Farrugia, Gianrico

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gut microbes play a significant role in development and maintenance of intestinal functions, but can contribute to pathogenesis of gastrointestinal (GI) disorders when host‐microbiome relationship is perturbed. In this study we sought to examine how constipation affects microbiome, and how these alterations in turn affect the host. Adult C57Bl/6 mice were treated with 0.1% Loperamide in drinking water for 7 days to induce constipation. Loperamide‐treated mice had increased GI transit time, indicating constipation. Bacterial DNA analysis by HiSeq sequencing showed decreased Firmicutes and increased Bacteroidetes in constipated mice. Microbiome metabolic potential was measured with tetrazolium dye assay and showed shift toward increased sugar metabolism in Loperamide‐treated mice. Microbial metabolites were measured by GC‐MS, and showed lower butyrate concentration in constipated mice. To determine constipation effects on the host, we introduced microbiota from constipated donors to germ‐free recipients. These mice had delayed GI transit time. To determine mechanism for delayed GI transit we analyzed colon tissues by qRT‐PCR and western blotting analysis, and determined lower expression of nNos mRNA and protein levels in these mice. Overall, these results suggest that delayed GI transit shifts microbiome taxonomic and metabolic profile, and these changes further contribute to development of constipation. Grant Funding Source : NIH NIDDK T32 DK07074

ABSTRACT Intestinal barrier function and microbiota are integrally related and play critical roles in maintenance of host physiology. Sex is a key biologic variable for several disorders. Our aim was to determine sex‐based differences in response to perturbation and subsequent recovery of intestinal barrier function and microbiota in healthy humans. Twenty‐three volunteers underwent duodenal biopsies, mucosal impedance, and in vivo permeability measurement. Permeability testing was repeated after administration of indomethacin, then 4 to 6 wk after its discontinuation. Duodenal and fecal microbiota composition was determined using 16S rRNA amplicon sequencing. Healthy women had lower intestinal permeability and higher duodenal and fecal microbial diversity than healthy men. Intestinal permeability increases after indomethacin administration in both sexes. However, only women demonstrated decreased fecal microbial diversity, including an increase in Prevotella abundance, after indomethacin administration. Duodenal microbiota composition did not show sex‐specific changes. The increase in permeability and microbiota changes normalized after discontinuation of indomethacin. In summary, women have lower intestinal permeability and higher microbial diversity. Intestinal permeability is sensitive to perturbation but recovers to baseline. Gut microbiota in women is sensitive to perturbation but appears to be more stable in men. Sex‐based differences in intestinal barrier function and microbiome should be considered in future studies.—Edogawa, S., Peters, S. A., Jenkins, G. D., Gurunathan, S. V., Sundt, W. J., Johnson, S., Lennon, R. J., Dyer, R. B., Camilleri, M., Kashyap, P. C., Farrugia, G., Chen, J., Singh, R. J., Grover, M. Sex differences in NSAID‐induced perturbation of human intestinal barrier function and microbiota. FASEB J. 32, 6615–6625 (2018). www.fasebj.org