The role of sarcomere length non-uniformities in residual force enhancement of skeletal muscle myofibrils

Abstract

The sarcomere length non-uniformity theory (SLNT) is a widely accepted explanation for residual force enhancement (RFE). RFE is the increase in steady-state isometric force following active muscle stretching. The SLNT predicts that active stretching of a muscle causes sarcomere lengths (SL) to become non-uniform, with some sarcomeres stretched beyond actin–myosin filament overlap (popping), causing RFE. Despite being widely known, this theory has never been directly tested. We performed experiments on isolated rabbit muscle myofibrils (n = 12) comparing SL non-uniformities for purely isometric reference contractions (I-state) and contractions following active stretch producing RFE (FE-state). Myofibrils were activated isometrically along the descending limb of the force–length relationship (mean ± 1 standard deviation (SD) = 2.8 ± 0.3 µm sarcomere−1). Once the I-state was reached, myofibrils were shortened to an SL on the plateau of the force–length relationship (2.4 µm sarcomere−1), and then were actively stretched to the reference length (2.9 ± 0.3 µm sarcomere−1). We observed RFE in all myofibrils (39 ± 15%), and saw varying amounts of non-uniformity (1 SD = 0.9 ± 0.5 µm) that was not significantly correlated with the amount of RFE, but through pairwise comparisons was found to be significantly greater than the non-uniformity measured for the I-state (0.7 ± 0.4 µm). Three myofibrils exhibited no increase in non-uniformity. Active stretching was accompanied by sarcomere popping in four myofibrils, and seven had popped sarcomeres in the I-state. These results suggest that, while non-uniformities are present with RFE, they are also present in the I-state. Furthermore, non-uniformity is not associated with the magnitude of RFE, and myofibrils that had no increase in non-uniformity with stretch still showed normal RFE. Therefore, it appears that SL non-uniformity is a normal associate of muscle contraction, but does not contribute to RFE following active stretching of isolated skeletal muscle myofibrils.

In vivo Sarcomere Lengths and Sarcomere Elongations Are Not Uniform across an Intact Muscle

“…Locally, we found that the standard deviation ( SD ) and coefficient of variation ( CV ) of sarcomere lengths were ~0.1 μm and 5%, respectively (Figure 4 ). These values are comparable to previous in vivo studies using whole muscle (Llewellyn et al, 2008 ; Cromie et al, 2013 ), ex vivo studies using muscle biopsies (Plotnikov et al, 2008 ), in vitro studies using muscle fibers (Infantolino et al, 2010 ) and in vitro studies using isolated myofibrils (Rassier and Pavlov, 2010 ; Johnston et al, 2016 ). It should be noted that even though the SD and CV -values are relatively small, the differences between the longest and the shortest sarcomeres from these small local regions can be as high as 1 μm, which corresponds to about 40% of the average sarcomere length (Table S1 , Figure 6 ).…”

“…The results of this study indicate that neither local nor global sarcomere lengths are uniform in the passive mouse TA muscle. The local non-uniformities in sarcomere lengths are similar to those observed in single fibers (Huxley and Peachey, 1961 ; Infantolino et al, 2010 ), isolated myofibrils (Rassier and Pavlov, 2010 ; Johnston et al, 2016 ), and locally, also in entire muscles (Llewellyn et al, 2008 ; Cromie et al, 2013 ). This result, combined with the findings from other studies, suggests that sarcomere lengths are naturally non-uniform in muscles, and that the results observed here are likely applicable across vertebrate skeletal muscles.…”

Skeletal muscle mechanics: questions, problems and possible solutions

“…A similar argument for instability on the descending limb of the force-length relationship has been made for entire muscle segments [ 21 ], and for single sarcomeres [ 22 ]. However, when stretching sarcomeres in a single myofibril to lengths on the descending limb of the force-length relationship, all sarcomeres undergo a (variable) stretch and remain at constant, but vastly different, (half-) sarcomere lengths after stretch, thereby demonstrating perfectly stable properties [ 23 , 24 ] (Fig. 4 ).…”

Force–length relation of skeletal muscles: from sarcomeres to myofibril

“…It provides a theoretical affirmation of the increased sarcomere length inhomogeneity observed through myofibril stretches (Rassier 2012). If such inhomogeneity during myofibril stretches cannot be observed (Johnston et al 2016), certainly none of these sarcomeres contains a force descending region or their force plateaus are of a same height and width, and their valleys are of a same depth. The seemingly inter-sarcomere coordination (Shimamoto et al 2009) ought to be a simple consequence of series connectivity of inhomogeneous sarcomeres having force descending regions.…”

“…Under consecutive discrete stretches, which is the condition to be assumed in the current study, increased sarcomere length inhomogeneity has been observed in a myofibril portion (Rassier 2012), which evidence the general 'popping' phenomenon. More recently, this phenomenon was also observed under isometric and eccentric contractions in a systematic experimental study (Johnston et al 2016).…”