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/reconstitu-tion technique. The effects of the Ca²⁺, ATP, phos-phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com-pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T(HC) (high Ca²⁺ ten-sion 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)-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 ten-sion 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 cooper-a-tivity 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.
Departments of Anatomy and Cell Biology, and Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America.
Fan
F
Bai
Heather L
HL
Groth
Masataka
M
Kawai
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002311:Cardiomyopathy, Dilated / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D002417:Cattle; D006321:Heart / Q000503:physiopathology*; D006801:Humans; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates; D013599:Systole; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D050505:Mutant Proteins; D010710:Phosphates; D014335:Tropomyosin; D000205:Actomyosin; D002118:Calcium