Gα13–TRPC6–NFAT signaling pathway negatively regulates cardiac myofibroblast formation

Abstract

Sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) has been implicated in many cellular events. We previously reported that α subunits of G12 family G proteins (Gα12/13) participate in sustained Ca2+ influx required for the activation of nuclear factor of activated T cells (NFAT) in rat neonatal cardiac fibroblasts. However, the molecular mechanism and the role of NFAT in cardiac fibroblasts are unknown. Here, we show that Gα13-mediated upregulation of canonical transient receptor potential 6 (TRPC6) channels participates in sustained Ca2+ influx and NFAT activation by endothelin (ET)-1 treatment. Expression of constitutively active (CA) Gα13 increased the expression of TRPC6 proteins, which was suppressed by TRPC6 siRNA. The treatment of ET-1 increased TRPC6 protein levels through Gα12/13-dependent pathways. NFAT is activated by sustained increase in [Ca2+]i through upregulated TRPC6. A Gα12/13-inhibitory polypeptide suppressed the ET-1-induced increase in α-smooth muscle actin expression and [3H]proline incorporation, suggesting that Gα12/13 mediate ET-1-induced transformation of cardiac fibroblasts to myofibroblasts. This myofibroblast formation was suppressed by overexpression of TRPC6 and CA-NFAT, while enhanced by TRPC6 siRNAs and cyclosporine A. These results suggest two opposite roles of Gα13 in cardiac fibroblasts. First, Gα13 mediates ET-1-induced myofibroblast formation. Second, Gα13 mediates TRPC6 upregulation and NFAT activation that work as a negative feedback regulator against ET-1-induced myofibroblast formation.