The LncRNA Carmn is a Critical Regulator for Gastrointestinal Smooth Muscle Contractile Function and Motility

Abstract

Background & aimsVisceral 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.MethodsAnalysis 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.ResultsUnbiased 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.ConclusionOur 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.

Coronary Artery Disease Risk GenePRDM16is Preferentially Expressed in Vascular Smooth Muscle Cells and a Potential Novel Regulator of Smooth Muscle Homeostasis

“…Interestingly, unlike other SMC-enriched genes that are expressed in both vascular and visceral SMCs of GI tissues such as MYOCD 32 , LMOD1 33 and CARMN 34 , PRDM16 exhibits negligible expression in GI tissues and visceral SMCs (Figure 1D-G & Online Figure S3-5). Previous studies showed that global or SMC-specific knockout of these pan SMC-enriched genes in mice often leads to lethal GI phenotypes [35][36][37] , which largely impedes the efforts for investigating their vascular function. Therefore, the VSMC-enriched expression pattern of PRDM16 offers a unique opportunity to study its function in VSMCs and vascular disorders by inherently circumventing GI phenotypes.…”