https://doi.org/10.1007/s00392-025-02625-4
1Medizinische Hochschule Hannover Institut für Molekulare und Translationale Therapiestrategien, OE-8886 Hannover, Deutschland; 2Fraunhofer-Institut für Toxikologie und Experimentelle Medizin ITEM Hannover, Deutschland; 3Universitätsklinikum Hamburg-Eppendorf Institut für Klinische Pharmakologie und Toxikologie Hamburg, Deutschland
Pathological cardiac remodeling and subsequent heart failure (HF) largely contribute to disease-related mortality in the world. Cardiac fibrosis (CF) is characterized as excessive extracellular matrix deposition, which is associated with mechanical stiffness and cardiac dysfunction - key components of the pathological remodelling process leading to HF development. This process is closely linked to the induction and activation of transforming growth factor beta 1 (TGFβ1) which plays a pivotal role in fibroblast activation. Despite numerous improvements in pharmacological treatment strategies, the prospects of cure for patients with advanced HF derived from CF remain poor, which indicates the urgent need for innovative therapeutic strategies specifically targeting CF.
Long non-coding RNAs (lncRNAs) represent promising therapeutic targets as they have already been proven to be involved in the progression of various diseases, including cardiovascular diseases. In this project, downstream effectors of TGFβ1 signaling in human cardiac fibroblasts (HCFs) were identified using a data mining approach based on a large patient cohort. First, 15 highly dysregulated lncRNAs were identified after TGFβ1 stimulation and after in vitro validation of expression and TGFβ1-responsiveness, two candidates were selected for further analysis: lncFIB#10 and lncFIB#11.
In order to test the antifibrotic effects of both lncRNAs, specific knockdown (KD) experiments were performed in HCFs combined with TGFß1 stimulation. Encouragingly, both, inhibition of lncFIB#10 and #11 significantly reduced expression of fibrosis-related markers at mRNA and protein level. Additionally, an impaired proliferation and migratory ability was observed after KD in primary HCFs derived from various donor patients. Based on these promising results, we proceeded with transcriptome analysis to elucidate the signaling roles of lncFIB#10 and #11. To this end, bulk RNA sequencing was conducted in HCFs treated with TGFβ1 in combination with transfection of lncRNA-specific GapmeRs. KEGG pathway analysis revealed an association of both lncRNAs with fibrosis-related pathways. Moreover, the translational potential of lncFIB#11 inhibition was further investigated in human engineered heart tissues (EHTs), representing a well-established multicellular 3D cardiac model that reflects the complexity of the in vivo heart. EHTs consisting of human induced pluripotent stem cell-derived cardiac fibroblasts and cardiomyocytes were utilized and transfected with GapmeR #11 upon TGFβ1 stimulation. Successful inhibition of lncFIB#11 and induction of a fibrotic response following TGFβ1 stimulation was confirmed, as evidenced by a significant upregulation of the fibrosis markers POSTN, COL1A1 and MMP2. Interestingly, lncFIB#11 reduction effectively abolished the fibrotic response confirming the antifibrotic potential of this candidate. Furthermore, significant improvements in various contractile parameters were observed upon lncFIB#11 KD, including force enhancement and increased fractional shortening as well as a reduced relaxation time and time to peak properties, indicating cardioprotective effects of lncFIB#11 inhibition.
Summarizing these results, specific KD of the TGFβ1-induced lncRNAs lncFIB#10 and #11 suppressed the major hallmarks of cardiac fibrosis in HCFs and EHTs and therefore, both lncRNAs represent promising targets for the treatment of CF, which strongly warrants further investigations.