GW24-e1418 Protective effect of bone morphogenetic protein 4(BMP4) on oxidative stress induced cardiomyocyte death

Objectives Bone morphogenetic proteins (BMP) may have multiple actions on cardiac cells. The aim of our study is to investigate the effect of BMP4 on oxidative stress-induced cardiomyocyte cell death and the possible signalling pathway. Methods All experiments were conducted using the immortalised cardiomyocyte-like HL-1 cells. 1, Oxidative stress was induced by hydrogen peroxide (H2O2). HL-1 cells were stimulated with incremental concentrations of H2O2 (10, 50, 100, 200, 300, 400, 500 µM) for 4 hours, and cell viability was evaluated to establish the optimal model of H2O2-induced injury. 2, To study the effect of BMP4 on cell viability, HL-1 cells were seeded on 96-well plates and pretreated for 24 hours with different concentrations of human recombinant BMP4 (0,10, 50, 100ng/ml) prior to the application of optimal concentrated H2O2 For 4 hours. 3, To define the signalling mechanism downstream to BMP4 effects, Proteins were isolated from cells 30min and 24h after treatment of BMP4, and subjected to western blotting with antibodies against phosphorylated Smad1/5/8 and the downstream protein-inhibitor of differentiation-1 (ID-1). The western blot analysis expression value was calculated relative to that of actin. Cell viability was detected by using Lactate dehydrogenase (LDH) cytotoxicity detection kit. Results 300uM H2O2 induces 57.8 ± 5.1% death of HL-1 cells, and was selected for further experiments. HL-1 cells pretreated with 100ng/ml recombinant BMP4 had a significiant reduction of H2O2-mediated cell death (38.4 ± 2.1%, P < 0.05), while the other concentrations did not protect. Western blot analysis showed that BMP4 increased phosphorylation of Smad1/5/8 after 30 min incubation, while ID1 protein expression increased after 24 hours. Conclusions Recombinant BMP4 protect HL-1 cells from oxidative stress. Smad1/5/8 and ID-1 may be involved in the protective BMP4 signalling pathway.