Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Depletion of the cardiac lncRNA Charme impairs the maturation and paracrine signaling of cardiac mesenchymal stromal cells

Background: The long non-coding RNA Charme is a key regulator of myogenesis and cardiac tissue development. In vivo knockout of Charme in mice causes dysregulation of many cardiac specific genes (among which many associated with different cardiomyopathies), with a phenotype of cardiac hyperplasia and structural anomalies, suggesting a potential role of the cardiac stroma as well in the alteration of tissue architecture. The cardiac stroma is composed of different cell types with a great heterogeneity. It plays key roles in tissue development and homeostasis, mainly regulating extracellular matrix (ECM) synthesis and remodeling, as well as through paracrine signalling which mediates responses in other cell types in the heart. The effects of in vivo Charme depletion on the cardiac stroma have never been investigated. Aims: This study focused on the impact of Charme depletion on cardiac mesenchymal stromal cells (CMSCs), with specific interest for the ECM composition and remodeling, cardioprotective paracrine functions, response to activation stimuli, and the influence on embryonic cardiomyocytes maturation. Results: Transcriptomic analysis revealed that Charme-/- CMSCs exhibit significant downregulation of ECM-related genes, particularly those involved in collagen I production and ECM organization. These changes were reflected in cardiac tissue, where Charme-/- hearts showed reduced collagen I protein content and an altered collagen I/collagen III ratio. Functional assays demonstrated that Charme-/- CMSCs have impaired ECM remodeling capacity, as evidenced by reduced matrix digestion activity. Additionally, Charme-/- CMSCs showed a diminished ability to differentiate into mature fibroblasts in response to TGFβ-1 stimulation, with lower αSMA levels and decreased collagen I gene expression. The paracrine function of Charme-/- CMSCs was also compromised, with a marked reduction in the secretion of cardioprotective cytokines, including those involved in the PI3K/Akt signaling pathway. Cardiomyocytes treated with conditioned media from Charme-/- CMSCs exhibited decreased Akt phosphorylation, confirming impaired signaling. Furthermore, Charme-/- CMSCs were less effective in promoting angiogenesis, as demonstrated by reduced endothelial tube formation. Charme-/- CMSC secretome showed also lower abundance of molecules involved in myoblast differentiation and fusion. In an in vitro model of early cardiac differentiation, murine embryonic stem cells (mESCs)-derived embryoid bodies co-cultured with Charme-/- CMSCs showed impaired cardiomyocyte maturation, with reduced expression of key cardiac differentiation markers, including Mef2c and GATA4, as well as a significantly reduced Myh6/Myh7 expression ratio indicating higher expression of embryonic isoforms versus adult ones. These findings suggest that Charme-/- CMSCs negatively influence the paracrine environment necessary for proper cardiomyocyte differentiation during development. We also showed that Charme expression levels in adult CMSCs are lower by two orders of magnitude compared to cardiomyocytes, suggesting that the effects observed in adult stromal cells are likely not dependent on Charme direct function on the chromatin. Nonetheless, during specific cardiac fibroblast differentiation from hiPSCs, hs-Charme levels are transiently increased. Conclusions: Data collected so far suggest that Charme depletion not only affects cardiomyocytes, but has also a profound impact on the function of the cardiac stroma. The impaired ECM remodeling, fibroblast differentiation, and paracrine signaling observed in Charme-/- CMSCs likely contribute to the structural and functional defects seen in the hearts of knock-out animals. These results highlight the importance of Charme in maintaining the integrity of the cardiac microenvironment, and suggest that its absence could exacerbate cardiac pathologies through multiple mechanisms involving both cardiomyocytes and stromal cells. The actual intracellular levels in adult CMSCs may not be sufficient to exert direct effects on chromatin and transcription. Nonetheless, the evidence of hs-Charme being expressed in human iPS-derived CFs lays the foundation for new studies on its role in stromal cells heterogeneity and maturation, and could open the way to new investigations on the role of this key lncRNA in human pathologies involving the cardiac stroma.