LAUSR

BACKGROUND & AIMS Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by post-translational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long non-coding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (Cardiac mesoderm enhancer-associated noncoding RNA), a SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. METHODS GTEx and publicly available single-cell RNA sequencing (scRNA-seq) datasets from embryonic, adult human and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel GFP knock-in (KI) reporter/knockout (KO) mice. Bulk RNA sequencing (RNA-seq) and single nucleus 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 highly expressed in GI SMCs in human and mouse. Premature lethality was observed in global Carmn KO (gKO) and inducible SMC-specific KO (iKO) mice due to GI pseudo-obstruction, severe distension of the GI tract with dysmotility in cecum and colon segments. Histology, GI transit and muscle myography analysis revealed severe dilation, significantly delayed GI transit and impaired GI contractility in Carmn KO versus control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching 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 KO not only compromised myogenic motility by reducing contractile gene expression 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. CONCLUSIONS 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.