Objectives Doxorubicin often causes damage to the heart, which may present as cardiomyopathy. However, the mechanisms by which doxorubicin induces cardiotoxicity remain not fully understood and effective prevention for doxorubicin cardiomyopathy is not available. Calpains, which are a family of calcium-dependent thiol-proteases, have been implicated in cardiovascular diseases. Their activities are tightly inhibited by calpastatin. This study used transgenic mice overexpressing calpastatin to study the role of calpain in doxorubicin-induced cardiotoxicity.
Methods The models of doxorubicin-induced cardiotoxicity were created by challenging the mice with 20 mg/kg doxorubicin intraperitoneally. Cardiomyocytes apoptosis was assayed by caspase-3 activity assay, DNA fragmentation ELISA and Annexin-V staining. Calpain activities in tissue and cultured cardiomyocytes in situ extracts were detected by using a fluorescence substrate Nsuccinyl-LLVY-AMC. ROS production was measured using DCF-DA molecule probe. Neonatal cardiomyocytes were infected with adenoviral vectors containing human calpain-1, calpain-2, calpastatin or beta-gal as a control at a multiplicity of infection of 100 PFU/cell. Cardiac function was determined by echocardiography and hemodynamic measurements.
Results Doxorubicin decreased calpain activities in cultured neonatal mouse cardiomyocytes and in vivomouse hearts, which correlated with down-regulation of calpain-1 and calpain-2 proteins. Over-expression of calpastatin or treatment with pharmacological calpain inhibitors aggravated apoptosis in neonatal and adult cardiomyocytes caused by doxorubicin. On the while, over-expression of calpain-2 but not calpain-1 decreased doxorubicin-induced apoptosis in cardiomyocytes. The pro-apoptotic effects of calpain inhibition were concerned with down-regulation of AKT protein and mRNA expression, and a concomitant reduction in GSK-3 beta phosphorylation (Ser9) in doxorubicin-treated cardiomyocytes. Inhibiting AKT further increased doxorubicin-induced cardiac injuries, suggesting the effects of calpain inhibition may be mediated through inactivating the AKT signalling. In an in vivomodel of doxorubicin-induced cardiotoxicity, overexpression of calpastatin aggravated myocardial dysfunction in transgenic mice 5 days after doxorubicin treatment. The fiveday mortality was much higher in transgenic mice (29.16%) compared with their wild-type littermates (8%) after doxorubicin treatment.
Conclusions Over-expression of calpastatin aggravates doxorubicin-induced cardiac injuries by calpain inhibition and thus, calpains may protect cardiomyocytes against doxorubicin-induced cardiotoxicity.