*Contributed equally
The underlying mechanism of cardiac hypertrophy has not yet been fully elucidated. The present study aimed to explore the function of transcription factor EC (TFEC) in mouse models of cardiac hypertrophy and to determine the underlying mechanism. Pressure-overload cardiac hypertrophy and angiotensin II (AngII) infusion-induced animal models of cardiac hypertrophy were established
Cardiac hypertrophy is an adaptive response to hemodynamic stress and is associated with impaired cardiac function, including an increase in cardiomyocyte size, higher sarcomere organization and enhanced protein synthesis for natriuretic peptide A (ANP), natriuretic peptide B (BNP) and β-myosin heavy chain (β-MHC), and higher sarcomere organization (
Transcription factor EC (TFEC) is a basic helix-loop-helix transcription factor that is a member of the MITF family (
The present study aimed to determine whether TFEC is dysregulated in mouse models of cardiac hypertrophy. In addition, this study aimed to explore the mechanism by which TFEC might be involved in the development of cardiac hypertrophy.
Eight-week-old male adult C57BL/6 mice weighing 20-25 g (n=45) were purchased from Charles River Laboratories. The mice were housed under a 12-h light/dark cycle and pathogen-free conditions and were given free access to standard mouse chow and tap water. This study was approved by the research ethics committee of Weapon Industry 521 Hospital (approval no. WI-J2018923A).
The pressure-overload model of cardiac hypertrophy was established by transverse aortic constriction (TAC) (
The mice that survived 24 h after operation (n=10 mice per group) were observed for 4 weeks. The survival rate of 10 C57BL/6 mice in sham group was 100%. Only one of the 10 C57BL/6 animals undergoing TAC operation died (survival rate for TAC, 90%). The heart and body weight (HW/BW) ratio was compared between the TAC group and sham group.
A mouse model (C57BL/6, 8 weeks old, weighing 20-25 g) of AngII (1.46 mg/kg/day for 28 days) infusion-induced cardiac hypertrophy was established (
For the control group, all mice (n=10) survived during the experiment, equating to a 100% survival rate in the 10 C57BL/6 mice of the control group. Only one of the 10 C57BL/6 animals undergoing AngII treatment died (survival rate for AngII treated mice, 90%).
One-day-old (n=4 in the same litter for each experiment) mice were born from the wide type 8-10 week-old female adult C57BL/6 mice (Charles River Laboratories). Pups were decapitated using sterile scissors (straight) without anaesthesia and the chest was open along the sternum to allow access to the chest cavity and the heart as previously described (
Primary cardiomyocytes were isolated as described above and previously (
Mice were anesthetized with isoflurane inhalation at the concentration of 2.5% for anesthetic induction and at 1% for anesthetic maintenance (
The mouse hearts were fixed in 4% formalin for 20 min at room temperature. The heart sections were embedded in paraffin and sectioned to a thickness of 5 µm for staining with hematoxylin and eosin (H&E). Slides were stained with hematoxylin (Beijing Solarbio Science & Technology Co., Ltd.) for 5 min and washed with tap water for 2 min at room temperature. The sides were differentiated with 1% alcohol for 2 min and washed with tap water for 5 min. Subsequently, the slides were stained with eosin at room temperature for 2 min (Beijing Solarbio Science & Technology Co., Ltd.). The slides were washed with tap water for 2 min and 70% alcohol twice, after which the slides were sealed and observed under a fluorescence microscope (magnification, x20; BX43; Olympus Corporation). For wheat germ agglutinin (WGA) staining of cardiac muscle cell membrane, the tissue sections were stained with 1.0 mg/ml Alexa FluorVR 488-conjugated WGA (Molecular Probes) at room temperature for 20 min to visualize the size of cardiomyocytes using a fluorescence microscope (magnification, x20; IX71; Olympus Corporation).
Adenovirus vectors carrying RNA for overexpression of TFEC (rAd-TFEC) or a short hairpin (sh) RNA targeting TFEC (rAd-sh-TFEC) or a negative control (rAd-NC) were constructed by Shanghai GeneChem Co., Ltd. C57BL/6 mice were injected once with vectors at a concentration of 1x109 U/ml (50 µl) through the left ventricular chamber after 28 days of treatment with AngII.
Total RNA was extracted from the heart tissues or primary cardiomyocytes using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using a One Step PrimeScriptTM RT-PCR Kit (Takara Bio, Inc.) according to the manufacturers' instructions. RT-qPCR analysis was performed using LightCycler 480 SYBR Green 1 Master Mix (No. 04707516001; Roche Diagnostics) according to the manufacturers' protocol. The relative mRNA expression was normalized to that of GAPDH using the 2-∆∆Cq method (
Primary cardiomyocytes were treated with compound C (cat. no. 62749-26-2; Sigma-Aldrich; Merck KGaA) at a concentration of 20 µM or PBS for 24 h at 37˚C. Subsequently, rAd-NC or rAd-sh-TFEC was transfected into primary caridomycytes for 24 h and cells were collected for further study.
Proteins isolated from heart tissue or primary cardiomyocytes were extracted using a total protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature. A BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. Proteins (40 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% fat-free milk (Pierce; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. Membranes were incubated with primary antibodies against GAPDH (cat. no. ab8245; 1:3,000), phosphorylated (p)-AMPK (cat. no. ab133448; 1:1,000), AMPK (cat. no. ab131357; 1:1,000), p-ACC (cat. no. ab173583; 1:1,000), ACC (cat. no. ab222774; 1:1,000), p-mTOR (cat. no. ab109268; 1:1,000), mTOR (cat. no. ab134903; 1:1,000) (all from Abcam) and TFEC (cat. no. ab116167; 1:1,000; Abcam) overnight at 4˚C. The membranes were then washed with 0.1% TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 2 h at room temperature. Enhanced chemiluminescence reagent (GE Healthcare) was used to detect the signal on the membrane. Signals were detected using a Super ECL Plus kit (Nanjing KeyGen Biotech Co., Ltd.) and quantitative analysis was performed using UVP 7.0 software (UVP, LLC). Relative protein expressions were normalized to GAPDH. All experiments were repeated three times. ImageJ 1.43b software (National Institutes of Health) was used for densitometry analysis.
Prism 7.0 (GraphPad Software, Inc.) was used for the quantification of all data. In all experiments, each measurement was performed at least in triplicate. The data were presented as the means ± the standard error of the mean. Two-tailed unpaired Student's t-test was used for comparisons between two groups. Comparisons between more than three groups were made by one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
Compared with the sham control, TAC increased the size of mouse hearts, but no significant changes in body weight were observed (
The weight of the hypertrophic myocardia was significantly increased in the AngII-treated group compared with the control group, but no significant changes were observed in the body weight of mice between the two groups (
The expression of TFEC was silenced in AngII-treated mice. As presented in
A cellular cardiomyocyte model was established. As demonstrated in
AMPK/mTOR signaling is suggested to play a key role in the AngII-induced cardiac hypertrophy model (
Cardiac hypertrophy and fibrosis can be induced by many factors, including mechanical pressure overload and AngII (
AngII is the most important constituent of the renin-angiotensin aldosterone system, which serves a key role in regulating cardiomyocyte growth, cardiac hypertrophy and extracellular matrix accumulation (
AMPK signaling is suggested to be an important regulator of different physiological and pathological cellular events that occur in cardiovascular diseases (
This study presented some limitations. It would be interesting to evaluate the serum concentration of cholesterol, triglyceride, HDL and LDL in mice, since these markers are known to be associated with heart function. In a future study, these biochemical parameters will be determined in the serum of pressure-overload cardiac hypertrophy and AngII infusion-induced cardiac hypertrophy animal models.
In conclusion, the findings from the present study demonstrated that TFEC was overexpressed in the hearts of mice with cardiac hypertrophy, and that silencing TFEC could improve AngII-induced cardiac hypertrophy and dysfunction by activating AMPK/mTOR signaling. These results suggested that TFEC may serve important roles in cardiac hypertrophy and dysfunction, providing potential therapeutic targets for heart failure.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
TZ and ZW performed the experiments, analyzed the data and wrote the paper. YC and CN performed some of the RT-qPCR experiments. XZ designed the experiments, analyzed the data and gave final approval of the version to be published. All authors read and approved the final manuscript. TZ and XZ confirm the authenticity of all the raw data.
The present study was approved by the Research Ethics Committee of Weapon Industry 521 Hospital (approval no. WI-J2018923A).
Not applicable.
The authors declare that they have no competing interests.
TFEC expression is increased in the hypertrophic myocardia of mice subjected to TAC. (A) Hematoxylin and eosin staining of heart tissues. (B) HW/BW was evaluated in the sham and TAC groups. (C) Wheat germ agglutinin staining showed that the cardiomyocyte size was significantly increased in the hypertrophic myocardia of mice subjected to TAC compared with control mice (magnification, x20). (D) Representative echocardiographic images. (E) LVPWd, (F) LVEF (%), (G) dp/dtmax and (H) dp/dtmin were quantified in the hearts of TAC and sham control groups. (I) Western blotting showed that TFEC expression was increased in the hypertrophic myocardia of mice subjected to TAC compared with control mice. **P<0.01 and ***P<0.001. TAC, transverse aortic constriction; TFEC, transcription factor EC; HW/BW, heart weight and body weight ratio; LVEF, left ventricular ejection fractions; LVPWd, left ventricular posterior wall diameter; dp/dtmax, maximum change in left ventricular pressure over time; dp/dtmin, minimum change in left ventricular pressure over time.
TFEC expression is elevated in the hearts of AngII-treated mice compared with control mice. (A) Hematoxylin and eosin staining of heart tissues. (B) HW/BW was evaluated in control and Ang-treated mice. (C) Wheat germ agglutinin staining was performed to evaluate the relative cardiomyocyte size (magnification, x20). (D) Representative echocardiographic images. (E) LVPWd, (F) LVEF (%), (G) dp/dtmax and (H) dp/dtmin were quantified in the hearts of AngII-treated mice and sham control mice. (I) Western blotting assay demonstrated that TFEC expression was increased in the hearts of AngII-treated mice compared with those of control mice. *P<0.05 and ***P<0.001. AngII, angiotensin II; TFEC, transcription factor EC; HW/BW, heart weight and body weight ratio; LVEF, left ventricular ejection fractions; LVPWd, left ventricular posterior wall diameter; dp/dtmax, maximum change in left ventricular pressure over time; dp/dtmin, minimum change in left ventricular pressure over time.
TFEC knockdown improves cardiac function in AngII-treated mice compared with NC mice. (A) Western blotting analysis showed that injection with rAd-sh-TFEC significantly decreased the expression of TFEC in heart tissues compared with injection with rAd-NC. (B) Knockdown of TFEC reduced the HW/BW compared with NC group. (C) Relative cardiomyocyte size was decreased in mice injected with rAd-sh-TFEC compared with those injected with rAd-NC (magnification, x20). (D) Transfection with rAd-sh-TFEC reduced the relative mRNA levels of ANP, BNP and β-MHC compared with transfection with rAd-NC. (E) Representative echocardiographic images. (F) LVPWd, (G) LVEF (%), (H) dp/dtmax and (I) dp/dtmin were quantified in the hearts of rAd-sh-TFEC and rAd-NC-treated mice. *P<0.05, **P<0.01 and ***P<0.001. TFEC, transcription factor EC; sh, short hairpin; NC, negative control; HW/BW, heart weight and body weight ratio; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain; LVEF, left ventricular ejection fractions; LVPWd, left ventricular posterior wall diameter; dp/dtmax, maximum change in left ventricular pressure over time; dp/dtmin, minimum change in left ventricular pressure over time.
Silencing TFEC abolishes AngII-induced cardiomyocyte hypertrophy. (A) Compared with the control, treatment with AngII increased the relative cardiomyocyte size (magnification, x40). (B) mRNA levels of ANP, BNP and β-MHC were significantly increased in primary cardiomyocytes treated with AngII compared with the control. (C) In primary cardiomyocytes, treatment with AngII significantly increased the expression of TFEC compared with treatment with the control. (D) Transfection with rAd-sh-TFEC significantly decreased the expression of TFEC even in primary cardiomyocytes treated with AngII. (E) AngII-induced increase in cardiomyocyte size could be reversed by TFEC knockdown in primary cardiomyocytes (magnification, x40). (F) Elevated mRNA levels of ANP, BNP and β-MHC induced by AngII could be partially abolished following TFEC knockdown in primary cardiomyocytes. *P<0.05, **P<0.01 and ***P<0.001. AngII, angiotensin II; Con, control; NC, negative control; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain; TFEC, transcription factor EC; sh, short hairpin.
TFEC activates AMPK/mTOR signaling in primary cardiomyocytes. (A) Western blotting showed that TFEC expression was significantly increased in primary cardiomyocytes transfected with Ad-TFEC compared with those transfected with Ad-NC. (B) Reverse transcription quantitative PCR showed that transfection with rAd-TFEC did not change the mRNA levels of AMPK, ACC and mTOR, compared with transfection with rAd-NC. (C) Western blotting showed that TFEC overexpression decreased the expression of p-AMPK and p-ACC but increased the level of p-mTOR. (D) Compound C significantly suppressed the activation of AMPK/ACC but increased the activation of mTOR, even in primary cardiomyocytes transfected with rAd-sh-TFEC. *P<0.05, **P<0.01 and ***P<0.001. AMPK, AMP-activated protein kinase; mTOR, mechanistic target of rapamycin; ACC, acetyl-CoA carboxylase; NC, negative control; p, phosphorylated; sh, short hairpin; TFEC, transcription factor EC.