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

ABSTRACT

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.