title: Identification of two new regions in the N-terminus of cardiac troponin T that have divergent effects on cardiac contractile function.
abstract: Abstract  Cardiac troponin T (cTnT) has a highly acidic extended N-terminus, the physiological role of which remains poorly understood. To decipher the physiological role of this unique region, we deleted specific regions within the N-terminus of mouse cTnT (McTnT) to create McTnT1-44 and McTnT45-74 proteins. Contractile function and dynamic force-length measurements were made after reconstituting the McTnT deletion proteins into detergent-skinned cardiac papillary fibres harvested from non-transgenic mice that expressed α-tropomyosin (Tm). To further understand how the functional effects of the N-terminus of cTnT are modulated by Tm isoforms, McTnT deletion proteins were reconstituted into detergent-skinned cardiac papillary fibres harvested from transgenic mice that expressed both α- and β-Tm. McTnT1-44, but not McTnT45-74, attenuated maximal activation of the thin filament. Myofilament Ca(2+) sensitivity, as measured by pCa50 (-log of [Ca(2+)]free required for half-maximal activation), decreased in McTnT1-44 (α-Tm) fibres. The desensitizing effect of McTnT1-44 on pCa50 was ablated in β-Tm fibres. McTnT45-74 enhanced pCa50 in both α- and β-Tm fibres, with β-Tm having a bigger effect. The Hill coefficient of tension development was significantly attenuated by McTnT45-74, suggesting an effect on thin-filament cooperativity. The rate of cross-bridge (XB) detachment and the strained XB-mediated impact on other XBs were augmented by McTnT1-44 in β-Tm fibres. The magnitude of the length-mediated recruitment of XBs was attenuated by McTnT1-44 in β-Tm fibres. Our data demonstrate that the 1-44 region of McTnT is essential for maximal activation, whereas the cardiac-specific 45-74 region of McTnT is essential for augmenting cooperativity. Moreover, our data show that α- and β-Tm isoforms have divergent effects on McTnT deletion mutant's ability to modulate cardiac thin-filament activation and Ca(2+) sensitivity. Our results not only provide the first explicit evidence for the existence of two distinct functional regions within the N-terminus of cTnT, but also offer mechanistic insights into the divergent physiological roles of these regions in mediating cardiac contractile activation.
authors.affiliation: Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA-99164, USA. murali@vetmed.wsu.edu
authors.forename: Ranganath
authors.initials: R
authors.lastname: Mamidi
authors.forename: Sri Lakshmi
authors.initials: SL
authors.lastname: Mallampalli
authors.forename: David F
authors.initials: DF
authors.lastname: Wieczorek
authors.forename: Murali
authors.initials: M
authors.lastname: Chandra
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009200:Myocardial Contraction; D010210:Papillary Muscles / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D014335:Tropomyosin; D020107:Troponin T; D002118:Calcium