Iron overload induces mitochondria-dependent apoptosis and calpain-dependent titin proteolysis of cardiomyocytes

Abstract

BACKGROUND & OBJECTIVES: Cardiomyopathy secondary to iron overload is well documented in patients with beta-thalassaemia major. Nonetheless, the underlying mechanisms of iron entry into cardiomyocytes and the subsequent detrimental effects on cardiac function remain to be defined. We hypothesized that iron overload contributes to cardiac dysfunction by inducing apoptosis via mitochondria-dependent pathway, and alters the properties of the giant myofilament protein titin. MATERIALS & METHODS: Iron-overloaded HL-1 cardiomyocytes, derived from a spontaneously contracting mouse atrial cell line, were used for in vitro studies; iron-overloaded B6D2F1 mice were used for in vivo studies. Using fluorescent probes, iron was visualized dynamically and quantitatively in living HL-1 cardiomyocytes by fluorescence microscopy and quantitative fluorimetry. Using flow cytometry, phosphatidylserine exposure, active caspase-3 expression and mitochondrial membrane potential were determined in Iron-overloaded HL-1 cardiomyocytes. Immunostaining and gel electrophoresis of titin were examined in the heart tissues of Iron-overloaded mice. Activation of the upstream calcium-dependent protease of titin, calpain, was detected by FRET substrate cleavage using quantitative fluorimetry. RESULTS: We demonstrated the uptake of labile iron from culture medium to HL-1 cardiomyocytes. With intracellular accumulation of iron, HL-1 cells showed dose-dependent lost of membrane integrity, dose-dependent increase of caspase-3 activity and dose-dependent drop in mitochondrial membrane potential. In the cardiac tissue of Iron-overloaded mice, calpain activity increased significantly and titin disarray and degradation were also observed. CONCLUSION: Our findings suggest that iron overloading, through activation of calpain, causes titin proteolysis, and induces mitochondria-mediated caspase-3 dependent apoptosis in cardiomyocytes, which results in cardiac dysfunction.