329 - Peroxynitrite Alters the Ryanodine Receptor Activity Enhancing Ca2+ Release in High Cytoplasmic [Ca2+

Abstract

Increased peroxynitrite (ONOO–) production has been implicated in heart disease. Several studies have demonstrated that cardiac contractility and intracellular [Ca2+] are diminished after ONOO- exposure. In isolated sarcoplasmic reticulum vesicles, the activity of Ca2+ release channel ryanodine receptor (RyR) was reduced in the presence of ONOO–. This result suggested that the negative inotropic ONOO–-dependent effect may be mediated through RyR. Thus, we hypothesized that ONOO– alters the RyR function affecting intracellular Ca2+ levels and myocyte contraction. To demonstrate this, Wistar rats cardiomyocytes were perfused with the ONOO– donor, SIN-1 (200 µM). Results showed that maximum amplitude of the Ca2+ transients and myocyte shortening were reduced by 60% and this effect was abolished by FeTMPyP, an ONOO– decomposition catalyst. In addition, the maximum amplitude of caffeine-induced Ca2+ transient was reduced 60% after ONOO– exposure, in comparison with the control group. In high levels of cytoplasmic [Ca2+], ONOO–-treated myocytes unraveled an immediate second spontaneous Ca2+ release event right at the beginning of the diastole process thus modifying the shape of the Ca2+ transient. To elucidate this event, cells were simultaneously perfused with caffeine (10 mM) and tetracaine (2 mM), a RyR antagonist. The modified shape of the Ca2+ transient was absent under these conditions. In conclusion, our data suggest that peroxynitrite alters the RyR gating in a high [Ca2+] environment.