Genomic profiling reveals that transient adipogenic activation is a hallmark of mouse models of skeletal muscle regeneration.

The marbling of skeletal muscle by ectopic adipose tissue is a hallmark of many muscle diseases, including sarcopenia and muscular dystrophies, and generally associates with impaired muscle regeneration. Although the etiology and the molecular mechanisms of ectopic adipogenesis are poorly understood, fatty regeneration can be modeled in mice using glycerol-induced muscle damage. Using comprehensive molecular and histological profiling, we compared glycerol-induced fatty regeneration to the classical cardiotoxin (CTX)-induced regeneration model previously believed to lack an adipogenic response in muscle. Surprisingly, ectopic adipogenesis was detected in both models, but was stronger and more persistent in response to glycerol. Importantly, extensive differential transcriptomic profiling demonstrated that glycerol induces a stronger inflammatory response, and promotes adipogenic regulatory networks while reducing fatty acid β-oxidation. Altogether, these results provide a comprehensive repository of gene expression changes during the time course of two muscle regeneration models, and strongly suggest that adipogenic commitment is a hallmark of muscle regeneration, which can lead to ectopic adipocyte accumulation in response to specific physiopathological challenge

Chemokine-like receptor 1 plays a critical role in modulating the regenerative and contractile properties of muscle tissue.

Musculoskeletal diseases are a leading contributor to mobility disability worldwide. Since the majority of patients with musculoskeletal diseases present with associated muscle weakness, treatment approaches typically comprise an element of resistance training to restore physical strength. The health-promoting effects of resistance exercise are mediated via complex, multifarious mechanisms including modulation of systemic and local inflammation. Here we investigated whether targeted inhibition of the chemerin pathway, which largely controls inflammatory processes via chemokine-like receptor 1 (CMKLR1), can improve skeletal muscle function. Using genetically modified mice, we demonstrate that blockade of CMKLR1 transiently increases maximal strength during growth, but lastingly decreases strength endurance. In-depth analyses of the underlying long-term adaptations revealed microscopic alterations in the number of Pax7-positive satellite cells, as well as molecular changes in genes governing myogenesis and calcium handling. Taken together, these data provide evidence of a critical role for CMKLR1 in regulating skeletal muscle function by modulating the regenerative and contractile properties of muscle tissue. CMKLR1 antagonists are increasingly viewed as therapeutic modalities for a variety of diseases (e.g., psoriasis, metabolic disorders, and multiple sclerosis). Our findings thus have implications for the development of novel drug substances that aim at targeting the chemerin pathway for musculoskeletal or other diseases

Genomic Profiling Reveals That Transient Adipogenic Activation Is a Hallmark of Mouse Models of Skeletal Muscle Regeneration

<div><p>The marbling of skeletal muscle by ectopic adipose tissue is a hallmark of many muscle diseases, including sarcopenia and muscular dystrophies, and generally associates with impaired muscle regeneration. Although the etiology and the molecular mechanisms of ectopic adipogenesis are poorly understood, fatty regeneration can be modeled in mice using glycerol-induced muscle damage. Using comprehensive molecular and histological profiling, we compared glycerol-induced fatty regeneration to the classical cardiotoxin (CTX)-induced regeneration model previously believed to lack an adipogenic response in muscle. Surprisingly, ectopic adipogenesis was detected in both models, but was stronger and more persistent in response to glycerol. Importantly, extensive differential transcriptomic profiling demonstrated that glycerol induces a stronger inflammatory response and promotes adipogenic regulatory networks while reducing fatty acid β-oxidation. Altogether, these results provide a comprehensive mapping of gene expression changes during the time course of two muscle regeneration models, and strongly suggest that adipogenic commitment is a hallmark of muscle regeneration, which can lead to ectopic adipocyte accumulation in response to specific physio-pathological challenges.</p></div

The inflammatory signature is stronger in response to glycerol than to CTX.

<p>qPCR analysis of the mRNA levels of various macrophage markers and cytokines. Data are expressed as mean ± s.e.m., n = 5–6/group. * p-value <0.05 <i>vs</i>. control, # p-value <0.05 in Glycerol <i>vs</i>. CTX at same time points. Emr1; EGF-like module containing mucin-like hormone receptor 1; TNFα, tumor necrosis factor alpha, IL, interleukin; TGF-β1, transforming growth factor beta 1.</p

Adipogenesis and β-oxidation are differentially regulated in muscle after glycerol or CTX injection.

<p>qPCR analysis of the mRNA levels of different adipogenic (A), or in fatty-acid oxidation (B) regulators. Data are expressed as mean ± s.e.m., n = 5–6/group. * p-value <0.05 <i>vs</i>. control, # p-value <0.05 in Glycerol <i>vs</i>. CTX at same time points. Acadm, acyl-CoA dehydrogenase medium; Acs/l, acyl-CoA synthesase short-/long-chain; Acss, Acetyl-coenzyme A synthetase; Acox, Acyl-coenzyme A oxidase, Palmitoyl; C/EBP: CCAAT/ Enhancer binding protein; Cpt, carnitine palmitoyltransferase; Hadh, hydroxyacyl-CoA dehydrogenase; PPAR, peroxisome proliferator activated receptor.</p

Gene set enrichment mapping of glycerol- <i>vs.</i> CTX-injected muscle.

<p>Gene set enrichment analysis was performed on glycerol-injected compared to CTX-injected muscles 3 and 7 days after injection, and clustered according to gene set ontology. The size of nodes is proportional to the number of genes contained in the gene set. Red nodes: gene sets upregulated in glycerol <i>vs</i>. CTX model, blue nodes: gene sest downregulated in glycerol <i>vs</i>. CTX model, green bar: link between two gene sets sharing regulated genes.</p

Ectopic adipogenesis occurs in both glycerol- and CTX-induced muscle regeneration.

<p>(A) qPCR analysis of the mRNA level of the platelet-derived growth factor receptor alpha (PDGFRα). (B) Cryosections were performed at the mid-belly part of TA and subjected to H&E and perilipin staining at each time points after injection. Representative perilipin (green) /DAPI (blue) fluorescent stainings at 21 dpi are shown next to an H&E staining of the same region. Scale bars, 50 μm. (C), Quantitative analysis of perilipin expression assessed by counting and measuring the area of all perilipin expressing cells per section. Data are expressed as mean ± s.e.m., n = 5–6/group. * p-value <0.05 <i>vs</i>. control, # p-value <0.05 in Glycerol <i>vs</i>. CTX at same time points.</p

Glycerol and CTX induce similar kinetics of degeneration and regeneration.

<p>Control uninjured tibialis anterior muscle, and tibialis anterior muscles injected with either 25 µl of 50% (v/v) glycerol or 10 µM CTX were sectioned and stained with laminin and DAPI 3, 7, 14 or 21 days after injection (dpi) (A), or with hematoxylin-eosin at 21 dpi (B). Cryosections were performed at the mid-belly part of tibialis anterior. Scale bars, 100 μm. Yellow arrow: immune cell nuclei, white arrow: central nuclei, red circle: fat cell-like structure. (C) Quantitative analysis of total myofibers and of myofibers with at least one central nuclei from laminin/DAPI stained sections. (D) qPCR analysis of the mRNA levels of different markers of muscle regeneration. Data are expressed as mean ± s.e.m., n = 5–6/group. * p-value <0.05 <i>vs</i>. control. MYH, Myosin Heavy Chain.</p

Blockade of metallothioneins 1 and 2 increases skeletal muscle mass and strength

Abstract Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. Here, we demonstrate that these genes are also induced coincident with aging. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo, and found that concomitant abrogation of metallothionein 1 and 2 activates the Akt pathway, promotes myotube growth, specifically drives type IIb fiber hypertrophy and ultimately increases muscle strength. Silencing of metallothioneins results in elevated cytosolic zinc; increasing intracellular zinc levels are sufficient to mimic the effects of metallothionein blockade on myotube hypertrophy. We thereby provide direct evidence of the mechanisms by which metallothioneins can modulate skeletal muscle mass. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function are preserved in the presence of a strong catabolic stimulus: treatment with glucocorticoids. Taken together, our results suggest that blockade of metallothioneins constitutes a promising approach for the treatment of muscle diseases

Modulation of Microglia by Voluntary Exercise or CSF1R Inhibition Prevents Age-Related Loss of Functional Motor Units

Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss