Novel interventions to recover the regenerative capacity of aged skeletal muscle by targeting the interactions in the stem cell niche

The remarkable ability of skeletal muscle to regenerate upon injury is conferred by tissue-resident stem cells called satellite cells. With age, the regenerative capacity of muscle stem cells (MuSCs) dramatically declines. Developing strategies to enhance muscle repair in elderly people is therefore required; in particular to accelerate their recovery from injuries following falls or from surgical interventions affecting muscle tissues. As the causes of MuSC dysfunction with age are multi-systemic, we decided to dissect age-related changes at different levels of the MuSC environment, in order to uncover synergistic ways to restore their regenerative capacities. This thesis describes three major interventions at the extracellular matrix, cell-cell interaction and tissue/systemic level that successfully restored skeletal muscle regeneration in aged mice. Using a proteomic screen, we identified fibronectin as a structural and signaling molecule of the extracellular matrix that is lost in the aged muscle niche. Loss of fibronectin in aged regenerating muscle primarily arises from perturbed cellular turnover of hematopoietic and endothelial cells which were shown to be the major fibronectin producers in muscle upon injury. Our work also uncovered that the function of old MuSCs is impaired by loss of adhesion and cell death by anoikis, and can be rescued by fibronectin treatment both ex vivo and in vivo. At the molecular level, loss of fibronectin is an upstream trigger leading to perturbed MuSC signaling. Fibronectin treatment rescues perturbations of pathways, such as p38 and ERK, that were previously known to be altered in aged MuSCs, as well as the newly discovered age-related perturbations of FAK signaling. Altogether we demonstrated that aging impairs the remodeling of the extracellular matrix of the MuSC niche, and thereby triggers multiple dysfunction of old MuSCs that can be rescued therapeutically. In a second project, we dissected the cellular cross-talk between MuSCs and non-myogenic support cells called Fibro-Adipogenic Progenitors (FAPs). We uncovered that the adipogenic fate of FAPs is tightly correlated to the muscle micro-environment and the myogenic regenerative capacity in different models of regeneration and aging. The function of FAPs and their cross-talk with MuSCs are impaired with age. We identified the secreted protein WISP1 as a paracrine communication factor between FAPs and MuSCs which is perturbed with age. Treating aged mice with WISP1 restored stem cell function and muscle regeneration, highlighting the possibility to target the cross-talk of MuSC with other cells of the niche to ameliorate their function. Our last project identified altered signaling of the small circulating peptide apelin in aged MuSCs through lowered circulating levels of apelin and down-regulation of its receptor APJ in aged MuSCs. We demonstrated that apelin treatment rescued muscle stem cell function and regeneration in aged mice, highlighting that MuSC dysfunction can also be targeted systemically. Taken together, these approaches reveal the multiple possibilities to ameliorate muscle repair by targeting MuSC interactions with their niche. Our results also pave the way to develop integrated therapeutic strategies to boost old MuSC function

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

A Novel, cost-effective and efficient chicken egg IgY purification procedure

Chicken IgY antibodies have been touted to be a superior alternative to mammalian antibodies for use in various immunological, molecular biology and proteomics applications for several reasons. These include, but are not limited to, improved specificity due to maximum phylogenetic distance between host and recipient, cost effectiveness in maintaining commercial numbers of hens, IgY yield and the use of non-invasive methods used to isolate IgY from eggs as opposed to blood. Despite this, the routine use of IgY-based methodologies in the laboratory is not widespread. One reason for this reluctance may be derived from the difficulties and expense of isolating IgY antibodies from egg yolk in sufficient yield, with high purity at a realistic reasonable price. Here, we describe an extremely cost-effective ($5USD per egg), rapid (within 5. h), efficient and optimised technique to isolate high yields (60. mg) of high purity (~. 80%) chicken IgY from egg yolks using the common plant gums pectin and κ-carrageenan in the presence of calcium chloride to delipidate egg yolk mixtures whilst maintaining IgY in solution and then ammonium sulphate to subsequently precipitate the resulting IgY antibodies to higher purity. Our data demonstrates that this technique results in a high yield and purity of IgY that is comparable (if not superior to) existing commercial IgY isolation kits. The method also allows the isolation of immunologically active IgY which can be used for further downstream immunotechnological processes. Furthermore, it can also be easily implemented in a standard well equipped laboratory, and may be scaled up to commercial quantities (i.e., thousands of eggs).4 page(s

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

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

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

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

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

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

Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro- adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis