F‑box and WD repeat‑containing protein 7 ameliorates angiotensin II‑induced myocardial hypertrophic injury via the mTOR‑mediated autophagy pathway

Figure 1. FBW7 expression is downregulated in Ang II-induced H9C2 cells and its upregulation activates mTOR-mediated cell autophagy. (A) mRNA expression of FBW7 in the control and Ang II (0.5, 1, 5 and 10 µM) groups. (B) Protein expression level of FBW7 in the control and Ang II (0.5, 1, 5 and 10 µM) groups. Overexpression efficiency of FBW7 in the control, Oe-NC and Oe-FBW7 groups was measured using (C) RT-qPCR and (D) western blotting. (E) Protein expression levels of LC3B-II/I, P62 and Beclin-1 related to autophagy in the control, Ang II, Ang II+Oe-NC and Ang II+Oe-FBW7 groups. (F) Protein levels of p-mTOR and mTOR related to mTOR signaling pathway in the control, Ang II, Ang II+Oe-NC and Ang II+Oe-FBW7 groups. *P<0.05, ***P<0.001. FBW7, F-box and WD repeat-containing protein 7; Ang, angiotensin; Oe, overexpression; NC, negative control; LC3B, microtubule-associated proteins 1A/1B light chain 3B; p-, phosphorylated; -, Ang II treatment without transfection.

Figure 2. FBW7 inhibits Ang II-induced apoptosis in H9C2 cells via autophagy. (A) Cell viability of H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. (B and C) Apoptosis of H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. Scale bar, 50 µm. (D) Protein levels of Bcl-2, Bax and cleaved caspase 3 associated with apoptosis in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. *P<0.05, **P<0.01, ***P<0.001. FBW7, F-box and WD repeat-containing protein 7; Ang, angiotensin; Oe, overexpression; NC, negative control; 3-MA, 3-methyladenine; -, Ang II treatment without transfection.

Figure 3. FBW7 inhibits Ang II-induced hypertrophy and fibrosis in H9C2 cells through autophagy. (A) Cell length of H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. Scale bar, 25 µm. (B) Protein levels of hypertrophic markers β-MHC, BNP and ANF in H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. (C) Protein levels of α-SMA, Fibronectin, Vimentin Collagen I and Collagen III associated fibrosis in H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. **P<0.01, ***P<0.001. FBW7, F-box and WD repeat-containing protein 7; Ang, angiotensin; Oe, overexpression; NC, negative control; 3-MA, 3-methyladenine; β-MHC, β-myosin heavy chain; BNP, brain natriuretic peptide; ANF, atrial natriuretic factor; atrial natriuretic factor; SMA, smooth muscle actin; -, Ang II treatment without transfection.

Figure 4. FBW7 suppresses Ang II-induced inflammation and oxidative stress in H9C2 cells via autophagy. Levels of inflammatory cytokines (A) TNF-α and (B) IL-1β in H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. (C) Protein levels of iNOS and COX-2 associated with inflammation in H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. Levels of (D) SOD and (E) MDA associated with oxidative stress in H9C2 cells in the control, Ang II, Ang II+Oe-NC, Ang II+Oe-FBW7 and Ang II+Oe-FBW7+3-MA groups. *P<0.05, **P<0.01, ***P<0.001. FBW7, F-box and WD repeat-containing protein 7; IL, interleukin; Ang, angiotensin; Oe, overexpression; NC, negative control; 3-MA, 3-methyladenine; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; SOD, superoxide dismutase; MDA, malondialdehyde; -, Ang II treatment without transfection.

Figure 5. Schematic diagram presenting the hypothesis of the present study. FBW7, F-box and WD repeat-containing protein 7; Ang, angiotensin.