Impaired non-canonical transforming growth factor-β signalling prevents profibrotic phenotypes in cultured peptidylarginine deiminase 4-deficient murine cardiac fibroblasts.

Transforming growth factor-β (TGF-β) becomes rapidly activated in the infarcted heart. Hence, TGF-β-mediated persistent activation of cardiac fibroblasts (CFs) and exaggerated fibrotic responses may result in adverse cardiac remodelling and heart failure. Additionally, peptidylarginine deiminase 4 (PAD4) was described to be implicated in organ fibrosis. Here, we investigated the impact of PAD4 on CF function and myofibroblast transdifferentiation in vitro. The expression of fibrosis-related genes was largely similar in cultured WT and PAD4 CFs of passage 3, although collagen III was reduced in PAD4 CFs. Exposure to TGF-β inhibited proliferation and increased contractile activity and migration of WT CFs, but not of PAD4 CFs. However, under baseline conditions, PAD4 CFs showed comparable functional characteristics as TGF-β-stimulated WT CFs. Although the SMAD-dependent TGF-β pathway was not disturbed in PAD4 CFs, TGF-β failed to activate protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3) in these cells. Similar results were obtained in WT CFs treated with the PAD4 inhibitor Cl-amidine. Abrogated Akt activation was associated with diminished levels of phosphorylated, inactive glycogen synthase kinase-3β (GSK-3β). Consequently, PAD4 CFs did not upregulate collagen I and α-smooth muscle actin (α-SMA) expression after TGF-β treatment. Thus, PAD4 is substantially involved in the regulation of non-canonical TGF-β signalling and may represent a therapeutic target for the treatment of adverse cardiac remodelling.