The sarcomere force-length relationship has been extensively used to predict muscle force potential. The common practice is to measure the mean sarcomere length (SL) in a relaxed muscle at a single location and at a given length, and this mean SL is assumed to represent the SLs at other locations across the muscle. However, in a previous study, we found that SLs are highly non-uniform across an intact passive muscle. Moreover, SL non-uniformity increases during activation in single myofibril experiments. Myofibrils lack some structural proteins that comprise an intact muscle, and therefore, the increased SL dispersion upon activation seen in myofibrils may not occur in intact whole muscle. The objectives of the current study were (i) to measure the distribution of SLs in an activated intact muscle; and (ii) to assess the feasibility of using the mean SL measured at a specific location of the muscle to predict muscle force. Using state-of-the-art multi-photon microscopy and a miniature tendon force transducer, in vivo sarcomeres in the mouse tibialis anterior were imaged simultaneously with muscle force during isometric tetanic contractions. We found that in vivo SL dispersion increased substantially during activation and reached average differences of ~1.0 μm. These differences in SL are associated with theoretical force differences of 70-100% of the maximal isometric force. Furthermore, SLs measured at a single location in the passive muscle were poor predictors of active force potential. Although mean SLs in the activated muscle were better predictors of force potential, predicted forces still differed by as much as 35% from the experimentally measured maximal isometric forces.

The instantaneous sarcomere length (SL) is regarded as an important indicator of the functional properties of striated muscle. Previously, we found greater sarcomere elongations at the distal end compared to the mid-portion in the mouse tibialis anterior (TA) when the muscle was stretched passively. Here, we wanted to see if SL dispersions increase with activation, as has been observed in single myofibrils, and if SL dispersions differ for different locations in a muscle. Sarcomere lengths were measured at a mid- and a distal location of the TA in live mice using second harmonic generation imaging. Muscle force was measured using a tendon force transducer. We found that SL dispersions increased substantially from the passive to the active state, and were the same for the mid- and distal portions of TA. Sarcomere length non-uniformities within a segment of ~30 serial sarcomeres were up to 1.0 µm. We conclude from these findings that passive, mean SLs obtained from a single location are not necessarily representative of the distribution of SL in active muscle, and thus may be misinterpreted when deriving muscle mechanical properties, such as the force-length relationship. In view of these findings, it seems crucial to determine how SL distributions within a muscle relate to the most fundamental properties of muscle, such as the maximal isometric force.

The seemingly uniform striation pattern of skeletal muscles, quantified in terms of sarcomere lengths (SLs), is inherently non-uniform across all hierarchical levels. The SL non-uniformity theory has been used to explain the force creep in isometric contractions, force depression following shortening of activated muscle, and residual force enhancement following lengthening of activated muscle. Our understanding of sarcomere contraction dynamics has been derived primarily from in vitro experiments using regular bright-field light microscopy or laser diffraction techniques to measure striation/diffraction patterns in isolated muscle fibers or myofibrils. However, the collagenous extracellular matrices present around the muscle fibers, as well as the complex architecture in the whole muscles may lead to different contraction dynamics of sarcomeres than seen in the in vitro studies. Here, we used multi-photon excitation microscopy to visualize in situ individual sarcomeres in intact muscle tendon units (MTUs) of mouse tibialis anterior (TA), and quantified the temporal changes of SL distribution as a function of SLs in relaxed and maximally activated muscles for quasi-steady state, fixed-end isometric conditions. The corresponding muscle forces were simultaneously measured using a force transducer. We found that SL non-uniformity, quantified by the coefficient of variation (CV) of SLs, decreased at a rate of 1.9-3.1%/s in the activated muscles, but remained constant in the relaxed muscles. The force loss during the quasi-steady state likely did not play a role in the decrease of SL non-uniformity, as similar force losses were found in the activated and relaxed muscles, but the CV of SLs in the relaxed muscles underwent negligible change over time. We conclude that sarcomeres in the mid-belly of maximally contracting whole muscles constantly re-organize their lengths into a more uniform pattern over time. The molecular mechanisms accounting for SL non-uniformity appear to differ in active and passive muscles, and need further elucidation, as do the functional implications of the SL non-uniformity.