Kinetics Of Force Generation Research Articles

The myofilament elasticity and its effect on kinetics of force generation by the myosin motor

The half-sarcomere is the functional unit of striated muscle, in which, according to a “linear” mechanical model, myosin motors are parallel force generators with an average strain s acting between the opposing myosin and actin filaments that behave as a series elastic element with compliance Cf. Thus the definition of the mechanism of force generation by myosin motors in muscle requires integration of the crystallographic model of the working stroke with the mechanical constraints provided by the organization of motors in the half-sarcomere. The relation between half-sarcomere compliance and force (Chs–T) during the development of isometric contraction deviates, at low forces, from that predicted by the linear model, indicating the presence of an elastic element in parallel with the myosin motors, which may influence the estimate of s. A working stroke model, kinetically constrained by the early phase of the isotonic velocity transient following a force step, predicts that the rate of quick force recovery following a length step is reduced to the observed value by a Cf of 12.6nm/MPa. With this value of Cf, the fit of Chs–T relation during the isometric force rise gives s=1.8–1.9nm, similar to the values estimated using the linear model.

Tropomyosin D137L of Reduced Flexibility Increases Submaximal Ca2+-Activated Tension in Skeletal Muscle Myofibrils after Troponin-Tropomyosin Removal and Reconstitution

Tropomyosin (Tm) is a coiled-coil alpha-helix regulating the cooperative activation of muscle contraction by the thin filament. Tm forms strands along actin filaments which azimuthally move between switched-on/off locations under the influence of Ca2+-troponin (Tn) and strong crossbridge formation. The flexibility of Tm strongly influences its movement so that the relaxation-activation mechanism within the thin filament may be critically modulated. Here we study the mechanical consequences of the presence of chicken recombinant alpha tropomyosin, D137L, in rabbit skeletal muscle myofibrils, previously characterized in vitro (Sumida et al., 2008). Endogenous Tm and Tn are replaced into rabbit skeletal muscle myofibrils (Siththanandan et al., 2009) with recombinant rabbit Tn (WT) and chicken alphaTm (WT and D137L). SDS-PAGE analyses of samples of myofibrils show that Tm-Tn replacement was about 90%. Force recordings from small bundles of myofibrils (Tesi et al., 2002) show that at saturating [Ca2+ ] (pCa 4.5), maximal tension was not affected by Tm flexibility nor were the rates of force activation (kACT) and force redevelopment (kTR). However, at submaximal [Ca2+ ] (pCa 5.9), the presence of D137L Tm significantly increases (about 15-20%) both force and kinetics of force generation. Consistently, force-pCa curves obtained from myofibrils replaced with chicken D137L Tm showed a 0.15 increase in pCa50 compared to WT. This result supports the hypothesis (Sumida et al., 2008) that the increased flexibility imparted to the Tm coiled-coiled structure by Asp at 137 avoids excessive turning-on of the system at the high physiological myosin concentrations. Supported by Telethon-Italy GGP07133, STREP Project “BIG-HEART” 241577 EC and NIH HL22461.

The influence of PKA treatment on the Ca2+ activation of force generation by trout cardiac muscle

β-Adrenergic stimulation of the mammalian heart increases heart rate, the strength of contraction as well as the kinetics of force generation and relaxation. These effects are due to the phosphorylation of select membrane and thin filament proteins by cAMP-activated protein kinase (PKA). At the level of the sarcomere, it is typically the phosphorylation of cardiac myosin binding protein C (cMyBP-C) and cardiac troponin I (cTnI) that is responsible for the change in the kinetics of contraction and relaxation. Trout cTnI (ScTnI) lacks two critical PKA targets within the N-terminus of the protein that, when phosphorylated in mammalian cTnI, cause a reduction in myofilament Ca(2+) affinity. To determine what role the contractile element plays in the response of the trout heart to β-adrenergic stimulation, we characterized the influence of PKA treatment on the Ca(2+) activation of skinned preparations dissected from ventricular trabeculae. In these experiments, isometric force generation and the rate of force development were measured over a range of Ca(2+) concentrations. The results demonstrate that PKA treatment does not influence the Ca(2+) sensitivity of force generation but it decreases maximum force generation by 25% and the rate of force re-development at maximal activation by 46%. Analysis of the trabeculae preparations for phosphoproteins revealed that PKA treatment phosphorylated myosin light chain 2 but not cTnI or cMyBP-C. These results indicate that the function of the trout cardiac contractile element is altered by PKA phosphorylation but in a manner different from that in mammalian heart.

Simultaneous Optical Mapping of Intracellular Free Calcium and Action Potentials from Langendorff Perfused Hearts

The cardiac action potential (AP) controls the rise and fall of intracellular free Ca2+ (Ca(i)), and thus the amplitude and kinetics of force generation. Besides excitation-contraction coupling, the reverse process where Ca(i) influences the AP through Ca(i)-dependent ionic currents has been implicated as the mechanism underlying QT alternans and cardiac arrhythmias in heart failure, ischemia/reperfusion, cardiac myopathy, myocardial infarction, congenital and drug-induced long QT syndrome, and ventricular fibrillation. The development of dual optical mapping at high spatial and temporal resolution provides a powerful tool to investigate the role of Ca(i) anomalies in eliciting cardiac arrhythmias. This unit describes experimental protocols to map APs and Ca(i) transients from perfused hearts by labeling the heart with two fluorescent dyes, one to measure transmembrane potential (Vm), the other Ca(i) transients. High spatial and temporal resolution is achieved by selecting Vm and Ca(i) probes with the same excitation but different emission wavelengths, to avoid cross-talk and mechanical components.

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T0) from 3.54 nm in the control solution (T0,NR) to ∼0.5 nm in 1 μM BTS, where T0 is reduced to ∼0.15 T0,NR. The quick force recovery after a step release (1–3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T0. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T0 is ∼6000/s in the absence of filament compliance.

Regulation of Force Development Studied by Photolysis of Caged ADP in Rabbit Skinned Psoas Fibers

The present study examined the effects of Ca 2+ and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca 2+] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is −log [Ca 2+]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca 2+ and increased recruitment of cross-bridges at submaximal Ca 2+. Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k ADP, which was similar in rate and Ca 2+ sensitivity to the rate constant of tension re-development, k tr, measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca 2+, reaching a maximum at intermediate Ca 2+ (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca 2+. The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca 2+. This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP.

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 microM ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 microM-1.s-1, followed by force generation at 45 s-1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.

Kinetics of force generation and Pi release in rabbit soleus muscle fibers.

Recent studies have shown that the release of Pi from the AM.ADP.Pi crossbridge occurs in two steps: an initial force-generating isomerization which is followed by the release per se of Pi. These two steps have been shown to be little affected by the presence or absence of Ca2+ but both steps are significantly altered by temperature. In this study, the kinetics of the force generating and Pi release steps of the actomyosin ATPase cycle have been compared in Ca(2+)-activated skinned fibers of rabbit soleus (slow twitch) and psoas (fast twitch) muscle. Pi was rapidly photogenerated within the fiber lattice by laser flash photolysis of caged Pi (1-(2-nitro)phenylethyl phosphate). Pi reduces isometric tension in the steady state, but is less effective in slow muscle than in fast (eg. 14 mM Pi reduces tension by 29 +/- 4.6% in slow muscle and by 47 +/- 5.3% in fast). As in fast muscle, the tension response to a sudden increase in [Pi] in slow muscle has four phases. As in fast muscle, only phase II (an exponential decline in force) appears to be caused by Pi binding to cross-bridges, while the other three phases are probably indirect effects caused by caged Pi photolysis. The amplitude of phase II is consistent with the steady state reduction in force by Pi. The rate of phase II (kpi) is 3.9 +/- 0.33 s-1 at 20 degrees C and 0.28 +/- 0.02 s-1 at 10 degrees C (1 mM Pi).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers. A steady-state and transient kinetic study

The effect of varying concentrations of Pi and Ca2+ on isometric force and on the rate of force development in skinned rabbit psoas muscle fibers has been investigated. Steady-state results show that the three parameters that define the force-pCa relation (Po, pK, and n) all vary linearly with log [Pi]. As [Pi] increases, Po and pK decrease while n increases. The kinetics of force generation in isometrically contracting fibers were studied by laser flash photolysis of caged phosphate. The observed rate of the resulting tension transient, kPi, is 23.5 +/- 1.7 s-1 at 10 degrees C, 0.7 mM Pi, and is independent of [Ca2+] over the range pCa 4.5-7.2. By contrast, kTR, the rate of tension redevelopment following a period of isotonic shortening, is sensitive to [Ca2+] and is slower than kPi (kTR = 13.6 +/- 0.2 s-1 at pCa 4.5, 0.7 mM Pi). The results show that [Ca2+] does not directly affect the Pi release or force-generating steps of the cross-bridge cycle and show that the observed rate of force development depends on how the measurement is made. The data can be interpreted in terms of a model in which strong cross-bridges activate the thin filament, this activation being modulated by Ca2+ binding to troponin.

Temperature effects on the kinetics of force generation in normal and dystrophic mouse muscles

The kinetics of isolated extensor digitorum longus and soleus muscles from normal and genetically dystrophic ( 129 ReJ dy dy ) mice were studied at temperatures from 8 to 38°C. The rate constants for the exponential rise of tetanic force and for the exponential decay of force during an isometric twitch or short tetanus were similar in normal and dystrophic soleus muscles, but the decay rates were significantly reduced in dystrophic extensor digitorum longus muscles. The temperature dependence for several rate constants for isometric twitches and tetani was similar in all muscles studied, suggesting that the same rate limiting processes apply to fast and slow, normal and dystrophic muscles. Thus, the contractile proteins and those in the sarcoplasmic reticulum of dystrophic muscle are probably normal. The slower relaxation phase in dystrophic extensor digitorum longus muscles is compatible with a reduction in Ca 2+-pumping sites in the sarcoplasmic reticulum, perhaps secondary to a change in motor unit composition. Some changes in the temperature dependence for measured times, toward those of soleus muscles, is consistent with the increased proportion of slow twitch motor units in dystrophic extensor digitorum longus muscles.

Relative resistance to dystrophy of slow skeletal muscle of the mouse.

Relative resistance to dystrophy of slow skeletal muscle of the mouse.