β3‐ AR /β‐arrestin2 Interaction Triggers Cardiac Fibrosis Through JNK /c‐Jun Pathway in Cardiac Fibroblasts

Myocardial fibrosis (MF) is a critical pathological substrate of heart failure (HF), and β3-adrenergic receptor (β3-AR) has been implicated in cardiac remodelling with controversial roles. This study aimed to clarify the pro-fibrotic mechanism of β3-AR in cardiac fibroblasts and its association with β-arrestin2-mediated biased activation. Primary cardiac fibroblasts were isolated from neonatal C57BL/6 mice and subjected to β3-AR overexpression, agonist (BRL37344) stimulation, antagonist (SR59230A) inhibition, JNK inhibitor (SP600125) treatment, or β-arrestin2 knockdown via siRNA. RNA sequencing, immunofluorescence, Western blotting, and coimmunoprecipitation assays were performed to explore molecular mechanisms. RNA sequencing identified 577 upregulated and 231 downregulated genes in β3-AR-activated fibroblasts, enriched in extracellular matrix organisation and inflammatory pathways. Functional experiments confirmed that BRL37344 significantly upregulated fibrosis markers (α-SMA, FN, collagen I/III) and TGF-β1 expression, which was reversed by SP600125. Notably, inhibition of Gi signalling by pertussis toxin (PTX) failed to block β3-AR-induced fibrosis, while β-arrestin2 knockdown abrogated JNK/c-Jun/TGF-β1 pathway activation. Immunofluorescence and coimmunoprecipitation verified colocalisation and direct interaction between β3-AR and β-arrestin2, which was enhanced by BRL37344. Our findings demonstrate that β3-AR promotes cardiac fibrosis through β-arrestin2-mediated biased activation of the JNK/c-Jun/TGF-β1 pathway, independent of classical Gi signalling. This study reveals a novel cell-specific signalling mechanism of β3-AR and provides a potential therapeutic target for developing precision drugs against MF and HF.