Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy. Cardiovascular diseases (CVD) such as heart failure and arrhythmia represent the leading cause of death worldwide [1,2]. A well-recognized concomitant of CVD is cardiac fibrosis [1], which is the structural manifestation of an imbalance in extracellular matrix (ECM) homeostasis. With nearly 45% of all deaths in the Western world attributable to fibroproliferative disease, the clinical relevance of fibrotic remodeling is enormous [3,4]. This is particularly true in the case of atrial fibrosis, associated with a detrimental clinical outcome of highly abundant supraventricular arrhythmias like atrial fibrillation (AF) [5]. However, due to

Cardiovascular diseases are exacerbated and driven by cardiac fibrosis. TGFβ induces fibroblast activation and differentiation into myofibroblasts that secrete excessive extracellular matrix proteins leading to stiffening of the heart, concomitant cardiac dysfunction, and arrhythmias. However, effective pharmacotherapy for preventing or reversing cardiac fibrosis is presently unavailable. Therefore, drug repurposing could be a cost- and time-saving approach to discover antifibrotic interventions. The aim of this study was to investigate the antifibrotic potential of mesalazine in a cardiac fibroblast stress model. TGFβ was used to induce a profibrotic phenotype in a human cardiac fibroblast cell line. After induction, cells were treated with mesalazine or solvent control. Fibroblast proliferation, key fibrosis protein expression, extracellular collagen deposition, and mechanical properties were subsequently determined. In response to TGFβ treatment, fibroblasts underwent a profound phenoconversion towards myofibroblasts, determined by the expression of fibrillary αSMA. Mesalazine reduced differentiation nearly by half and diminished fibroblast proliferation by a third. Additionally, TGFβ led to increased cell stiffness and adhesion, which were reversed by mesalazine treatment. Collagen 1 expression and deposition—key drivers of fibrosis—were significantly increased upon TGFβ stimulation and reduced to control levels by mesalazine. SMAD2/3 and ERK1/2 phosphorylation, along with reduced nuclear NFκB translocation, were identified as potential modes of action. The current study provides experimental pre-clinical evidence for antifibrotic effects of mesalazine in an in vitro model of cardiac fibrosis. Furthermore, it sheds light on possible mechanisms of action and suggests further investigation in experimental and clinical settings.

Rationale: Fibrosis promotes the maintenance of atrial fibrillation (AF), making it resistant to therapy. Improved understanding of the molecular mechanisms leading to atrial fibrosis will open new pathways towards effective antifibrotic therapies. Objective: This study aims to decipher the mechanistic interplay between polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of atrial fibrosis and atrial fibrillation. Methods and Results: Atrial PLK2 mRNA expression was 10-fold higher in human fibroblasts than in cardiomyocytes. Compared to sinus rhythm (SR), right atrial appendages and isolated right atrial fibroblasts from AF patients showed downregulation of PLK2 mRNA and protein, along with increased PLK2 promotor methylation. Genetic deletion as well as pharmacological inhibition of PLK2 induced pro-fibrotic phenotype conversion in cardiac fibroblasts and led to a striking de novo secretion of OPN. Accordingly, PLK2-deficient (PLK2 KO) mice showed cardiac fibrosis and were prone to experimentally induced AF. In line with these findings, OPN plasma levels were significantly higher only in AF patients with atrial low-voltage zones (surrogates of fibrosis) compared to SR controls. Mechanistically, we identified ERK1/2 as the relevant downstream mediator of PLK2 leading to increased OPN expression. Finally, oral treatment with the clinically-available drug mesalazine, known to inhibit ERK1/2, prevented cardiac OPN overexpression and reversed the pathological PLK2 KO phenotype in PLK2 KO-mice. Conclusions: In summary, abnormal PLK2/ERK1/2/OPN axis function critically contributes to AF-related atrial fibrosis, suggesting reinforcing PLK2 activity and/or OPN inhibition as innovative targets to prevent fibrosis progression in AF. Mesalazine derivatives may be used as lead compounds for the development of novel anti-AF agents targeting fibrosis.