Cardiac fibrosis is closely associated with cardiovascular diseases (CVDs) and critically contributes to the progression to heart failure. Diverse stress responses can initiate the activation of quiescent fibroblasts, leading to their differentiation into so-called myofibroblasts. One major driver initiating the pro-fibrotic gene program is the transforming growth factor beta 1 (TGFβ1), which is secreted by numerous cell types within the heart and thereby combines diverse cardiac remodeling processes. Nevertheless, TGFβ1 functions in growth, differentiation and apoptosis are not restricted to the heart limiting the potential as a suitable therapeutic target to modulate fibroblast phenotypes. In contrast, long non-coding RNAs (lncRNAs) emerged as promising, most often cell type- or tissue-specific regulators of cellular functions and dysfunctions. Targeting lncRNAs downstream of the potent TGFβ1-signaling pathway may, therefore, help to circumvent undesired side effects. A datamining approach from a large patient cohort identified potential effectors of the TGFβ1 signaling in primary human cardiac fibroblasts (HCFs). Initially, 15 highly deregulated lncRNA candidates were identified. Validating their expression and TGFβ1 responsiveness in HCFs helped to narrow down the number to two highly promising candidates, here referred to as LncFIB #10 and #11. Specific knockdown experiments via modified locked nucleic acids named GapmeRs confirmed a pronounced anti-fibrotic effect by determining expression levels of common fibrosis marker genes such as ACTA2, POSTN, CTGF, MMP2 and COL1A1. LncFIB #10 and #11 inhibition additionally resulted in an impaired metabolic activity, proliferation and migratory ability in commercial HCFs and primary cardiac fibroblasts derived from end-stage heart failure biopsies. Similar effects were observed in human lung and liver fibroblasts, but the maximum TGFβ1 induction occurred in HCFs indicating a superior relevance in the heart. Furthermore, the characteristic contractility of myofibroblasts that is crucial for wound closure was assessed via an imaging-based gel contraction assay and the results further reinforced the anti-fibrotic actions of both lncRNA GapmeRs.

In conclusion, this comprehensive in vitro characterization comprised the major aspects of the pathological remodeling mediated by fibroblasts. Specific knockdown of the TGFβ1-induced lncRNAs LncFIB#10 and LncFIB#11 potently suppressed the major hallmarks of fibrosis in human cardiac fibroblasts. Although, lncRNA conservation is poor and respective homologues could not be identified to date, we want to highlight the great importance of this study, since lncRNA functions are still far from being fully understood. Novel prediction approaches and deeper understandings of the lncRNA complexity might help to find genomic counterparts within relevant model organisms. Thus, LncFIB#10 and #11 can be considered as highly interesting targets for the treatment of cardiac fibrosis and heart failure, which strongly warrants further investigations including lncRNA pulldown, a global secretome analysis and translational studies in organoids or engineered heart tissues.