Variability of in vivo Sarcomere Length Measures in the Upper Limb Obtained With Second Harmonic Generation Microendoscopy

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.

Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging

“…Optical imaging is the only available tool for characterizing the sarcomeric striation pattern and the corresponding average SL, which is highly informative for understanding the functional relationships between actual SL and the contractile state of the cell ( Bub et al, 2010 ; Botcherby et al, 2013 ; Aguirre et al, 2014 ; Guo and Song, 2014 ; Pasqualin et al, 2016 ; Kobirumaki-Shimozawa et al, 2018 ; Varga et al, 2020 ; Dowrick et al, 2021 ). Recent studies have also focused on the existence of intracellular SL heterogeneity in striated muscle ( Rassier, 2017 ; Haeger et al, 2020 ; Adkins et al, 2022 ) and, importantly, its alteration upon the transition of myofilaments from the inactivated to the activated state ( Johnston et al, 2016 ; de Souza Leite et al, 2017 ; Moo et al, 2017 ; Haeger and Rassier, 2020 ; Lookin et al, 2022 ). Therefore, special care is needed to choose a proper method for SL measurements.…”

Load sharing between synergistic muscles characterized by a ligand-binding approach and elastography

“…In addition, we are aware that additional evidence from more direct characterization methods is needed to confirm the predictions of the present work regarding the contractile dynamics at the sarcomere level. We believe the microendoscopy could be helpful in this regard 75 – 77 . Beyond these considerations, the data and methods described here could be the basis to continue delving into the implications of the ligand-binding behaviors in skeletal muscle biomechanics.…”