MATERIALS AND METHODS

Reagents: This study was conducted from July, 1, 2015 to July, 1, 2017 in the Central Laboratory of Huaihe Hospital of Henan University. Visfatin was bought from PeproTech (Catalog number, 130-09, Rocky Hill, NJ, USA). PD98059 was ordered from Cayman (Catalog number, 10006726, Ann Arbor, MI, USA). The FBS (Sijiqing) was ordered from Zhejiang Tianhang Biological Polytron Technologies Inc. (Huzhou, Zhejiang, China). Rat cardiac muscle cell line H9c2 was ordered from American Type Culture Collection (Manassas, VA, USA). High glucose DMEM was ordered from GE Healthcare Life Sciences (Catalog number, SH30022.01, Logan, Utah, USA). RIPA lysis buffer was bought from Beyotime (Catalog number, P0013B, Beijing, China). BeyoECL Plus was bought from Beyotime (Catalog number, P0018, Beijing, China). BCA protein assay kit was bought from CWBIO (Catalog number, CW0014S, Beijing, China). About 0.45 μm of PVDF membranes was purchased from EMD Millipore (Catalog number, HVHP14250, Billerica, MA, USA). p-ERK1/2 was purchased from Cell Signaling Technology (Catalog number, 9101S, Danvers, MA, USA). T-ERK1/2 was purchased from Beyotime (Catalog number, AM076, Beijing, China). BNP (Q-16) was purchased from Santa Cruz Biotechnology Inc. (Catalog number, sc-18817, Dallas, TX, USA). α-SMA (EPR5368) was purchased from abcam (Catalog number, ab124964, Cambridge, MA, USA). β-actin (I102) was ordered from Bioworld (Catalog number, AP0060, Nanjing, China). Goat anti-Rabbit IgG (H+L) was obtained from Jackson (Catalog number, 111-035-003, West Grove, PA, USA). Donkey anti-Goat IgG (H+L) was bought from Sangon Biotech Co., Ltd., (Catalog number, D110115-0100, Shanghai, China).

Cell culture: The H9c2 rat cardiomyoblast cells were seeded into 10 cm dishes and cultured in DMEM medium containing 10% FBS, 1% penicillin and 1% streptomycin at a temperature of 37°C with a 5% CO₂ atmosphere. Cells were assigned to three groups: Control, visfatin and PD98059+visfatin. The medium was changed every 2-3 days. Cells were grown up to about 70-80% and passaged to 3.5 cm dishes and then cultured for 24 h. Cells were changed with serum-free medium for 24 h and administered with visfatin for indicated time.

Determination of cell surface area: The 6×10⁴ cells were seeded into 3.5 cm dishes. Cells were pre-treated¹⁷ with PD98059 at 50 μM for 2 h and then treated with visfatin¹⁸ at 100 ng mL^–1 for 48 h. Cells were used to observe morphology under the inverted microscope by randomly selected 5 horizons of photographs per dish, each field arbitrarily selected 10 of cells. Cell surface area was determined using NIH ImageJ software (NIH, Bethesda, MD, USA)^19,20.

Determination of protein synthesis: The 1×10⁵ cells were seeded into 3.5 cm culture dishes. After cells were pre-treated with PD98059 at 50 μM for 2 h, cells were treated with visfatin at 100 ng mL^–1 for 48 h. Cells were then trypsined and centrifuged. About 2 mL of fresh DMEM medium containing 10% FBS was added to the supernatant and the cell suspension was made into a single cell suspension. About 20 μL of cell suspension was mixed with 20 μL of 0.2% of the trypan blue solution and 20 μL of mixture was added to the automatic counting board to count cell number using Countstar counter. Each hole was counted 3 fields of view at least 5 times. The remaining cells were lysed with RIPA lysis buffer and protein concentration was measured with the BCA protein assay kit. Cell protein synthesis was determined by the protein content divided by the cell number^19,21.

Western blot analysis: Cells were lyses with RIPA lysis buffer and protein concentration was quantified with the BCA protein assay kit. The loading samples were 10-120 μg (BNP, 120 μg, α-SMA, ERK1/2, p-ERK1/2, 10 μg). The electrophoresis for α-SMA, BNP, ERK1/2 and p-ERK1/2 was used with 10% denatured SDS-PAGE gels. The proteins were transferred to 0.45 μm PVDF membranes and the membranes were blocked with 5% non-fat milk in TBST buffer at room temperature (BNP, 2 h; p-ERK1/2, α-SMA, ERK1/2 and β-actin 1 h). The primary antibodies (BNP, 1:100, α-SMA, 1:50000, ERK1/2, 1:2000, p-ERK1/2, 1:2000 and β-actin, 1:5000) were diluted with 5% non-fat milk TBST buffer and added to the membranes shaking at 4°C overnight. The secondary antibodies were diluted to 1:5000 and incubated with membranes at room temperature for 1 h. The bands were detected by BeyoECL Plus and analyzed with the NIH ImageJ software. The ratio of phosphorylated protein antibodies over corresponding total protein antibodies was calculated.

Statistical analysis: All experiment data were expressed as means±SD. The difference between the two groups was analyzed by using independent samples t-test and the SPSS17.0 statistical software was used for the analysis of the experimental data. The difference was considered statistically significant if p<0.05.

RESULTS

Visfatin induces cardiomyocyte hypertrophy that is inhibited by PD98059: In order to observe the effects of visfatin and PD98059 inhibitor of ERK1/2 signaling pathway on cardiomyocyte hypertrophy, cells were pre-treated with PD98059 and then stimulated with visfatin. The results showed that visfatin induced H9c2 cell surface area increase significantly. After pre-treatment of PD98059, cell surface area was decreased significantly as compared with the visfatin group (Fig. 1a). Visfatin induced protein synthesis increase, while PD98059 pre-treatment caused protein synthesis reduction significantly (Fig. 1b). Furthermore visfatin induced α-SMA and BNP protein expression increase obviously, whereas, PD98059 pre-treatment caused α-SMA (Fig. 1c, d) and BNP (Fig. 1e, f) protein expression decrease significantly compared to the visfatin group.

Visfatin increases phosphorylation of ERK1/2 protein that is decreased by PD98059: The inhibitor PD98059 of ERK1/2 signaling pathway was used to treat cells and visfatin was again employed. The results displayed that visfatin induced p-ERK1/2 protein expression increase significantly. After pre-treatment with PD98059, p-ERK1/2 protein expression was significantly decreased compared to the visfatin group (Fig. 2a, b) suggesting that visfatin can activate ERK1/2 signaling pathway in cardiomyocytes.

DISCUSSION

Myocardial hypertrophy is a basic adaptive response to the hemodynamic overload caused by various kinds of cardiac muscle, which is mainly characterized by the increase of intracellular protein synthesis and the change of cell volume. The experiment results showed: (1) Visfatin induced cardiomyocyte hypertrophy, (2) Visfatin activated ERK1/2 signaling pathway in cardiac myocytes and (3) Inhibition of ERK1/2 signaling pathway attenuated visfatin-induced cardiomyocyte hypertrophy.

Visfatin was discovered by Fukuhara et al.²², which is a cDNA fragment identical to the 5' non-coding region of PBEF (pre-B cell colony-enhancing factor). Visfatin was subsequently found in human and mouse abdominal visceral fat. So it is called visfatin^23,24. Some studies have indicated that visfatin has a close relationship with atherosclerosis, cardiac hypertrophy and acute myocardial infarction^10,25. Visfatin induces cardiac hypertrophy via activation of MAPK pathway. The amount of visfatin is significantly increased in the plasma of patients with acute heart failure, which is positively correlated with NT-proBNP²⁵. In the experiments, visfatin induced increased myocyte surface area and protein synthesis, enhanced hypertrophic marker BNP¹⁹ and α-SMA²⁶ protein expression, which are consistent with previous studies²⁷.

Many studies have shown that ERK1/2 signaling pathway is involved in the cardiac hypertrophy^28-30. Yu et al.⁸ showed that visfatin can cause myocardial fibrosis via the activation of p38MAPK and ERK1/2 pathways. Kim et al.^31,32 found that visfatin not only promotes the migration of human umbilical vein endothelial cells (HUVECs) by activating ERK1/2 signaling pathway but also accelerates the formation of new blood vessels.

Fig. 1(a-f):	PD98059 attenuates visfatin-induced cardiomyocyte hypertrophy, (a) Measurement of cell surface area, (b) Changes of protein synthesis in H9c2 cells, (c, e) Western blot analysis of α-SMA and BNP protein expression and (d, f) Quantitative results of α-SMA and BNP protein expression
	*p<0.05 vs. the control group, #p<0.05 vs. the visfatin group, Con: Control group, Vis: Visfatin group, PD+Vis: PD98059+visfatin group

Fig. 2(a-b):	PD98059 decreases p-ERK1/2 that is increased by visfatin in cardiomyocytes, (a) Western blot analysis of p-ERK1/2 protein expression and (b) Quantification of p-ERK1/2 protein expression
	*p<0.05 vs. the control group, #p<0.05 vs. the visfatin group, Con: Control group, Vis: Visfatin group, PD+Vis: PD98059+visfatin group

PD98059 is a specific inhibitor of the ERK1/2 signaling pathway, which exerts its effect by inhibiting the upstream kinase²⁷ of ERK1/2. The experiments showed that visfatin causes increased phospho-ERK1/2 protein expression, which is decreased by PD98059. PD98059 could significantly reduce myocardial cell surface area and protein synthesis. Western blotting also showed that PD98059 decreased cardiomyocyte BNP and α-SMA protein expression, suggesting that ERK1/2 signaling pathway is involved in the regulation of myocardial hypertrophy.

CONCLUSION

In general, the experiments displayed that visfatin induced cardiomyocyte hypertrophy, increased ERK1/2 signaling pathway. The inhibitor of ERK1/2 signaling pathway could inhibit visfatin-induced cardiomyocyte hypertrophy. These results suggested that visfatin induces cardiomyocyte hypertrophy by the activation of ERK1/2 signaling pathway. This will undoubtedly provide a new target for the treatment of cardiac hypertrophy.

SIGNIFICANCE STATEMENT

This study showed that visfatin induces cardiomyocyte hypertrophy via ERK1/2 signaling pathway. It will help the researchers to explore the underlying molecular mechanisms of visfatin-induced cardiomyocyte hypertrophy and provides the new insight that inhibition of visfatin expression may be beneficial for treatment of cardiac hypertrophy.

ACKNOWLEDGMENTS

This work was supported by the Key Research and Development Program of Shanxi Province, China (Grant No. 201603D321057), Scientific Research Foundation of Health and Family Planning Commission of Shanxi Province, China (Grant No. 201601093), Natural Science Foundation of Shanxi, China (Grant No: 201701D121162), Zhejiang Medical and Health Research Project, China (Grant No: 2018KY915) and Wenling Science and Technology Project, China (Grant No: 2017C311105).