Abstract 299: Engineering Rodent TRPV1 to Mimic Chicken TRPV1 Reduces Capsaicin-induced Calcium Influx in H9C2 Cells

Background and Aim: Birds, unlike mammals, are resistant to the spicy hot sensation when consuming chili peppers. The inability for birds to sense the hot sensation is accredited to genetic divergence in the transient receptor potential vanilloid 1 (TRPV1) when compared to mammals. Recently, we described how TRPV1 is important for regulating myocardial ischemia-reperfusion injury in rodents. Here, we tested our hypothesis that K710 is a crucial mediator of capsaicin-induced calcium influx in myocardial H9C2 cells. Methods and Results: Sequence alignment of the C-terminus TRP domain of TRPV1 among species was performed. Unlike mammals having K710 TRPV1, birds instead have N710 TRPV1 (Fig 1A). Site-directed mutagenesis of K710N was performed on wild type (WT) rTRPV1 with a pCMV6-entry vector. The empty vector, WT and K710N plasmids were transfected into H9C2 cells and the cellular localization of TRPV1 channel were detected by immunofluorescent staining and channel activity by capsaicin challenge with live cell calcium imaging with Fura-2AM. Interestingly, the K710N mutation caused the TRPV1 channel to be retained intracellularly and co-localized with PDI, an endoplasmic reticulum marker (Fig 1B, Bar=25μM). K701N cells treated with 1μM capsaicin showed a reduced response to capsaicin compared to wild type TRPV1 transfected cells (Fig 1C-E, 011±0.01 in WT vs . 0.03±0.01 in K710N, n=10, P<0.01). Conclusions: Site-directed mutagenesis of K710N caused rTRPV1 to be insensitive to capsaicin and suggests a potential target for limiting calcium influx from myocardial ischemia-reperfusion injury which could be a novel protective strategy to prevent damage from a heart attack.