Thin Filament Interaction and Ca ²⁺ -Desensitization Effect of the C-Terminal End Peptide of Cardiac Troponin T Where Loss of Function Mutations Cause Hypertrophic Cardiomyopathy

Figure 1.. The C-terminal end segment of mammalian cardiac TnT in comparison with skeletal muscle isoforms and cardiomyopathy mutations. (A) Primary structure map of cardiac TnT is aligned with the cTnT-C14 peptide to show their structural relationships with the segments encoded by each exon and the binding sites for TnI, TnC and Tm in relation to the T1 and T2 chymotryptic and CB2 CNBr fragments indicted. (B) The amino acid sequence alignment of the C-terminal end segment of TnT along with the average isoelectric point (pI) and net charge at pH 7.0 showed that structure of the C-terminal end segment encoded by the last exon of TnT genes is highly conserved and has an identical amino acid sequence in all mammalian species from platypus to human. The C-terminal end segments of human fast and slow skeletal muscle TnT are aligned as examples to show the variation among muscle type isoforms. The three cTnT-C14 peptides containing hypertrophic cardiomyopathy (HCM) mutation R278C, K280N or R286C are compared to implicate that the loss of a positive charge may be a pathogenic factor shared by these mutations. The NCBI GenBank accession numbers of the TnT sequences analyzed are: Platypus cardiac,…

Figure 2.. Purified cardiac α-tropomyosin and striated muscle actin used in the present study. The SDS-gel images show the tropomyosin and actin preparations of good purity and anticipated molecular weights (43-kDa and 32.5-kDa, respectively).

Figure 3.. LSPR analysis of the binding of cTnT-C14 peptides and tropomyosin. WT human cTnT-C14 peptide or cTnT-C14 peptides containing HCM mutations R278C, K280N and R286C at 2 μM concentration was flow through an LSPR sensor chip with saturated coating of Tm. In comparison with the negative control peptide cTnT-E16, the representative LSPR traces from three independent experiments demonstrate that cTnTC-14WT produced a saturable binding to immobilized Tm with fast association and dissociation rates. In contrast, the three HCM mutant peptides all showed weakened Tm-binding with significantly decreased association rates. While HcTnT-C14K280N shows much diminished binding affinity for Tm, HcTnT-C14R278C and HcTnT-C14R286C show decreased maximum binding but notably slower dissociation rates. The results demonstrate that the C-terminal end segment of cardiac TnT functions as a Tm-binding site in the form of isolated free peptide at physiologically relevant affinity, which is significantly impaired by the HCM mutations.

Figure 4.. LSPR analysis of the binding of cTnT-C14 peptides to tropomyosin-actin composite filament. The sensor chip was coated with a low density of F-action (at the flow concentration of 30 μg/mL). After a saturating addition of Tm (at the flow concentration of 10 μM) to the immobilized F-actin and a brief wash to remove free Tm, WT or HCM mutant human cTnT-C14 peptides was injected at 2 μM concentration to measure the association to and dissociation from the Tm-F-actin filaments. In comparison with the negative control peptide cTnT-E16, the LSPR profile shows an initial peak representing the binding of Tm to F-actin followed by the binding peak of cTnT-C14 peptides. The representative LSPR traces from at least three independent experiments demonstrate that WT human cTnT-C14 peptide had rapid association and saturable binding to Tm-F-actin filament followed by a rapid dissociation, similar to its interaction to Tm alone (Fig. 3). Also similar to the binding patterns to Tm alone, the K280N mutant showed drastically diminished binding affinity to the Tm-F-actin filament while the R278C and R286C mutants showed decreased maximum binding with slowed dissociation rates. The results confirm…

Figure 5.. LSPR analysis of the direct binding of cTnT-C14 peptides to F-actin. LSPR sensor chip was coated with F-action (at the flow concentration of 30 μg/mL) to test the interactions of WT and mutant human cTnT-C14 peptides with F-actin. The representative LSPR traces show that cTnT-C14WT produces the strongest binding in contrast to the weaker bindings of the mutants. Distinct from the binding kinetics to Tm of Tm-F-actin thin filaments, the binding of cTnT-C14 WT was slower and did not reach a plateau in 300 s. The binding of cTnT-R278C and cTnT-R286C showed lower maximum binding to F-action with slower rates of dissociation similar to that from Tm alone (Fig. 3). The binding of cTnT-C14K280N to F-actin exhibits fast association and dissociation rates but a significantly lower level of maximum binding. The similarities and differences of WT and HCM mutant cTnT-C14 peptides’ binding kinetics with Tm and F-actin suggest that the interaction of TnT C-terminal end segment with Tm and actin are via different mechanism involving different amino acid residues.

Figure 6.. Effect of cTnT-C14 peptide on Ca2+-activation of permeabilized cardiac muscle strips. The force-pCa curves and inset tables and bar graphs show that (A) the addition of WT cTnT-C14 peptide to skinned strips of WT mouse left ventricular papillary muscle decreased Ca2+-sensitivity. At sarcomere lengths 2.0 μm and 2.3 μm, the treatment with 20 μM cTnT-C14 peptide had a notable Ca2+-desensitization effect on cardiac muscle strips (*P < 0.05, **P < 0.01 vs control at the same sarcomere length, SL), and (B) the adding of WT cTnT-C14 peptide to skinned strips of cTnT-ND mouse left ventricular papillary muscle in force-pCa measurements did not produce the Ca2+-desensitization effect while the expected positive response to increasing SL from 2.0 μm to 2.3 μm was retained (*P < 0.05). cTnT-C14 peptide did not decrease maximum force development (Fmax dev) of the cardiac muscle strips in both experiments. N = 5 fibers in each group.

Figure 7.. HCM mutations in cTnT-C14 peptide cause a loss of function in modulating myofilament Ca2+-sensitivity. Consistent with their diminished Tm- and thin filament-bindings, the force-pCa curves and inset tables and bar graphs show that the addition of 20 μM cTnT-C14 peptides containing R278C (A), K280N (B) or R286C (C) mutations to skinned strips of WT mouse left ventricular papillary muscle did not produce any detectable effect on Ca2+-activation of contraction, indicating a loss of function that underlies the pathophysiology of HCM caused by these cardiac TnT point mutations. The addition of mutant cTnT-C14 peptide did not decrease the maximum force development (Fmax dev) of the cardiac muscle strips. N = 4 strips for R278C, N = 5 strips for R286C and N = 3 strips for K280N studies.