Abstract
The lengths of a muscle’s sarcomeres are a primary determinant of its ability to contract and produce force. In addition, sarcomere length is a critical parameter that is required to make meaningful comparisons of both the force-generating and excursion capacities of different muscles. Until recently, in vivo sarcomere length data have been limited to invasive or intraoperative measurement techniques. With the advent of second harmonic generation microendoscopy, minimally invasive measures of sarcomere length can be made for the first time. This imaging technique expands our ability to study muscle adaptation due to changes in stimulus, use, or disease. However, due to past inability to measure sarcomeres outside of surgery or biopsy, little is known about the natural, anatomical variability in sarcomere length in living human subjects. To develop robust experimental protocols that ensure data provide accurate representations of a muscle’s sarcomere lengths, we sought to quantify experimental uncertainty associated with in vivo measures of sarcomere lengths. Specifically, we assessed the variability in sarcomere length measured (1) within a single image, along a muscle fiber, (2) across images captured within a single trial, across trials, and across days, as well as (3) across locations in the muscle using second harmonic generation in two upper limb muscles with different muscle architectures, functions, and sizes. Across all of our measures of variability we estimate that the magnitude of the uncertainty for in vivo sarcomere length is on the order of ∼0.25 μm. In the two upper limb muscles studied we found larger variability in sarcomere lengths within a single insertion than across locations. We also developed custom code to make measures of sarcomere length variability across a single fiber and determined that this codes’ accuracy is an order of magnitude smaller than our measurement uncertainty due to sarcomere variability. Together, our findings provide guidance for the development of robust experimental design and analysis of in vivo sarcomere lengths in the upper limb.
Highlights
Whole muscle is made up of hundreds of thousands of sarcomeres arranged in series and parallel
Any experimental uncertainty in measurements of fascicle length or muscle volume. We investigated both anatomical and experimental sources of variability in sarcomere length measures obtained in vivo, in the upper limb, using second harmonic generation microendoscopy
Based on assessments completed in two muscles in the upper limb that have different functions, size, and architectures, we estimate the magnitude of the uncertainty in such measures to be on the order of ∼0.25 μm, which we conclude is dominated by the anatomical variability of sarcomere lengths in these muscles, under these conditions, rather than from random error associated with our methods
Summary
Whole muscle is made up of hundreds of thousands of sarcomeres arranged in series and parallel. Serial sarcomere number (SSN) is a important architectural parameter, as it describes the range of lengths over which a muscle can actively generate force and, given optimal sarcomere length, a measure of the fascicle length at which the muscle will produce its maximum isometric force. The number of sarcomeres in parallel is characterized by a muscle’s physiological crosssectional area (PCSA), which is calculated from the ratio of the muscle’s volume and OFL, with a correction for the pennation angle of the fibers to estimate how much of the force-generating capacity is transmitted by the tendon. The novel capacity to make minimally invasive measures of sarcomere length in vivo provides new opportunities to study functionally meaningful muscle parameters (OFL and PCSA) and how they vary in living subjects across muscles, individuals, and due to alterations in muscle stimulus or use
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