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

Key pointsTo 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): McTnT 1‐44Δ and McTnT 45‐74Δ . 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 McTnT 1‐44Δ irrespective of the type of Tm background. Cooperativity was decreased by McTnT 45‐74Δ , 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 McTnT 1‐44Δ and McTnT 45‐74Δ 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. McTnT 1‐44Δ , but not McTnT 45‐74Δ , attenuated maximal activation of the thin filament. Myofilament Ca 2+ sensitivity, as measured by pCa 50 (−log of [Ca 2+ ] free required for half‐maximal activation), decreased in McTnT 1‐44Δ (α‐Tm) fibres. The desensitizing effect of McTnT 1‐44Δ on pCa 50 was ablated in β‐Tm fibres. McTnT 45‐74Δ enhanced pCa 50 in both α‐ and β‐Tm fibres, with β‐Tm having a bigger effect. The Hill coefficient of tension development was significantly attenuated by McTnT 45‐74Δ , 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 McTnT 1‐44Δ in β‐Tm fibres. The magnitude of the length‐mediated recruitment of XBs was attenuated by McTnT 1‐44Δ 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 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.