Tripartite motif‑containing 14 may aggravate cardiac hypertrophy via the AKT signalling pathway in neonatal rat cardiomyocytes and transgenic mice

Abstract

Tripartite motif‑containing 14 (TRIM14) is an E3 ubiquitin ligase that primarily participates in the natural immune response and in tumour development via ubiquitination. However, the role of TRIM14 in cardiac hypertrophy is not currently clear. The present study examined the role of TRIM14 in cardiac hypertrophy and its potential molecular mechanism. TRIM14 was overexpressed in neonatal rat cardiomyocytes using adenovirus and cardiomyocyte hypertrophy was induced using phenylephrine (PE). Cardiomyocyte hypertrophy was assessed by measuring cardiomyocyte surface area and markers of hypertrophy. In addition, TRIM14‑transgenic (TRIM14‑TG) mice were created and cardiac hypertrophy was induced using transverse aortic constriction (TAC). Cardiac function, heart weight‑to‑body weight ratio (HW/BW), cardiomyocyte cross‑sectional area, cardiac fibrosis and hypertrophic markers were further examined. The expression of AKT signalling pathway‑related proteins was detected. TRIM14 overexpression in cardiomyocytes promoted PE‑induced increases in cardiomyocyte surface area and hypertrophic markers. TRIM14‑TG mice developed worse cardiac function, greater HW/BW, cross‑sectional area and cardiac fibrosis, and higher levels of hypertrophic markers in response to TAC. TRIM14 overexpression also increased the phosphorylation levels of AKT, GSK‑3β, mTOR and p70S6K in vivo and in vitro. To the best our knowledge, the present study was the first to reveal that overexpression of TRIM14 aggravated cardiac hypertrophy in vivo and in vitro, which may be related to activation of the AKT signalling pathway.

Keywords: AKT signalling pathway; cardiac hypertrophy; tripartite motif‑containing 14.

Figure 1.

TRIM14 is increased in TAC-induced cardiac hypertrophy. (A) Western blot analysis of TRIM14 protein in the heart tissues of the sham and TAC groups. (B) Semi-quantification of TRIM14 protein expression in the heart tissues of the sham and TAC groups. **P<0.01 vs. sham; n=6. TRIM14, tripartite motif-containing 14; TAC, transverse aortic constriction; 4W, 4 weeks.

Figure 2.

TRIM14 aggravates PE-induced cardiomyocyte hypertrophy. (A) Representative images of cardiomyocytes infected with AdGFP or AdTRIM14 and treated with PBS or PE (50 µmol/l). (B) Quantitative results of cardiomyocyte surface area. **P<0.01 vs. AdGFP PBS; ^##P<0.01 vs. AdGFP PE; ≥100 cells in each experimental group; scale bar, 20 µm; n=3. The relative mRNA expression levels of (C) TRIM14, (D) ANP and MYH7 in neonatal rat cardiomyocytes after infection with AdGFP or AdTRIM14 and treatment with PBS or PE. **P<0.01, ***P<0.001 vs. AdGFP PBS; ^##P<0.01, ^###P<0.001 vs. AdGFP PE; n=3. TRIM14, tripartite motif-containing 14; PE, phenylephrine; AdGFP, adenovirus GFP; AdTRIM14, adenovirus TRIM14.

Figure 3.

TRIM14 overexpression exacerbates cardiac hypertrophy and dysfunction caused by pressure overload. A total of 4 weeks after sham or TAC surgery, the statistical results of (A) LVEDd, (B) LVESd and (C) FS were obtained in 10 NTG mice and 10 TRIM14-TG mice. A total of 4 weeks after sham or TAC surgery, the (D) HW/BW, (E) LW/BW and (F) HW/TL ratios of 10 NTG mice and 10 TRIM14-TG mice were analysed. **P<0.01 vs. NTG sham; ^##P<0.01 vs. NTG TAC. TRIM14, tripartite motif-containing 14; TAC, transverse aortic constriction; LVEDd, left ventricular end diastolic diameter; LVESd, left ventricular end systolic diameter; FS, fractional shortening; NTG, non-transgenic; TG, transgenic; HW, heart weight; BW, body weight; LW, lung weight; TL, tibia length.

Figure 4.

Overexpression of TRIM14 promotes cardiac hypertrophy and fibrosis. (A) Representative H&E staining images of cardiac and cardiomyocyte cross-sectional area; n=5. (B) Statistical results of cardiomyocyte cross-sectional area; ≥100 cells per group; scale bar, 25 µm; n=5. (C) Representative PSR staining images of cardiac perivascular and interstitial collagen fibres; scale bar, 100 µm; n=5. (D) Statistical results of cardiac interstitial fibrosis; ≥40 fields per group. The relative mRNA expression levels of (E) TRIM14, (F) ANP, BNP and MYH7, and (G) collagen Iα, collagen III and CTGF were determined using reverse transcription-quantitative PCR. n=6; **P<0.01, ***P<0.001 vs. NTG sham; ^##P<0.01, ^###P<0.001 vs. NTG TAC. TRIM14, tripartite motif-containing 14; PSR, picrosirius red; NTG, non-transgenic; TAC, transverse aortic constriction; TG, transgenic; LV, left ventricular.

Figure 5.

TRIM14 enhances activation of the AKT signalling pathway in response to hypertrophic stimulation. (A) Western blotting of total and p-AKT, p-GSK-3β, p-mTOR and p-P70S6K in NRCMs after infection with AdGFP or AdTRIM14 and treatment with PBS or PE. (B) Semi-quantification of the protein expression levels of p-AKT, p-GSK-3β, p-mTOR and p-P70S6K in NRCMs. **P<0.01 vs. AdGFP PE; n=3. (C) Western blotting of total and p-AKT, p-GSK-3β, p-mTOR and p-P70S6K in NTG and TRIM14-TG mice with TAC surgery. (D) Semi-quantification of the protein expression levels of p-AKT, p-GSK-3β, p-mTOR and p-P70S6K in mice. **P<0.01 vs. NTG TAC; n=6. TRIM14, tripartite motif-containing 14; NRCMs, neonatal rat cardiomyocytes; AdGFP, adenovirus GFP; AdTRIM14, adenovirus TRIM14; PE, phenylephrine; p, phosphorylated; NTG, non-transgenic; TAC, transverse aortic constriction; TG, transgenic.