Poster - #UCLA-49 - Keywords: intestine bowel motility dysmotility muscle contractility smooth muscle cell Piezo1 SIP syncytium Cre

Smooth Muscle Cell Piezo1 is Critical for Maintenance of the SIP Syncytium

Michael Matthews, Allison Flores, Geoanna Bautista, Yingjie Du, Nicolle Martin, Nam Phuong Nguyen, Elmira Tokhtaeva, RS Solorzano-Vargas, James C.Y. Dunn, Martin G. Martin
1.Department of Pediatrics, Division of Neonatal-Perinatal Medicine, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA 2. Department of Pediatrics, Division of Neonatology, University of California Davis Children’s Hospital, Sacramento, CA 95817 USA 3. Department of Materials Science and Engineering, University of California Los Angeles, Los Angeles, CA 90095, USA 4. University of California, Davis, School of Veterinary Medicine, Davis, CA 95616, USA 5.Department of Pathology, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA 6.Division of Pediatric Surgery, Departments of Surgery and Bioengineering, Stanford University School of Medicine, Stanford, CA, USA

ABSTRACT:
Introduction Proper assimilation of nutrients in the small intestine requires proper contraction of smooth muscle cells (SMCs) within the intestinal muscularis in order to efficiently propel digestive content. The SIP syncytium is critical for bowel motility and it consists of Interstitial cells of Cajal, platelet-derived growth factor receptor α cells (PDGFRα), and SMCs. SIP cells are electrically coupled to each other and work in an orchestrated manner to allow for normal contraction and relaxation of the small bowel. SIP cells respond to stretch, but the molecular mechanisms used to sense mechanical force remain obscure. Piezo1 is a mechanosensitive cation channel that responds to stretch and it is expressed in all layers of the gastrointestinal tract, including SMCs1-4. We hypothesized that Piezo1 in the SMCs of the intestinal muscularis plays an important role in gut motility, and that loss of Piezo1 in the SMCs will lead to altered expression of cell populations within the SIP syncytium. Materials and Methods Using the Piezo1/Myh11-ERT2/Cre-LoxP system, we generated a mouse model with a tamoxifen (Tam) inducible-Piezo1 knockout in the SMCs of the intestine (Piezo1∆SMC). We performed histologic and immunofluorescence staining of distal small intestinal segments to assess changes in the cell populations of the SIP syncytium in both Piezo1∆SMC and wild type (WT) control mice. Furthermore, we isolated distal small intestinal segments from both groups of mice and used wire myography to record isometric force tracings generated by the myogenic or peristaltic reflex at baseline and with augmented stretch5. Results Piezo1∆SMC leads to thinning of the muscularis When we performed histological analyses of the intestinal muscularis, we found that muscularis thickness was comparable between the two groups when analyzed at 10 days following tamoxifen treatment, but at 21-days post-treatment we found a significant decrease in the muscularis thickness of Piezo1∆SMC mice compared to controls. Piezo1∆SMC alters cell populations of the SIP syncytium We quantified the cell populations of the SIP syncytium at 10 days and 21 days following tamoxifen treatment. We found that both Interstitial cells of Cajal and PDGFR-α were significantly decreased in the knockout group starting at 10 days which is prior to the onset of muscularis thinning that occurs at 21 days. Furthermore, there was a continued decline of these cell populations to near cessation at 21 days post tamoxifen treatment. Piezo1∆SMC leads to impaired contractility Our ex vivo studies utilized isometric force measurements of isolated distal intestinal segments. Tracings of both groups were obtained at baseline and with acute stretch. Isolated intestinal segments of Piezo1ΔSMC mice had reduced amplitude (contractile strength) and area under the curve (AUC, integral force) at baseline (p<0.001) and with augmented stretch (p<0.0001) when compared to WT control mice. In Piezo1ΔSMC mice, the period between contractions was augmented (p<0.0001), and there was notable period irregularity and dysrhythmia that was associated with a greater coefficient of variation (29%) compared to controls (6.2%). Furthermore, the average duration of contraction at baseline and following acute mechanical stretching were also reduced in Piezo1ΔSMC mice compared to WT control mice (p<0.001). These findings suggest that loss of Piezo1 in the SMCs of the small intestine leads to an impaired myogenic response resulting in shorter, irregular and weaker contractions when compared to control mice. Conclusions Piezo1 deficient SMCs had decreased contractile strength that was asynchronous, dysrhythmic, and less frequent compared to control samples at baseline and following stretch. Moreover, ablation of SMC Piezo1 led to thinning of the muscularis and a decline in pacemaker cells within the muscularis, among other changes. Thus, SMC Piezo1 is important for the maintenance of normal, rhythmic gut motility and may have important implications for future therapies in patients with dysmotility resulting from a wide range of pathologies References 1. Sanders, K.M., Koh, S.D., Ro, S. & Ward, S.M. Regulation of gastrointestinal motility--insights from smooth muscle biology. Nat Rev Gastroenterol Hepatol 9, 633-645 (2012). 2. Joshi, V., Strege, P.R., Farrugia, G. & Beyder, A. Mechanotransduction in gastrointestinal smooth muscle cells: role of mechanosensitive ion channels. Am J Physiol Gastrointest Liver Physiol 320, G897-G906 (2021). 3. Gnanasambandam, R., Bae, C., Gottlieb, P.A. & Sachs, F. Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels. PLoS One 10, e0125503 (2015). 4. Nourse, J.L. & Pathak, M.M. How cells channel their stress: Interplay between Piezo1 and the cytoskeleton. Semin Cell Dev Biol 71, 3-12 (2017). 5. Daoud, F., et al. Inducible Deletion of YAP and TAZ in Adult Mouse Smooth Muscle Causes Rapid and Lethal Colonic Pseudo-Obstruction. Cell Mol Gastroenterol Hepatol 11, 623-637 (2021).