ABSTRACT
Objective Bone morphogenetic protein 4 ( BMP4) induces rat myocardial hypertrophy in H9c2 cells, but the spe-cific mechanism remains unclear .The study aimed to elucidate the role of autophagy in myocardial hypertrophy and its relationship with extracellular signal regulating kinase ( ERK1/2) signal transduction pathway . Methods H9c2 cardiomyocytes were randomly divided into 4 groups:control group, BMP4 group, BMP4+PD98059 (ERK1/2 signal pathway inhibitor) group and BMP4+3MA (autophagy inhibitor) group.With PD98059(50μmol/L) and 3MA(5mmol/L) blocking for 30min, BMP4 (50μg/L) were added.Expressions of tiny tube related proteins 1 light chain 3 (LC3) and p-ERK1/2 were detec-ted after culturing for 30min.48h later, measurements were made on cell surface area , average protein content and α-smooth muscle actin (α-SMA ) protein expression level .Cell morphology and size were measured respectively by inverted microscope and Image J software .Total protein content was detected by BCA , and western blot was used to measure the expressions of LC3, ERK1/2, p-ERK1/2 and α-SMA. Resul ts 30min later, the expressions of LC3 and p-ERK1/2 were significantly higher in BMP4 group than in control group [(1.54 ±0.05) vs (1.95 ±0.11),(0.94 ±0.04) vs (1.33 ± 0.06),P0.05). Conclusion Autophagy may participate in BMP4-induced rat myo-cardial hypertrophy in H9c2 cells through ERK1/2 signal transduction pathway .The clinical treatment of myocardial hypertrophy may benefit from the blocking of autophagy or ERK 1/2 signal transduction pathway .
ABSTRACT
Objective The stimulating factors and etiology of myocyte hypertrophy are not yet clear.We aim to investigate the mechanisms of bone morphogenetic protein 4 (BMP4)-induced H9C2 myocyte hypertrophy in rats by activating the extracellular signal regulating kinase ( ERK1/2) signaling pathway. Methods Rat H9C2 myocytes were cultured, the model of BMP4-induced H9C2 myocyte hypertrophy was established, and the cells were randomly di-vided into control A, BMP4 A, and angiotensinⅡ( AngⅡ) groups to be treated for 48 hours.The changes in the expression of ERK1/2 protein phosphorylation were observed at 0, 10, 30, 60, and 120 min after treated with BMP4 at the concentration of 50 μg/L and at 30 min after treated with BMP4 at 0, 10, 50, and 100 μg/L.The changes of the H9C2 myocytes were also observed after blocking the ERK1/2 signaling pathway.The cells were divided into control B, BMP4 B, BMP4+PD98059, and PD98059 groups to be cultured with 50μg/L BMP4 for another 48 hours after 30 min blockage with PD98059 at 50 ×10 -6 mol/L.The size and area of the cells were measured by inverted microscopy and image J Software, their total protein content detected by BCA, and the expressions of p-ERK1/2 and brain natriuretic peptide ( BNP) determined by Western blot. Results Compared with control A, the BMP4 A, and AngⅡ groups showed markedly larger cell areas ([649.74 ±2.03] vs [744.85 ±1.31] and [748.39 ±1.54] μm2), higher protein contents ([243.18 ±3.69] vs [340.09 ±2.14] and [347.43 ± 3.30] pg/cell), and higher expressions of the BNP protein ([0.72 ±0.44] vs [0.96 ±0.55] and [1.07 ±0.55]/GAPDH), with statistically significant differences between any two groups (P>0.05).After BMP4 intervention, the expression of p-ERK1/2 manifes-ted a time-dependent trend of first going up and then coming down, reaching the peak at 30 min, with the ERK1/2 protein phosphoryl-ation significantly higher at 30 min than at 10 min (P>0.05), especially than at 0, 60, and 120 min (P>0.01).The level of p-ERK1/2 protein phosphorylation in the mytocytes was elevated with the increased concentration of BMP4, remarkably higher at 50μg/L than at 0 and 10μg/L, but lower than at 100μg/L ( P>0.01) .The cell area, protein content, and BNP protein expression of the H9C2 myocytes were significantly lower in the BMP4+PD98059 group ([650.49 ±1.28] μm2, [244.06 ±3.48] pg/cell, and [0.55 ±0.15]/GAPDH) than in the BMP4 B group ([746.04 ±1.45] μm2, [343.57 ±4.11] pg/cell, and [0.79 ±0.55]/GAPDH) (P>0.01). Conclusion BMP4 may induce hypertrophy of rat H9C2 myocytes by activating the ERK1/2 signaling path-way.Early blocking of ERK1/2 activation may contribute to the management of myocardial hypertrophy.