Atrial Fibrillation Is Associated with Altered miRNA Expressional Profiles and Results in Major Functional Changes in Human Atrial Fibroblasts

Objectives: The development and maintenance of interstitial fibrosis, which is considered the basis for structural remodeling in atrial fibrillation (AF), involves expressional changes in cardiac microRNAs (miRNAs). Our previous investigations showed that various pro-fibrotic miRNAs are upregulated in LA and RA of AF patients compared with SR patients while anti-fibrotic miRNAs are downregulated. Furthermore, anti-fibrotic miRNAs are significantly different between paroxysmal AF than in persistent AF patients with lowest level in persistent atrial fibrillation.

Methods: Pro- and anti-fibrotic miRNAs were overexpressed by lentiviral transfection in primary human cardiac fibroblasts. Cellular effects with relevance for structural remodeling of the identified, differentially expressed pro- and anti-fibrotic miRNAs were analyzed.

Results: In AF patients, pro-fibrotic miRNAs (miR-199a-5p, miR-21–5p, miR-216a-5p, miR-217, miR-208a-3p) were upregulated and anti-fibrotic miRNAs (miR-133a, −26a-5p, 29b-3p) were downregulated compared with SR patients. Moderate (2–3fold) overexpression, comparable to the in vivo levels observed in AF patients, of single miRNAs in RA fibroblasts from SR patients resulted only in minor effects on proliferation (BrdU incorporation), migration (wound healing assay) and synthesis of extracellular matrix proteins. However, combined moderate overexpression of these pro-fibrotic miRNAs resulted in a strong increase in proliferation and migration. Furthermore, it increased the expression of α SM actin, a commonly used marker to identify myofibroblasts. Additionally, a strong increase in COL1A1, COL3A1 and fibronectin mRNA and protein expression was observed in these cells, suggesting a hypersecretory phenotype. The supernatant of these fibroblasts contained up to 10-fold higher concentrations of Connective Tissue Growth Factor (CTGF) and Transforming growth factor-β (TGF-β), which may have contributed to the activation of TGF-β downstream signals (pSmad2/3) and TGF-β target gene expression. Inhibition of TGF-β signaling by Smad3siRNA or TGF-β receptor inhibition was sufficient to partially rescue the FB phenotype.

Conclusion: The increase in different pro-fibrotic miRNA induces a phenotype switch in cardiac fibroblasts toward hypersecretory myofibroblasts, which can be mimicked in vitro in cardiac fibroblasts from SR patients. These miRNA patterns may offer therapeutic options for anti-fibrotic, atrial re-remodeling.