PT - JOURNAL ARTICLE AU - Xiangqin He AU - Kunzhe Dong AU - Jian Shen AU - Guoqing Hu AU - James D. Mintz AU - Reem T. Atawia AU - Juanjuan Zhao AU - Xiuxu Chen AU - Robert W. Caldwell AU - Meixiang Xiang AU - David W. Stepp AU - David J. Fulton AU - Jiliang Zhou TI - The LncRNA <em>Carmn</em> is a Critical Regulator for Gastrointestinal Smooth Muscle Contractile Function and Motility AID - 10.1101/2022.06.28.498024 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.06.28.498024 4099 - http://biorxiv.org/content/early/2022/06/30/2022.06.28.498024.short 4100 - http://biorxiv.org/content/early/2022/06/30/2022.06.28.498024.full AB - Background &amp; aims Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract and are critical for regulating motility. SMC contraction is regulated by changes in post-translational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality but the mechanisms regulating the expression levels of SMC-specific contractile proteins, including the role of long non-coding RNAs (lncRNAs), remains largely unexplored. Herein, we have uncovered an important role of Carmn (Cardiac mesoderm enhancer-associated noncoding RNA), a SMC-specific lncRNA, in regulating the phenotype of visceral SMCs of the GI tract.Methods Analysis of GTEx and publicly available single-cell RNA sequencing (scRNA-seq) datasets from embryonic, adult human and mouse GI tissues were used to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using a novel GFP knock-in (KI) reporter/knockout (KO) mouse model. Bulk RNA sequencing (RNA-seq) and single nuclei RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms.Results Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is specifically expressed in SMCs in human and mouse GI tract. Premature lethality was observed in global Carmn KO (gKO) and inducible SMC-specific KO (iKO) mice due to colonic pseudo-obstruction, severe distension of the GI tract with blockages in cecum and colon segments. Histology, whole-gut GI transit time and muscle myography analysis revealed severe dilation, significantly delayed GI transit and impaired GI contractility in Carmn KO mice versus control mice. Bulk RNA-seq of colonic muscularis revealed that Carmn deficiency promotes SMC de-differentiation as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes including Mylk, a key regulator of SMC contraction. SnRNA-seq further revealed SMC Carmn deficiency not only compromised myogenic motility by reducing expression of contractile genes but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance as silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression including MYLK and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program.Conclusion Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice, and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.Competing Interest StatementThe authors have declared no competing interest.