Maresin 1 repletion improves muscle regeneration after volumetric muscle loss.

The acute traumatic or surgical loss of skeletal muscle, known as volumetric muscle loss (VML), is a devastating type of injury that results in exacerbated and persistent inflammation followed by fibrosis. The mechanisms that mediate the magnitude and duration of the inflammatory response and ensuing fibrosis after VML remain understudied, and as such, the development of regenerative therapies has been limited. To address this need, we profiled how lipid mediators, which are potent regulators of the immune response after injury, varied with VML injuries that heal or result in fibrosis. We observed that non-healing VML injuries displayed increased pro-inflammatory eicosanoids and a lack of pro-resolving lipid mediators. Treatment of VML with a pro-resolving lipid mediator synthesized from docosahexaenoic acid, called Maresin 1, ameliorated fibrosis through reduction of neutrophils and macrophages and enhanced recovery of muscle strength. These results expand our knowledge of the dysregulated immune response that develops after VML and identify a novel immuno-regenerative therapeutic modality in Maresin 1.

Neutrophil and natural killer cell imbalances prevent muscle stem cell-mediated regeneration following murine volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell­ and progenitor-mediated myogenic repair. However, how immune cell infiltration and intercellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in the fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observed the heightened infiltration of natural killer (NK) cells as well as the persistence of neutrophils beyond 2 wk postinjury. Functional validation of NK cells revealed an antagonistic role in neutrophil accumulation in part via inducing apoptosis and CCR1-mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFß1). Blocking TGFß signaling reduced neutrophil accumulation and fibrosis and improved muscle-specific force. Collectively, these results enhance our understanding of immune cell­stem cell cross talk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

Sestrins regulate muscle stem cell metabolic homeostasis.

The health and homeostasis of skeletal muscle are preserved by a population of tissue-resident muscle stem cells (MuSCs) that maintain a state of mitotic and metabolic quiescence in adult tissues. The capacity of MuSCs to preserve the quiescent state declines with aging and metabolic insults, promoting premature activation and stem cell exhaustion. Sestrins are a class of stress-inducible proteins that act as antioxidants and inhibit the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling complex. Despite these pivotal roles, the role of Sestrins has not been explored in adult stem cells. We show that SESTRIN1,2 loss results in hyperactivation of the mTORC1 complex, increased propensity to enter the cell cycle, and shifts in metabolic flux. Aged SESTRIN1,2 knockout mice exhibited loss of MuSCs and a reduced ability to regenerate injured muscle. These findings demonstrate that Sestrins help maintain metabolic pathways in MuSCs that protect quiescence against aging.

Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging.

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.

Metabolipidomic profiling reveals an age-related deficiency of skeletal muscle pro-resolving mediators that contributes to maladaptive tissue remodeling.

Specialized pro-resolving mediators actively limit inflammation and support tissue regeneration, but their role in age-related muscle dysfunction has not been explored. We profiled the mediator lipidome of aging muscle via liquid chromatography-tandem mass spectrometry and tested whether treatment with the pro-resolving mediator resolvin D1 (RvD1) could rejuvenate the regenerative ability of aged muscle. Aged mice displayed chronic muscle inflammation and this was associated with a basal deficiency of pro-resolving mediators 8-oxo-RvD1, resolvin E3, and maresin 1, as well as many anti-inflammatory cytochrome P450-derived lipid epoxides. Following muscle injury, young and aged mice produced similar amounts of most pro-inflammatory eicosanoid metabolites of cyclooxygenase (e.g., prostaglandin E2 ) and 12-lipoxygenase (e.g., 12-hydroxy-eicosatetraenoic acid), but aged mice produced fewer markers of pro-resolving mediators including the lipoxins (15-hydroxy-eicosatetraenoic acid), D-resolvins/protectins (17-hydroxy-docosahexaenoic acid), E-resolvins (18-hydroxy-eicosapentaenoic acid), and maresins (14-hydroxy-docosahexaenoic acid). Similar absences of downstream pro-resolving mediators including lipoxin A4 , resolvin D6, protectin D1/DX, and maresin 1 in aged muscle were associated with greater inflammation, impaired myofiber regeneration, and delayed recovery of strength. Daily intraperitoneal injection of RvD1 had minimal impact on intramuscular leukocyte infiltration and myofiber regeneration but suppressed inflammatory cytokine expression, limited fibrosis, and improved recovery of muscle function. We conclude that aging results in deficient local biosynthesis of specialized pro-resolving mediators in muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on tissue regeneration of non-steroidal anti-inflammatory drugs.

Resolvin D1 supports skeletal myofiber regeneration via actions on myeloid and muscle stem cells.

Specialized proresolving mediators (SPMs) actively limit inflammation and expedite its resolution by modulating leukocyte recruitment and function. Here we profiled intramuscular lipid mediators via liquid chromatography-tandem mass spectrometry-based metabolipidomics following myofiber injury and investigated the potential role of SPMs in skeletal muscle inflammation and repair. Both proinflammatory eicosanoids and SPMs increased following myofiber damage induced by either intramuscular injection of barium chloride or synergist ablation-induced functional muscle overload. Daily systemic administration of the SPM resolvin D1 (RvD1) as an immunoresolvent limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokine expression, enhanced polymorphonuclear cell clearance, modulated the local muscle stem cell response, and polarized intramuscular macrophages to a more proregenerative subset. RvD1 had minimal direct impact on in vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs can modulate both infiltrating myeloid and resident muscle cell populations. These data reveal the efficacy of immunoresolvents as a novel alternative to classical antiinflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.