Decrease in RGS4 Expression via miR‐1 and Increase in CPI‐17 Expression via miR‐145 in Diabetes Lead to Augmentation of Ca 2+ ‐Dependent Initial Contraction and Ca 2+ ‐ Independent Sustained Contraction, Respectively, in Gastrointestinal Smooth Muscle

In gastrointestinal smooth muscle, contraction in response to G protein‐coupled receptor agonists is biphasic. Initial phase is mediated by Ga q /PLC‐β1/IP 3 /Ca 2+ pathway involving Ca 2+ /calmodulin‐dependent activation of myosin light chain kinase (MLCK) and phosphorylation of MLC 20 . Sustained phase is mediated by RhoA‐dependent pathway involving Rho kinase‐mediated phosphorylation of MYPT1, a targeting subunit of myosin light chain phosphatase (MLCP), and PKC‐mediated phosphorylation of CPI‐17, an endogenous inhibitor of MLCP. Both lead to inhibition of MLCP activity and thus, an increase in MLC 20 phosphorylation. Previous studies in gastrointestinal smooth muscle showed that the Ga q /PLC‐b1 signaling is rapidly terminated by the regulator of G protein signaling 4 (RGS4). Studies also showed that the expression of RhoA was increased and acetylcholine (ACh)‐induced contraction was augmented in gastric and colonic smooth muscle of diabetic mice. However, the effect of diabetes on the expression of RGS4 and CPI‐17 is unknown. Recent studies showed that microRNAs (miRs) play a key role in the pathogenesis of diabetes by regulating the expression of signaling molecules. RNA hybrid analysis showed binding sites for miR‐1 in the 3′ UTR of RGS4 mRNA and for miR‐145 in the 5′UTR of CPI‐17 mRNA. Aim To test the hypothesis that decrease in RGS4 expression via miR‐1 and increase in CPI‐17 expression via miR‐145 in diabetes leads to increase in Ga q /PLC‐b1/IP 3 /Ca 2+ /MLCK and CPI‐17/MLCP pathways and augmentation of both initial and sustained contraction. Methods Smooth muscle of stomach and colon from control and diabetic (ob/ob and db/db) mice, and from diabetic patients were used to measure: i) expression of miR‐1, miR‐145, RGS4 and CPI‐17 by real‐time RT‐PCR; ii) PLC‐b1 activity by ion exchange chromatography; iii) cytosolic Ca 2+ levels by image analysis in fura‐2 loaded cells; and iv) muscle contraction by organ bath experiments and scanning micrometry. Results Expression of miR‐1, miR‐145 and CPI‐17 was increased and RGS4 was decreased in gastric and colonic smooth muscle from diabetic mice, and from diabetic patients compared to control. Similar results were obtained in smooth muscle of stomach and colon from control mice treated with 30 mM glucose for 48h. Expression of RGS4 was decreased in cells transfected with pre‐miR‐1 and increased with antag‐miR‐1, suggesting that RGS4 expression is negatively regulated by miR‐1. ACh‐induced PLC‐b1 activity and Ca 2+ release were increased in muscle cells treated with 30 mM glucose. ACh‐induced initial and sustained contraction were also augmented in gastric and colonic smooth muscle from diabetic mice. Conclusion In smooth muscle, RGS4 expression is negatively regulated by miR‐1 and CPI‐17 expression is positively regulated by miR‐145. In diabetic smooth muscle, a decrease in RGS4 expression and an increase in CPI‐17 expression lead to upregulation of Gα q /PLC‐β1/IP 3 /Ca 2+ and PKC/CPI‐17 pathways, respectively, and augmentation of MLCK‐dependent initial contraction and MLCP‐dependent sustained contraction. Support or Funding Information R01DK28300