The force-length relation is one of the most defining features of the sliding filament theory of muscle contraction, and yet a topic of debate in the literature. The force-length relation predicts that the force produced by muscle fibers is directly proportional to the degree of overlap between myosin and actin filaments, but several studies have shown forces that are larger than predicted, especially at long sarcomere lengths. The reason for such discrepancies may be due to technical difficulties – studies have been conducted with muscle fibers, a preparation containing thousands of sarcomeres that makes accurate length measurements challanging. The aim of this study was to directly evaluate force production and sarcomere dynamics in isolated myofibrils and single sarcomeres to enhance our understanding about mechanisms governing deviations from the theoretically predicted force-length relation. Contractions at varying sarcomere lengths (SLs) along the plateau (SL=2.25m-2.39m) and the descending limb (SL>2.39m) of the force-length relation were induced by solution switching, and different modes of force measurements were used. Our results show that single sarcomeres and extrapolated forces resemble the theoretical force-length relation closely, while myofibrils with sarcomere length dispersions show an extension of the plateau region and forces elevated above predicted levels. We also found an increase in sarcomere length non-uniformity and slowed rate of force production at long lengths in myofibrils with sarcomeres in series but not in single sarcomere preparations. We conclude that the deviation of the descending limb of the force-length relation obtained from plateau forces is correlated with the degree of sarcomere length non-uniformity and slowed force development.

Two needles were tracked during experiments and their displacement was measured using Fiji software. The needle tracks (.xls) of the video analysis are provided as well as sheets for further analysis (macro and R code file). The needle stiffness was measured by the cross-bending method as described in the manuscript. 

In each xls file a single track of displacement of one needle is found for further calculation of total displacement of two needles.

Given displacement, force can be calculated as follows:

F = delta(displacement) * stiffness