Mechanisms Regulating Muscle Regeneration: Insights into the Interrelated and Time-Dependent Phases of Tissue Healing

Abstract

Despite a massive body of knowledge which has been produced related to the mechanisms guiding muscle regeneration, great interest still moves the scientific community toward the study of different aspects of skeletal muscle homeostasis, plasticity, and regeneration. Indeed, the lack of effective therapies for several physiopathologic conditions suggests that a comprehensive knowledge of the different aspects of cellular behavior and molecular pathways, regulating each regenerative stage, has to be still devised. Hence, it is important to perform even more focused studies, taking the advantage of robust markers, reliable techniques, and reproducible protocols. Here, we provide an overview about the general aspects of muscle regeneration and discuss the different approaches to study the interrelated and time-dependent phases of muscle healing.

Keywords: cell precursors; experimental methods; inflammatory response; muscle homeostasis; muscle regeneration; satellite cells; stem cell markers.

Figure 1

A simplified “wave on wave” model of skeletal muscle healing: The regenerative program activated by muscle tissue in response to damage can be outlined in five interrelated and time-dependent waves, namely degeneration, inflammation, regeneration, maturation-remodelling, and functional recovery, which can be highlighted by using different methodologies. Tissue injury leads to myofiber degeneration/necrosis. Damage stimuli activate the so-called sterile inflammation, characterized by the infiltration of different immune cells dominating in succession the lesion. Inflammation triggers also the regenerative stage, in which satellite cells, along with the support of other stem cells and precursors, undergo activation, expansion, and differentiation. The maturation of myofibers is accompanied by the fine remodelling of tissue architecture, with matrix rearrangement and angiogenesis. The last step of the healing process is characterized by the reconstitution of neuromuscular connections, necessary to regain tissue functionality. DAMPs: Damage-associated molecular patterns; EBD: Evans Blue Dye; IgG: Immunoglobulin G; ALB: Albumin; NEU: neutrophils; MAC: macrophages; MPO: myeloperoxidase; SCs: satellite cells; Activ.SCs: activated SCs; Prolif.SCs: proliferating SCs; Diff.SCs: differentiating SCs; BrdU: 5-bromo-2′-deoxyuridine; EdU: 5-ethynyl-2′-deoxyuridine; MFs: myogenic factors; eMyHC: embryonal myosin heavy chain; H&E: Haematoxylin and Eosin; AchR: Acetylcholine receptor; BTX: Bungarotoxin.

Figure 2

Skeletal muscle regeneration upon acute injury: The upper panel shows a schematic representation of relevant biological responses activated in muscle tissue following damage. Lower panel reports haematoxylin and eosin images of muscle sections, representative of each step of muscle degeneration and regeneration after cardiotoxin (CTX) injection. Early, after the injection (1 day), necrotic myofibers are evident in damaged muscle. During the second day after damage, the lesion is dominated by inflammatory infiltrated cells. Activated satellite cells undergo active proliferation, and newly regenerating fibers appears within the first week. Ten days after injection, the overall tissue architecture is restored and most of myofibers display centrally located nuclei. Regenerated myofibers then undergo progressive growth and maturation, highlighted by the increasing cross-sectional area and the nuclear relocation towards the periphery.