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 (cTnT77-289) 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 cTnT77-289. After reconstituting the cTnT77-289 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 cTnT77-289 were exchanged into skinned fiber bundles. Prior to reconstitution with cTnI-cTnC, the Ca2+-independent maximal force developed by the cTnT77-289 containing fiber was 45% of the force developed by the cTnT containing fiber. After reconstituting with cTnI-cTnC, the Ca2+-activated maximal force of the cTnT77-289 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 cTnT77-289 to Tm was 3-4 fold stronger than that of cTnT. Our results suggest that strong interactions between cTnT77-289 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.
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000737:chemistry / Q000502:physiology; D009200:Myocardial Contraction / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*