Effect of the cardiac long non-coding RNA Charme depletion on the maturation and paracrine signaling of resident cardiac fibroblasts

Abstract Charme is a murine long non-coding RNA necessary for the embryonic development of the heart. In vivo Charme knock-out causes prominent alterations of tissue structure, due to cardiac hyperplasia, and leads to the development of cardiac dysfunctions. Cardiac fibroblasts (CFs) play pivotal roles in both the development and homeostatic maintenance of the cardiac tissue, and the effect of Charme depletion on this cell compartment has not been addressed. This study investigated the phenotype and function of resident CFs isolated from Charme knockout (Charme KO ) mice and revealed their impaired maturation and functionality. Charme KO hearts show decreased levels of collagen I content in the extracellular matrix, associated with reduced extracellular matrix-related gene expression and matrix remodeling ability of CFs. Charme KO CFs show impaired phenotypic conversion into myofibroblasts and reduced responsiveness to activation stimuli, accompanied by the retention of features proper of unactivated mesenchymal cells. Charme depletion also affects CF paracrine function, determining an impoverishment in cardioprotective cytokines in their secretome, with consequent reduced ability to mediate PI3K/Akt pathway activation in cardiomyocytes and to induce the angiogenic process in endothelial cells. Charme KO CFs also proved to be less supportive of cardiomyocyte maturation in an in vitro model of cardiac differentiation, thus indicating a potential contribution to the impairment of cardiomyocyte maturation in vivo. Overall, the evidence collected suggests that Charme depletion in the heart impairs CF maturation capacity and ECM deposition function, which can contribute to the alterations observed in the Charme KO mice. These findings pave the way to deeper investigations on the intercellular signaling occurring in the heart upon Charme ablation to identify how microenvironment homeostasis contributes to cardiomyocyte maturation and is potentially involved in cardiac diseases.