Abstract 14797: Fyn is a Sensor and Regulator of Nox4-derived ROS in the Heart
Introduction: NADPH oxidases (Noxes) are enzymes that purposefully produce reactive oxygen species, including O2− and H2O2. Nox4 facilitates the oxidation of intracellular signaling molecules, thereby regulating cell growth and death. So far, the modulation of Nox4 through posttranslational mechanisms is poorly understood.
Purpose: The purpose of this study was to identify novel mechanisms that regulate the O2−-producing activity of Nox4.
Methods and Results: Using yeast two-hybrid screening, we found that Fyn, a Src family tyrosine kinase, interacted with the C-terminal cytoplasmic domain of Nox4. In vitro binding assays showed that Nox4 and Fyn directly interacted with one another. Nox4 and Fyn were co-localized in the peri-nuclear region, including mitochondria, in cardiomyocytes (CMs). Overexpression of Fyn significantly inhibited Nox4-induced O2− production and apoptosis in CMs. Conversely, downregulation of Fyn exacerbated Nox4-induced O2− production and apoptosis, suggesting that endogenous Fyn negatively regulates Nox4 activity. Fyn was activated by H2O2 treatment or overexpression of Nox4. Amino acids 1-80 of Fyn, containing the unique domain, but not amino acids 81-537 of Fyn, containing SH3, SH2 and kinase domains, interacted with Nox4. In vitro kinase assay and mass spectrometry analysis showed that Fyn directly phosphorylated tyrosine-566 of Nox4. In Nox4 Y566A-overexpressing CMs, mitochondrial O2− production was not suppressed by Fyn overexpression, indicating that phosphorylation of tyrosine-566 is critical for regulation of Nox4 activity by Fyn. Transverse aortic constriction (TAC) activated Fyn in the left ventricle (LV). Two weeks after TAC, Fyn knockout (KO) mice showed exacerbated LV remodeling compared to wild-type mice, accompanied by increases in O2− production in the mitochondrial fraction and apoptosis in the heart. Deletion of Nox4 rescued this exacerbation of LV remodeling in Fyn KO mice. Fyn expression was significantly decreased in failing human hearts compared to in control patient hearts.
Conclusions: Fyn is activated by oxidative stress and serves as a negative feedback regulator of Nox4 in CMs. Fyn negatively regulates cell death in the failing heart as a sensor and regulator of Nox4-derived ROS.
Author Disclosures: S. Matsushima: None. J. Kuroda: None. P. Zhai: None. T. Liu: None. H. Li: None. S. Kinugawa: None. H. Tsutsui: None. J. Sadoshima: None.
- © 2014 by American Heart Association, Inc.