Temperature dependence of RLC N- and C-lobe orientation in relaxed trabeculae with unphosphorylated RLC. (A) Orientation of E (black) and BC (red) probes in the C- and N-lobe of RLC, respectively, in the blocked (orange) and free (yellow) head in the human β-cardiac myosin IHM (PDB 5TBY, essential light chains not shown for clarity), with respect to the filament axis (dashed line). (B) <P2> for E (black; mean ± SEM, n = 3) and BC (red; mean ± SEM, n = 3) probes in the absence (open circles) and in the presence (filled circles) of 3% Dextran T-500 in relaxed trabeculae at pCa 9 at 2.1 µm SL, fitted with Boltzmann curves (SI Appendix, Table S1 and Materials and Methods). Filled and open diamonds: values of <P2> (mean ± SEM, n = 3) at 26 °C and pCa 4.7 at the plateau of contraction in the presence (active isometric force per CSA [T0] = 84 ± 7 kPa; mean ± SEM, n = 6) and in the absence of Dextran (T0 = 78 ± 5 kPa; mean ± SEM, n = 6), respectively. Before activation, SL was set at 2.1 µm. (C) Mean angle (θME) and dispersion (σME) of the maximum entropy orientation distribution for E and BC probes in the presence of Dextran before RLC phosphorylation under relaxing conditions (pCa 9) at 34 °C (pink) and 5 °C (blue) and during active contraction (pCa 4.7) at 26 °C (green). Bars are mean ± SEM.