Mechanosensitive long non-coding RNAs as upstream modulators of pro-fibrotic activation in human cardiac fibroblasts

Abstract Mechanical signaling cooperates with pro-fibrotic stimuli in driving the transition of cardiac fibroblasts (CFs) into myofibroblasts in vitro and contributes to post-myocardial infarction remodeling in vivo. This process depends on the mechanically regulated activity of the YAP/TEAD transcriptional network, as demonstrated by treating cells and animals with Verteporfin (VTP), a specific inhibitor of the complex. RNA sequencing of human CFs treated or not with VTP identified a signature of 222 differentially expressed Poly-A+ long non coding-RNAs (lncRNAs). Correlation analysis identified clusters of lncRNAs putatively co-regulated with genes involved in profibrotic regulation and Hippo signaling. Pearson’s r correlation highlighted a signature of 8 lncRNAs common to both pathways, representing potential regulators of the fibrotic process in CFs. To further characterize the potential role of this signature of lncRNAs, CFs were exposed to 11% uniaxial deformation for 72 hours (FlexCell) or seeded onto substrates with controlled mechanical compliance (Young’s modulus 12 and 100 kPa). Mechanical stimulation significantly modulated 3 of these lncRNAs, and this correlated with increased expression of canonical fibrosis markers and enhanced YAP nuclear translocation/activity. Silencing YAP with siRNA suppressed the expression of these 3 lncRNAs, indicating a YAP-dependent mechanosensitive regulation. Isoform-specific expression and subcellular localization of each lncRNA were characterized to guide gain- and loss-of-function studies. Single-cell RNA sequencing of mechanically stimulated versus control CFs was performed to assign these lncRNAs to distinct fibroblast subpopulations. UMAP visualization showed two clearly separated cell populations corresponding to mechanically stimulated and control cells, indicating distinct transcriptional responses. Strained cells expressed markers of activated fibroblasts, while controls displayed a quiescent phenotype. This was further validated by RT-PCR analysis of myofibroblast activation genes. Additionally, single-cell RNA sequencing was performed on CFs cultured on substrates of different stiffness, allowing the assessment of lncRNA expression patterns and fibroblast state transitions in response to matrix mechanical compliance. These results suggest, for the first time, the existence of a mechanosensitive lncRNA signature functionally connected to the transcriptional control of Hippo and pro-fibrotic pathways. This mechanosensitive regulation points to these lncRNAs as upstream modulators of fibroblast activation. These findings open the way to RNA-based therapeutic strategies aimed at limiting pro-fibrotic remodeling in cardiac fibroblasts and ultimately reducing the burden of cardiac fibrosis and heart failure.