Du, Peng

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl–channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Flmice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+transients.NEW & NOTEWORTHY The Ca2+-activated Cl−channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+transients in myenteric ICC but Ca2+transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4 .

Key points Ano1, a Ca2+‐activated Cl− channel, is expressed in interstitial cells of Cajal (ICC) throughout the gut. We report here that it is required to maintain coordinated Ca2+ transients within myenteric ICC of mouse small intestine. Ca2+ transients in Ano1 WT mice were rhythmic and coordinated whereas uncoordinated Ca2+ transients were seen in knockout mice. Ca2+ transients were un‐coordinated following pharmacological block of Ano1 in WT mice using niflumic acid, 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid and 4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid disodium salt. Transient knockdown of Ano1 in organotypic cultures with short hairpin RNA to Ano1 in WT tissues also caused loss of coordinated Ca2+ transients. Contractility of Ano1 knockout mouse intestinal segments in organ bath experiments was significantly decreased, less coordinated and non‐rhythmic. Spatiotemporal maps from knockout mouse small intestine also showed loss of phasic contractile activity. This study provides important information on the basic mechanisms driving coordinated contractile activity in the gastrointestinal tract. AbstractInterstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca2+‐activated Cl− channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca2+ transients between myenteric ICC (ICC‐MY) of small intestine. First, we found spontaneous Ca2+ transients in ICC‐MY in both Ano1 WT and knockout (KO) mice. However, Ca2+ transients within the ICC‐MY network in Ano1 KO mice were uncoordinated, while ICC‐MY Ca2+ transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca2+ transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA‐mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca2+ transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca2+ transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non‐rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca2+ transients in ICC‐MY.

Depletion of interstitial cells of Cajal (ICC) networks is known to occur in several gastrointestinal motility disorders. Although confocal microscopy can effectively image and visualize the spatial distribution of ICC networks, current descriptors of ICC depletion are limited to cell numbers and volume computations. Spatial changes in ICC network structural properties have not been quantified. Given that ICC generate electrical signals, the organization of a network may also affect physiology. In this study, six numerical metrics were formulated to automatically determine complex ICC network structural properties from confocal images: density , thickness , hole size , contact ratio , connectivity and anisotropy . These metrics were validated and applied in proof-of-concept studies to quantitatively determine jejunal ICC network changes in mouse models with decreased ( 5-HT 2 B receptor knockout (KO)) and normal ( Ano1 KO) ICC numbers, and during post-natal network maturation. Results revealed a novel remodelling phenomenon occurring during ICC depletion, namely a spatial rearrangement of ICC and the preferential longitudinal alignment. In the post-natal networks, an apparent pruning of the ICC network was demonstrated. The metrics developed here enabled the first detailed quantitative analyses of structural changes that may occur in ICC networks during depletion and development.