Myofilament calcium sensitization in non-human primate lower esophageal sphincter and esophagus

Background/Objectives: Peristalsis in the esophagus (ESO) moves ingested contents into the stomach. The lower esophageal sphincter (LES) is located at the distal end of the ESO and generates tone to prevent reflux of gastric contents. Previous studies suggest that the expression and/or phosphorylation of proteins involved in myofilament Ca2+ sensitization differs between phasic and tonic smooth muscles (Brozovich et al., 2016; PMID: 27037223). We hypothesize that differences in expression and/or phosphorylation of these proteins contributes to differences in contractile activity between LES and ESO. The aims were to 1) characterize contractile activity in Cynomolgus monkey LES and ESO, 2) evaluate myofilament Ca2+ sensitization protein expression and phosphorylation, 3) examine changes in phosphorylation when contractile activity is enhanced or inhibited pharmacologically. Methods: Contraction was assessed in strips of LES and ESO by isometric tension recordings. Carbachol (CCh, 1 μM) was used to enhance contractions. ROCK2 (SR3677; 2 μM) or CavL (nifedipine; 1 μM) antagonists were used to inhibit contractions. Tissues were snap frozen in liquid nitrogen after peak response and prepared for protein expression analysis using Jess immunoelectrophoresis. Results: Under basal conditions, the LES exhibited tone whereas the ESO had phasic activity. CCh significantly increased contractions. Nifedipine and SR3677 significantly decreased basal and CCh-induced contractions with nifedipine having the greater effect. MLCK and MYPT1 expression was significantly greater in LES than ESO while LC20 and CPI-17 expression was greater in ESO. ROCK2 expression was similar in LES and ESO. Under basal conditions MYPT1 and LC20 phosphorylation was greater in LES. MYPT1 and LC20 phosphorylation was decreased by SR3677 but not nifedipine in both tissues. CCh increased MYPT1 and LC20 phosphorylation in both tissues though was greater in LES. SR3677 inhibited phosphorylation of both proteins in LES and ESO. Discussion/Conclusions: The differences in expression and phosphorylation of key proteins involved in myofilament Ca2+ sensitization likely contribute to differences in contractile activity. Notably, there is greater LC20 phosphorylation in LES than ESO under basal conditions and with CCh. Furthermore, there is an important role for ROCK2 in regulating basal contractile activity in LES and ESO as both contraction and phosphorylation of MYPT1 and LC20 are reduced by SR3677. While nifedipine significantly reduced contractile activity in LES and ESO, it did not reduce phosphorylation of MYPT1 indicating that the ROCK2 pathway is independent of Ca2+ influx. These findings may improve understanding of the pathophysiology associated with esophageal dysmotility. NIH DK129528 to CAC. 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.