TPO exerts a protective effect on iron-overload induces apoptosis in cardiomyocytes via mitochondrial pathways

Thalassaemia companied with iron-overload is common in Hong Kong. Iron overload induced cardiomyopathy is the commonest cause of morbidity and mortality in b-thalassaemia patients. One of the causes of cardiac failure is chronic iron overload of blood transfusion. Some studies showed that iron overload can cause toxic effect in heart cells. Iron-overload induced cardiomyopathy damages are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes. Our previous study showed TPO has cardiac protective effect (Li et al, Circulation, 2007). In this study, we demonstrated firstly that iron induced oxidative stress can cause apoptosis in cardiomyocytes. By using H9C2 cells, we showed that iron increased reactive oxygen species (ROS) production (n=3) and reduced cell viability in a dose-dependent manner (0-0.6 mM) (n=6). Apoptotic cells were found to be significantly increased under iron treatment (0.3 mM, 72 hrs) in the AnnexinV/PI assay (n=6). The expression of active caspase-3 significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/mL). The ROS production was significantly decreased with the addition of TPO at 50 ng/mL (n=3). Dot-plot analysis of AnnexinV/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6), indicating a trend in attenuation of the drop of mitochondrial membrane potential. Our findings suggest that iron-overload lead to generation of ROS which further induces apoptosis in cardiomyocytes via mitochondrial pathways and TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and mitochondrial pathway in cardiomyocytes.