Transient Receptor Potential Channel Canonical Type 3 Deficiency Antagonizes Myofibroblast Transdifferentiation In Vivo

Abstract EN

Objective.

Myofibroblast transformation has been shown to be associated with the reactive oxygen species- (ROS-) producing enzyme NADPH oxidase (Nox4).

Inhibition of transient receptor potential channel canonical type 3 (TRPC3) attenuates mitochondrial calcium handling and ROS production in the vasculature of hypertensive rats.

However, it remains elusive whether TRPC3 regulates mitochondrial calcium and ROS production and participates in myofibroblast transdifferentiation during wound healing.

Methods and Results.

In this study, we demonstrated that activation of TRPC3 by transforming growth factor β (TGFβ1) elevated myofibroblast transdifferentiation by upregulating the myofibroblast marker alpha smooth muscle actin (αSMA).

Inhibition of TRPC3 with its specific inhibitor, Pyr3, significantly decreased TGFβ1-induced αSMA expression, as demonstrated by immunofluorescence.

Real-time PCR and immunohistochemistry revealed higher TRPC3 and TGFβ1 mRNA expression levels in fibroblasts from hypertrophic scar (HTS) tissue than in those from normal skin tissue.

TGFβ1 treatment increased TRPC3-mediated mitochondrial calcium uptake and ROS production but decreased ATP content in human fibroblasts, whereas inhibition of TRPC3 significantly reversed these effects.

The beneficial effects were associated with improvements in mitochondrial respiratory function mediated by recovery of the activity of pyruvate dehydrogenase (PDH).

In vivo, Trpc3-/- mice exhibited significantly attenuated myofibroblast transdifferentiation, as demonstrated by decreased αSMA, TGFβ1, fibronectin, and collagen-1 (Col1a1) protein expression in wound granulation tissues.

Furthermore, TGFβ1-induced store-operated calcium entry (SOCE) was significantly decreased in fibroblasts from Trpc3-/- mice compared with those from Trpc3+/+ mice.

In addition, Trpc3-/- mice exhibited significantly decreased Nox4 and phosphorylated Smad2/3 protein expression in wound granulation tissues.

Conclusions.

Our data indicate that TGFβ1-mediated activation of TRPC3 enhances mitochondrial calcium and ROS production, which promotes myofibroblast transdifferentiation and HTS formation.

Inhibition of the TRPC3-mediated Nox4/pSmad2/3 pathway may be a useful strategy to limit HTS formation after injury.