Edges vs. Centers: Variability of Sarcomere Lengths Depends on What You Choose as “Sarcomere Ends”

In our recent experimental study we found that variability in sarcomere lengths (SLs) is lower if we get them from A-band centers (visualized by Second Harmonic Generation microscopy, SHG) vs. Z-disks (ANEPPS-staining of t-tubules). In the present study we demonstrate, using a simulation approach, that the extent of variability and “randomness” in SL are directly affected by what “sarcomeric structure” is used to calculate the SL. We simulated randomly dispersed positions of Z-disks and calculated the center points between two Z-disks as A-band centers. We found that variance in SL, if calculated from A-band centers (A-SL), was twice lower vs. variance in Z-disk-based SL (Z-SL), regardless the predefined dispersion of Z-disks. Furthermore, A-SLs were “non-randomly” interrelated – each next A-SL tended to be longer if the preceding one was longer – in contrast to Z-SLs which were truly random. Next, we demonstrated the presence and prevalence of this “non-random” effect for A-SL vs. Z-SL in our previously acquired experimental records. The rationale for this artifact is the “mathematical dependence” of sarcomere center on its edges, and it must always be taken into account in the interpretation of smaller variability of the distance between sarcomeric A-band centers vs. the distance between Z-disks.