Angiogenesis precedes myogenesis during regenerationfollowing biopsy injury in skeletal muscle

Abstract

BackgroundAcute injury to skeletal muscle damages myofibers and fragments capillaries, impairing contractile function and local perfusion. Myofibers and microvessels regenerate from satellite cells and from surviving microvessel fragments, respectively, to restore intact muscle. However, it is unknown whether myofiber regeneration and microvascular regeneration reflect interdependent processes or may proceed sequentially. MethodsTo investigate the temporal relationship between myogenesis and angiogenesis during regeneration, a punch biopsy (diameter, 2 mm) was performed through the center of the gluteus maximus (GM) muscle. Complete removal of all tissue components created a void into which regeneration was evaluated through 21 days post injury (dpi). Confocal imaging and histological analyses of whole-mount GM preparations and GM cross sections assessed the growth of microvessels and myofibers into the wound. Regeneration of perfused microvessels was evaluated in vivo by injecting fluorescent dextran into the circulation during intravital imaging. ResultsA provisional matrix filled with PDGFR+ and CD45+ cells spanned the wound within 1 dpi. Regenerating microvessels advanced into the matrix by 7 dpi. At 10 dpi, sprouting and intussusceptive angiogenesis produced disorganized microvascular networks and spanned the wound with perfusion by 14 dpi. In striking contrast, the wound remained devoid of myofibers at 7 and 10 dpi. Myogenesis into the wound began by 14 dpi with nascent myofibers traversing the wound by 21 dpi. Regenerating myofibers and microvessels were less well organized than in the surrounding (uninjured) muscle. ConclusionsAngiogenesis precedes myogenesis following punch biopsy injury of adult skeletal muscle. Regenerating microvessels encompass the wound and become perfused with blood prior to colocalization with regenerating myofibers. These findings infer that a microvascular supply supports the metabolic demands of regenerating skeletal muscle. Finding that regenerated microvascular networks and myofibers are disorganized within the biopsy site suggests that loss of guidance cues upon complete tissue removal impairs re-establishment of canonical skeletal muscle structure.