Tripartite motif 27 promotes cardiac hypertrophy via PTEN/Akt/mTOR signal pathways

Abstract

Tripartite motif-containing 27 (Trim27) is highly expressed in tumor cells and regulates natural immunity and apoptosis. However, the effects of Trim27 in cardiac hypertrophy are not fully elucidated. In this study, we tried to explore the potential role of Trim27 in pressure overload-induced cardiac hypertrophy and the underlying mechanism. The results indicated that compared to sham operation (Sham) group, transverse aortic constriction (TAC) group showed significantly up-regulated Trim27 protein expression (P < 0.05). The neonatal rat cardiomyocytes (NRCMs) were isolated and stimulated with PBS, angiotensin (AngII) and phenylephrine (PE). NRCMs were collected to detect the protein expression of Trim27. The results were consistent with the results in vivo. Compared to PBS treatment, the expression of Trim27 protein in NRCMs was significantly increased after PE or AngII stimulation (P < 0.05, respectively). Knockout of Trim27 can reduce the size of cardiomyocytes and reduce the proteins expression of ANP, BNP, and β-MHC, improve cardiac function, and reverse myocardial hypertrophy (P < 0.05). Trim27 may be involved in regulating the development of cardiac hypertrophy. Further results showed that Trim27 can increase the protein expression of phosphorylation of Akt, GSK3β, mTOR, and P70s6k by interacting with PTEN (phosphatase tensin homolog). These findings revealed that Trim27 can promote cardiac hypertrophy by activating PTEN/Akt/GSK3β/mTOR signaling pathway.

Keywords: Tripartite motif27; cardiac hypertrophy; signal pathways.

Figure 1.

Tripartite motif 27 (Trim27) is induced by hypertrophic stimuli. (a) Experimental protocol of this study. (b) Protein expression of Tripartite motif 27 (Trim27) in transverse aortic constriction (TAC)-induced cardiac hypertrophy. n = 6 mice per group, * P < 0.05 vs. sham. (c) Protein expression of Trim27 in extracts from NRCMs treated with PE or phosphate-buffered solution (PBS). n = 6 per group, (d) Protein expression of Trim27 in extracts from NRCMs treated with AngII or PBS. ** P < 0.01 vs. PBS.

Figure 2.

Tripartite motif 27 (Trim27) deficiency in the heart attenuates transverse aortic constriction (TAC)-induced cardiac hypertrophy. (a-c) The ratios of heart weight to body weight (HW/BW), lung weight (LW) /BW, and HW/tibia length (TL) in WT and Trim27- KO mice after sham treatment or TAC for four weeks. n = 8 for each group. (d-f) Left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), and short-axis shortening rate (FS%) in WT and Trim27- KO mice after sham treatment or TAC. n = 8 for each group. **P < 0.01 vs. WT sham; ## P < 0.01 vs. WT TAC.

Figure 3.

Trim27-Deficient Mice modulates TAC-induced hypertrophy, fibrosis, and heart function in vivo. (a) Sections of hearts from WT and Trim27-KO mice subjected to TAC or Sham treatment were stained with H&E and WGA to analysis heart and cardiomyocyte size and quantification analysis (n = 10 mice per group). (b) Picrosirius red (PSR) staining of histological sections of hearts from sham- and TAC-treated WT or Trim27-KO mice and quantification analysis (n = 10 mice per group). Scale bars, 100 μm. ** P < 0.01 vs. WT sham; ## P < 0.01 vs. WT TAC. (c, d) Hypertrophic markers atrial natriuretic peptide (Anp), B-type natriuretic peptide (Bnp), and β-myosin heavy chain (Myh7) mRNA levels and myocardial fibrosis of the Collagen I, Collagen III and Ctgf in vivo (n = 4 mice per group). ** P < 0.01 vs. WT sham; ## P < 0.01 vs. WT TAC.

Figure 4.

Trim27 promotes phenylephrine (PE)-induced cardiomyocyte hypertrophy. (a) Representative images of cardiomyocytes (immunostained with the α-actinin antibody) infected with Ad-shTrim27 or Ad-shRNA after treatment with PE (100 μM) for 24 hours and quantification analysis (n = 4 independent experiments). Scale bars, 20 μm. **P < 0.01 vs. PBS- AdshRNA, ##P < 0.01 vs. PE- AdshRNA. (b) Atrial natriuretic peptide (ANP) and Myh7 mRNA levels in NRCMs after PE treatment. The real-time polymerase chain reaction analysis was performed. n = 4 per group, **P < 0.01 vs. PBS- AdshRNA, ##P < 0.01 vs. PE- AdshRNA. (c) Representative images of cardiomyocytes (immunostained with the α-actinin antibody) infected with AdTrim27 or AdGFP after treatment with PE (100 μM) for 24 hours and quantification analysis (n = 4 independent experiments). Scale bars, 20 μm. **P < 0.01 vs. PBS-AdGFP, ##P < 0.01 vs. PE-AdGFP. (d) Atrial natriuretic peptide (ANP) and Myh7 mRNA levels in NRCMs after PE treatment. The real-time PCR analysis was performed. n = 4 per group, **P < 0.01 vs. PBS-AdGFP, ##P < 0.01 vs. PE-AdGFP.

Figure 5.

Effects of Trim27 on the phosphorylation levels of Akt, GSK3β, mTOR, and p70s6k. (a) Representative Western blots of the phosphorylated and the total level of Akt, GSK3β, mTOR, and p70s6k in WT cardiomyocytes hypertrophy treated by TAC and Trim27-KO group. Quantification of the relative changes in phosphorylation of Akt, GSK3β, mTOR, and p-p70s6k (n = 4 mice per group). ** P < 0.01 vs. WT-TAC. (b) Representative Western blots of the phosphorylated and the total level of Akt, GSK3β, mTOR, and p70s6k in neonatal rat cardiomyocytes with PBS-and PE-induced cardiomyocytes. Quantification of the relative changes in phosphorylation of Akt, GSK3β, mTOR, and p-p70s6k. GAPDH was used as a loading control. ** P < 0.01 vs. AdshRNA/PE (n = 4 per group).

Figure 6.

Trim 27 interacted with PTEN in NRCMs. Co-IP assay was performed to examine the interaction between Trim27 and PTEN in NRCMs.