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.
Recombinant BMP4 protect HL-1 cells from oxidative stress.
Smad1/5/8 and ID-1 may be involved in the protective BMP4 signalling pathway.