Residual force enhancement has been observed following active stretch of skeletal muscles and single fibres. However, there has been intense debate whether force enhancement is a sarcomeric property, or is associated with sarcomere length instability and the associated development of non-uniformities. Here, we studied force enhancement for the first time in isolated myofibrils (nZ18) that, owing to the strict in series arrangement, allowed for evaluation of this property in individual sarcomeres (nZ79). We found consistent force enhancement following stretch in all myofibrils and each sarcomere, and forces in the enhanced state typically exceeded the isometric forces on the plateau of the force-length relationship. Measurements were made on the plateau and the descending limb of the force-length relationship and revealed gross sarcomere length non-uniformities prior to and following active myofibril stretching, but in contrast to previous accounts, revealed that sarcomere lengths were perfectly stable under these experimental conditions. We conclude that force enhancement is a sarcomeric property that does not depend on sarcomere length instability, that force enhancement varies greatly for different sarcomeres within the same myofibril and that sarcomeres with vastly different amounts of actin-myosin overlap produce the same isometric steady-state forces. This last finding was not explained by differences in the amount of contractile proteins within sarcomeres, vastly different passive properties of individual sarcomeres or (half-) sarcomere length instabilities, suggesting that the basic mechanical properties of muscles, such as force enhancement, force depression and creep, which have traditionally been associated with sarcomere instabilities and the corresponding dynamic redistribution of sarcomere lengths, are not caused by such instabilities, but rather seem to be inherent properties of the mechanisms of contraction.

The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils ( n = 6) were 35.0 ± 2.9 nN/ μm2 when stretched to an average sarcomere length of 3.4 μm in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only ∼25% of the passive force enhancement observed in intact myofibrils. Therefore, ∼75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation.

In the cross-bridge theory, contractile force is produced by crossbridges that form between actin and myosin filaments. However, when a contracting muscle is stretched, its active force vastly exceeds the force that can be attributed to cross-bridges. This unexplained, enhanced force has been thought to originate in the giant protein titin, which becomes stiffer in actively compared with passively stretched sarcomeres by an unknown mechanism. We investigated this mechanism using a genetic mutation (mdm) with a small but crucial deletion in the titin protein. Myofibrils from normal and mdm mice were stretched from sarcomere lengths of 2.5 to 6.0 μm. Actively stretched myofibrils from normal mice were stiffer and generated more force than passively stretched myofibrils at all sarcomere lengths. No increase in stiffness and just a small increase in force were observed in actively compared with passively stretched mdm myofibrils. These results are in agreement with the idea that titin force enhancement stiffens and stabilizes the sarcomere during contraction and that this mechanism is lost with the mdm mutation.

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The purpose of this study was to gain further insight into passive force enhancement by testing whether passive force enhancement occurs in single myofibrils. Myofibrils (n = 6) isolated from rabbit psoas muscle were fixed at a sarcomere length of 2.4 microm, and then stretched passively and actively to a sarcomere length of 3.4 microm. Passive force after deactivation of the myofibrils was increased after active compared to passive stretching. Therefore, passive force enhancement, previously observed in muscle and fiber preparations, also occurs in single myofibrils. Passive force enhancement in myofibrils ranged from 86 to 145% of the steady-state force observed after passive stretch. Because titin is the main source of passive force in myofibrils, we propose that titin might be responsible for passive force enhancement observed in myofibrils. We propose that this might occur through an increase in stiffness when calcium concentration increases upon activation.

According to the cross-bridge theory, the steady-state isometric force of a muscle is given by the amount of actin-myosin filament overlap. However, it has been known for more than half a century that steady-state forces depend crucially on contractile history. Here, we examine history-dependent steady-state force production in view of the cross-bridge theory, available experimental evidence, and existing explanations for this phenomenon. This is done on various structural levels, ranging from the intact muscle to the myofibrillar and isolated contractile protein level, so that advantages and limitations of the various preparations can be fully exploited and overcome. Based on experimental evidence, we conclude that steady-state force following active muscle stretching is enhanced, and this enhancement has a passive and an active component. The active component is associated with the cross-bridge kinetics, and the passive component is associated with a calcium-dependent increase in titin stiffness.

SUMMARYForce depression observed following active shortening is not well understood. Previous research suggested that force depression might be associated with a stress-induced inhibition of cross-bridges in the newly formed overlap zone following shortening. Our aim was to investigate this theory in skinned fibres and determine whether there was an inhibition of the attachment of cross-bridges or a decrease in the force produced per cross-bridge. The stress-induced inhibition of cross-bridge theory gives testable predictions, including: (1) skinned fibres should show proportional force and stiffness depression, (2) force after shortening should not be lower than force before shortening, (3) stiffness following shortening should not be lower than stiffness before shortening and (4) force depression should decrease when the stress during shortening is decreased. In agreement with these predictions, force and stiffness depression were approximately proportional, and force depression decreased with decreasing stress during shortening. However, in contrast to the predictions of the stress-induced inhibition of cross-bridge theory, force after shortening from sarcomere lengths of 2.8 and 3.0m to a sarcomere length of 2.4m was smaller than force before shortening, and this was not accompanied by a corresponding decrease in stiffness. We conclude that the stress-induced inhibition of cross-bridge theory, as proposed previously, cannot be the only mechanism for force depression, but that there is an additional, stress-induced inhibition of cross-bridges in the old overlap zone. Furthermore, both mechanisms, inhibition of cross-bridge attachment and reduction of force produced per cross-bridge, contribute to force depression. Inhibition and/or reduction of force depend(s) on the amount of stress imposed on actin during the shortening phase.

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