SLIT۳ promotes cardiac fibrosis and differentiation of cardiac fibroblasts by RhoA/ROCK۱ signaling pathway

Objective(s): Slit guidance ligand ۳ (SLIT۳) has been identified as a potential therapeutic regulator against fibroblast activity and fibrillary collagen production in an autocrine manner. However, this research aims to investigate the potential role of SLIT۳ in cardiac fibrosis and fibroblast differentiation and its underlying mechanism.Materials and Methods: C۵۷BL/۶ mice (male, ۸-۱۰ weeks, n=۴۷) were subcutaneously infused with Ang II (۲.۰ mg/kg/day) for ۴ weeks. One to two-day-old Sprague-Dawley (SD) rats were anesthetized by intraperitoneal injection of ۱% pentobarbital sodium (۶۰ mg/kg) and ketamine (۵۰ mg/kg) and the cardiac fibroblast was isolated aseptically. The mRNA and protein expression were analyzed using RT-qPCR and Western blotting.Results: The SLIT۳ expression level was increased in Ang II-induced mice models and cardiac fibroblasts. SLIT۳ significantly increased migrated cells and α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts. Ang II-induced increases in mRNA expression of collagen I (COL۱A۱), and collagen III (COL۳A۱) was attenuated by SLIT۳ inhibition. SLIT۳ knockdown attenuated the Ang II-induced increase in mRNA expression of ACTA۲ (α-SMA), Fibronectin, and CTGF. SLIT۳ suppression potentially reduced DHE expression and decreased malondialdehyde (MDA) content, and the superoxide dismutase (SOD) and catalase (CAT) levels were significantly increased in cardiac fibroblasts. Additionally, SLIT۳ inhibition markedly decreased RhoA and ROCK۱ protein expression, whereas ROCK inhibitor Y-۲۷۶۳۲ (۱۰ μM) markedly attenuated the migration of cardiac fibroblasts stimulated by Ang II and SLIT۳.Conclusion: The results speculate that SLIT۳ could significantly regulate cardiac fibrosis and fibroblast differentiation via the RhoA/ROCK۱ signaling pathway.

Keywords:

α-Smooth muscle actin , Cardiac fibroblast , Cardiac fibrosis , Collagen I , Collagen III , Slit guidance ligand ۳