TRPV1 inhibition supports cell survival during hypoxia/reoxygenation in the rat cardiomyoblast H9C2 cell line

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ion channel that gated calcium (Ca2 + ). Over the last decade, several TRPV1-targeted strategies based on remote, pre- and post-conditioning in order to decrease infarct size have been successful. In fact, during myocardial infarction, Ca2+ dysregulation occurs leading to cell death and causing irreversible myocardial injury. TRPV1 modulation would counteract these deadly effects. The aim of this study is to decipher molecular mechanisms implicated in TRPV1 modulation to better understand the role of TRPV1 in hypoxia-reoxygenation (H/R). Here, we used H9C2 cell line as an alternative model to adult cardiomyocytes in order to perform live imaging with genetic probes. First, using RT-PCR, Western blot and confocal microscopy, we demonstrated that TRPV1 is expressed in H9C2 and seems to be localized at endoplasmic reticular (ER) membrane. Secondly, thanks to cytosolic and reticular Ca2 + imaging (respectively with Fura-2 and ErGAP1), we showed that TRPV1 is a functional ER Ca2 + leak channel. Since ATP synthesis and cell fate are dependent on Ca2 + exchanges between ER and mitochondria, we then analyzed the role TRPV1 on the mitochondrial [Ca2 + ] using R-GECO probe. We observed that pharmacological TRPV1 modulation increases both cytosolic and mitochondrial Ca2 + contents by at least 20%. Finally, cell death was evaluated by flow cytometry after H/R sequences. In particular, we showed that TRPV1 inhibition improves cell survival (at least by 22%). Many physiological processes (including cell death/survival balance) rely on precise and spatiotemporal changes in the intracellular Ca2 + concentration. In this study, we show: – that TRPV1 could precisely regulate Ca2 + signals in a localization-specific way, (i.e. between ER and mitochondria); – that H9C2 is a valuable model to evaluate the role of TRPV1 in Ca2 + fluxes during H/R.