Background: Recently, our lab was the first to identify the functional presence of transient receptor potential (TRP) channels of the ankyrin 1 subtype (TRPA1) in mouse cardiomyocytes (CMs). However, the extent to which TRPA1 mediates signaling events in cardiac tissue has yet to be fully determined. Based upon investigations completed in other tissue types in the body, we hypothesized that TRPA1 activation will lead to Akt and eNOS activation in CMs.

Methods: CMs were untreated or treated with the TRPA1-specific agonist, allyl isothiocyanate (AITC), in the presence of absence of Akt inhibitor (LY294002) or eNOS inhibitor (L-NAME) and prepared for immunoblot analysis. Antibodies recognizing Akt phosphorylation at serine 473 (pAkt-S 473 ) and eNOS phosphorylation at serine 1177 (peNOS-S 1177 ) were utilized, as these sites are regularly used as indicators of protein activation. Results were obtained using an ImageQuant and quantified utilizing ImageJ software.

Results: Wild-type CMs treated with AITC demonstrated a significant increase in phosphorylated Akt and eNOS compared to the untreated control. These effects were absent in CMs obtained from TRPA1 null mice. Furthermore, CMs pretreated with LY294002 before AITC addition demonstrated a significant lowering of peNOS-S 1177 ., whereas pretreatment of CMs with L-NAME had no effect on the AITC-induced increase in pAkt-S 473 levels.

Conclusions: Herein, we identify a novel signaling cascade in adult mouse cardiomyocytes whereby TRPA1 stimulation elicits Akt phosphorylation followed by eNOS phosphorylation. As Akt and eNOS activation have direct implications in a myriad of cardiovascular physiological and pathophysiological events, the current results have potential for significant clinical applications.