Cytotoxicity of non-myofilament-incorporated troponin T fragments

Abstract

Troponin T (TnT) is a striated muscle-specific protein and an abundant component in skeletal and cardiac myofilaments. Forced expression of TnT protein in non-muscle cells or undifferentiated myoblasts in the absence of myofibrils indicated cytotoxicity (Wang et al., J. Biol. Chem. 280:13241-9, 2005). To investigate the cytotoxic effect of non-myofibril-incorporated TnT, we constructed non-fusion co-expression vectors encoding green fluorescence protein tracer and different regions of the TnT polypeptide chain. Transient transfection in culture was studied in HEK293 non-muscle cells and undifferentiated C2C12 myoblasts. Cytotoxicity of the TnT fragments was examined by the viability of the transfected cells. The results revealed distinct toxic effects of different regions of TnT. The evolutionarily conserved middle and C-terminal segments of TnT were highly toxic to cells whereas the N-terminal variable region was not. The cytotoxicity of the middle and C-terminal regions of TnT was associated with apoptotic cell death. Although muscle cells have high capacity of proteolysis to rapidly remove non-myofilament incorporated TnT protein, peak releases of TnT or TnT fragments from myofibrils may occur in the events of myocardial ischemia reperfusion and skeletal muscle fatigue or injuries. When the level of non-myofilament-associated TnT and TnT fragments exceeds the protective capacity of proteolytic removal in the muscle cell, they may impose cytotoxic effect and cause cell death. Therefore, the activity of non-myofilament-associated TnT or TnT fragments in inducing apoptosis and cell death is a potential pathogenic factor, particularly important in adult cardiac myocytes that lack the ability of regeneration.