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

Abstract

The second phase of the biphasic force decay upon release of phosphate from caged phosphate was previously interpreted as a signature of kinetics of the force-generating step in the cross-bridge cycle. To test this hypothesis without using caged compounds, force responses and individual sarcomere dynamics upon rapid increases or decreases in concentration of inorganic phosphate [P i ] were investigated in calcium-activated cardiac myofibrils. Rapid increases in [P i ] induced a biphasic force decay with an initial slow decline (phase 1) and a subsequent 3-5-fold faster major decay (phase 2). Phase 2 started with the distinct elongation of a single sarcomere, the so-called sarcomere ''give''. ''Give'' then propagated from sarcomere to sarcomere along the myofibril. Propagation speed and rate constant of phase 2 (k þPi(2) ) had a similar [P i ]-dependence, indicating that the kinetics of the major force decay (phase 2) upon rapid increase in [P i ] is determined by sarcomere dynamics. In contrast, no ''give'' was observed during phase 1 after rapid [P i ]-increase (rate constant k þPi (1) ) and during the single-exponential force rise (rate constant k ÀPi ) after rapid [P i ]-decrease. The values of k þPi(1) and k ÀPi were similar to the rate constant of mechanically induced force redevelopment (k TR ) and Ca 2þ -induced force development (k ACT ) measured at same [P i ]. These results indicate that the major phase 2 of force decay upon a P i -jump does not reflect kinetics of the force-generating step but results from sarcomere ''give''. The other phases of P i -induced force kinetics that occur in the absence of ''give'' yield the same information as mechanically and Ca 2þ -induced force kinetics (k þPi(1)~k-Pi~kTR~kACT ). Model simulations indicate that P i -induced force kinetics neither enable the separation of P i -release from the rate-limiting transition f into force states nor differentiate whether the ''forcegenerating step'' occurs before, along, or after the P i -release.

Kinetic coupling of phosphate release, force generation and rate-limiting steps in the cross-bridge cycle

“…The arrow marks the duration of phase 1 (t +Pi(1) = 57 msec) obtained by fitting the black transient with the biphasic function used in (Stehle et al 2002a;Stehle 2017). The similar slopes of the normalized force changes before t +Pi(1) illustrate the symmetry of k +Pi(1) and k −Pi and similarity with k TR (for more data, see (Stehle 2017) and Fig. 3c).…”

“…In cardiac myofibrils, there is initial symmetry during upon the [P i ] change (best seen by the magnification of the initial part in the inlet), turning to asymmetry at the time of the transition from the first, minor force decay to the second, major force decay upon increase of [P i ] (black lined transient). The arrow marks the duration of phase 1 (t +Pi(1) = 57 msec) obtained by fitting the black transient with the biphasic function used in (Stehle et al 2002a;Stehle 2017). The similar slopes of the normalized force changes before t +Pi(1) illustrate the symmetry of k +Pi(1) and k −Pi and similarity with k TR (for more data, see (Stehle 2017) and Fig.…”

“…This behavior closely matches the sarcomere dynamics taking place in guinea pig cardiac myofibrils during the fast phase of force relaxation following calcium removal (Stehle et al 2002a;Telley et al 2006), both for the absolute kinetics and [P i ] dependence. Interestingly, the force kinetics of the other phases ('truly isometric') were found reasonably symmetric, with the rate constant of the initial slow decline upon rapid increase in [P i ] k +Pi(1) as slow as k −Pi or k TR and dependent only on the final [P i ] (Stehle 2017). These results strongly suggest that, at least in cardiac myofibrils, the asymmetry arises from sarcomere dynamics, speeding up the force decrease transient by the superimposition of a relaxationlike process (Stehle et al 2002a;Tesi et al 2002b;Poggesi et al 2005).…”

“…a Rate constants obtained in experiments on fast skeletal rabbit psoas myofibrils (fs-mf) at 5 °C. c Rate constants obtained in experiments on cardiac myofibrils (c-mf) from guinea pig left ventricle at 10 °C (Stehle 2017). Experimentally observed rate constants in a and c were determined at full Ca 2+ activation (pCa 4.5).…”

“…Recently, a possible resolution of the puzzle posed by the asymmetry in P i jumps came from the investigation of force responses and individual sarcomere dynamics upon rapid increase or decrease in P i in maximally Ca 2+ activated guinea pig cardiac myofibrils (Stehle 2017). This study showed that in this experimental model (10 °C), the force decay upon rapid increase in [P i ] is clearly biphasic with an initial slow decline [phase 1; k +Pi(1) ] and a subsequent three to fivefold faster major decay [phase 2; k +Pi(2) ].…”