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 1

FYN interacts with NOX4 in CMs.

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FYN interacts with NOX4 in CMs. (A) Interaction between the NOX4 C-termi...
(A) Interaction between the NOX4 C-terminus (NOX4-CT) and FYN was examined by pull-down assays with His-tagged NOX4-CT recombinant protein and whole cell lysates. IB, immunoblot. The experiment was conducted 3 times. (B) Interaction between the NOX4-CT and FYN was examined by pull-down assays using indicated recombinant proteins. (C) Coimmunoprecipitation assays using lysates of CMs transduced with Ad-NOX4 WT and heart homogenates. After immunoprecipitation with control IgG or a FYN antibody, immunoblotting for NOX4 was performed. Immunoblots of input controls (5% lysates) are also shown. IP, immunoprecipitation. The experiment was conducted 3 times. (D) Cultured CMs were costained with anti-NOX4 antibody (red), anti-FYN antibody (green), MitoTracker (blue), and DAPI. Scale bar: 10 μm. The experiment was conducted 3 times. (E) Mitochondrial, ER, and nuclear fractions were prepared from cultured CMs. Coimmunoprecipitation assays in each fraction from CMs transduced with Ad-NOX4 WT. After immunoprecipitation with control IgG or a FYN antibody, immunoblotting for NOX4 was performed. Immunoblots of input controls (5% lysates) are also shown. The purity of each fraction was confirmed by the absence or presence of GAPDH, cytochrome C oxidase IV (COX IV), histone H3, and binding immunoglobulin protein (Bip). The experiment was conducted 3 times.