Upregulation of Transient Receptor Potential Canonical Type 3 Channel via AT1R/TGF-β1/Smad2/3 Induces Atrial Fibrosis in Aging and Spontaneously Hypertensive Rats

Fibroblast proliferation and migration are central in atrial fibrillation (AF) promoting structure remodeling, which is strongly associated with aging and hypertension. Transient receptor potential canonical-3 channel (TRPC3) is a key mediator of cardiac fibrosis and the pathogenesis of AF. Here, we have observed the increased TRPC3 expression that induced atrial fibrosis which possibly is either mediated by the aging process or related to hypertensive progression. In this study, we measured the pathological structure remodeling by H&E staining, Masson staining, and transmission electron microscope (TEM). The protein expression levels of fibrotic biomarkers and TRPC3 were measured by Western blotting with atrial tissues from normotensive Wistar Kyoto rats (WKY 4m-o (4 months old)), old WKY (WKY 24m-o (24 months old)), spontaneously hypertensive rat (SHR 4m-o (4 months old)), and old SHR (SHR 24m-o (24 months old)). To illuminate the molecular mechanism of TRPC3 in atrial fibrosis of aging rats and SHR, we detected the inhibited role of TRPC3 selective blocker ethyl-1-(4-(2,3,3-trichloroacrylamide) phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate,pyrazole-3 (Pyr3) on angiotensin II (Ang II) induced fibrosis in neonatal rat atrial fibroblasts. The pathological examination showed that the extracellular matrix (ECM) and collagen fibrils were markedly increased in atrial tissues from aged and hypertensive rats. The protein expressions of fibrotic biomarkers (collagen I, collagen III, and transforming growth factor-β1 (TGF-β1)) were significantly upregulated in atrial tissues from the WKY 24m-o group, SHR 4m-o group, and SHR 24m-o group compared with the WKY 4m-o group. Meanwhile, the expression level of TRPC3 was significantly upregulated in WKY 24m-o and SHR 4m-o atrial tissues compared to WKY 4m-o rats. In isolated and cultured neonatal rat atrial fibroblasts, Ang II induced the atrial fibroblast migration and proliferation and upregulated the expression levels of TRPC3 and fibrotic biomarkers. TRPC3 selected blocker Pyr3 attenuated the migration and proliferation in neonatal rat atrial fibroblasts. Furthermore, Pyr3 significantly alleviated Ang II-induced upregulation of TRPC3, collagen I, collagen III, and TGF-β1 through the molecular mechanism of the TGF-β/Smad2/3 signaling pathway. Similarly, knocking down TRPC3 using short hairpin RNA (shTRPC3) also attenuated Ang II-induced upregulation of TGF-β1. Pyr3 preconditioning decreased Ang II-induced intracellular Ca(2+) transient amplitude elevation. Furthermore, AT1 receptor was involved in Ang II-induced TRPC3 upregulation. Hence, upregulation of TRPC3 in aging and hypertension is involved in an atrial fibrosis process. Inhibition of TRPC3 contributes to reverse Ang II-induced fibrosis. TRPC3 may be a potential therapeutic target for preventing fibrosis in aging and hypertension.