Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling
Shouji Matsushima, … , Hiroyuki Tsutsui, Junichi Sadoshima
Shouji Matsushima, … , Hiroyuki Tsutsui, Junichi Sadoshima
Published August 15, 2016
Citation Information: J Clin Invest. 2016;126(9):3403-3416. https://doi.org/10.1172/JCI85624.
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Research Article Cardiology

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling

Abstract

NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2– production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling.

Authors

Shouji Matsushima, Junya Kuroda, Peiyong Zhai, Tong Liu, Shohei Ikeda, Narayani Nagarajan, Shin-ichi Oka, Takashi Yokota, Shintaro Kinugawa, Chiao-Po Hsu, Hong Li, Hiroyuki Tsutsui, Junichi Sadoshima

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Figure 6

NOX4 and FYN affect the activity of one another.

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NOX4 and FYN affect the activity of one another. Cultured neonatal rat C...
Cultured neonatal rat CMs were transduced with indicated adenoviruses. (A, B, and D) CMs were treated with or without 100 μM H2O2 for 1 hour. (A, C, and D) expression levels of phosphorylated FYN, total FYN, NOX4, and GAPDH were evaluated with immunoblotting. After immunoprecipitation (IP) with an anti-FYN antibody, immunoblot analyses (IB) for phospho-Src (S416) were performed to detect FYN phosphorylated at the tyrosine in the activation loop of the kinase domain (p-FYN). In the quantitative analysis, the level of p-FYN detected by the anti–phospho-Src antibody/total FYN in control samples is expressed as 1 (n = 5). (B) Apoptosis in CMs transduced with indicated adenoviruses was evaluated with TUNEL staining (n = 6). (E) Coimmunoprecipitation assays with lysates of CMs treated with or without H2O2. After immunoprecipitation with control IgG and an anti-FYN antibody, immunoblot for endogenous NOX4 was performed. Immunoblots of NOX4 and GAPDH in input controls (5% lysates) are also shown. The experiment was conducted 3 times (D and E). Statistical analyses between groups were done by unpaired Student’s t test (A and C) or 1-way ANOVA followed by a post-hoc Fisher’s comparison test (B). *P < 0.05, **P < 0.01.