Force Kinetics and Individual Sarcomere Dynamics in Cardiac Myofibrils after Rapid Ca2+ Changes

Abstract

Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).

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

“…Myofibrils were prepared from left ventricles of the guinea pig as in Stehle et al (30). Relaxing/activating standard solutions without added P i contained either 3 mM K 4 Cl 2 CaEGTA (activating) or 3 mM K 4 Cl 2 EGTA (relaxing solution), 10 mM imidazole, 1 mM K 2 Cl 2 Na 2 MgATP, 3 mM MgCl 2 , 47.7 mM Na 2 CrP, 2 mM DTT, pH 7.0 at 10 C, ionic strength (m) ¼ 0.17 M. The standard solutions contained 0.16 5 0.04 mM (mean 5 SD) contaminant P i as determined by a phosphate assay kit (E-6646; Molecular Probes, Eugene, OR).…”

“…IX-70 microscope (Olympus, Melville, NY) and previously described in detail in Stehle et al (25,30). All experiments were performed at 10 C using thin bundles of few myofibrils with bundle diameters ranging from 1.5 to 3.0 mm and bundle lengths from 28 to 96 mm.…”

“…One reason could be that the force decay upon [P i ]-increase is accelerated by organized sarcomere dynamics similar to the ''give'' that we had previously shown to occur during the fast phase of muscle relaxation. This sarcomere ''give'' is responsible for the asymmetric force kinetics observed with myofibrils when free Ca 2þ -ion concentration is stepped up and stepped down in a contraction-relaxation cycle (30,31). Thus, together with the force changes, the length of individual sarcomeres was monitored in response to rapid changes in [P i Here, it is found that the force decay upon an increase in [P i ] consists of two phases, an initial slow force decay (phase 1) followed by a major fast force decay (phase 2).…”

A New and Improved View of Force Production

“…With reductions in the [Pi] from 20 mM Pi to as low as 0.16 mM Pi, the rise in isometric force is a single exponential (with no sarcomere give) and whose rate (k ÀPi ) is proportional to the final Pi. Stehle shows that these initial slow phase rates of force transients at given final levels of [Pi], (k þPi(1) and K ÀPi ), are the same as the rates of force changes produced by sudden rapid short releases and restretches (k TR ) in maximal isometric contractions and those produced by maximal step increases in Ca þ2 (k ACT ) and thus correspond to the rate-limiting transitions in the cross-bridge cycle (8,9). Finally, in model calculations, Stehle shows that that Pi transients cannot directly provide information about steps in the cross-bridge cycle before or after the rate-limiting transition.…”

SPontaneous Oscillatory Contraction (SPOC): auto-oscillations observed in striated muscle at partial activation

“…propagated sarcomeric lengthening) was observed by Stehle et al (2002) when Ca 2+ was rapidly removed in activated cardiac myofibrils, leading these authors to suggest that SPOC might facilitate the rapid relaxation of the myocardium in vivo. Sasaki et al (2006) proposed that this propagation phenomenon and SPOC might share a fundamental molecular mechanism.…”