Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C

Abstract

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.

Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca2+-sensitizing mutations

“…The L29Q mutant was the least open; interhelical angles below 115°were not observed and had the highest closed probability. The closed state was least frequently observed in the A8V construct, which is consistent with paramagnetic NMR data showing that A8V cTnC opened more readily than WT N-cTnC (33) and contribute to the increased affinity between A8V TnC and TnI SW (31). These data suggest that the molecular etiology is different for each mutation, despite producing a similar disease phenotype.…”

“…In this study, we report the ITC-derived Ca 2ϩ -binding affinity of N-cTnC mutant constructs and their calculated effects on the dynamics, Ca 2ϩ interaction, and TnI SW interaction strengths. The N-cTnC mutations selected for analysis in this work are the FHC-associated mutations A8V (31)(32)(33), L29Q (12,19,35,36), A31S (13), and C84Y (14); the engineered Ca 2ϩsensitizing mutation L48Q (11,15,16,37,38); and the dilated cardiomyopathy-associated mutation Q50R (39) (Fig. 1).…”

Modeling Ca2+-Bound Troponin in Excitation Contraction Coupling

“… a Shown previously to fit steady-state Ca 2+ -dependent tension of skinned fibers of fast twitch (Zot et al, 2009 ) and slow twitch (Zot et al, 2016b ) muscle . …”

“…Thermodynamic principles dictate that K 1 / K 3 = K 3 / K 5 = K 2 / K 4 , which allows , K 1 , K 2 , and K 4 to be selected independently. The values used here for these four parameters, α, and n (Table 1 ) are the same shown elsewhere to fit steady-state activation of skinned fibers of fast muscle (Zot et al, 2009 ) and cardiac muscle (Zot et al, 2016b ) by Ca 2+ . Component rate constants (lower case, “ k ”) are varied to fit transient data.…”

Myofilament modulation of contraction

“…A collection of original works in this Highlight Issue has therefore focused on the structure-function effects of myofilament protein mutations linked to cardiomyopathy. Work from the laboratory of Jose Pinto sought to identify the mechanism by which the cardiomyopathy linked A8V TnC mutation results in increased myofilament calcium sensitivity (11). Results demonstrated that although the TnC A8V mutation does not directly alter calcium binding to isolated TnC, the measurement of TnC-TnI binding and modeling identified the A8V mutation sensitized the myofilament to calcium by increasing the affinity of the TnI C-terminus for TnC.…”