Linden, David R.

Age-related gastrointestinal dysfunction is common, but the mechanisms of aging-associated smooth muscle failure remain unclear. We show that aging in mice slows whole gut and colonic transit, increases regional stiffness, and reduces smooth muscle contractility. Inducible smooth muscle cell (SMC)– specific deletion of Piezo1 preserved youthful transit and force generation, whereas Piezo1 activation in young mice phenocopied aging-associated transit delay. Single-cell transcriptomics, RNA velocity, stiffness-controlled cell and tissue cultures, and pharmacologic studies revealed that Piezo1 couples increased stiffness to Ca 2+ –calcineurin–NFAT signaling, loss of contractile gene programs, leading to age- related contractile loss and contractile-to-synthetic SMC remodeling and gut wall stiffening. Human intestinal SMCs supported conservation of this pathway, and PIEZO1 gain-of-function carriers showed a trend toward delayed colonic transit. Thus, maladaptive SMC Piezo1 mechanotransduction is a targetable mechanism of aging-associated gut dysmotility. Aging alters organ structure, yet how these changes drive functional decline remains poorly defined. We show that progressive stiffening of the gut with age activates the mechanosensitive ion channel Piezo1 in smooth muscle cells, initiating a maladaptive response that impairs contractility and slows intestinal transit. On the other hand, deletion of smooth muscle Piezo1 prevents age-related gut dysmotility. These findings reveal that age-related physical shifts actively drive functional decline through mechanotransduction pathways. By identifying Piezo1 as a critical link between tissue mechanics and organ aging, this work provides a framework for understanding how mechanical cues contribute to age-related dysfunction in the gut and possibly other smooth muscle organs.

Abstract Interstitial cells of Cajal (ICC) regulate gastrointestinal motility by generating electrical slow waves that orchestrate downstream calcium (Ca 2 + ) waves and coordinate smooth muscle contractions. A reduced ICC number has been reported in diabetic gastroparesis patients and in mouse models of delayed gastric emptying (DGE). However, functional changes in ICC propagating across the gastric regions in intact stomach are not well understood. The aim of this study was to develop an imaging approach to visualize ICC Ca 2+ waves in mouse stomach and determine spatiotemporal changes in mice with DGE. Tamoxifen‐treated B6.GCaMP5‐KitCreERT2 female mice were used as non‐diabetic controls (Non‐Db), while mice treated with streptozocin to induce diabetes were categorized as having normal (Db‐NGE) or delayed (Db‐DGE) gastric emptying based on emptying of solids. The ICC Ca 2+ network was imaged from excised intact mouse stomachs and subsequently opened along the lesser curvature and pinned flat. ICC Ca 2+ waves typically propagated along the greater curvature from the fundus–corpus line toward the antrum. The propagation length of ICC Ca 2+ activity was significantly decreased in Db‐DGE compared to Non‐Db and Db‐NGE and correlated to longer GE times. There was also a decrease in frequency, and the velocity was significantly faster in stomachs from Db‐DGE compared to Non‐Db and Db‐NGE. All experimental groups exhibited similar proportions of aberrant Ca 2+ propagation patterns (e.g., retrograde waves), but ectopic initiation only occurred in Db‐DGE stomachs. Thus, DGE is possibly driven by dysfunction in Ca 2+ activity in the ICC network that propagates rapidly over short distances, resulting in uncoordinated, non‐phasic muscle activity. image Key points Dysfunction of the interstitial cells of Cajal (ICC) has been implicated in diabetic gastroparesis but the changes in spatiotemporal properties of ICC network activity are not known. We developed an ex vivo Ca 2+ imaging approach to visualize ICC network activity across the entire stomach and determined changes in ICC Ca 2+ activity in a diabetic mouse model of delayed gastric emptying (GE). Decreased frequency and increased velocity of ICC Ca 2+ activity was observed in stomachs of diabetic mice with delayed GE. Ectopic initiation was a key feature associated with delayed GE. The propagation length of ICC Ca 2+ activity was inversely correlated to GE times and significantly reduced in stomach preparations from diabetic mice with delayed GE.

ABSTRACT Background The circadian rhythm regulates gastrointestinal motility. In humans and preclinical models, such as rodents, whole gut transit (WGT) is slower during the rest phase compared to the active phase. Investigators typically study GI transit in rodents during the day, which is their rest phase, rather than during the night, which is their active phase. A circadian rhythm reversal in which mice are in a dark room during the working day (reverse light) allows studies on nocturnal animals during their active phase and has been previously shown to reduce WGT time. GI motility is often disrupted in individuals with disorders of gut–brain interaction (DGBI), which are female predominant. However, the effect of circadian rhythm on regional transit and sex dependence of the differences is not known, as most motility studies looking at circadian rhythm reversal are done in male mice. Methods We tested C57BL/6 wild‐type male and female mice in rest (12 h of light during the day) and active (reverse cycle for 2 weeks: 12 h of dark during the day) phases. We noted female estrous cycle by visual inspection. We performed carmine WGT by monitoring time‐lapse videos of pellet production. We performed fluorescence imaging of excised intestines 30 min after gavage to assess percent fluorescence for each GI region and then examined small intestinal transit (SIT) by measuring geometric center and leading edge. For colonic transit, we monitored bead expulsion time from distal colon to anus. Key Results Compared to rest phase, in the active phase, like male mice, female mice had (1) faster WGT, (2) increased frequency of pellet expulsion in the first 3 h of transit, (3) and greater total pellet production. Both male and female mice in their active phase exhibited (4) more contrast emptied from the stomach and they had (5) further leading edge of fluorescence and (6) geometric center, in SIT, and (7) faster colonic bead expulsion times. There were no significant sex differences in the active phase of WGT. In SIT, male mice had further leading edge in the rest phase than female mice, but this difference was not seen in the active phase, and in colonic transit, male mice in both the active and rest phases had faster bead expulsion than female mice. Conclusions Mice in the active phase have faster regional transit in small and large bowel than mice in the rest phase that collectively contributes to faster WGT times in the active phase of both male and female mice. These findings highlight the importance of circadian biology in sex‐dependent rodent GI transit.

Using 32 immune cell surface markers, we identified 23 monocyte clusters in murine blood. Diabetic gastroparesis was associated with a significant decrease in two circulating classical monocyte-like clusters and an upregulation of the Ccr2-Ccl2 axis in the gastric muscularis propria, suggesting increased tissue monocyte migration. This study offers new targets by pointing to a possible role for two classical monocyte subsets connected to the Ccr2- Ccl2 axis.

AbstractBackgroundIn preclinical studies whole gut transit (WGT) in mice is a gold‐standard “leading‐edge” approach that measures the time between orogastric gavage of carmine red and defecation of the first carmine red pellet. Transit studies in humans are performed during the active day because GI motility and transit are suppressed during the night. Since mice are nocturnal, WGT studies traditionally done during the day occur during their rest phase. How circadian rhythm affects WGT in mice is not known.MethodsWe used an automated approach for high temporal resolution uninterrupted testing of mouse WGT and activity. We housed wild‐type Bl6/C57 mice under the standard 12 h light–dark cycles. At 8 weeks, we performed carmine red orogastric gavage and assessed WGT during Light (rest) conditions. Then, we exposed mice to a reverse 12 h light–dark cycle for 2 weeks and tested them in the Dark (active) under red light conditions. Timelapse videos were analyzed to quantify activity and to timestamp all pellets, and multiple parameters were analyzed.Key ResultWhen complementary light cycle reversal experiments were performed, we found a significant increase in mouse activity when mice were tested during their Dark (active) phase, compared to their Light (rest) phase. In mice tested in the Active phase compared to the Rest phase, we found a significant acceleration in WGT, increased rate and total number of pellets produced, and more pellet clustering. These data show that the mice tested in the Active phase have important differences in activity that correlate with multiple alterations in gastrointestinal transit.Conclusion & InferencesDuring the Active phase mice have faster WGT, produce more pellets, and cluster their output compared to testing in the Rest phase. Like in humans, circadian rhythm is an important consideration for transit studies in mice, and a simple reverse light cycle approach facilitates further studies on the role of circadian rhythm in GI motility.

The human gastrointestinal (GI) tract is home to trillions of microorganisms. There is dynamic interaction between the gut microbiota and the human host, but how gut microbiota modulates GI physiology is largely unknown. A recent study from our laboratory showed that human gut microbiota increases host serotonin (5‐hydroxytryptamine, 5‐HT) biosynthesis. 5‐HT is an important neurotransmitter and a paracrine messenger in the GI tract that increases intestinal secretion primarily through 5‐HT 3 and 5‐HT 4 receptors. In this study we investigated whether gut microbiota modulate 5‐HT 3 R and 5‐HT 4 R expression and host secretory response to 5‐HT. Methods We used proximal colon mucosa‐submucosa preparations from germ free (GF) and humanized (ex‐GF colonized with human bacteria for 4–5 weeks; HM) male mice. The change in short circuit current (ΔIsc) was determined in response to increasing concentrations of 5‐HT (0.003–300μM) on the submucosal side either alone or in the presence of 5‐HT 3 R antagonist ondansetron (100nM) in an Ussing chamber. Receptor expression was quantified using rt‐PCR (normalized to L32) and western blot (normalized to GAPDH). Immunohistochemistry (IHC) was used to determine localization of 5‐HT 3 R in the paraformaldehyde‐fixed 10μm sections from proximal colonic epithelium. Differences between groups were tested using Mann‐Whitney U (Mdn; IQ range) with P ‐value lower than 0.05 considered significant. Results 5‐HT induced ΔIsc was significantly higher in GF than HM mice (218; 184–274 vs 163; 132–182 μA/cm 2 , n=6, P = 0.04). To determine if this change was due to an alteration in 5‐HTR, we quantified receptor expression. We found significantly higher 5‐HT 3 R mRNA expression (1.82; 1.08–2.80 vs 1.00; 0.33–1.38, n=8–9, P = 0.046) in GF compared to HM mice, but no change in 5‐HT 4 R expression. 5‐HT 3 R protein expression was also significantly higher in GF mice compared to HM (4.62; 1.06–6.14 vs 1.00; 1–1.3, n= 11–12, P = 0.01) mice. There was a significantly higher ΔIsc in GF compared to HM (87.1; 51.7–149 vs 37.0; 19.5–44 μA/cm 2 , n=5, P = 0.02) mice, following application of 2‐methyl 5‐hydroxytryptamine hydrochloride (selective 5‐HT 3 R agonist) suggesting increased receptor expression augments secretory response. Furthermore, ondansetron significantly blocked 5‐HT induced maximum ΔIsc in GF mice (201; 189–260 vs 117.6; 92–149 μA/cm 2 , n=6–8, P = 0.001) but not in HM mice (163; 132–182 vs 120; 107–150 μA/cm 2 , n=6, P = 0.24). To determine the expression of 5‐HT 3 R in mouse colon mucosa‐submucosa we used 5‐HT 3 –tagged GFP mice. IHC identified 5‐HT 3 R‐GFP positive cells in the mouse colonic epithelium. To ascertain if the significant difference in 5‐HT‐induced ΔIsc between GF and HM mice (188; 152–200 vs 125; 99–139 μA/cm 2 , n=5, P = 0.008) is driven by epithelial 5‐HT 3 R, we measured the response in the presence of TTX (block neuronal transmission). TTX (500 nM) had no effect of ondansetron inhibition of 5‐HT‐induced ΔIsc, suggesting a role for epithelial 5HT 3 R. Conclusion Gut microbial colonization decreases host secretory response to 5‐HT by downregulating 5‐HT 3 R expression. Epithelial 5‐HT 3 R may be important in gut microbiota modulation of intestinal secretion Support or Funding Information NIH K08 DK

AbstractBackgroundGastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase‐1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin‐10 (IL‐10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL‐10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno‐associated viruses (AAVs) in diabetic mice with delayed GE.MethodsC57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9‐cre, scAAV9‐GFP, or scAAV9‐HO1 particles. Changes to GE were assessed weekly utilizing our [13C]‐octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression.Key ResultsDelayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9‐cre and in 4/5 mice that received the scAAV9‐GFP, whereas mice that received scAAV9‐HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4–6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9‐GFP‐treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE.Conclusions and InferencesInjection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

Abstract Organs that face external environments, such as skin and gut, are lined by epithelia, which have two functions – to provide a semi-permeable barrier and to sense stimuli. The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections between intestinal epithelial cells and enteric neurons remain poorly resolved, which leaves us with limited mechanistic understanding of their function. We describe the development of a two-compartment microfluidic device for modeling neuro-epithelial interactions, and apply it to form the gut’s neuro-epithelial connections. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform for dissecting structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.