Crossbridge Interaction During Isometric Loading

To better understand the processes at work under isometric loading the interaction of crossbridges for two parameter settings was examined in more detail: x4 = 10nm, x-4 =-

75nm and x4 = 40nm, x-4 =-25nm. The right end of the filament position was held and

the left was free to move.

The two results started with similar numbers of crossbridges in similar states but generated different peak forces. In Figure 4.7.5, A, loading on the filament began with nine crossbridges in a pre-lever state (black) and 3 levering (red) resulting in a peak force of 24pN (Figure 4.7.5, A) while the second example (Figure 4.7.6, A) also had

nine pre-lever crossbridges (black) and 3 levering (red), but generates 5.6pN at most (Figure 4.7.6, C).

In both examples, there was very little movement of the filament, which limits the forming of new crossbridges and the generation of more force. The crossbridges present release and are not replenished. A single new crossbridge formed at actin bond site 8, Figure 4.7.5, A.

In plot A Figure 4.7.5, the individual reaction cycles show most of the crossbridges are released during or linger in the levering stage (red) strongly influencing the expression of the stored crossbridge energy. More crossbridges with the lower peak force result, Figure 4.7.6, complete the reaction cycle, being released by ATP (blue) demonstrating a much lower tension within the filament.

Some crossbridges, in both examples, contribute little or nothing to the overall force production e.g. actin site 4 in Figure 4.7.6, A, C and actin sites 13,16,17,19 Figure 4.7.5, A, C. At the onset of loading they have already expressed their strain energy or the crossbridge has moved so much since forming that its strain energy straightens the myosin arm it is attached to diverting the energy to the cofilament. The lower value of

x-4 =-75nm would have made the filament in Figure 4.7.5 more sensitive to negative

loading than that in Figure 4.7.6 (x-4 =-25nm) removing crossbridges that had travelled

excessively more rapidly.

Crossbridges occurred which acted as blocks in the tension distribution along the filament’s length. The most notable of these was at actin site 15, Figure 4.7.5, A, which remained in the levering state throughout the study period. In plot B it can be seen to have caused a disruption in the filament tension; the tension to the right changed but was consistently lower than the tension to the left. Such behaviour could prevent the filament from slipping if a sudden loss in tension occurred.

Examination of the initial length of the filaments showed the first, fast filament was under extension and the second under compression. This is highlighted by the negative force detected in Figure 4.7.6, C after the right hand crossbridges are released.

Figure 4.7.5, The isometric loading of an actin filament with parameter settings x4 = 10nm, x-4 =-75nm A: the reaction state, position and timing of crossbridge interactions (see Figure 4.3.2 for a detailed description), B: the change over time of tension between bond sites along the length of the actin filament and C: the force level at the right-hand end of the actin filament

Figure 4.7.6, The isometric loading of an actin filament with parameter settings x4 = 40nm, x-4 =-25nm A: the reaction state, position and timing of crossbridge interactions (see Figure 4.3.2 for a detailed description), B: the change over time of tension between bond sites along the length of the actin filament and C: the force level at the right-hand end of the actin filament

against time.

4.7.5

Summary.

Examination of the results of Section 4.7 show there were two key stages to the development of isometric force. The first was the number and state of the filament crossbridges at the onset of loading and the second was the expression of those crossbridges as force began to develop.

present with stored strain energy with an increased potential for higher peak forces and impulses. However, once formed, a crossbridge that travelled too far released strain energy into the cofilament rather than the actin filament and could inhibit the release of strain energy from other crossbridges. The benefits were observed in impulse results of removing these crossbridges by reducing the value of the reverse reaction, increasing the rate of removal of crossbridges loaded in the direction of contraction. There was a balance demonstrated between the forward and reverse reaction strain sensitivity in order to maintain a higher number of crossbridges in readiness for the load onset.

After the onset of loading, force developed but was not sustained. Due to a lack of movement in the filament, few new crossbridges were formed; the recycling of the initial crossbridges seems necessary to maintain force for longer periods of time as observed in vitro [30,57]. However it is notable, that the filaments in vitro tend to be much longer, have more crossbridges and may experience more internal tension and shifting bond site positions due to the higher number of crossbridges. In the

experiment, a perfect equilibrium in force and displacement was imposed. In a sarcomere and fibres, more movement may be expected as filaments potentially jostle one another generating more bonding opportunities. After the onset of force, a fast turn-over in response to filament tension may generate more force. This may be in contradiction to the pre-lever parameter settings for the initial onset of force, but without the formation of new crossbridges, cannot be gauged here.

A large proportion of crossbridges was held in the levering stage highlighting the importance of this reaction stage in force development and indicating, that further investigation of it is required. Again, greater movement in the sarcomere through the interaction of filaments may reduce this occurrence. There is some experimental evidence for the increased duration and detachment during the levering stage: In in vitro experiments at higher loads, the rate of ATP utilisation in fibres declines [101,102] and the lever stroke is shorter and slower in in vitro filament studies [103].

In comparison to in vitro data, the peak, forces for a single filament under isometric loading generated by the model (~37pN) were higher by a factor of 3. In vitro the substrate against which the filament force was reacted has been demonstrated to be more compliant than the cofilament in the model (Section 3.2.6), which may account for the difference. The model’s stiffer cofilament was more representative of the stiffness in the sarcomere. The distribution of peak forces in time, demonstrated here,

may be beneficial in a fibre, avoiding a sharp, potentially damaging stab of force under initial loading.