Sarcomere lengths have been a crucial outcome measure for understanding and explaining basic muscle properties and muscle function. Sarcomere lengths for a given muscle are typically measured at a single spot, often in the mid-belly of the muscle, and at a given muscle length. It is then assumed implicitly that the sarcomere length measured at this single spot represents the sarcomere lengths at other locations within the muscle, and force-length, force-velocity, and power-velocity properties of muscles are often implied based on these single sarcomere length measurements. Although, intuitively appealing, this assumption is yet to be supported by systematic evidence. The objective of this study was to measure sarcomere lengths at defined locations along and across an intact muscle, at different muscle lengths. Using second harmonic generation (SHG) imaging technique, sarcomere patterns in passive mouse tibialis anterior (TA) were imaged in a non-contact manner at five selected locations (“proximal,” “distal,” “middle,” “medial,” and “lateral” TA sites) and at three different lengths encompassing the anatomical range of motion of the TA. We showed that sarcomere lengths varied substantially within small regions of the muscle and also for different sites across the entire TA. Also, sarcomere elongations with muscle lengthening were non-uniform across the muscle, with the highest sarcomere stretches occurring near the myotendinous junction. We conclude that muscle mechanics derived from sarcomere length measured from a small region of a muscle may not well-represent the sarcomere length and associated functional properties of the entire muscle.

The periodic striation pattern in skeletal muscle reflects the length of the basic contractile unit: the sarcomere. More than half a century ago, Gordon, Huxley and Julian provided strong support for the 'sliding filament' theory through experiments with single muscle fibres. The sarcomere force-length (FL) relationship has since been extrapolated to whole muscles in an attempt to unravel in vivo muscle function. However, these extrapolations were frequently associated with nontrivial assumptions, such as muscle length changes corresponding linearly to SL changes. Here, we determined the in situ sarcomere FL relationship in a whole muscle preparation by simultaneously measuring muscle force and individual SLs in an intact muscle-tendon unit (MTU) using state-of-the-art multi-photon excitation microscopy. We found that despite great SL non-uniformity, the mean value of SLs measured from a minute volume of the mid-belly, equivalent to about 5×10 −6 % of the total muscle volume, agrees well with the theoretically predicted FL relationship, but only if the precise contractile filament lengths are known, and if passive forces from parallel elastic components and activation-associated sarcomere shortening are considered properly. As SLs are not uniformly distributed across the whole muscle and changes in SL with muscle length are location dependent, our results may not be valid for the proximal or distal parts of the muscle. The approach described here, and our findings, may encourage future studies to determine the role of SL non-uniformity in influencing sarcomere FL properties in different muscles and for different locations within single muscles.

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.

In actively stretched skeletal muscle sarcomeres, titin-based force is enhanced, increasing the stiffness of active sarcomeres. Titin force enhancement in sarcomeres is vastly reduced in mdm, a genetic mutation with a deletion in titin. Whether loss of titin force enhancement is associated with compensatory mechanisms at higher structural levels of organization, such as single fibres or entire muscles, is unclear. The aim of this study was to determine whether mechanical deficiencies in titin force enhancement are also observed at the fibre level, and whether mechanisms compensate for the loss of titin force enhancement. Single skinned fibres from control and mutant mice were stretched actively and passively beyond filament overlap to observe titin-based force. Mutant fibres generated lower contractile stress (force divided by cross-sectional area) than control fibres. Titin force enhancement was observed in control fibres stretched beyond filament overlap, but was overshadowed in mutant fibres by an abundance of collagen and high variability in mechanics. However, titin force enhancement could be measured in all control fibres and most mutant fibres following short stretches, accounting for ∼25% of the total stress following active stretch. Our results show that the partial loss of titin force enhancement in myofibrils is not preserved in all mutant fibres and this mutation likely affects fibres differentially within a muscle. An increase in collagen helps to reestablish total force at long sarcomere lengths with the loss in titin force enhancement in some mutant fibres, increasing the overall strength of mutant fibres.

Experimental findings indicate that in-situ chondrocytes die readily following impact loading, but remain essentially unaffected at low (non-impact) strain rates. This study was aimed at identifying possible causes for cell death in impact loading by quantifying chondrocyte mechanics when cartilage was subjected to a 5% nominal tissue strain at different strain rates. Multi-scale modelling techniques were used to simulate cartilage tissue and the corresponding chondrocytes residing in the tissue. Chondrocytes were modelled by accounting for the cell membrane, pericellular matrix and pericellular capsule. The results suggest that cell deformations, cell fluid pressures and fluid flow velocity through cells are highest at the highest (impact) strain rate, but they do not reach damaging levels. Tangential strain rates of the cell membrane were highest at the highest strain rate and were observed primarily in superficial tissue cells. Since cell death following impact loading occurs primarily in superficial zone cells, we speculate that cell death in impact loading is caused by the high tangential strain rates in the membrane of superficial zone cells causing membrane rupture and loss of cell content and integrity.

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