Fig. 1: Intrasarcomere cross-sectional area heterogeneity is dependent on muscle cell type.
a Longitudinal raw focused ion beam scanning electron microscopy (FIB-SEM) image of a mouse fast-twitch glycolytic gastrocnemius muscle showing two parallel myofibrillar segments comprised of three sarcomeres each (cyan and yellow). Diameter of the A-band (A) is larger than the diameter of the Z-disk (Z, red). Mitochondria (magenta), sarcoplasmic reticulum (SR, green), and t-tubules (T, blue) are also shown. Representative of four fast glycolytic FIB-SEM datasets. b 3D rendering of the sarcomere boundaries from a (translucent cyan and yellow) and their Z-disk structures (red). White arrows highlight gaps between in parallel sarcomeres. c Same 3D rendering as b but also showing sarcomere adjacent mitochondria (magenta), and partial sarcoplasmic reticulum (green) and t-tubule (blue) structures. d–h Raw FIB-SEM cross-sectional views at the Z-disk, I-band, and A-band regions indicated by dotted lines in b highlighting the varying diameters of the mitochondrial and SR/T structures located between in parallel sarcomeres (cyan and yellow). Black arrows highlight SR doublets. Yellow arrow highlights longitudinal SR. Blue arrow highlights regions without SR between sarcomeres. Representative of four fast glycolytic FIB-SEM datasets. i 3D rendering of the sarcomere boundaries (translucent cyan and yellow) for six sarcomeres from Drosophila indirect flight muscle and their Z-disk structures (red). j 90° rotation of the indirect flight muscle sarcomeres from i also showing adjacent mitochondria which do not run perpendicular to the contractile axis. k Percent difference in sarcomere cross-sectional area at the A-band relative to the Z-disk per half sarcomere sheet across eleven muscle cell types. Large shapes represent individual cell values, smaller shapes represent values per individual half sarcomere sheet. Black lines represent cell Mean ± SD. Graph made with SuperPlotsofData web app117. l Stimulated emission depletion (STED) microscopy image of a live mouse FDB fiber stained with SPY555-FastAct dye. Red arrow highlights intersarcomere space between Z-disks. Images representative of 9 cells, 2 mice. m Corresponding mitochondrial (genetically encoded TOMM20-mNeon) image from (l). n Corresponding live cell actin and mitochondria merged image from (l, m). White dotted lines correspond to locations of intensity profiles in (o). o Fluorescent intensity profile along laterally adjacent sarcomeres corresponding to the Z-disk (cyan dashed line), A-band (black solid line), and mitochondria (magenta dotted line). *significantly different from Cardiac. %significantly different from Early Postnatal (postnatal day 1). #significantly different from Late Postnatal (postnatal day 14). ^significantly different from Fast Glycolytic. $significantly different from Fast Oxidative. @significantly different from Slow Oxidative. +significantly different from Indirect Flight. >significantly different from Jump. <significantly different from Direct Flight. Two-sided, one-way ANOVA with a Tukey’s HSD post hoc test. N values: Cardiac-5 mice, 5 cells, 122 half sarcomere sheets; Early Postnatal-5 mice, 5 cells, 106 half sarcomere sheets; Late Postnatal-4 mice, 4 cells, 70 half sarcomere sheets; Fast Glycolytic-4 mice, 4 cells, 86 half sarcomere sheets; Fast Oxidative-4 mice, 4 cells, 86 half sarcomere sheets; Slow Oxidative-4 mice, 4 cells, 84 half sarcomere sheets; Indirect Flight-3 flies, 3 cells, 56 half sarcomere sheets; Jump-1 fly, 3 cells, 84 half sarcomere sheets; Direct Flight-1 fly, 5 cells, 80 half sarcomere sheets; Leg-1 fly, 5 cells, 21 half sarcomere sheets; Human-3 subjects, 3 cells, 80 half sarcomere sheets.
