OP0186 LIN-GP38+ STROMAL CELLS ARE KEY EFFECTOR CELLS IN MYOCARDIAL FIBROSIS AND DEFECTS OF THE CONDUCTION SYSTEM
Authors: Mara Stellato, Michal Rudnik, Florian Renoux, Przemyslaw Blyszczuk, Elena Osto, Matthias Dewenter, Petra Seebeck, Elena Pachera, Oliver Distler, Gabriela Kania
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background</h3> Cardiac dysfunction is a significant cause of mortality in SSc due to inflammation, vasculopathy and fibrosis. In fibrotic conditions, the excessive number of fibroblasts, myofibroblasts and extracellular matrix in the myocardium may directly or indirectly affect the cardiac conduction system by different mechanisms. Myofibroblasts are the main players in cardiac fibrogenesis, but their origin in SSc remains unknown. <h3>Objectives</h3> To unravel the role of Fos-related antigen 2 (Fra2) in stromal cell activation, cardiac fibrosis and alteration of the conduction system. <h3>Methods</h3> Fra2 transgenic (tg) mice and Rag2<sup>-/-</sup> Fra2 tg mice were studied. Hearts from Fra2 tg and WT mice were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). Cardiac function was assessed by echocardiography, electrocardiogram (ECG) and telemetry. Myocardial stromal (Ter119<sup>-</sup>CD45<sup>-</sup>CD31<sup>-</sup>, hereafter Lin<sup>-</sup>) gp38<sup>+</sup> stromal cells were sorted and cultured <i>in vitro</i>. The cellular phenotype was evaluated by qPCR, α-smooth muscle actin (αSMA) and stress fibre staining, secreted collagens and contraction assay. <h3>Results</h3> Echocardiography of 20-week-old Fra2 tg mice displayed increased ejection fraction (p=0.02) and fractional shortening (p=0.02) with decreased left ventricle end-diameter and end-volume in systole (p=0.006, p=0.008) and in diastole (p=0.007, p=0.008). ECG in conscious mice revealed lower heart rate (HR) (WT 741 ± 33 BPM, tg 644 ± 37 BPM, p&lt;0.0001) and prolonged QRS, ST and QTc parameters (p=0.001, p=0.01, p=0.01), which may predispose to arrhythmias. Telemetry recording showed the presence of atrio-ventricular (AV) blocks that correlated with disease severity (R=0.9, p=0.02). The myocardium of Fra2 tg mice presented mild inflammation and fibrosis with higher expression of collagen (p=0.03, n=7) and periostin (p=0.001, n=7), increased number of αSMA<sup>+</sup> myofibroblasts and expansion of stromal Lin-gp38<sup>+</sup> cells compared to WT mice (1.4% ± 1.4, n=15 and Fra-2 tg: 6% ± 4.5, n=14, p=0.001). Fibrosis disrupts the conductivity of cardiac tissue. Therefore, we investigated whether these stromal cells might be responsible for fibrosis. The majority of Lin<sup>-</sup>gp38<sup>+</sup> cells co-expressed αSMA, collagen, ADAM12 and periostin, indicating that myocardial Lin<sup>-</sup>gp38<sup>+</sup> cells might acquire a myofibroblast-like phenotype. Moreover, gp38 expression correlated with collagen deposition (R=0.9, p&lt; 0.0001). The comparison between Lin<sup>-</sup>gp38<sup>+</sup> cells isolated from WT and Fra2 tg showed that both αSMA total protein and αSMA fibres were increased in Fra2 cells. Importantly, αSMA fibres co-localized with stress fibres, resulting in a faster and stronger contraction capacity of Fra2 cells (p&lt;0.0001, n=3). Proliferation of Fra2 cells was increased compared to WT (p=0.007, n=5), while apoptosis was unchanged (p=0.335, n=5). Interestingly, Rag2<sup>-/-</sup>Fra2 tg mice showed no fibrosis or Lin<sup>-</sup>gp38<sup>+</sup> cell expansion. ECG parameters of Rag2<sup>-/-</sup>Fra2 tg mice were not changed compared to control mice, indicating that inflammation is necessary to acquire Fra2-driven fibrotic phenotype and defects in the conduction system. <h3>Conclusion</h3> Fra2 overexpression and inflammation foster stromal cell-to-myofibroblast differentiation, leading to cardiac fibrosis and defects of the conduction system. Targeting this process might be a therapeutic strategy for SSc patients with disorders of cardiac involvement. <h3>Disclosure of Interests</h3> &nbsp;Mara Stellato: None declared, Michal Rudnik: None declared, Florian Renoux: None declared, Przemyslaw Blyszczuk: None declared, Elena Osto: None declared, Matthias Dewenter: None declared, Petra Seebeck: None declared, Elena Pachera: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Gabriela Kania: None declared