DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges |
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Authors: | Fan Bai Heather L Groth Masataka Kawai |
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Institution: | Departments of Anatomy and Cell Biology, and Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America.; University of Minnesota, United States of America, |
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Abstract: | Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitution technique. The effects of the Ca2+, 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 Ca2+ 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- Ca2+ 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 (Ca2+ activatable tension) (T
act = T
HC–TLC, 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 Ca2+ sensitivity (pCa50) 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. |
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