Abstract 18005: Fyn Plays a Critical Role in Regulation of Cell Death in Heart Failure Through Tyrosine Phosphorylation of Nox4
NADPH oxidases (Noxes) are enzymes which purposefully produce reactive oxygen species, including O2- and H2O2. Cardiomyocytes (CMs) primarily express Nox2 and Nox4. The O2- producing activity of Nox2 is regulated by cytosolic factors, including p47phox and Rac. However, the modulation of Nox4 through posttranslational mechanisms is poorly understood. The purpose of this study was to identify novel mechanisms that regulate the O2- producing activity of Nox4. Using yeast two hybrid screening, we found that Fyn, a Src family tyrosine kinase, interacts with the C-terminal cytoplasmic domain of Nox4. In vitro binding assays showed that Nox4 and Fyn directly interact with one another. Nox4 and Fyn are co-localized in the perinuclear area, including mitochondria, in CMs. Fyn tyrosine-phosphorylated Nox4 in its C-terminus. Fyn is activated by H2O2 (100 μM, 5 min) in CMs (1.8 fold, p<0.05). Adenovirus-mediated overexpression of wild type Fyn in CMs did not change the protein level of Nox4 but significantly inhibited O2- production (10092±639 vs. 3092±532 RLU, p<0.05) from the mitochondrial fraction, as evaluated with lucigenin assays. Overexpression of Fyn significantly inhibited Nox4-induced apoptosis in CMs (9.6±0.7 vs. 5.5±0.6 %, p<0.05). On the other hand, downregulation of Fyn with adenovirus harboring anti-Fyn short hairpin RNA exacerbated Nox4-induced O2- production (8142±521 vs. 11054±1202 RLU, p<0.05) and apoptosis (8.3±0.4 vs. 10.4±0.4 %, p<0.05), suggesting that endogenous Fyn negatively regulates Nox4. Transverse aortic constriction (TAC) activated Fyn (2.0 fold, p<0.05) in the left ventricle. Two weeks after TAC, Fyn knockout mice showed a lower ejection fraction (49±10 vs. 80±3 %, p<0.05) and a higher lung weight/tibial length (7.6±0.1 vs. 6.1±0.6 %, p<0.05), than wild type (WT) mice, accompanied by increases in O2- production (10712±941 vs. 5739±832 RLU, p<0.05) in the mitochondrial fraction and apoptosis (0.08±0.02 vs. 0.05±0.01%, p<0.05) in the left ventricle. In conclusion, Fyn physically interacts with and phosphorylates Nox4 at tyrosine residue(s) located in the C-terminus, thereby inhibiting the O2- producing activity of Nox4. Fyn is activated by oxidative stress and serves as a negative feedback regulator of Nox4 in CMs.
- © 2012 by American Heart Association, Inc.