Identification of two new regions in the N-terminus of cardiac troponin T that have divergent effects on cardiac contractile function

Authors


M. Chandra: PO Box 646520, 210 Wegner Hall, Department of VCAPP, Washington State University, Pullman, WA-99164, USA. Email: murali@vetmed.wsu.edu

Key points

  • To elucidate the cardiac-specific role of the highly acidic extended N-terminus of cardiac troponin T (cTnT), the following deletions were made in the N-terminus of mouse cTnT (McTnT): McTnT1-44Δ and McTnT45-74Δ.

  • Thin-filament activation was assessed after reconstituting the deletion proteins into skinned non-transgenic mouse cardiac fibres expressing α-tropomyosin (Tm).

  • Because the N-terminus of cTnT interacts with the overlapping ends of Tm, we also sought to understand how Tm isoforms modulate the functional effects of the N-terminus of cTnT. Thus, the deletion proteins were reconstituted into skinned transgenic mouse cardiac fibres expressing β-Tm.

  • Maximal activation was decreased by McTnT1-44Δ irrespective of the type of Tm background. Cooperativity was decreased by McTnT45-74Δ, an effect that was more pronounced under β-Tm background.

  • We provide the first explicit evidence to show that the cardiac-specific extended N-terminus of cTnT contains two distinct regions that have divergent physiological roles in modulating cardiac thin-filament activation.

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 McTnT1-44Δ and McTnT45-74Δ 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. McTnT1-44Δ, but not McTnT45-74Δ, attenuated maximal activation of the thin filament. Myofilament Ca2+ sensitivity, as measured by pCa50 (−log of [Ca2+]free required for half-maximal activation), decreased in McTnT1-44Δ (α-Tm) fibres. The desensitizing effect of McTnT1-44Δ on pCa50 was ablated in β-Tm fibres. McTnT45-74Δ 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 McTnT45-74Δ, 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 McTnT1-44Δ in β-Tm fibres. The magnitude of the length-mediated recruitment of XBs was attenuated by McTnT1-44Δ 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 Ca2+ 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.

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