X-ray Diffraction Studies on the Structural Origin of Dynamic Tension Recovery Following Ramp-Shaped Releases in High-Ca Rigor Muscle Fibers

It is generally believed that during muscle contraction, myosin heads (M) extending from myosin filament attaches to actin filaments (A) to perform power stroke, associated with the reaction, A-M-ADP-Pi → A-M + ADP + Pi, so that myosin heads pass through the state of A-M, i.e., rigor A-M complex. We have, however, recently found that: (1) an antibody to myosin head, completely covering actin-binding sites in myosin head, has no effect on Ca2+-activated tension in skinned muscle fibers; (2) skinned fibers exhibit distinct tension recovery following ramp-shaped releases (amplitude, 0.5% of Lo; complete in 5 ms); and (3) EDTA, chelating Mg ions, eliminate the tension recovery in low-Ca rigor fibers but not in high-Ca rigor fibers. These results suggest that A-M-ADP myosin heads in high-Ca rigor fibers have dynamic properties to produce the tension recovery following ramp-shaped releases, and that myosin heads do not pass through rigor A-M complex configuration during muscle contraction. To obtain information about the structural changes in A-M-ADP myosin heads during the tension recovery, we performed X-ray diffraction studies on high-Ca rigor skinned fibers subjected to ramp-shaped releases. X-ray diffraction patterns of the fibers were recorded before and after application of ramp-shaped releases. The results obtained indicate that during the initial drop in rigor tension coincident with the applied release, rigor myosin heads take up applied displacement by tilting from oblique to perpendicular configuration to myofilaments, and after the release myosin heads appear to rotate around the helical structure of actin filaments to produce the tension recovery.

The rate of tension recovery in cardiac muscle correlates with the relative residual tension prevailing after restretch

Isolated cardiac muscles generate tension more quickly at higher levels of Ca2+ activation. We investigated the molecular mechanisms underlying this effect in permeabilized rat myocardial preparations by measuring the rate of tension recovery following brief shortening/restretch perturbations. Separate series of experiments used Ca2+-activating solutions with different pH values (pH 6.75, 7.00, and 7.25) and different phosphate (Pi) concentrations (0, 2.5, and 5.0 mM added Pi) to modulate the recovery kinetics. Subsequent analysis showed that the rate of tension recovery correlated ( P < 0.001) with the relative residual tension, that is, the minimum tension measured immediately after restretch normalized to the steady-state isometric tension for the experimental condition. This new finding suggests that the rate at which cardiac muscles develop force increases with the proportion of cross bridges bound to the thin filament and is strong evidence of cooperative contractile activation.

Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 μM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa–tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release–restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.