Fig. 9: AnkB regulates SR-mitochondria coupling and calcium homeostasis in SKM fibers.

a Protein-protein interaction map of differentially expressed genes (DEGs) associated with Ca²⁺ signaling derived from RNA-seq analysis. Blue and red denote downregulated and upregulated genes, respectively. Nodes are presented based on fold change (FC), indicated by color intensity and on p-value, shown as node size. Triad- or SR-localized DEGs are indicated. Cytosolic (b) and mitochondrial (c) Ca²⁺ measured at baseline in 4-mo FDB fibers. Data in b was collected from n = 53 WT and n = 42 AnkB-KO fibers. Data in c was collected from n = 57 WT and n = 39 AnkB-KO fibers. d TEM images of longitudinal GC sections from 4-mo mice (representative of sections collected from n = 3 mice/genotype in one experiment). Scale bar, 1 µm. Insets show higher magnification of yellow dotted boxed regions. Scale bar, 500 nm. Arrows indicate the sarcoplasmic reticulum (SR). e FDB fibers stained with ER-tracker (representative of n = 3 independent experiments). Scale bar, 5 µm. Insets show higher magnification of yellow dotted boxed regions. White dashed lines show regions used for line intensity profile analysis shown in (g). Scale bar, 1 µm. f Ryanodine receptor (RyR) distribution in FDB fibers from 4-mo mice. Scale bar, 5 µm. Insets sh≤ow higher magnification of yellow dotted boxed regions. Arrowheads indicate RyR puncta spacing. Scale bar, 1 µm. g Line intensity plot of ER-tracker signal in (e). h Average distance between triads (n = 17 WT, n = 27 AnkB-KO fibers). i Quantification of triad spacing based on RyR distribution in (f) (n = 19 WT, n = 33 AnkB-KO fibers). Western blot analysis of levels of the muscle-specific AnkR isoform sAnk1.5 in GC (j) and quantification (k). Protein levels were normalized to total protein stain and graphed as relative fold-change over WT (control n = 7, KO = 6). l Representative images show ER-tracker and PKmito distribution in FDB fibers from 4-mo mice. Scale bar, 5 µm. Insets show higher magnification of the yellow dotted boxed regions. Scale bar, 1 µm. m-o, Mander’s overlap coefficient (m) and channel-specific overlap coefficients for mitochondria (n) and ER tracker (o). Data in m was computed from n = 46 WT and n = 81 AnkB-KO fibers. Data in n, o was computed from n = 22 WT and n = 29 AnkB-KO fibers. Data show mean ± S.D. Statistical differences were determined by two-tailed unpaired t-test with a significance threshold of p < 0.05.