Smoothelin-A Is Essential for Functional Intestinal Smooth Muscle Contractility in Mice
2005; Elsevier BV; Volume: 129; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.08.018
ISSN1528-0012
AutoresPetra Niessen, Sander S. Rensen, Jan van Deursen, Joris De Man, Ann De Laet, Jean‐Marie Vanderwinden, Thilo Wedel, Darren J. Baker, Pieter A. Doevendans, Marten H. Hofker, Marion J. Gijbels, Guillaume van Eys,
Tópico(s)Thyroid and Parathyroid Surgery
ResumoBackground & Aims: In patients with chronic intestinal pseudo-obstruction, intestinal motility is disturbed by either nervous or myogenic aberrations. The cause of the myogenic form is unknown, but it is likely to originate in the contractile apparatus of the smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Experimental data indicate a role for smoothelins in smooth muscle contraction. A smoothelin-deficient mouse model may help to establish the role of smoothelin-A in intestinal contraction and provide a model for myogenic chronic intestinal pseudo-obstruction. Methods: We used gene targeting to investigate the function of smoothelin-A in intestinal tissues. By deletion of exons 18, 19, and 20 from the smoothelin gene, the expression of both smoothelin isoforms was disrupted. The effects of the deficiency were evaluated by pathologic and physiologic analyses. Results: In smoothelin-A/B knockout mice, the intestine was fragile and less flexible compared with wild-type littermates. The circular and longitudinal muscle layers of the intestine were hypertrophic. Deficiency of smoothelin-A led to irregular slow wave patterns and impaired contraction of intestinal smooth muscle, leading to hampered transport in vivo. This caused obstructions that provoked intestinal diverticulosis and occasionally intestinal rupture. Conclusions: Smoothelin-A is essential for functional contractility of intestinal smooth muscle. Hampered intestinal transit in smoothelin-A/B knockout mice causes obstruction, starvation, and, ultimately, premature death. The pathology of mice lacking smoothelin-A is reminiscent of that seen in patients with chronic intestinal pseudo-obstruction. Background & Aims: In patients with chronic intestinal pseudo-obstruction, intestinal motility is disturbed by either nervous or myogenic aberrations. The cause of the myogenic form is unknown, but it is likely to originate in the contractile apparatus of the smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Experimental data indicate a role for smoothelins in smooth muscle contraction. A smoothelin-deficient mouse model may help to establish the role of smoothelin-A in intestinal contraction and provide a model for myogenic chronic intestinal pseudo-obstruction. Methods: We used gene targeting to investigate the function of smoothelin-A in intestinal tissues. By deletion of exons 18, 19, and 20 from the smoothelin gene, the expression of both smoothelin isoforms was disrupted. The effects of the deficiency were evaluated by pathologic and physiologic analyses. Results: In smoothelin-A/B knockout mice, the intestine was fragile and less flexible compared with wild-type littermates. The circular and longitudinal muscle layers of the intestine were hypertrophic. Deficiency of smoothelin-A led to irregular slow wave patterns and impaired contraction of intestinal smooth muscle, leading to hampered transport in vivo. This caused obstructions that provoked intestinal diverticulosis and occasionally intestinal rupture. Conclusions: Smoothelin-A is essential for functional contractility of intestinal smooth muscle. Hampered intestinal transit in smoothelin-A/B knockout mice causes obstruction, starvation, and, ultimately, premature death. The pathology of mice lacking smoothelin-A is reminiscent of that seen in patients with chronic intestinal pseudo-obstruction. The principal function of smooth muscle cells (SMCs) in the intestinal tract is to enable propulsion and mixing of food,1Kunze W.A. Furness J.B. The enteric nervous system and regulation of intestinal motility.Annu Rev Physiol. 1999; 61: 117-142Crossref PubMed Scopus (327) Google Scholar which improves the digestion of complex molecules and the absorption of nutrients. Coordinated contractions of the circular and longitudinal smooth muscle layers are responsible for peristalsis of the gastrointestinal tract. Contractions of SMCs are slower than those of skeletal and cardiac myocytes, but are more sustained. Hence, the composition of the contractile apparatus of SMCs differs from that of the striated muscle cells. In both cell types, actin–myosin interactions are at the basis of contraction. The contraction of striated muscle is well understood and accessory proteins, such as troponins, are known to be part of the organization of the contractile apparatus and determine the mode of contraction.2Herzog W. Ait-Haddou R. Considerations on muscle contraction.J Electromyogr Kinesiol. 2002; 12: 425-433Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 3Gordon A.M. Homsher E. Regnier M. Regulation of contraction in striated muscle.Physiol Rev. 2000; 80: 853-924Crossref PubMed Scopus (1316) Google Scholar The architecture and composition of contractile elements in SMCs, however, still is not understood fully. The contractile apparatus of SMCs is connected with the cytoskeleton via dense bodies. It consists of an actin–myosin axis complemented with structural muscle proteins, including α-actinin and tropomyosin, and more smooth muscle–specific proteins such as calponin, caldesmon, and smoothelin.4Owens G.K. Kumar M.S. Wamhoff B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol Rev. 2004; 84: 767-801Crossref PubMed Scopus (2556) Google Scholar, 5Szymanski P.T. Calponin (CaP) as a latch-bridge protein—a new concept in regulation of contractility in smooth muscles.J Muscle Res Cell Motil. 2004; 25: 7-19Crossref PubMed Scopus (33) Google Scholar Based on their expression pattern, smoothelins have been described as proteins specific for fully differentiated smooth muscle. The 2 major isoforms are smoothelin-A in visceral tissues such as the digestive tract, bladder, and prostate, and smoothelin-B in vascular tissues.6van Eys G.J. Voller M.C. Timmer E.D. Wehrens X.H. Small J.V. Schalken J.A. Ramaekers F.C. van der Loop F.T. Smoothelin expression characteristics development of a smooth muscle cell in vitro system and identification of a vascular variant.Cell Struct Funct. 1997; 22: 65-72Crossref PubMed Scopus (54) Google Scholar, 7Rensen S. Thijssen V. De Vries C. Doevendans P. Detera-Wadleigh S. Van Eys G. Expression of the smoothelin gene is mediated by alternative promoters.Cardiovasc Res. 2002; 55: 850-863Crossref PubMed Scopus (53) Google Scholar Both are found only in actively contracting smooth muscle tissues. Under pathologic conditions with impaired function of smooth muscle, such as aneurysms and restenosis, expression of smoothelins rapidly decreases.8Christen T. Verin V. Bochaton-Piallat M.L. Popowski Y. Ramaekers F. Debruyne P. Camenzind E. van Eys G. Gabbiani G. Mechanisms of neointima formation and remodeling in the porcine coronary artery.Circulation. 2001; 103: 882-888Crossref PubMed Scopus (145) Google Scholar, 9van der Loop F.T. Gabbiani G. Kohnen G. Ramaekers F.C. van Eys G.J. Differentiation of smooth muscle cells in human blood vessels as defined by smoothelin, a novel marker for the contractile phenotype.Arterioscler Thromb Vasc Biol. 1997; 17: 665-671Crossref PubMed Scopus (147) Google Scholar In cultured SMCs, smoothelins colocalize with smooth muscle α-actin (α-SMA) stress fibers.8Christen T. Verin V. Bochaton-Piallat M.L. Popowski Y. Ramaekers F. Debruyne P. Camenzind E. van Eys G. Gabbiani G. Mechanisms of neointima formation and remodeling in the porcine coronary artery.Circulation. 2001; 103: 882-888Crossref PubMed Scopus (145) Google Scholar, 10van der Loop F.T. Schaart G. Timmer E.D. Ramaekers F.C. van Eys G.J. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.J Cell Biol. 1996; 134: 401-411Crossref PubMed Scopus (214) Google Scholar Recently, we showed in vitro that smoothelins can bind physically to α-SMA under normal physiologic conditions.11Niessen P. Clement S. Fontao L. Chaponnier C. Teunissen B. Rensen S. van Eys G. Gabbiani G. Biochemical evidence for interaction between smoothelin and filamentous actin.Exp Cell Res. 2004; 292: 170-178Crossref PubMed Scopus (39) Google Scholar These findings point toward a direct role of smoothelin in contraction. If SMCs are brought into culture, smoothelin expression is down-regulated rapidly,6van Eys G.J. Voller M.C. Timmer E.D. Wehrens X.H. Small J.V. Schalken J.A. Ramaekers F.C. van der Loop F.T. Smoothelin expression characteristics development of a smooth muscle cell in vitro system and identification of a vascular variant.Cell Struct Funct. 1997; 22: 65-72Crossref PubMed Scopus (54) Google Scholar, 10van der Loop F.T. Schaart G. Timmer E.D. Ramaekers F.C. van Eys G.J. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.J Cell Biol. 1996; 134: 401-411Crossref PubMed Scopus (214) Google Scholar concomitant with their modulation toward a synthetic phenotype. This hampers in vitro investigations of the function of smoothelin in smooth muscle contraction. Therefore, assessment of the function of smoothelins in intestinal contractility requires an in vivo approach. Here, we report the interruption of the smoothelin gene in mice, leading to elimination of both smoothelin-A and smoothelin-B. The smoothelin knockout mice (Smtn-A/B−/−) show dysfunction of intestinal motility and contractility and die prematurely. The observed phenotype displays pathologies reminiscent of intestinal diverticulosis, chronic intestinal pseudo-obstruction (CIP), and hollow visceral myopathy in humans. To generate Smtn-A/B−/− mice, we replaced part of exon 18 and exon 19–20 with a neomycin resistance gene under the control of the thymidine kinase promoter in reverse orientation (Figure 1A). The targeting vector contained the thymidine kinase gene for negative selection. After electroporation of the PvuII-linearized construct into mouse L129/Sv embryonic stem cells, we selected neomycin-resistant clones with G418 (Invitrogen, Carlsbad, CA) and 1-[2-deoxy]2-fluoro-β-D-arabinofurasonyl (Invitrogen). DNA from resistant clones was screened by Southern blotting after PstI restriction digestion, using the 3′ probe indicated in Figure 1A. Embryonic stem cells from 2 independent targeted clones were injected into C57Bl/6 blastocysts and implanted into pseudopregnant C57Bl/6 females. Mating of the resulting chimeric males to C57Bl/6 females led to germline transmission of the targeted allele as detected by Southern blotting (Figure 1B). Because the mice had a mixed background (L129/Sv and C57Bl/6), we used littermates as controls. In the food transit experiment we used age- and sex-matched controls. The generation of smoothelin-B knockout mice (Smtn-B−/−) is described elsewhere (Rensen et al, unpublished data). In these mice, exons 3–6 of the smoothelin gene are removed and smoothelin-B synthesis is absent; however, smoothelin-A synthesis is not affected. All animal studies were performed according to protocols approved by the Committee on Animal Experimentation of the University of Maastricht. Total RNA was extracted from various tissues with Tri reagent (Sigma-Aldrich, Zwijndrecht, The Netherlands). Reverse transcription was performed using 1 μg of RNA in the RevertAid First-Strand complementary DNA synthesis kit (Fermentas, St. Leon-Rot, Germany). Expression of smoothelin messenger RNA in Smtn-A/B−/− mice was investigated by reverse-transcription polymerase chain reaction using smoothelin-B–specific primers 1F 5′-CCAGGGGGCAGTATGAAGAC-3′ and 1R 5′-CGCAGGTGGTTGTAGAGCGA-3′ and common smoothelin primers 2F 5′-GAGGAGCGCAAGCTGATCA-3′ and 2R 5′-CTGCTGGTGCTGAGAAGGGT-3′. Reverse-transcription polymerase chain reaction products were cloned and sequenced. Intestinal tissue homogenates (n = 5) were prepared in buffer (.25 mol/L sucrose, .01 mol/L Tris-HCl pH 7.4, 2 mmol/L ethylenediaminetetraacetic acid) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentration was measured with the BCA Protein Assay Kit (Pierce, Rockford, IL) and 15 μg was loaded onto a 9% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. Proteins were blotted on a polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Roosendaal, The Netherlands) and blocked overnight in phosphate-buffered saline containing .2% Tween-20 and 5% Marvel at 4°C. α-SMA was detected using the monoclonal antibody 1A4 (DAKO, Glostrup, Denmark) and a secondary rabbit anti-mouse antibody conjugated with horseradish peroxidase (DAKO). Smooth muscle myosin heavy chain was detected with the rabbit polyclonal immunoglobulin G bt-562 (Campro Scientific, Veenendaal, The Netherlands) followed by donkey-anti-rabbit horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Bands were visualized by enhanced chemiluminescence. Subsequently, signals were digitized and analyzed with Quantity One software (Bio-Rad Laboratories, Hercules, CA). Organs from mice aged from 0 days to 6 months were fixed overnight in 3.7% formaldehyde in phosphate-buffered saline, embedded in paraffin, sectioned, and stained with H&E. Sirius Red staining was performed for the detection of collagen. Samples of intestine (and several other tissues) were snap-frozen in liquid nitrogen–precooled isopentane and embedded in OCT Tissue Tek compound (Sakura, Chicago, IL). Cryostat sections were stained with biotinylated mouse monoclonal R4A specific for smoothelin.12van der Heijden O.W. Essers Y.P. Fazzi G. Peeters L.L. De Mey J.G. van Eys G.J. Uterine artery remodeling and reproductive performance are impaired in endothelial nitric oxide synthase-deficient mice.Biol Reprod. 2005; 72: 1161-1168Crossref PubMed Scopus (82) Google Scholar The ABC-peroxidase kit (Vector Laboratories, Inc, Burlingame, CA) was used for detection, followed by diaminobenzidine tetrahydrochloride staining and hematoxylin counterstaining. Interstitial cells of Cajal (ICCs) were identified by antibody against c-kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).13Rumessen J.J. Vanderwinden J.M. Interstitial cells in the musculature of the gastrointestinal tract Cajal and beyond.Int Rev Cytol. 2003; 229: 115-208Crossref PubMed Scopus (92) Google Scholar Full-thickness biopsy specimens from ileum were obtained from a patient with CIP. Age-matched control specimens were obtained from patients with diseases unrelated to gastrointestinal motility disorders. The use of human tissues was approved by the Medical Institutional Ethics Committee of the Faculty of Medicine, University of Luebeck, Germany. Tissues were processed as described by Vanderwinden et al.14Vanderwinden J.M. Rumessen J.J. Liu H. Descamps D. De Laet M.H. Vanderhaeghen J.J. Interstitial cells of Cajal in human colon and in Hirschsprung’s disease.Gastroenterology. 1996; 111: 901-910Abstract Full Text PDF PubMed Scopus (270) Google Scholar For electron microscopy, tissues were fixed in 3% phosphate-buffered glutaraldehyde for 24 hours and postfixed for 1 hour in 1% osmium tetroxide. Tissues then were dehydrated through a graded ethanol series and routinely embedded in Epon (SPI Supplies, West Chester, PA). Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined in a Philips CM100 electron microscope (Philips, Eindhoven, The Netherlands). To determine the intestinal function, a whole-gut transit time test was used as previously described.15van der Zee R. Welling G.W. A simple method to determine the transit time in mice.Z Versuchstierkd. 1983; 25: 233-237PubMed Google Scholar We injected 100 μL of 6% carmine (Sigma-Aldrich C1022) in phosphate-buffered saline (pH 7.0) into the stomachs of 6 Smtn-A/B+/+, Smtn-A/B+/−, and Smtn-A/B−/−, and 5 Smtn-B+/+ and Smtn-B−/− mice, and monitored their feces for the first appearance of red dye. After sedation with .15 μL Nembutal (Ceva Sante Animale BV, Maassluis, The Netherlands) by intraperitoneal injection, .3 mL of barium sulfate suspension (polibar 1 g/mL, E-Z-EM Inc, Lake Success, NY; diluted 1:4 with water) was injected into the stomachs of 2 Smtn-A/B−/− and 2 Smtn-A/B+/+ mice (12 weeks old). Progression through the gastrointestinal tract was followed by continuous radiographic examination (60 kV, 1.6 mA). Digital radiographic images were taken every 10 minutes with a Philips Diagnost 1997 device (Philips Medical Systems, Best, The Netherlands). Intestinal contractility was studied on 4–6-week-old mice (n = 6 for each group). Before anesthetizing mice with diethyl ether, we fasted them for 24 hours with free access to water. The small intestine of Smtn-A/B−/− and Smtn-A/B+/+ littermates (and of Smtn-B−/− and Smtn-B+/+ littermates) was removed and put in ice-cold aerated Krebs–Ringer solution. A 10-cm segment of the jejunum was opened along the longitudinal axis and the mucosa was removed. Longitudinal muscle strips of 6.0 mm of the jejunum were mounted in organ baths (5 mL) filled with Krebs–Ringer solution, maintained at 37°C, and aerated with a mixture of 5% CO2 and 95% O2. The muscle strips were positioned between 2 platinum ring electrodes (distance, 10 mm; diameter of rings, 3 mm) that were mounted on a Plexiglas (or Perspex) rod. The lower end of the muscle strip was fixed and the other end of the muscle strip was connected to a strain gauge transducer (Scaime, Annemasse, France) for continuous recording of isometric tension. After an equilibration period of 30 minutes during which the strips were washed every 5 minutes, the muscle strips were contracted with .1 μmol/L carbachol. After washout of carbachol, the strips were stretched (increments of 5 mN). After stabilization of the basal tone, muscle strips again were contracted with .1 μmol/L carbachol. This procedure was repeated until the contraction to .1 μmol/L carbachol was maximal. This point was taken as the point of the optimal length–tension relation.16Pelckmans P.A. Boeckxstaens G.E. Van Maercke Y.M. Herman A.G. Verbeuren T.J. Acetylcholine is an indirect inhibitory transmitter in the canine ileocolonic junction.Eur J Pharmacol. 1989; 170: 235-242Crossref PubMed Scopus (38) Google Scholar Muscle strips then were allowed to equilibrate for 60 minutes before starting the experiment. During the equilibration period, the muscle strips were washed every 15 minutes with Krebs–Ringer solution. The contractile effect of electrical field stimulation (.5–8 Hz, 40 V, pulse width, 1 ms; pulse train, 10 s), enteric nerves, carbachol (1 nmol/L to 1 μmol/L), prostaglandin F2α (1 nmol/L to 10 μmol/L), substance-P (.1–100 nmol/L), serotonin (1 nmol/L to 10 μmol/L), and KCl (50 mmol/L) was investigated. To block inhibitory responses to nitric oxide, contractions were studied in the presence of L-nitroarginine, a blocker of nitric oxide synthase. An adjacent jejunal segment was fixed in 4% formaldehyde for histologic examination and determination of the cross-sectional area. Contractions were normalized to the cross-sectional area of the longitudinal jejunal muscle layer. A standard microelectrode technique was used to record slow waves from the SMCs of the small intestine. Pieces of duodenum (2 cm beyond the pyloric sphincter) from the same mice as used for the intestinal contractility studies were used. The segments were opened along the mesenteric border and the mucosa and submucosa were removed. A muscle strip (15 × 6 mm) then was pinned, serosal side down, to the Sylgard floor (Dow Corning Europe, La Hulper, Belgium) of a recording chamber that was placed on the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan). The tissue was superfused continuously (10 mL/min; temperature, 36.5°C–37°C) with oxygenated Krebs–Ringer solution. Throughout the experiment, the L-type Ca2+ blocker nicardipine (1 μmol/L) (Sigma Chemical, St. Louis, MO) was present in the superfusion solution to reduce contraction of the intestinal smooth muscle.17Kunze W.A. Bertrand P.P. Furness J.B. Bornstein J.C. Influence of the mucosa on the excitability of myenteric neurons.Neuroscience. 1997; 76: 619-634Crossref PubMed Scopus (61) Google Scholar, 18Brookes S.J. Ewart W.R. Wingate D.L. Intracellular recordings from myenteric neurones in the human colon.J Physiol. 1987; 390: 305-318PubMed Google Scholar Intracellular recordings of the SMCs were made with borosilicate glass microelectrodes (1-mm outer diameter; Clarc Electromedical Instruments, Reading, UK) pulled on a P-97 Brown–Flaming micropipette puller (Sutter Instrument Co., Novato, CA). The electrodes were back-filled with 1 mol/L KCl (resistance, 50–70 mol/LΩ). The electrode was positioned by a micromanipulator (Narishige MO388; Narishige Scientific Instrument Lab, Toyko, Japan). Passive electrical events were measured with an Axoclamp 2A current-voltage amplifier (headstage HS-2 L, gain 0.1) connected to a Labmaster TL-1 DMA interface (Axon Instruments, Foster City, CA). The amplifier bridge circuit was balanced for each electrode before impalement, and capacitance was compensated for during injection of rectangular electrical current pulses (−.2 nA, 7 ms) through the microelectrode. After amplification and low-pass filtering (3 kHz), the signal was digitized at a sample rate of 5 kHz and stored on a computer using the pClamp 6.0.2 software (Axon Instruments). To determine the cross-sectional area of the longitudinal and circular muscle layer of the jejunum, cross-sections were stained with H&E. Images were captured using a Zeiss Axioscope (Zeiss, Göttingen, Germany) and a standard CCD camera (Stemmer Imaging, Puchheim, Germany), and analyzed with Leica QWin image analysis software (Leica Microsystems, Cambridge, UK). To determine the number of nuclei of the longitudinal and circular muscle layers of these sections, 2 opposite angular segments (15°) of the intestinal wall were selected and counted manually. Statistical significance was calculated by repeated-measures 1-way ANOVA followed by Bonferroni’s multiple comparison test, or 2-tailed (paired) Student t tests using Graphpad Prism software (version 4.0; GraphPad Software, Inc, San Diego, CA). Groups were considered significantly different when the P value was less than .05. Values are expressed as mean ± standard error of the mean. Smoothelin-A/B−/− mice were generated by removal of exons 18–20 (Figure 1A and B). These exons code for the calponin homology domain that is involved in actin binding.11Niessen P. Clement S. Fontao L. Chaponnier C. Teunissen B. Rensen S. van Eys G. Gabbiani G. Biochemical evidence for interaction between smoothelin and filamentous actin.Exp Cell Res. 2004; 292: 170-178Crossref PubMed Scopus (39) Google Scholar The deletion resulted in smaller smoothelin transcripts with an intact reading frame, corresponding to variants with exons 16 or 17 spliced to exon 21 (Figure 1C). However, smoothelin protein could not be detected in smooth muscle tissues of Smtn-A/B−/− mice using an antibody against an epitope upstream of the deletion (Figure 1D), indicating that the targeting resulted in a null-mutation. Western blot analyses showed that α-SMA was down-regulated in intestines of Smtn-A/B−/− mice and smooth muscle myosin heavy chain concentration was similar (Figure 1E). Smtn-A/B−/− pups were born at the expected Mendelian ratio (Smtn-A/B+/+ 30%; Smtn-A/B+/− 46%; Smtn-A/B−/− 24%, n = 415). At birth, Smtn-A/B−/− mice had a size and weight comparable with their wild-type littermates, but their postnatal growth was retarded overtly and surviving animals reached only approximately 80% of normal body weight. In the Smtn-A/B−/− mice, a remarkable absence of visceral and subcutaneous fat was observed. About 50% of Smtn-A/B−/−mice died before weaning at 3 weeks of age (Figure 1F). Smtn-A/B−/− mice that developed into adulthood were infertile. In contrast, Smtn-A/B+/− (and also Smtn-B−/− mice) had no overt abnormalities, were fertile, and had a normal life span. Motility of the intestine was determined by functional assays. Carmine, a red dye, was injected into the stomach and stool was monitored. Despite a comparable length of the intestinal tract, the whole-gut transit time of Smtn-A/B−/− mice was about twice as long as that of wild-type littermates (Figure 2A). Smtn-B−/− mice showed no increase in whole-gut transit time (Figure 2A). Contrast radiography after loading barium sulfate into the stomach of Smtn-A/B−/− mice confirmed the slower food transport in Smtn-A/B−/− mice and showed a greatly dilated proximal duodenum (Figure 2B), which was confirmed by macroscopic and microscopic observations. Twenty minutes after injection of barium sulfate, hardly any barium had passed the duodenum in Smtn-A/B−/− mice, whereas in Smtn-A/B+/+ mice barium had proceeded several centimeters into the jejunum. Isolated jejunal smooth muscle strips showed spontaneous spike activity in wild-type Smtn-A/B−/− and Smtn-B−/− mice. However, the amplitude and frequency of these spontaneous spikes were significantly lower in muscle strips of Smtn-A/B−/− mice (amplitude: Smtn-A/B+/+ 8.57 ± 1.23, Smtn-A/B−/− 2.44 ± .39; frequency: Smtn-A/B+/+ 43.09 ± 1.04, Smtn-A/B−/− 31.30 ± 1.70). When subjected to several contractile agonists, the forces generated by these strips were in Smtn-A/B−/− mice at least 4 times smaller than in control mice (Figure 3A). Electrical stimulation of excitatory enteric nerves showed at least a 5-fold stronger contraction in wild-type mice compared with Smtn-A/B−/− mice (Figure 3A). Also, receptor-independent contractions to KCl and receptor-dependent contractions to carbachol, prostaglandin F2α, substance P, and serotonin were significantly lower for Smtn-A/B−/− mice (Figure 3A). In the same experimental set-up, contractility of jejunal strips of Smtn-B−/− mice was comparable with Smtn-A/B+/+ mice. The slow waves measured in the SMCs of Smtn-A/B−/− mice were very irregular and variable, whereas in Smtn-A/B+/+ littermates, regular slow waves were observed in all cells tested (Figure 3B–D). The amplitude of the slow waves in Smtn-A/B−/− mice was significantly smaller (10 ± 1 mV) compared with Smtn-A/B+/+ mice (24 ± 1 mV), and the frequency of the slow waves was diminished significantly in Smtn-A/B−/− vs Smtn-A/B+/+ mice (33.7 ± 2.0 cycles/min vs 39.8 ± .6 cycles/min). In addition, there was a much higher variability in frequency in Smtn-A/B−/− (0–53 cycles/min) compared with Smtn-A/B+/+ littermates (35–46 cycles/min). In the same experimental set-up, the slow waves measured in the SMCs of Smtn-B−/− mice did not differ from wild-type mice. To ascertain whether the intestinal contractile dysfunction of Smtn-A/B−/− mice led to development of intestinal pathologies, we examined the morphology and histology of the digestive tract. No changes in the structure of the stomach were observed, although the structure of the intestine was compromised in Smtn-A/B−/− mice. The intestinal wall of Smtn-A/B−/− mice was stiff and fragile. The duodenum often was dilated (see also Figure 2B) and the mass of the jejunal smooth muscle was increased significantly compared with wild-type littermates (Table 1). The cross-sectional area of both longitudinal and circular muscle layers was approximately 4-fold greater in Smtn-A/B−/− mice than in wild-type mice (Figure 4A and B), which could be attributed largely to SMC hypertrophy (Table 1). Nuclei and cytoplasm of the intestinal SMCs were larger in smoothelin-deficient mice (Figures 4C, D, and 5). Extracellular matrix volume did not increase significantly as deduced from Sirius red staining and the distribution and appearance of the ICCs were normal (data not shown). Electron microscopy showed subtle differences in the ultrastructural organization of the intestinal SMCs. In Smtn-A/B−/− mice, the distribution of mitochondria and dense bodies appeared to be organized less strictly (Figure 5A and B). In addition, the micropinocytotic vesicles that are characteristic of smooth muscle were common in Smtn-A/B+/+ mice but almost absent in Smtn-A/B−/− mice (Figure 5C and D). Hypertrophy was less common and less extensive in the more distal part of the gut. Numerous diverticula were observed in all parts of the intestine with the exception of the cecum (Figure 4E and F). The diverticula were located predominantly on the mesenteric side. In 50% of the Smtn-A/B−/− mice, the diverticula already were present 24 hours after birth. The diverticula increased in number and size as the mice aged (Figure 4F). Inflammation of the diverticula and the mesentery was common, and perforations leading to sepsis and death were observed (Figure 4G). In addition, the intestinal villi in the affected areas were irregular and atrophic, which is probably a consequence of the disturbed motility and may be due to inflammation of the affected areas. The absence of fat deposition in Smtn-A/B−/− mice may be the consequence of diminished absorption as a result of the deteriorated structure of the villi. Inspection of the intestine of Smtn-B−/− mice did not show any abnormalities. Also, these mice had normal fat disposition, indicating sufficient food adsorption from the intestine. Furthermore, there are no morphologic changes in the intestinal vasculature of both Smtn-A/B−/− and Smtn-B−/− mice at this age.Table 1Smtn-A/B−/− Mice Show Jejunal Smooth Muscle HypertrophySmtn-A/B+/+ (n = 5)Smtn-A/B−/− (n = 4)P valueIntestinal CSAaValues shown in mm2.4.533 ± 1.2467.582 ± 1.780<.05Intestinal muscle CSAaValues shown in mm2..376 ± .1301.684 ± .381<.001Muscle area/intestinal area9% ± 3%23% ± 3%<.001Nuclei longitudinal musclebNumber of nuclei per 10% of the intestinal circumference.71 ± 1281 ± 20NSNuclei circular musclebNumber of nuclei per 10% of the intestinal circumference.51 ± 1379 ± 15<.05CSA, cross-sectional area; NS, not significant.a Values shown in mm2.b Number of nuclei per 10% of the intestinal circumference. Open table
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