Higher affinity for TnI explains how troponin C (TnC) carrying a causative hypertrophic cardiomyopathy mutation, TnCA8V, sensitizes muscle cells to Ca2+. Muscle fibers reconstituted with TnCA8V require ~2.3-fold less [Ca2+] to achieve 50% maximum-tension compared to fibers reconstituted with wild-type TnC (TnCWT). Binding measurements rule out a significant change in N-terminus Ca2+-affinity of isolated TnCA8V, and TnCA8V binds the switch-peptide of troponin-I (TnIsp) ~1.6-fold more strongly than TnCWT; thus we model the TnC-TnIsp interaction as competing with the TnI-actin interaction. Tension data are well-fit by a model constrained to conditions in which the affinity of TnCA8V for TnIsp is 1.5-1.7-fold higher than that of TnCWT at all [Ca2+]. Mean ATPase rates of reconstituted cardiac myofibrils is greater for TnCA8V than TnCWT at all [Ca2+], with statistically significant differences in the means at higher [Ca2+]. To probe TnC-TnI interaction in low Ca2+, displacement of bis-ANS from TnI was monitored as a function of TnC. Whereas Ca2+-TnCWT displaces significantly more bis-ANS than Mg2+-TnCWT, Ca2+-TnCA8V displaces probe equivalently to Mg2+-TnCA8V and Ca2+-TnCWT, consistent with stronger Ca2+-independent TnCA8V-TnIsp. A Matlab program for computing theoretical activation is reported. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnCA8V.
Mutations in TNNC1—the gene encoding cardiac troponin C (cTnC)—that have been associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction may also affect Ca2+-regulation and function of slow skeletal muscle since the same gene is expressed in both cardiac and slow skeletal muscle. Therefore, we reconstituted rabbit soleus fibers and bovine masseter myofibrils with mutant cTnCs (A8V, C84Y, E134D, and D145E) associated with HCM to investigate their effects on contractile force and ATPase rates, respectively. Previously, we showed that these HCM cTnC mutants, except for E134D, increased the Ca2+ sensitivity of force development in cardiac preparations. In the current study, an increase in Ca2+ sensitivity of isometric force was only observed for the C84Y mutant when reconstituted in soleus fibers. Incorporation of cTnC C84Y in bovine masseter myofibrils reduced the ATPase activity at saturating [Ca2+], whereas, incorporation of cTnC D145E increased the ATPase activity at inhibiting and saturating [Ca2+]. We also tested whether reconstitution of cardiac fibers with troponin complexes containing the cTnC mutants and slow skeletal troponin I (ssTnI) could emulate the slow skeletal functional phenotype. Reconstitution of cardiac fibers with troponin complexes containing ssTnI attenuated the Ca2+ sensitization of isometric force when cTnC A8V and D145E were present; however, it was enhanced for C84Y. In summary, although the A8V and D145E mutants are present in both muscle types, their functional phenotype is more prominent in cardiac muscle than in slow skeletal muscle, which has implications for the protein-protein interactions within the troponin complex. The C84Y mutant warrants further investigation since it drastically alters the properties of both muscle types and may account for the earlier clinical onset in the proband.
laser beam, which momentarily generated a concentric temperature gradient with the center reaching 50 C. Because the pulse duration was brief (2 s), this procedure did not denature proteins. This area was viewed in a microscope field, which allowed observations of gliding thin filaments at different temperatures in the same field. We found that the gliding occurred on elevating the temperature even in the absence of Ca (Oyama et al., BBRC, 2012; see also Ishiwata, BBA, 1978). The thermally-activated association of HMM to actin results in a temporary opening of the thin filament, where it is likely that Tm/ Tn is partially dissociated from the actin filament, which causes a partial activation of the thin filament. We found that the thin filament reconstituted with V95A was more difficult to become thermally activated at around physiological temperature than that with WT or D175N. This result suggests that the critical temperature for activation is higher for V95A than WT and D175N. These results further suggest that the thermal stability of the association of V95A Tm to the actin filament is higher than that of WT Tm. Our results indicate that extra stability of the actin-Tm association may cause HCM.
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