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Introduction Skin fibrosis, the hallmark of systemic sclerosis (SSc), is characterized by excessive extracellular matrix deposition and increased dermal stiffness. Emerging research implicates dysregulated mechanotransduction, the process by which cells convert mechanical cues into biochemical signals, as a contributing factor in SSc pathogenesis (1). Central to this process is the Rho/ROCK signalling pathway, which activates non-muscle myosin II (NMII), a motor protein essential for cellular contractility and implicated in fibrosis (2,3). However, the specific roles of individual isoforms NMIIA and NMIIB, and their relation to the pro-fibrotic cytokine TGFβ, remain to be defined. This study investigates the role of NMII isoform regulation in the development of skin fibrosis.Material and Methods We investigated the NMII-related pathway expression in SSc and healthy donor (HD) skin by ccomparative RNA-seq analysis of publicly available databases. The expression and activation (T18/S19 phosphorylation) of NMIIA and NMIIB were evaluated by immunofluorescence ( IF) in SSc and HD skin biopsies (n >/= 3 per group) and in SSc and HD fibroblasts (n >/= 7 per group), stimulated with rh-TGFβ. Fluorescence Recovery After Photobleaching (FRAP) was used to quantify NMII dynamics in healthy fibroblasts stimulated with rh-TGFβ. NMII contractility was inhibited in HD and SSc fibroblasts stimulated with rh- TGFβ (n=10) using the NMII inhibitor blebbistatin, the pan-ROCK inhibitor Y-27632, and the ROCK2-selective inhibitor KD025. Furthermore, NMIIB was depleted with siRNA to assess its specific role. The production of type-I collagen was assessed by ELISA and qPCR. Golgi structure and TANGO/COPII vesicle morphology were examined by immunofluorescence in NMIIB-depleted fibroblasts primed with rh-TGFβ.Results RNA-seq analysis and IF revealed increased activation of NMII via the Rho-ROCK signaling pathway in SSc skin. This correlated with a cytoskeletal redistribution of NMII isoforms, significantly increased T18/S19 phosphorylation, and higher expression in SSc fibroblasts. HD fibroblasts primed with rh-TGFβ exhibited a partially similar pattern. FRAP analysis further confirmed an altered NMII dynamics in rh-TGFβ-stimulated fibroblasts. Treatment of HD fibroblasts with blebbistatin, Y-27632, and KD025 substantially reduced type-I collagen production at both RNA and protein level (p<0.05). Furthermore, NMIIB depletion reproduced the inhibitory effects of the small molecules (p<0.05). Preliminary data also showed altered Golgi and TANGO/COPII vesicle morphology in NMIIB-depleted fibroblasts stimulated with rh- TGFβ, suggesting a disruption in secretory pathway dynamics.Conclusions Our data point to an altered actomyosin cytoskeleton dynamics in SSc fibroblasts and indicate that particularly NMIIB is required for the deposition by fibroblasts being involved in the secretory pathway.