DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca(2+), ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T (HC) (high Ca(2+) ten­sion at pCa 4.66) (E40K: 1.21±0.06 T (a), ±SEM, N = 34; E54K: 1.24±0.07 T (a), N = 28), a significantly lower T (LC) (low- Ca(2+) tension at pCa 7.0) (E40K: 0.07±0.02 T (a), N = 34; E54K: 0.06±0.02 T (a), N = 28), and a significantly lower T (act) (Ca(2+) activatable tension) (T (act) = T (HC)–T(LC,) E40K: 1.15±0.08 T (a), N = 34; E54K: 1.18±0.06 T (a), N = 28) than WT (T (HC) = 1.53±0.07 T (a), T (LC) = 0.12±0.01 T (a), T (act) = 1.40±0.07 T (a), N = 25). All tensions were normalized to T (a) ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca(2+) sensitivity (pCa(50)) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.