Inhibition of shortening velocity of skinned skeletal muscle fibers in conditions that mimic fatigue

The mechanisms responsible for the inhibition of shortening velocity that occurs during muscle fatigue have not been completely elucidated. Phosphorylation of the myosin regulatory light chain (RLC) occurs during heavy use; however, previous reports on its role in affecting velocity have been equivocal. To further understand the process of fatigue, we varied the levels of myosin RLC phosphorylation (from 10 to >50%) and the concentrations of protons (from pH 7 to 6.2) and phosphate (from 5 to 30 mM), all of which change during fatigue. We measured the mechanics of permeable rabbit psoas fibers at a temperature closer to physiological (30 degrees C), using a temperature jump protocol to briefly activate the fibers at the higher temperature to preserve sarcomere homogeneity. Although lowered pH alone had an effect on velocity, it was the three factors together, i.e., high phosphorylation, low pH, and high phosphate, that acted synergistically to inhibit fiber velocity by similar to 40%. Our data demonstrate that in conditions that simulate physiological muscle fatigue, myosin phosphorylation does contribute to the inhibition of contraction velocity of fully activated fast muscle fibers

Depletion of phosphate in active muscle fibers probes actomyosin states within the powerstroke.

Variation in the concentration of orthophosphate (Pi) in actively contracting, chemically skinned muscle fibers has proved to be a useful probe of actomyosin interaction. Previous studies have shown that isometric tension (Po) decreases linearly in the logarithm of [Pi] for [Pi] > or = 200 microM. This result can be explained in terms of cross-bridge models in which the release of Pi is involved in the transition from a weakly bound, low-force actin x myosin x ADP x Pi state to a strongly bound, high-force, actin x myosin x ADP state. The 200 microM minimum [Pi] examined results from an inability to buffer the intrafiber, diffusive buildup of Pi resulting from the fiber ATPase. In the present study, we overcome this limitation by employing the enzyme purine nucleoside phosphorylase with substrate 7-methylguanosine to reduce the calculated internal [Pi] in contracting rabbit psoas fibers to < 5 microM. At 10 degrees C we find that Po continues to increase as the [Pi] decreases for [Pi] > or = 100 microM. Below this [Pi], Po is approximately constant. These results indicate that the free energy drop in the cross-bridge powerstroke is approximately 9 kT. This value is shown to be consistent with observations of muscle efficiency at physiological temperatures

The effect of polyethylene glycol on the mechanics and ATPase activity of active muscle fibers.

We have used polyethylene glycol (PEG) to perturb the actomyosin interaction in active skinned muscle fibers. PEG is known to potentiate protein-protein interactions, including the binding of myosin to actin. The addition of 5% w/v PEG (MW 300 or 4000) to active fibers increased fiber tension and decreased shortening velocity and ATPase activity, all by 25-40%. Variation in [ADP] or [ATP] showed that the addition of PEG had little effect on the dissociation of the cross-bridge at the end of the power stroke. Myosin complexed with ADP and the phosphate analog V(i) or AlF(4) binds weakly to actin and is an analog of a pre-power-stroke state. PEG substantially enhances binding of these states both in active fibers and in solution. Titration of force with increasing [P(i)] showed that PEG increased the free energy available to drive the power stroke by about the same amount as it increased the free energy available from the formation of the actomyosin bond. Thus PEG potentiates the binding of myosin to actin in active fibers, and it provides a method for enhancing populations of some states for structural or mechanical studies, particularly those of the normally weakly bound transient states that precede the power stroke

Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers

NatuurwetenskappeFisiologiese WetenskappePlease help us populate SUNScholar with the post print version of this article. It can be e-mailed to: [email protected]

The effect of polyethene glycol on the mechanics and ATPase activity of active muscle fibres.

NatuurwetenskappeFisiologiese WetenskappePlease help us populate SUNScholar with the post print version of this article. It can be e-mailed to: [email protected]