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.

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca 2+ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2 −/−) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2 −/−-iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca 2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca 2+ transients in RYR2 −/−-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca 2+ ([Ca 2+ ] o) and caffeine in a concentration-dependent manner, while RYR2 −/−-iPSC-CMs showed inconsistent reactions to [Ca 2+ ] o and were insensitive to caffeine, indicating there is no RYR2mediated Ca 2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2 −/−-iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca 2+ refilling in RYR2 −/−-iPSC-CMs is mediated by SERCA. Additionally, RYR2 −/−-iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca 2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca 2+ release is one of the major compensatory mechanisms for Ca 2+ cycling in human CMs with the RYR2 deficiency.

Skin fibrosis is a complex biological remodeling process occurring in disease like systemic sclerosis, morphea, or eosinophilic fasciitis. Since the knowledge about the underlying pathomechanisms is still incomplete, there is currently no therapy, which prevents or reverses skin fibrosis sufficiently. The present study investigates the role of polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of cutaneous fibrosis and demonstrates the antifibrotic effects of systemic mesalazine treatment in vivo. Isolated primary dermal fibroblasts of PLK2 wild-type (WT) and knockout (KO) mice were characterized invitro. Skin thickness and histoarchitecture were studied in paraffin-embedded skin sections. The effects of mesalazine treatment were examined in isolated fibroblasts and PLK2 KO mice, which were fed 100 µg/g mesalazine for 6 months via the drinking water. Compared to WT, PLK2 KO fibroblasts displayed higher spontaneous myofibroblast differentiation, reduced proliferation rates, and overexpression of the fibrotic cytokine OPN. Invitro, 72 h of treatment with 10 mmol/L mesalazine induced phenotype conversion in PLK2 KO fibroblasts and attenuated OPN expression by inhibiting ERK1/2. In vivo, dermal myofibroblast differentiation, collagen accumulation, and skin thickening were prevented by mesalazine in PLK2 KO. Plasma creatinine levels indicated good tolerability of systemic long-term mesalazine treatment. The current study reveals a spontaneous fibrotic skin phenotype and ERK1/2-dependent OPN overexpression in PLK2 KO mice. We provide experimental evidence for the antifibrotic effectiveness of systemic mesalazine treatment to prevent fibrosis of the skin, suggesting further investigation in experimental and clinical settings.

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