The Role And Mechanism Of Tumor Necrosis Factor Receptor-Associated Factor 3 On Cardiac Remodelling In Mice

Aims Cardiac remodeling is the basic pathophysiologic process of chronic heart failure and the ultimate common pathway of many forms of heart diseases. However, the underlying mechanisms of cardiac remodeling have not been clearly elucidated, that make the prevention and therapy of this disease be an enormous difficulty. In this research, we discuss the key role of tumor necrosis factor receptor-associated factor 3(TRAF3) on pressure overload-induced cardiac remodeling and the underlying mechanisms.Methods In our study, cardiac-specific TRAF3 conditional knockout mice and cardiac-specific TRAF3 conditional transgenic mice subjected to aortic banding for indicated times were applied to the following experiment. The weight of mice body, heart, lung and the length of tibia were recorded. Then we used these data to calculate the indices of heart weight/body weight (HW/BW), lung weight/body weight (LW/BW) and heart weight/tibia length (HW/TL) in order to assess the degree of cardiac hypertrophy. The left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic diameter (LVEDd) and fractional shortening (FS) were measured using echocardiography to assess myocardial hypertrophy and heart function. Hematoxylin-eosin staining (H&E), FITC-conjugated wheat germ agglutinin staining (WGA) and picrosirius red staining (PSR) were performed to evaluate the cross-sectional area of cardiomyocytes and cardiac fibrosis. The mRNA expression levels of markers associated with cardiac hypertrophy and fibrosis were determined by real-time PCR (RT-PCR). In cellular experiments, neonatal rat cardiomyocytes (NRCMs) extracted from 1-2 days old Sprague-Dawley rats were treated with Ang II for 48 hours, then the cross-sectional area of cardiomyocytes were evaluated using immunofluorescence staining and the mRNA expression levels of hypertrophic markers were determined by RT-PCR. To explore the mechanism, we performed western blotting to screen the downstream signaling pathways which mediated the pro-hypertrophic function of TRAF3 and concentrated on the AKT signaling pathway. Thereafter, MK-2206(the inhibitor of AKT) was used to block the enhancement of the hypertrophic response caused by TRAF3 overexpression in NRCMs which confirmed the above speculation. Finally, glutathione S-transferase (GST)-pull-down assay and Co-immunoprecipitation (Co-IP) proved the directly interaction between TRAF3 and TBK1, and mapping experiment uncovered the responsible domains.Results TRAF3 expression was upregulated in hypertrophied mice hearts and failing human hearts. Four weeks after aortic banding, cardiac-specific conditional TRAF3-knockout mice exhibited significantly reduced cardiac hypertrophy, fibrosis, and dysfunction. Conversely, transgenic mice which overexpressed TRAF3 in heart developed deteriorative cardiac remodeling under pressure overload. TRAF3 also promoted an angiotensin ?-induced hypertrophic response in isolated cardiomyocytes. Mechanistically, western blotting screened out the AKT signaling pathway which mediated the pro-hypertrophic impact of TRAF3, and the treatment with MK-2206 confirmed this speculation. Eventually, we proved that TRAF3 can directly bound to TBK1, while the internal construct 267-376aa of TRAF3 and the C-terminus residues 384-729aa of TBK1 mediated this interaction.Conclusion This study demonstrated that TRAF3 is a positive regulator of pathological cardiac remodeling. Mechanistically, TRAF3 directly bound to TBK1, causing increased TBK1 phosphorylation in response to pro-hypertrophic stimuli. This interaction between TRAF3 and TBK1 further activates AKT signaling, which ultimately promotes the development of cardiac remodeling. Our findings revealed a key role of TRAF3 in regulating the pathological cardiac remodeling and also uncover TRAF3-TBK1-AKT as a novel signaling pathway associated with the development of pathological cardiac remodeling and heart failure.