The N-terminal region of troponin T is essential for the maximal activation of rat cardiac myofilaments

Troponin T (TnT) is an essential protein in the transduction of the Ca2+-binding signal that triggers striated muscle contraction, Functional diversity among various TnT isoforms found in cardiac and skeletal muscles has been correlated with the sequence heterogeneity at the amino (N-) and the carboxyl (C-) terminal regions. The most striking difference between cardiac TnT (cTnT) and skeletal TnT (sTnT) is that cTnT has an extended N-terminus, which is rich in negatively charged amino acids. To investigate the role of this region in cTnT, we deleted the first 76 amino acids in rat cTnT (cTnT(77-289)) by site-directed mutagenesis. We exchanged the native troponin complex in rat cardiac myofibrillar preparations and detergent skinned cardiac fiber bundles by treatment with excess cTnT or cTnT(77-289). After reconstituting the cTnT(77-289) containing myofibrils with cardiac troponin I-cardiac troponin C (cTnI-cTnC), the MgATPase activity was 70% of the cTnT treated myofibrils in the relaxed state and 83% of the cTnT treated myofibrils in the maximal Ca2+-activated state. These observations were supported by force measurements in which cTnT and cTnTi(77-289) were exchanged into skinned fiber bundles, Prior to reconstitution with cTnI-cTnC, the Ca2+-independent maximal force developed by the cTnT(77-289) containing fiber was 45% of the force developed by the cTnT containing fiber. After reconstituting with cTnT-cTnC, the Ca2+-activated maximal force of the cTnT(77-289) containing fiber was 62% of the force developed by the cTnT containing + cTnI-cTnC reconstituted fiber. In both assays, no significant, changes in the normalized Ca2+-activity relation or in co-operativity were observed. Fluorescence experiments using pyrene-labeled Tm demonstrated that the binding of cTnT(77-289) to Tm was 3-4 fold stronger than that of cTnT. Our results suggest that strong interactions between cTnT(77-289) and Tm stabilize cardiac myofilaments in a sub-maximally activated state. Our findings also indicate that the N-terminus of cTnT is essential for maximal activation of cardiac myofilaments. (C) 1999 Academic Press.