D.A. Martyn, P.B. Chase, and A.M. Gordon
Center for Bioengineering and Dept. of Physiology and Biophysics, University of Washington, Seattle, WA 98195
The fluorescence (FL) polarization of tetramethylrhodamine labeled troponin C (TnC-Rh; labeled at Cys 98) was measured to monitor changes in probe orientation or order during Ca2+ and rigor activation of TnC reconstituted skinned rabbit psoas fibers. To test for the role of crossbridges (XBrs) in thin filament activation, force was inhibited by aluminofluoride (0.05 mM AlF4-). Dichroism was 0.14 ± 0.01 (± SEM, n = 5) in relaxing solution and decreased to 0.004 ± 0.004 (± SEM, n = 5) when force was maximally activated at pCa 4.0. During rigor contraction at pCa 9.2 dichroism decreased to 0.086 ± 0.01 (± SEM, n = 5), while force was 0.40 ± 0.05 (± SEM, n = 5) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased to 0.025 ± 0.002 (± SEM, n = 5), slightly above the pCa 4.0 control level; force was similar to pCa 9.2 rigor. Thus, rigor XBrs are able to effect a change in the orientation or order of TnC-Rh. Inhibition of cycling XBrs at pCa 4.0 with AlF4- decreased force to 0.02 ± 0.01 of pCa 4.0 control (± SEM, n = 3), but caused dichroism to increase only slightly to 0.02 ± 0.01 (± SEM, n = 3), relative to pCa 4.0 control (0.014 ± 0.01; ± SEM, n = 3). During recovery from inhibition dichroism remained at the same level as during AlF4- inhibition at pCa 4.0. In fibers reconstituted with dansylaziridine labeled TnC (sTnC-DANZ) to monitor TnC conformation changes, pCa 4.0 increased steady FL to 1.56 ± 0.21 (± SEM, n = 5 fibers) times the value at pCa 9.2. During inhibition by AlF4- (pCa 4.0) force was 0.07 ± 0.06 and sTnC-DANZ FL was 1.46 ± 0.07 (± SEM, n = 5 fibers) times control. Thus, cycling XBrs contribute less to changes in TnC structure than do Ca2+ binding or rigor Xbrs in skeletal muscle fibers.
Supported by NIH Grants HL-51277, HL-52558 and NS 08384.
D.A. Martyn, P.B. Chase, A.M. Gordon and L.L. Huntsman
Center for Bioengineering, Dept. of Physiology and Biophysics and Dept. of Radiology, University of Washington, Seattle, WA 98195
Recent evidence indicates that 50-70% of sarcomeric compliance in muscle fibers resides in structures other than crossbridges, rather than the 20% or less previously thought. Myofilament compliance, on the order of the fiber compliance due to cycling crossbridges, could explain the difference in timecourse of stiffness and force during the rise of tension in a tetanus, as well as the difference in Ca2+ sensitivity of force and stiffness, and more rapid phase 2 tension recovery at low Ca2+ activation observed in skinned skeletal fibers. For fibers in rigor, thin filament compliance decreases as sarcomere length (SL) is decreased (Higuchi et al., Biophys. J. 69, 1000, 1995). To characterize the effects of thin filament compliance on sarcomere stiffness and isometric force kinetics of cycling crossbridges, we measured the activation and SL (2.5 to 2.1 mm) dependence of sarcomere stiffness in single glycerinated rabbit psoas fibers, in the presence of ATP (5.0 mM). Sarcomere stiffness was measured using rapid length steps. At each steady SL, the ratio of stiffness/force was higher at lower force (low Ca2+) levels and phase 2 tension transients were faster, compared to maximum activation, as we previously reported (Martyn and Gordon, J. Gen. Physiol. 99, 795, 1992; Martyn and Chase, Biophys. J. 68, 235, 1995). If thin filament compliance decreases as SL decreases, the stiffness/force ratio and rate of phase 2 tension recovery should increase at short SL. However, at maximal and submaximal force, neither the stiffness/force ratio or rate of phase 2 tension recovery changed at 2.2 mm compared to 2.5 mm SL. Thus, a simple myofilament compliance cannot adequately describe results obtained from Ca2+ activated fibers.
Supported by NIH Grants HL-51277 and HL-52558.