[Show abstract][Hide 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 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 nontransgenic mice that expressed α-tropomyosin (Tm). To further understand how the functional effects of 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 crossbridge (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 demonstrates 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 shows 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.
The Journal of Physiology 12/2012; 591(5). DOI:10.1113/jphysiol.2012.243394 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a ∼32 amino acid N-terminal extension (NTE), the function of which remains unknown. To understand its function, we generated a transgenic (TG) mouse line that expressed a recombinant chimeric cTnT in which the NTE of mouse cTnT was removed by replacing its 1-73 residues with the corresponding 1-41 residues of mouse fast skeletal TnT. Detergent-skinned papillary muscle fibers from non-TG (NTG) and TG mouse hearts were used to measure tension, ATPase activity, Ca(2+) sensitivity (pCa(50)) of tension, rate of tension redevelopment, dynamic muscle fiber stiffness, and maximal fiber shortening velocity at sarcomere lengths (SLs) of 1.9 and 2.3 μm. Ca(2+) sensitivity increased significantly in TG fibers at both short SL (pCa(50) of 5.96 vs. 5.62 in NTG fibers) and long SL (pCa(50) of 6.10 vs. 5.76 in NTG fibers). Maximal cross-bridge turnover and detachment kinetics were unaltered in TG fibers. Our data suggest that the NTE constrains cardiac thin filament activation such that the transition of the thin filament from the blocked to the closed state becomes less responsive to Ca(2+). Our finding has implications regarding the effect of tissue- and disease-related changes in cTnT isoforms on cardiac muscle function.
[Show abstract][Hide abstract] ABSTRACT: Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~36% decrease in Ca(2+)-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics.
Archives of Biochemistry and Biophysics 06/2012; 525(1):1-8. DOI:10.1016/j.abb.2012.05.024 · 3.02 Impact Factor