The highly conserved C-terminal end segment of troponin T binds tropomyosin and actin to function in modulating contractile kinetics

Fig. 1.. Restrictive cleavage of the evolutionarily added N-terminal hypervariable region of cardiac TnT conformationally configures a TnI-like inhibitory function to sustain cardiac pumping in acute contractility-afterload mismatch. (A) TnT and TnI evolved from a TnI-like ancestral gene (10) and restrictive proteolysis to remove the evolutionarily added N-terminal hypervariable region of cardiac TnT in adaptation to contractility-afterload mismatch (8) reconfigures an epitope structure recognized by mAb TnI-1 that was originally raised against the C-terminal end segment of TnI (11), corresponding to a Tm-binding inhibitory structure (12–14). (B) Ex vivo working heart of transgenic mouse expressing cTnT-ND shows moderately slower left ventricular systolic velocity with maintained peak systolic pressure, which effectively elongates the rapid ejection time to increase stroke volume (9).

Fig. 2.. TnT protein constructs, Tm and F-actin used in the present study. (A) Primary structure maps of intact cardiac TnT, mini-TnT, and C-terminal 14 amino acids-deleted mini-TnT-CD14 are aligned to show their structural relationships with the segments encoded by each exon (the three alternatively spliced exons are highlighted). The binding sites for TnI, TnC, and the previously identified Tm-binding sites 1 and 2 in relation to the T1 and T2 chymotryptic fragments and CB2 CNBr fragment, and the site of the restrictive proteolytic cleavage to produce cTnT-ND are indicted. The mini-TnT is constructed to exclude the known Tm-binding sites 1 and 2. (B) The Tris-Tricine 15% small pore SDS-gels show the bacterial expression of mini-TnT and mini-TnT-CD14 proteins in inclusion bodies and their purification profiles. (C) The Tris-Tricine 15% small pore SDS-gel (Left) shows the mini-TnT and mini-TnT-CD14 stocks used in the present study. The 14% Laemmli SDS-gel (Right) shows the purified rabbit cardiac α-tropomyosin and rabbit skeletal muscle F-actin used in the present study.

Fig. 3.. Identification of an mAb TnI-1 epitope conformationally reconfigured in the C-terminal end segment of cardiac TnT by removing the evolutionarily added N-terminal variable region. (A) The ELISA titration curves of mAb TnI-1 against intact cardiac TnT (cTnT), N-terminal truncated cardiac TnT (cTnT-ND) and cTnT-C14 peptide together with mouse cardiac TnI (cTnI) and human cardiac TnI C-terminal peptide (cTnI-C27) positive controls demonstrate that mAb TnI-1 originally raised against the C-terminal end segment of TnI has no detectable cross-reaction with intact cardiac TnT whereas cTnT-ND becomes strongly recognized by mAb TnI-1 with an affinity very close to that for TnI and cTnI-C27 peptide. This reconfigured TnI C terminus-like epitope is also detectable in the C-terminal 14 amino acid peptide (n = 3, *P < 0.001 vs. intact cTnT). (B) The ELISA affinity titration curves of anti-cardiac TnT mAb 1G9 located its epitope in mini-TnT and the C-terminal end 14 amino acid peptide of cardiac TnT as compared to the mini-TnT-CD14 negative control (n = 3, *P < 0.01). (C) The normalized ELISA affinity titration curves show a higher affinity of mAb 1G9 for cTnT-ND than that for intact cTnT,…

Fig. 4.. The C-terminal end segment of TnT is a Tm-binding site. (A) The ELISA Tm-binding curves show that mini-TnT produces a saturable strong binding to Tm, which is completely abolished by deletion of the C-terminal 14 amino acids in mini-TnT-CD14. The data demonstrate that the C-terminal end 14 amino acid segment encoded by the last exon of the TNNT2 gene is a third Tm-binding site. (B) LSPR characterization of the third Tm-binding site of cardiac TnT confirms the high Tm-binding affinity of mini-TnT and cTnT-C14 peptide whereas mini-TnT-CD14 lost this function. A peptide corresponding to the segment encoded by exon 16 of human TNNT2 gene (E16) was used as negative control. The apparently biphasic binding of cTnT-C14 peptide may reflect a heterogeneity in the conformation of Tm immobilized on the LSPR chip. The faster initial association rate and slower dissociation rate of cTnT-C14 peptide than that of mini-TnT may be due to higher degree of flexibility of the shorter cTnT-C14 peptide structure.

Fig. 5.. Validation of the presence of three Tm-binding sites in TnT. (A) The linear structural maps show intact and two truncated mouse slow skeletal muscle TnT (ssTnT) constructs corresponding to the deletions of Tm-binding site 3 in ssTnT1-204 and Tm-binding sites 2 and 3 in ssTnT1-179. (B) The LSPR-binding traces show that ssTnT1-204 exhibits a notably weaker binding to Tm than that of intact ssTnT whereas ssTnT1-179 shows the weakest Tm-binding. The significantly different and clearly additive maximum Tm-binding signals of the three TnT constructs demonstrates the presence of three Tm-binding sites in TnT.

Fig. 6.. cTnT-C14 peptide reduces Ca2+ sensitivity of skinned cardiac muscle. The normalized isometric force–pCa curves demonstrate that 12 μM cTnT-C14 peptide produced a right-shift, corresponding to a decrease in myofilament Ca2+-sensitivity at SL of 2.3 µm and 2.0 µm. The force–pCa relationship data were fit using the Hill exponential equation. The Inset bar graph shows that the pCa for 50% maximal force activation (pCa50) was statistically lower in the presence of cTnT-C14 peptide. N = 6 for SL of 2.0 μm and n = 4 for SL of 2.3 μm. Statistical analysis was performed by paired Student’s t test. *P < 0.05 and **P < 0.01 vs. baseline. To validate the repeated force measurement protocol, parallel experiments did not detect any run-down effect (SI Appendix, Fig. S1).

Fig. 7.. Binding of mini-TnT to Tm–F-actin and F-actin alone. (A) F-actin-Tm—mini-TnT binding kinetics were analyzed in LSPR. 10 µM rabbit cardiac α-Tm was first injected at a flow rate of 20 µL/min for 240 s to bind F-actin immobilized on the chip. Free Tm was removed by 120 s washing with the binding buffer at the same flow rate. Mini-TnT or mini-TnT-CD14 was then injected at 20 µL/min for 240 s for binding to the F-actin-Tm filament followed by washing with the binding buffer at 20 µL/min for 540 s. Mini-TnT showed clear binding to the F-actin-Tm filament while mini-TnT-CD14 did not produce a detectable signal. (B) The binding of Mini-TnT and mini-TnT-CD14 to only F-actin immobilized on LSPR chip was further measured to show that Mini-TnT but not mini-TnT-CD14 also binds F-actin. (C) The binding kinetics of mini-TnT to F-actin-Tm filament are compared with that to Tm alone and F-actin alone. The data indicated that the association rate (Ka) is faster for Tm–F-actin and Tm alone than that for F-actin alone whereas the dissociation rate (Kd) is faster for Tm–F-actin and F-actin alone than that for Tm alone. The corresponding equilibrium dissociation constants (Kd) are shown in the Left…

Fig. 8.. The C-terminal end segment of TnT is a conserved structure among muscle type isoforms across vertebrate phylum with an identical sequence in cardiac TnT of all mammals. (A) The phylogenetic tree was generated by aligning amino acid sequences of the C-terminal end segment of cardiac, fast, and slow skeletal muscle TnTs from representative vertebrate species using DNAStar computer software with maximum likelihood algorithm. The evolutionary lineage indicates that this segment is a largely conserved homologous structure. (B) Similar to mini-cardiac TnT, mini-TnT constructs derived from fast and slow skeletal muscle TnTs also bound Tm in ELISA protein binding studies, demonstrating a conserved function of the third Tm-binding site in the three TnT isoforms. The affinity titration curves show that the mini-slow skeletal muscle TnT has a significantly higher affinity for Tm than that of the cardiac and fast skeletal counterparts. Mini-cTnT-CD14 was used as background control. (C) The amino acid sequence alignment of the last exon-encoded C-terminal end segment of TnT isoforms of representative vertebrate species using the DNAStar Clustal W method shows an identical sequence in all…

Fig. 9.. Myopathic mutations in the C-terminal 14 amino acid segment impair the ability of Tm-binding. (A) Mini-TnTs containing hypertrophic cardiomyopathy mutations R278C, R286C, or W287* (CD2) showed significantly diminished binding to Tm in LSPR. The results indicate that defects in the third Tm-binding site are pathogenic due to a loss of its Tm-binding and inhibitory function, consistent with the overcontractile phenotype of hypertrophic cardiomyopathies. (B) The negative impacts of the hypertrophic cardiomyopathy mutations are confirmed by LSPR Tm-binding analysis of wild type and mutant cTnT-C14 peptides. The C-terminal peptides containing mutations near the end (CD2 and R286C) produced more notable decreases in Tm-binding, consistent with the data that the C-terminus is the most conserved structure (Fig. 8C). While the R278C mutation resulted in significantly decreased Tm-binding of mini-TnT (A), its presence in C14 peptide slowed both association and dissociation rates. The difference effects of R278C in mini-TnT vs. in C14 peptide may be from the presence or absence of the upstream structure as this mutant is closer to the N-terminal direction of the C14 segment than the other…

Fig. 10.. Modulation of Tm-binding of TnT by restrictive truncation of the N-terminal variable region. (A) Intact cardiac TnT, cTnT-ND, and cTnT-C14 peptide are aligned with the locations of the three Tm-binding sites indicated. (B) Comparison of cTnT-ND with intact cardiac TnT for Tm-binding in LSPR showed that removal of the N-terminal variable region altered the Tm-binding kinetics. cTnT-ND showed a slower association rate that may indicate a compound conformational effect on the affinities of all three binding sites. Its significantly slower dissociation rate on the other hand may reflect the enhanced function of Tm-binding site 3.

Fig. 11.. N-terminal variations modulate TnT conformation and function. The structural model summarizes that the restrictive N-terminal truncation in response to contractility-afterload mismatch modulates the molecular conformation of cTnT-ND, including the reconfiguration of the C-terminal end C14 segment into a TnI C-terminus-like structure with enhanced Tm-binding affinity. Function of the conformationally modulated Tm-binding site 3 may regulate myocardial kinetics as shown by the slowing of systolic velocity to elongate ejection time and sustain stroke volume against afterload (Fig. 1B).