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

摘要

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.