The role of tropomyosin isoforms and phosphorylation in force generation in thin-filament reconstituted bovine cardiac muscle fibres
- First Online:
- Cite this article as:
- Lu, X., Heeley, D.H., Smillie, L.B. et al. J Muscle Res Cell Motil (2010) 31: 93. doi:10.1007/s10974-010-9213-x
The thin filament extraction and reconstitution protocol was used to investigate the functional roles of tropomyosin (Tm) isoforms and phosphorylation in bovine myocardium. The thin filament was extracted by gelsolin, reconstituted with G-actin, and further reconstituted with cardiac troponin together with one of three Tm varieties: phosphorylated αTm (αTm.P), dephosphorylated αTm (αTm.deP), and dephosphorylated βTm (βTm.deP). The effects of Ca, phosphate, MgATP and MgADP concentrations were examined in the reconstituted fibres at pH 7.0 and 25°C. Our data show that Ca2+ sensitivity (pCa50: half saturation point) was increased by 0.19 ± 0.07 units when βTm.deP was used instead of αTm.deP (P < 0.05), and by 0.27 ± 0.06 units when phosphorylated αTm was used (P < 0.005). The cooperativity (Hill factor) decreased (but insignificantly) from 3.2 ± 0.3 (5) to 2.8 ± 0.2 (7) with phosphorylation. The cooperativity decreased significantly from 3.2 ± 0.3 (5) to 2.1 ± 0.2 (9) with isoform change from αTm.deP to βTm.deP. There was no significant difference in isometric tension or stiffness between αTm.P, αTm.deP, and βTm.deP muscle fibres at saturating [Ca2+] or after rigor induction. Based on the six-state cross-bridge model, sinusoidal analysis indicated that the equilibrium constants of elementary steps differed up to 1.7× between αTm.deP and βTm.deP, and up to 2.0× between αTm.deP and αTm.P. The rate constants differed up to 1.5× between αTm.deP and βTm.deP, and up to 2.4× between αTm.deP and αTm.P. We conclude that tension and stiffness per cross-bridge are not significantly different among the three muscle models.