Figure 2

The collapse of myofibers induces the proliferation of myogenic progenitor cells in damaged muscle in vivo . (A) Representative photomicrographs of freshly isolated intact myofibers and collapsed myofibers from a C57Bl/6 WT mouse (n = 3 to 4). After 0 or 6 days of culture, the myofibers were incubated with Evans blue dye (EBD) to stain collapsed myofibers. The myofibers were then immunostained with fluorochrome-conjugated phalloidin to visualize F-actin and with anti-α-actinin to visualize both cytoskeleton proteins. Unlike collapsed myofibers, the intact myofibers displayed periodicity with the phalloidin and α-actinin staining in both the 0- and 6-day cultures. (B) Representative image of BF muscle longitudinal sections from 8- to 12-month-old mdx mice that had received an intraperitoneal injection of EBD 24 h prior to euthanasia (left panels). The sections were stained with anti-MyoD (green) and DAPI (blue). The actin cytoskeleton was stained with phalloidin (green) and DAPI (blue). Damaged myofibers were positive for EBD⁺ (red) whereas intact myofibers were EBD⁻ (n = 15 for intact myofibers and n = 4 for damaged myofibers; n = 3 mice). Arrows indicate degenerative EBD⁺ myofibers, and arrowheads indicate MyoD⁺ cells (activated/proliferative MPCs). Asterisks indicate intact myofibers with the striated pattern of actin. Scatter dot plot showing the number of MyoD⁺ cells per myofiber (right panel). Overlay images were mounted in Image Pro and were used to identify MyoD⁺ cells. ****P < 0.00001 versus control (intact myofibers). (C) Bar graph representing the distance of MPCs from EBD⁺ myofibers. After counting the number of cells, distance measurements were obtained by counting the number of MyoD⁺ cells in four different regions per muscle (n = 6 muscles). All results are expressed as means ± SEM.