Myonuclear Dynamics After Skeletal Muscle Surgical Injury

Figures

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  FIGURE 1. Fiber type‐specific analysis of resident versus non‐resident displaced myonuclei in response to surgical injury. (A) Study design where 10‐month‐old HSA‐GFP mice (M/F = 6/6) were treated with doxycycline to label myonuclei for 5 days followed by a 14‐day wash‐out. The distal ~1/3 of the gastrocnemius‐soleus complex was excised and mice were treated with 5‐ethynyl‐2′‐deoxyuridine (EdU) in drinking water to assess DNA synthesis following surgical resection injury. Resected gastrocnemius muscles were collected at 3 days (n = 4; M/F = 2/2) and 7 days (n = 4; M/F = 2/2) postsurgery, with sham controls collected at 7 days (N = 4; M/F = 2/2). (B–B‴) Show representative images of the gastrocnemius muscle for fiber type‐specific quantification of displaced myonuclei. Images show dystrophin (pink), myosin heavy chain IIB (MyHC IIB, red), GFP‐labeled resident myonuclei (green), and DAPI nuclei (blue). Green arrows = GFP+ resident myonuclei, white arrows = GFP− non‐resident myonuclei. B‴ is a degenerating area with a higher prevalence of displaced myonuclei. (C) Quantification of relative number of IIB+ or IIB− fibers containing one or more displaced myonuclei. Data are presented as mean ±…

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  FIGURE 2. Displaced myonuclei in eMyHC‐expressing muscle fibers and muscle fibers with multiple displaced resident myonuclei after surgical injury. (A) Z‐stack image from 30 μm section of 7 day gastrocnemius showing regional eMyHC abundance and prevalence of several displaced resident myonuclei within a single fiber. (B) Representative images of emybronic myosin positive fibers with displaced resident myonuclei. Green arrows = GFP+ resident myonuclei. Degenerating areas were selected to demonstrate the prevalence of displaced myonuclei in those regions. (C) Quantification of number of satellite cells at each timepoint. Data are presented as mean ± SD and are reported relative to the number of fibers. (D) Representative images of satellite cells in the gastrocnemius muscle at sham (D and D′) and 3 day timepoints (D″ and D‴). Laminin (red), PAX7 (white), and DAPI (nuclei, blue). (E) Representative image of satellite cells (white arrow) in the gastrocnemius muscle at 7 day timepoint. Scale bar is 50 μm in panels (D) and (E). All data are from whole gastrocnemius muscle cross‐sections, but images represent regions of interest.