Analysis of the molecular pathogenesis of cardiomyopathy-causing cTnT mutants I79N, ΔE96, and ΔK210

Abstract

Three troponin T (TnT) mutants that cause hypertrophic, restrictive, and dilated cardiomyopathy (I79N, ΔE96, and ΔK210, respectively), were examined using the thin-filament extraction/reconstitution technique. Effects of Ca(2+), ATP, phosphate, and ADP concentrations on force and its transients were studied at 25°C. Maximal Ca(2+) tension (THC) and Ca(2+)-activatable tension (Tact), respectively, were similar among I79N, ΔE96, and WT, whereas ΔK210 led to a significantly lower THC (∼20% less) and Tact (∼25% less) than did WT. In pCa solution containing 8 mM Pi and ionic strength adjusted to 200 mM, the Ca(2+) sensitivity (pCa50) of I79N (5.63 ± 0.02) and ΔE96 (5.60 ± 0.03) was significantly greater than that of WT (5.45 ± 0.04), but the pCa50 of ΔK210 (5.54 ± 0.04) remained similar to that of WT. Five equilibrium constants were deduced using sinusoidal analysis. All three mutants showed significantly lower K0 (ADP association constant) and larger K4 (equilibrium constant of force generation step) relative to the corresponding values for WT. I79N and ΔK210 were associated with a K2 (equilibrium constant of cross-bridge detachment step) significantly lower than that of ΔE96 and WT. These results demonstrated that at pCa 4.66, the force/cross-bridge is ∼18% less in I79N and ∼41% less in ΔK210 than that in WT. These results indicate that the molecular pathogenesis of the cardiac TnT mutation-related cardiomyopathies is different for each mutation.

Figure 1

pCa-tension plot comparing native cardiac muscle fibers (dashed curves with open circles) and fibers reconstituted with bovine cardiac actin, Tm, and Tn (curves with solid squares). Normalized tension is plotted against pCa. The symbols represent the data points and the curves represent the best fit to Eq. 1. Error bars represent the mean ± SE, but most are smaller than the symbol size. The pCa₅₀ value was identical between native fibers (5.69 ± 0.01, N=14) and fibers reconstituted with bovine cardiac actin, Tm, and Tn (5.68 ± 0.02, N=9). Although the cooperativity of actin, Tm, and Tn-reconstituted myocardium (2.71 ± 0.23, N=9) was slightly less than that of native fibers (3.22 ± 0.18, N=14), this difference was not statistically significant.

Figure 2

pCa-tension plots comparing WT and three mutant forms of TnT. The mean ± SE values for 12–20 experiments are shown. (A–C) pCa-tension curves for the mutants and WT at IS 200 mM and 8 mM Pi. (D) Curves for ΔK210 and WT at IS 150 mM and 0 mM Pi. Tension is normalized to T_a = 13.79 ± 0.73 kPa (n = 63).

Figure 3

Results of pCa-tension studies. (A) Isometric tension: T_HC, T_act, and T_LC are as defined in Eq. 1. (B) Stiffness. (C) Cooperativity. (D) pCa₅₀. Tension and stiffness were normalized to T_a. ^*p < 0.05.

Figure 4

Apparent rate constants. (A–C) 2πc is plotted against [MgATP]. (D–F) 2πb is plotted against [Pi]. Mean ± SE values are shown for 9–19 experiments. Solid curves are the best fit of the data to Eqs. S2–S4 (69).

Figure 5

Comparison of the kinetic constants. (A) Rate constants. (B) Equilibrium constants.

Figure 6

Cross-bridge distribution. The kinetic constants were used to calculate the cross-bridge distribution over six states under the standard activating condition (5 mM MgATP, 8 mM Pi, 0.02 mM MgADP, and pCa 4.66). Att indicates the sum of all strongly attached (force-generating) cross-bridges. The distribution was calculated individually and averaged for 9–13 fibers.