Abstract
BackgroundWhile muscle regeneration has been extensively studied in animal and cell culture models, in vivo myogenesis in adult human skeletal muscle has not been investigated in detail.MethodsUsing forced lengthening contractions induced by electrical stimulation, we induced myofibre injury in young healthy males. Muscle biopsies were collected from the injured leg 7 and 30 days after muscle injury and from the uninjured leg as a control. Immuno-stained single muscle fibres and muscle cross sections were studied by wide-field and confocal microscopy. Samples were also studied at the ultra-structural level by electron microscopy.ResultsMicroscopy of single muscle fibres in 3 dimensions revealed a repeating pattern of necrotic and regenerating zones along the length of the same myofibre, characterised by extensive macrophage infiltration alongside differentiating myogenic progenitor cells and myotubes: the hallmarks of myogenesis. The myofibre basement membrane was preserved during these processes and interestingly was seen at a later stage as a second basement membrane surrounding the regenerating fibres.ConclusionsThis is the first work to document in vivo myogenesis in humans in detail and highlights the importance of the basement membrane in the process of regeneration. In addition, it provides insight into parallels between the regeneration of adult skeletal muscle in mouse and man, confirming that this model may be a useful tool in investigating myofibre and matrix formation, as well as specific cell types, during regeneration from the perspective of human muscle.
Highlights
While muscle regeneration has been extensively studied in animal and cell culture models, in vivo myogenesis in adult human skeletal muscle has not been investigated in detail
Portions of the biopsy were prepared for preservation in three ways: (1) a small part of the sample was immersed in 2% glutaraldehyde in 0.05 M sodium phosphate buffer for transmission electron microscopy (TEM). (2) Parts of the biopsy suited for histology were aligned in parallel, embedded in Tissue-Tek (Sakura Finetek Europe, Zoeterwoude, The Netherlands), frozen in isopentane, pre-cooled by liquid nitrogen and stored at − 80 °C until analysis
Some fibres contained many Pax7 cells in contrast to a complete lack of Pax7+ cells in many fibre fragments. While this may represent an actual finding, this was not studied in a systematic manner in this paper and it is likely that enumeration of specific cell populations is best performed on fixed or frozen tissue cross sections, e.g. as we recently reported for satellite cells and fibroblasts [22]
Summary
While muscle regeneration has been extensively studied in animal and cell culture models, in vivo myogenesis in adult human skeletal muscle has not been investigated in detail. The study of muscle regeneration is largely based on animal studies, taking advantage of the possibilities for invasive interventions and where whole muscles can be excised for analyses These mostly include focal muscle injury [1, 2], or various types of toxin injection into the muscle, as recently studied in detail [3]. Mackey and Kjaer Skeletal Muscle (2017) 7:24 damage such as delayed onset muscle soreness and increased circulating levels of creatine kinase While these pseudomarkers of muscle damage are common and can reach substantial levels, studies of muscle biopsies from such studies only occasionally report evidence of myofibre necrosis [6,7,8], suggesting a process of remodelling of the muscle and its associated extracellular matrix rather than true regeneration, as recently discussed [9, 10]. We recently confirmed a large activation and proliferation of the satellite cell pool with this model and extended the time course of study to 30 days, where a large proportion of fibres demonstrated neonatal myosin expression [12], indicating the potential of this injury model to induce largescale muscle regeneration, how alike this process is to rodent muscle regeneration has rarely been addressed in the context of healthy human adult muscle
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