Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle.

Abstract

The Ca 2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83: 3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC~) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca 2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (< 1 ms) released to a shorter length and then reextended by ~200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 -e -~) to obtain the value of kt~. In control fibers, ktr increased sigmoidally with increases in [Ca 2+] ; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 + 1.2 s-1 at 15~ than in rabbit psoas fibers (18.7 _+ 1.0 s-l). Phosphorylation of LC~ was accomplished by repeated Ca 2+ activations (pCa 4.5) of the fibers in solutions containing 6 #M calmodulin and 0.5/~M myosin light chain kinase, a protocol that resulted in an increase in LC~ phosphorylation from ~ 10% in the control fibers to >80% after treatment. After phosphorylation, kt~ was unchanged at maximum or very low levels of Ca 2+ activation. However, at intermediate levels of Ca ~ § activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC~ phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca 2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology.86:585-600), the effect of LC2 phosphorylation to increase the Ca ~+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver. Readdition of purified troponin C to the extracted fibers restored the effect of phosphorylation to potentiate force at submaximal levels of Ca ~+ activation. Thus, the mechanism of phosphorylation-dependent increases in steady-state isometric force appears to involve modulation of the effect of cross-bridges bound to actin to cooperatively enhance the Ca ~+ activation of the thin filament. The observation of a phosphorylationdependent increase in the Ca 2+ sensitivity of k,r likely has important implications in terms of dynamic contractile funct...

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