The Effect of Inorganic Phosphate on Force Generation in Single Myofibrils from Rabbit Skeletal Muscle

Abstract

In striated muscle, force generation and phosphate (P(i)) release are closely related. Alterations in the [P(i)] bathing skinned fibers have been used to probe key transitions of the mechanochemical coupling. Accuracy in this kind of studies is reduced, however, by diffusional barriers. A new perfusion technique is used to study the effect of [P(i)] in single or very thin bundles (1-3 microM in diameter; 5 degrees C) of rabbit psoas myofibrils. With this technique, it is possible to rapidly jump [P(i)] during contraction and observe the transient and steady-state effects on force of both an increase and a decrease in [P(i)]. Steady-state isometric force decreases linearly with an increase in log[P(i)] in the range 500 microM to 10 mM (slope -0.4/decade). Between 5 and 200 microM P(i), the slope of the relation is smaller ( approximately -0.07/decade). The rate constant of force development (k(TR)) increases with an increase in [P(i)] over the same concentration range. After rapid jumps in [P(i)], the kinetics of both the force decrease with an increase in [P(i)] (k(Pi(+))) and the force increase with a decrease in [P(i)] (k(Pi(-))) were measured. As observed in skinned fibers with caged P(i), k(Pi(+)) is about three to four times higher than k(TR), strongly dependent on final [P(i)], and scarcely modulated by the activation level. Unexpectedly, the kinetics of force increase after jumps from high to low [P(i)] is slower: k(Pi(-)) is indistinguishable from k(TR) measured at the same [P(i)] and has the same calcium sensitivity.

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi]

“…1 A). This result is in agreement with previous studies on skinned fibers (13,14,27) and skeletal myofibrils (26). Concerning detail, however, the P i -induced force decay appears biphasic, starting with a slow decline (called here ''phase 1'') followed by a major, fast decay (i.e., ''phase 2'') ( Fig.…”

“…In some studies, the minor phase 1 was not examined (15,26). In this study, it is found that phase 1 also yields a rate constant (k þPi(1) ), thus the rate constant of the second phase is termed k þPi (2) .…”

“…In this study, the P i -induced force kinetics of cardiac myofibrils were investigated to test whether the asymmetry between k þPi and k ÀPi is similar to the one reported for skeletal myofibrils (26). In addition, the reason for the previously observed asymmetry was explored.…”

“…k TR is determined by rate-limiting transitions in the cross-bridge cycle (22,23). In experiments with cardiac trabeculae and with myofibrils, k TR is similar to k ACT , the rate constant of force development upon a stepwise increase in free Ca 2þ -ion concentration (24)(25)(26). k Pi being larger than k ACT and k TR had important implications for the assignment of the first force-generating step in the cross-bridge cycle.…”

“…Furthermore, k ÀPi was similar to k TR , indicating that a force rise upon a [P i ]-change yields similar kinetics as the classical mechanically induced force redevelopment. The authors (26) noted that the difference between k þPi and k ÀPi , and the similarity of k ÀPi and k TR , are unexpected from the models evolved from P i -jump studies on muscle fibers. However, the reason for the asymmetry of k þPi and k ÀPi remained unexplained.…”