The role of the long non-coding RNA Charme in the phenotype and function of resident cardiac mesenchymal stromal cells

Background: Charme is a murine lncRNA expressed in striated muscles, presenting a human orthologue with 45% identity. Charme-/-mice display incomplete muscle differentiation and altered cardiac phenotype with increased wall thickness due to cardiomyocyte hyperplasia and present impaired cardiac function at adult age (Taliani, Buonaiuto, eLife 2023). Cardiac mesenchymal stromal cells (CMSCs) play key roles in tissue pathophysiology, by maintaining the extracellular matrix (ECM) and by paracrine action. Purpose: To elucidate the role of Charme in the phenotype, paracrine function, and activation of resident CMSCs. Results: Charme-/-hearts showed reduced content of collagen-I (0.16±0.05 vs WT:0.83±0.18 normalized OD, N=4, p<0.005). Charme-/-CMSCs revealed reduced expression of genes involved in “Extracellular matrix organization” (GO:0030198) and “Cardiac muscle tissue development” (GO:0048738), enhanced spheroid growth (123.6±11.9 vs WT: 51.0±6.4 spheroids/well; N=8, p<0.0001), clonogenesis efficiency (2.7±0.5-fold; N=7, p<0.05), migration ability (68.5±2.7% vs WT: 80.2±0.7% wound area; N=3, p<0.005), and reduced ECM digestion capacity (74.03±2.31% vs WT 56.15±5.65% collagen area at 96h, N=3, p<0.05). Charme-/-CMSCs showed reduced lin-/Sca1+/CD90+ primitive mesenchymal cells (7.4±1.6% vs WT:56.9±6.5%; N=6, p<0.05). Their secretome was depleted of cytokines involved in PI3K-Akt signalling pathway (FDR<0,0005) and in myoblast differentiation (GO:1901741, GO:0045663; FDR<0.05), and mediated reduced Akt-phosphorylation in neonatal rat cardiomyocytes (0.08±0.01 vs WT:0.23±0.12 normalized OD; N=2), as well as reduced tube formation in an angiogenesis assay (fold change vs WT: 0.39±0.07 meshes, 0.60±0.06 nodes; N=3, p<0.01). Stimulation with TGFb-1 revealed lower expression of fibroblast activation markers in Charme-/- compared to WT CMSCs (aSMA fluorescence intensity: 0.5±0.1 vs 0.8±0.1, N=3, p<0.05; collagen I/III mRNA expression ratio: 1.3±0.1 vs 2.2±0.4 N=3, p<0.01), and their secretome was less effective in sustaining cardiomyocyte survival (1.7±0.1 vs WT:1.9±0.1 normalized absorbance; N=5, p<0.005). Conclusions: Charme-/-CMSCs show a less mature phenotype associated to ECM alteration, reduced activation, and impaired cardioprotective paracrine functions, suggesting a key role of Charme in cardiac stroma physiology.