic growth factor TGF-β1 is upregulated in pathologies of other smooth muscle tissues and may contribute to pathological remodeling outcomes. In the present study, we utilized an ex vivo organ culture system to investigate the response of UBW tissue under various strain-based mechanical stimuli and exogenous TGF-β1 to assess extracellular matrix (ECM) synthesis, mechanical responses, and bladder smooth muscle cell (BSMC) phenotype. Results indicated that a 0.5-Hz strain frequency triangular waveform stimulation at 15% strain resulted in fibrillar elastin production, collagen turnover, and a more compliant ECM. Further, this stretch regime induced changes in cell phenotype while the addition of TGF-β1 altered this phenotype. This phenotypic shift was further confirmed by passive strip biomechanical testing, whereby the bladder groups treated with TGF-β1 were more compliant than all other groups. TGF-β1 increased soluble collagen production in the cultured bladders. Overall, the 0.5-Hz strain-induced remodeling caused increased compliance due to elastogenesis, similar to that seen in early SCI bladders. Thus, organ culture of bladder strips can be used as an experimental model to examine ECM remodeling and cellular phenotypic shift and potentially elucidate BMSCs ability to produce fibrillar elastin using mechanical stretch either alone or in combination with growth factors.     

Structural and functional evaluation of branched myofibers lacking intermediate filaments

Intermediate filaments (IFs), composed of desmin and keratins, link myofibrils to each other and to the sarcolemma in skeletal muscle. Fast-twitch muscle of mice lacking the IF proteins, desmin and keratin 19 (K19), showed reduced specific force and increased susceptibility to injury in earlier studies. Here we tested the hypothesis that the number of malformed myofibers in mice lacking desmin (Des(-/-)), keratin 19 (K19(-/-)), or both IF proteins (double knockout, DKO) is increased and is coincident with altered excitation-contraction (EC) coupling Ca(2+) kinetics, as reported for mdx mice. We quantified the number of branched myofibers, characterized their organization with confocal and electron microscopy (EM), and compared the Ca(2+) kinetics of EC coupling in flexor digitorum brevis myofibers from adult Des(-/-), K19(-/-), or DKO mice and compared them to age-matched wild type (WT) and mdx myofibers. Consistent with our previous findings, 9.9% of mdx myofibers had visible malformations. Des(-/-) myofibers had more malformations (4.7%) than K19(-/-) (0.9%) or DKO (1.3%) myofibers. Confocal and EM imaging revealed no obvious changes in sarcomere misalignment at the branch points, and the neuromuscular junctions in the mutant mice, while more variably located, were limited to one per myofiber. Global, electrically evoked Ca(2+) signals showed a decrease in the rate of Ca(2+) uptake (decay rate) into the sarcoplasmic reticulum after Ca(2+) release, with the most profound effect in branched DKO myofibers (44% increase in uptake relative to WT). Although branched DKO myofibers showed significantly faster rates of Ca(2+) clearance, the milder branching phenotype observed in DKO muscle suggests that the absence of K19 corrects the defect created by the absence of desmin alone. Thus, there are complex roles for desmin-based and K19-based IFs in skeletal muscle, with the null and DKO mutations having different effects on Ca(2+) reuptake and myofiber branching.     

M2 receptors in genito-urinary smooth muscle pathology

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats which had their major pelvic ganglion bilaterally removed (denervation, DEN) or from rats in which the spinal cord was injured (SCI) via compression. DEN induced both hypertrophy (505+/-51 mg bladder weight) and a supersensitivity of the bladders to carbachol (EC50=0.7+/-0.1 uM). Some of the SCI rats regained the ability to void spontaneously (SPV). The bladders of these animals weighed 184+/-17 mg, significantly less than the bladders of non voiding rats (NV, 644+/-92 mg). The potency of carbachol was greater in bladder strips from NV SCI animals (EC50=0.54+/-0.1 uM) than either bladder strips from SPV SCI (EC50=0.93+/-0.3 microM), DEN or control (EC50=1.2+/-0.1 microM) animals. Antagonist affinities in control bladders for antagonism of carbachol induced contractions were consistent with M3 mediated contractions. Antagonist affinities in DEN bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.5) and para fluoro hexahydrosilodifenidol (p-F-HHSiD, 6.6); were consistent with M2 mediated contractions, although the methoctramine affinity (6.5) was consistent with M3 mediated contractions. p-F-HHSiD inhibited carbachol induced contraction with an affinity consistent with M2 receptors in bladders from NV SCI (pKb=6.4) animals and M3 receptors in bladders from SPV SCI animals (pKb=7.9). Subtype selective immunoprecipitation of muscarinic receptors revealed an increase in total and an increase in M2 receptor density with no change in M3 receptor density in bladders from DEN and NV SCI animals compared to normal or sham operated controls. M3 receptor density was lower in bladders from SPV SCI animals while the M2 receptor density was not different from control. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediate smooth muscle contraction in bladders from DEN and NV SCI rats.     

Diffusable growth factors induce bladder smooth muscle differentiation

Bladder smooth muscle differentiation is dependent on the presence of bladder epithelium. Previously, we have shown that direct contact between the epithelium and bladder mesenchyme (BLM) is necessary for this interaction. In this study, we tested the hypothesis that bladder smooth muscle can be induced via diffusable growth factors. Fourteen-day embryonic rat bladders were separated into bladder mesenchyme (prior to smooth muscle differentiation) and epithelium by enzymatic digestion and microdissection. Six in vitro experiments were performed with either direct cellular contact or no contact (1) 14-d embryonic bladder mesenchyme (BLM) alone (control), (Contact) (2) 14-d embryonic bladders intact (control), (3) 14-d embryonic bladder mesenchyme combined with BPH-1 cells (an epithelial prostate cell line) in direct contact, (4) 14-d embryonic bladder mesenchyme with recombined bladder epithelium (BLE) in direct contact, (No Contact) (5) 14-d embryonic bladder mesenchyme with BPH-1 prostatic epithelial cells cocultured in type 1 collagen gel on the bottom of the well, and (6) 14-d embryonic bladder mesenchyme with BPH-1 epithelium cultured in a monolayer on a transwell filter. In each case the bladder tissue was cultured on Millicell-CM 0.4-microm membranes for 7 d in plastic wells using serum free medium. Growth was assessed by observing the size of the bladder organoids in histologic cross section as well as the vertical height obtained in vitro. Immunohistochemical analysis of the tissue explants was performed to assess cellular differentiation with markers for smooth muscle alpha-actin and pancytokeratin to detect epithelial cells. Control (1) bladder mesenchyme grown alone did not exhibit growth or smooth muscle and epithelial differentiation. Contact experiments (2) intact embryonic bladder, (3) embryonic bladder mesenchyme recombined with BPH-1 cells, and (4) embryonic bladder mesenchyme recombined with urothelium each exhibited excellent growth and bladder smooth muscle and epithelial differentiation. Both noncontact experiments (5) and (6) exhibited growth as well as bladder smooth muscle and epithelial differentiation but to a subjectively lesser degree than the contact experiments. Direct contact of the epithelium with bladder mesenchyme provides the optimal environment for growth and smooth muscle differentiation. Smooth muscle growth and differentiation can also occur without direct cell to cell contact and is not specific to urothelium. This data supports the hypothesis that epithelium produces diffusable growth factors that induce bladder smooth muscle.     

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.