Bernard, Cheryl E.

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.

AbstractBackgroundAnimal models and human data have suggested macrophage‐driven immune dysregulation in diabetic gastroparesis (DG). Translocator protein (TSPO) upregulation has been suggested to indicate activated state of macrophages and ER176 is a high affinity third generation TSPO‐specific radioligand. The aim of this study was to determine feasibility of dynamic 11C‐ER 176 PET to identify macrophage activation in DG.MethodsTwelve patients, all females, were recruited (4 DG, 4 diabetics, and 4 healthy volunteers) for 11C‐ER 176 PET/CT scanning. The standardized uptake value (SUVmax) in the gastric fundus, body, pylorus, and descending part of the duodenum were compared between three groups using Kruskal‐Wallis test to perform the comparisons, and a p‐value of 0.05 was considered statistically significant.Key ResultsAge was comparable among the three groups with a median of 53 years. The uptake was higher in pylorus in diabetics compared to DG and healthy (SUVmax healthy 4.6 ± 0.2, diabetics 8.4 ± 4.1, DG 5.5 ± 1.0, p = 0.04). The uptake was similar in gastric fundus (9.0 ± 1.6, 13.1 ± 8.3, 7.8 ± 1.9 respectively, p = 0.3), body (7.7 ± 1.9, 13 ± 9.2, 7.8 ± 1.9 respectively, p = 0.8), and duodenum (6.2 ± 2.1, 9.5 ± 6.8, 7.0 ± 1.8 respectively, p = 0.6). No correlation was observed between SUVmax uptake and either HbA1C or fasting blood glucose.Conclusions and InferencesFemale diabetic gastroparesis patients did not demonstrate increased TSPO ligand 11C‐ER 176 uptake in the stomach. Possible explanations include lack of specificity of ligand for specific macrophage phenotypes in DG, sex effect, or small sample size. Further studies investigating non‐invasive ways of analyzing immune dysregulation in neurogastrointestinal disorders are warranted.

BACKGROUND Enterochromaffin (EC) cells are enteroendocrine cells in the gastrointestinal (GI) epithelium that release serotonin (5‐HT) in response to a variety of stimuli including mechanical force. In turn, EC cell 5‐HT affects GI secretion, motility and sensation. The molecular mechanism of EC cell mechanotransduction is unknown. The recently discovered mechanosensitive ion channel Piezo2 is important in several mechanosensory cell types. AIM to determine if the mechanosensitive ion channel Piezo2 is expressed and functional in EC cells. METHODS Murine small bowel was collected, fixed in 4% paraformaldehyde, and frozen. Human and mouse RNA from human jejunum and mouse duodenum, jejunum and ileum was analyzed by quantitative RT‐PCR for Piezo2 mRNA. Piezo2 and 5‐HT were also identified by fluorescence immunolabeling. QGP‐1, a human enteroendocrine cell model, was analyzed by RT‐PCR for the presence of Piezo2 and immunolabeling was done to detect Piezo2 and 5‐HT. Piezo2 mechanically activated currents were recorded from QGP‐1 cells in whole‐cell configuration. Voltage‐clamped QGP‐1 cells were subjected to a series of mechanical perturbations using a piezo‐electrically driven glass probe at steps of 0.3 μm for stimulation. RESULTS Human jejunum and all segments of mouse small bowel mucosa (duodenum, jejunum, ileum) expressed Piezo2 mRNA. In human jejunum and all segments of mouse small bowel Piezo2 co‐localized with 5‐HT in >90% of 5‐HT containing cells. No Piezo2 immunoreactivity was seen outside of 5‐HT positive enterochromaffin cells. The enteroendocrine cell model QGP‐1 expressed both Piezo2 and 5‐HT by immunohistochemistry and RT‐PCR showed message for Piezo2. Application of force to the surface of voltage‐clamped QGP‐1 cells elicited whole cell fast inward non‐selective cation currents of ~200 pA (at −60 mV) that were blocked by Gd 3+ with kinetic properties compatible with Piezo2 channels. CONCLUSIONS Piezo2 is specifically expressed in human and murine small bowel EC cells and in the enteroendocrine cell model QGP‐1. QGP‐1 cells have a mechanosensitive inward cation current with kinetic and pharmacological properties similar to Piezo2. Our data suggest that Piezo2 is involved in EC cell mechanotransduction. Support or Funding Information Supported by NIH DK106456 , DK84567, DK052766

Background SCN5A ‐encoded Na V 1.5 is a mechanosensitive voltage‐gated sodium channel expressed in human gastrointestinal smooth muscle cells. In the gastrointestinal tract, alterations in Na V 1.5 current density by naturally occurring mutations or by pharmaceutical block of channel function are associated with constipation. In the heart loss of Na V 1.5 current is associated with cardiac contractility disorders such as dilated cardiomyopathy. Ion channel expression level is regulated by miRNAs, and Na V 1.5 is known to be regulated by miRNA in heart. However , it is not known if miRNAs regulate Na V 1.5 expression in GI smooth muscle. Aim To identify miRNAs that regulate Na V 1.5 expression in gastrointestinal smooth muscle in health and disease. Methods A qPCR array was used to screen for differential expression of 372 of the most common miRNAs in the miRBase database to test the differential expression of miRNA between control tissue and tissues from patients with slow transit constipation. qRT‐PCR was done to determine expression levels of SCN5A mRNA. A cell line (HuJej) was derived from human jejunum smooth muscle cells by immortalization using the SV40 large T antigen. Whole cell voltage clamp experiments were done by standard patch clamp techniques. Results SCN5A mRNA expression was reduced by 66±0.08% in patients with slow transit constipation (57.5±1.8 years) compared to age matched controls (53±1.7 years). qPCR array screening showed that 12 miRNAs were significantly upregulated in colonic muscularis externa from patients with STC compared to controls. Let‐7f miRNA was increased 6.46‐fold in slow transit constipation compared to controls and was the only miRNA with strong negative correlation to SCN5A mRNA expression in the same tissues. HuJej cells express Na V 1.5; therefore, we investigated the effect of let‐7f on Na V 1.5 currents by transfection with a stable miRNA mimic. 48 hours after transfection with let‐7f mimic, Na V 1.5 currents were significantly decreased compared to untransfected cells or to cells transfected with non‐targeting miRNA (let‐7f, −0.6±0.1 pA/pF versus untransfected, −3.9±0.6 pA/pF or non‐targeting, −2.3±0.6 pA/pF). Summary and Conclusions SCN5A mRNA is decreased in slow transit constipation. Expression levels of let‐7f and Na V 1.5 RNA are negatively correlated in human colonic smooth muscle. miRNA let‐7f regulates Na V 1.5 current density. Regulation of Na V 1.5 current density may have relevance in health and disease. Support or Funding Information This work was supported by grant DK52766.

AbstractBackgroundGastroparesis (GP) is characterized by delayed gastric emptying in the absence of mechanical obstruction.ObjectiveGenetic predisposition may play a role; however, investigation at the genome‐wide level has not been performed.MethodsWe carried out a genome‐wide association study (GWAS) meta‐analysis on (i) 478 GP patients from the National Institute of Diabetes and Digestive and Kidney Diseases Gastroparesis Clinical Research Consortium (GpCRC) compared to 9931 population‐based controls from the University of Michigan Health and Retirement Study; and (ii) 402 GP cases compared to 48,340 non‐gastroparesis controls from the Michigan Genomics Initiative. Associations for 5,811,784 high‐quality SNPs were tested on a total of 880 GP patients and 58,271 controls, using logistic mixed models adjusted for age, sex, and principal components. Gene mapping was obtained based on genomic position and expression quantitative trait loci, and a gene‐set network enrichment analysis was performed. Genetic associations with clinical data were tested in GpCRC patients. Protein expression of selected candidate genes was determined in full thickness gastric biopsies from GpCRC patients and controls.ResultsWhile no SNP associations were detected at strict significance (p ≤ 5 × 10−8), nine independent genomic loci were associated at suggestive significance (p ≤ 1 × 10−5), with the strongest signal (rs9273363, odds ratio = 1.4, p = 1 × 10−7) mapped to the human leukocyte antigen region. Computational annotation of suggestive risk loci identified 14 protein‐coding candidate genes. Gene‐set network enrichment analysis revealed pathways potentially involved in immune and motor dysregulation (pFDR ≤ 0.05). The GP risk allele rs6984536A (Peroxidasin‐Like; PXDNL) was associated with increased abdominal pain severity scores (Beta = 0.13, p = 0.03). Gastric muscularis expression of PXDNL also positively correlated with abdominal pain in GP patients (r = 0.8, p = 0.02). Dickkopf WNT Signaling Pathway Inhibitor 1 showed decreased expression in diabetic GP patients (p = 0.005 vs. controls).ConclusionWe report preliminary GWAS findings for GP, which highlight candidate genes and pathways related to immune and sensory‐motor dysregulation. Larger studies are needed to validate and expand these findings in independent datasets.

In this study, we show that the electrogenic Na+/HCO3− cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A , the gene that encodes the Na + channel Na V 1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na + current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na + channel Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Macrophage-based immune dysregulation plays a critical role in development of delayed gastric emptying in diabetic mice. Loss of anti-inflammatory macrophages and increased expression of genes associated with pro-inflammatory macrophages has been reported in full-thickness gastric biopsies from gastroparesis patients. We aimed to determine broader protein expression (proteomics) and protein-based signaling pathways in gastric biopsies of diabetic (DG) and idiopathic gastroparesis (IG) patients. Additionally, we determined correlations between protein expressions, gastric emptying, and symptoms. Full-thickness gastric antrum biopsies were obtained from nine DG patients, seven IG patients, and five nondiabetic controls. Aptamer-based SomaLogic tissue scan that quantitatively identifies 1,305 human proteins was used. Protein fold changes were computed, and differential expressions were calculated using Limma. Ingenuity pathway analysis and correlations were carried out. Multiple-testing corrected P < 0.05 was considered statistically significant. Seventy-three proteins were differentially expressed in DG, 132 proteins were differentially expressed in IG, and 40 proteins were common to DG and IG. In both DG and IG, “Role of Macrophages, Fibroblasts and Endothelial Cells” was the most statistically significant altered pathway [DG false discovery rate (FDR) = 7.9 × 10−9; IG FDR = 6.3 × 10−12]. In DG, properdin expression correlated with GCSI bloating ( r = −0.99, FDR = 0.02) and expressions of prostaglandin G/H synthase 2, protein kinase C-ζ type, and complement C2 correlated with 4 h gastric retention ( r = −0.97, FDR = 0.03 for all). No correlations were found between proteins and symptoms or gastric emptying in IG. Protein expression changes suggest a central role of macrophage-driven immune dysregulation in gastroparesis, specifically, complement activation in diabetic gastroparesis. NEW & NOTEWORTHY This study uses SOMAscan, a novel proteomics assay for determination of altered proteins and associated molecular pathways in human gastroparesis. Seventy-three proteins were changed in diabetic gastroparesis, 132 in idiopathic gastroparesis compared with controls. Forty proteins were common in both. Macrophage-based immune dysregulation pathway was most significantly affected in both diabetic and idiopathic gastroparesis. Proteins involved in the complement and prostaglandin synthesis pathway were associated with symptoms and gastric emptying delay in diabetic gastroparesis.

The SCN5A-encoded voltage-gated mechanosensitive Na+channel NaV1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. NaV1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2–3% of patients have SCN5A missense mutations that alter NaV1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting NaV1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. NaV1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated NaV1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered NaV1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in NaV1.5 channels with abnormal voltage-dependent and mechanosensitive function.NEW & NOTEWORTHY The voltage-gated Na+channel NaV1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the NaV1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant NaV1.5 channels were loss of function in voltage dependence or mechanosensitivity.