Multimodal cell atlas of the ageing human skeletal muscle.

Muscle atrophy and functional decline (sarcopenia) are common manifestations of frailty and are critical contributors to morbidity and mortality in older people1. Deciphering the molecular mechanisms underlying sarcopenia has major implications for understanding human ageing2. Yet, progress has been slow, partly due to the difficulties of characterizing skeletal muscle niche heterogeneity (whereby myofibres are the most abundant) and obtaining well-characterized human samples3,4. Here we generate a single-cell/single-nucleus transcriptomic and chromatin accessibility map of human limb skeletal muscles encompassing over 387,000 cells/nuclei from individuals aged 15 to 99 years with distinct fitness and frailty levels. We describe how cell populations change during ageing, including the emergence of new populations in older people, and the cell-specific and multicellular network features (at the transcriptomic and epigenetic levels) associated with these changes. On the basis of cross-comparison with genetic data, we also identify key elements of chromatin architecture that mark susceptibility to sarcopenia. Our study provides a basis for identifying targets in the skeletal muscle that are amenable to medical, pharmacological and lifestyle interventions in late life.

Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration.

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.

Role of UCP2 in the Energy Metabolism of the Cancer Cell Line A549.

The uncoupling protein UCP2 is a mitochondrial carrier for which transport activity remains controversial. The physiological contexts in which UCP2 is expressed have led to the assumption that, like UCP1, it uncouples oxidative phosphorylation and thereby reduces the generation of reactive oxygen species. Other reports have involved UCP2 in the Warburg effect, and results showing that UCP2 catalyzes the export of matrix C4 metabolites to facilitate glutamine utilization suggest that the carrier could be involved in the metabolic adaptations required for cell proliferation. We have examined the role of UCP2 in the energy metabolism of the lung adenocarcinoma cell line A549 and show that UCP2 silencing decreased the basal rate of respiration, although this inhibition was not compensated by an increase in glycolysis. Silencing did not lead to either changes in proton leakage, as determined by the rate of respiration in the absence of ATP synthesis, or changes in the rate of formation of reactive oxygen species. The decrease in energy metabolism did not alter the cellular energy charge. The decreased cell proliferation observed in UCP2-silenced cells would explain the reduced cellular ATP demand. We conclude that UCP2 does not operate as an uncoupling protein, whereas our results are consistent with its activity as a C4-metabolite carrier involved in the metabolic adaptations of proliferating cells.

Mfn2 deficiency links age-related sarcopenia and impaired autophagy to activation of an adaptive mitophagy pathway.

Mitochondrial dysfunction and accumulation of damaged mitochondria are considered major contributors to aging. However, the molecular mechanisms responsible for these mitochondrial alterations remain unknown. Here, we demonstrate that mitofusin 2 (Mfn2) plays a key role in the control of muscle mitochondrial damage. We show that aging is characterized by a progressive reduction in Mfn2 in mouse skeletal muscle and that skeletal muscle Mfn2 ablation in mice generates a gene signature linked to aging. Furthermore, analysis of muscle Mfn2-deficient mice revealed that aging-induced Mfn2 decrease underlies the age-related alterations in metabolic homeostasis and sarcopenia. Mfn2 deficiency reduced autophagy and impaired mitochondrial quality, which contributed to an exacerbated age-related mitochondrial dysfunction. Interestingly, aging-induced Mfn2 deficiency triggers a ROS-dependent adaptive signaling pathway through induction of HIF1α transcription factor and BNIP3. This pathway compensates for the loss of mitochondrial autophagy and minimizes mitochondrial damage. Our findings reveal that Mfn2 repression in muscle during aging is a determinant for the inhibition of mitophagy and accumulation of damaged mitochondria and triggers the induction of a mitochondrial quality control pathway.

Mitofusin 2 (Mfn2) links mitochondrial and endoplasmic reticulum function with insulin signaling and is essential for normal glucose homeostasis.

Mitochondria are dynamic organelles that play a key role in energy conversion. Optimal mitochondrial function is ensured by a quality-control system tightly coupled to fusion and fission. In this connection, mitofusin 2 (Mfn2) participates in mitochondrial fusion and undergoes repression in muscle from obese or type 2 diabetic patients. Here, we provide in vivo evidence that Mfn2 plays an essential role in metabolic homeostasis. Liver-specific ablation of Mfn2 in mice led to numerous metabolic abnormalities, characterized by glucose intolerance and enhanced hepatic gluconeogenesis. Mfn2 deficiency impaired insulin signaling in liver and muscle. Furthermore, Mfn2 deficiency was associated with endoplasmic reticulum stress, enhanced hydrogen peroxide concentration, altered reactive oxygen species handling, and active JNK. Chemical chaperones or the antioxidant N-acetylcysteine ameliorated glucose tolerance and insulin signaling in liver-specific Mfn2 KO mice. This study provides an important description of a unique unexpected role of Mfn2 coordinating mitochondria and endoplasmic reticulum function, leading to modulation of insulin signaling and glucose homeostasis in vivo.

Brain-muscle communication prevents muscle aging by maintaining daily physiology.

A molecular clock network is crucial for daily physiology and maintaining organismal health. We examined the interactions and importance of intratissue clock networks in muscle tissue maintenance. In arrhythmic mice showing premature aging, we created a basic clock module involving a central and a peripheral (muscle) clock. Reconstituting the brain-muscle clock network is sufficient to preserve fundamental daily homeostatic functions and prevent premature muscle aging. However, achieving whole muscle physiology requires contributions from other peripheral clocks. Mechanistically, the muscle peripheral clock acts as a gatekeeper, selectively suppressing detrimental signals from the central clock while integrating important muscle homeostatic functions. Our research reveals the interplay between the central and peripheral clocks in daily muscle function and underscores the impact of eating patterns on these interactions.

A form of mitofusin 2 (Mfn2) lacking the transmembrane domains and the COOH-terminal end stimulates metabolism in muscle and liver cells.

Mitofusin 2 (Mfn2), a protein that participates in mitochondrial fusion, is required to maintain normal mitochondrial metabolism in skeletal muscle and liver. Given that muscle Mfn2 is repressed in obese or type 2 diabetic subjects, this protein may have a potential pathophysiological role in these conditions. To evaluate whether the metabolic effects of Mfn2 can be dissociated from its function in mitochondrial dynamics, we studied a form of human Mfn2, lacking the two transmembrane domains and the COOH-terminal coiled coil (ΔMfn2). This form localized in mitochondria but did not alter mitochondrial morphology in cells or in skeletal muscle fibers. The expression of ΔMfn2 in mouse skeletal muscle stimulated glucose oxidation and enhanced respiratory control ratio, which occurred in the absence of changes in mitochondrial mass. ΔMfn2 did not stimulate mitochondrial respiration in Mfn2-deficient muscle cells. The expression of ΔMfn2 in mouse liver or in hepatoma cells stimulated gluconeogenesis. In addition, ΔMfn2 activated basal and maximal respiration both in muscle and liver cells. In all, we show that a form of Mfn2 lacking mitochondrial fusion activity stimulates mitochondrial function and enhances glucose metabolism in muscle and liver tissues. This study suggests that Mfn2 regulates metabolism independently of changes in mitochondrial morphology.

Polycomb Ezh1 maintains murine muscle stem cell quiescence through non-canonical regulation of Notch signaling.

Organismal homeostasis and regeneration are predicated on committed stem cells that can reside for long periods in a mitotically dormant but reversible cell-cycle arrest state defined as quiescence. Premature escape from quiescence is detrimental, as it results in stem cell depletion, with consequent defective tissue homeostasis and regeneration. Here, we report that Polycomb Ezh1 confers quiescence to murine muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence. Following repeated injuries, the MuSC pool is progressively depleted, resulting in failure to sustain proper muscle regeneration. Rather than regulating repressive histone H3K27 methylation, Ezh1 maintains gene expression of the Notch signaling pathway in MuSCs. Selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs.

Mitochondrial fusion proteins: dual regulators of morphology and metabolism.

Mitochondria in mammalian cells are visualized as a network or as filaments that undergo continuous changes in shape and in localization within the cells. These changes are a consequence of the activity of different processes such as mitochondrial fusion and fission, and mitochondrial remodelling. In all, these processes are referred to as mitochondrial dynamics, and relevant questions, still unexplained, are why cells require such an active dynamics, or why mitochondria move to specific cellular regions. In this review we will summarize some of the biological functions assigned to the proteins identified as participating in mitochondrial fusion, namely mitofusin 1, mitofusin 2 and OPA1. In addition to the capacity of these proteins to promote fusion, mitofusin 2 or OPA1 regulate mitochondrial metabolism and loss-of-function reduces oxygen consumption and the capacity to oxidize substrates. We propose that mitochondrial fusion proteins operate as integrators of signals so they regulate both mitochondrial fusion and metabolism.

Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy.

Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.

Muscle repair after physiological damage relies on nuclear migration for cellular reconstruction.

Regeneration of skeletal muscle is a highly synchronized process that requires muscle stem cells (satellite cells). We found that localized injuries, as experienced through exercise, activate a myofiber self-repair mechanism that is independent of satellite cells in mice and humans. Mouse muscle injury triggers a signaling cascade involving calcium, Cdc42, and phosphokinase C that attracts myonuclei to the damaged site via microtubules and dynein. These nuclear movements accelerate sarcomere repair and locally deliver messenger RNA (mRNA) for cellular reconstruction. Myofiber self-repair is a cell-autonomous protective mechanism and represents an alternative model for understanding the restoration of muscle architecture in health and disease.

CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.

Regeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.

Sestrin prevents atrophy of disused and aging muscles by integrating anabolic and catabolic signals.

A unique property of skeletal muscle is its ability to adapt its mass to changes in activity. Inactivity, as in disuse or aging, causes atrophy, the loss of muscle mass and strength, leading to physical incapacity and poor quality of life. Here, through a combination of transcriptomics and transgenesis, we identify sestrins, a family of stress-inducible metabolic regulators, as protective factors against muscle wasting. Sestrin expression decreases during inactivity and its genetic deficiency exacerbates muscle wasting; conversely, sestrin overexpression suffices to prevent atrophy. This protection occurs through mTORC1 inhibition, which upregulates autophagy, and AKT activation, which in turn inhibits FoxO-regulated ubiquitin-proteasome-mediated proteolysis. This study reveals sestrin as a central integrator of anabolic and degradative pathways preventing muscle wasting. Since sestrin also protected muscles against aging-induced atrophy, our findings have implications for sarcopenia.

The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment.

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through ß1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/ß1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.

Regulation of Muscle Stem Cell Functions: A Focus on the p38 MAPK Signaling Pathway.

Formation of skeletal muscle fibers (myogenesis) during development and after tissue injury in the adult constitutes an excellent paradigm to investigate the mechanisms whereby environmental cues control gene expression programs in muscle stem cells (satellite cells) by acting on transcriptional and epigenetic effectors. Here we will review the molecular mechanisms implicated in the transition of satellite cells throughout the distinct myogenic stages (i.e., activation from quiescence, proliferation, differentiation, and self-renewal). We will also discuss recent findings on the causes underlying satellite cell functional decline with aging. In particular, our review will focus on the epigenetic changes underlying fate decisions and on how the p38 MAPK signaling pathway integrates the environmental signals at the chromatin to build up satellite cell adaptive responses during the process of muscle regeneration, and how these responses are altered in aging. A better comprehension of the signaling pathways connecting external and intrinsic factors will illuminate the path for improving muscle regeneration in the aged.

Chromatin-wide and transcriptome profiling integration uncovers p38α MAPK as a global regulator of skeletal muscle differentiation.

BACKGROUND: Extracellular stimuli induce gene expression responses through intracellular signaling mediators. The p38 signaling pathway is a paradigm of the mitogen-activated protein kinase (MAPK) family that, although originally identified as stress-response mediator, contributes to establishing stem cell differentiation fates. p38α is central for induction of the differentiation fate of the skeletal muscle stem cells (satellite cells) through not fully characterized mechanisms. METHODS: To investigate the global gene transcription program regulated by p38α during satellite cell differentiation (myogenesis), and to specifically address whether this regulation occurs through direct action of p38α on gene promoters, we performed a combination of microarray gene expression and genome-wide binding analyses. For experimental robustness, two myogenic cellular systems with genetic and chemical loss of p38α function were used: (1) satellite cells derived from mice with muscle-specific deletion of p38α, and (2) the C2C12 murine myoblast cell line cultured in the absence or presence of the p38α/ß inhibitor SB203580. Analyses were performed at cell proliferation and early differentiation stages. RESULTS: We show that p38α binds to a large set of active promoters during the transition of myoblasts from proliferation to differentiation stages. p38α-bound promoters are enriched with binding motifs for several transcription factors, with Sp1, Tcf3/E47, Lef1, FoxO4, MyoD, and NFATc standing out in all experimental conditions. p38α association with chromatin correlates very well with high levels of transcription, in agreement with its classical function as an activator of myogenic differentiation. Interestingly, p38α also associates with genes repressed at the onset of differentiation, thus highlighting the relevance of p38-dependent chromatin regulation for transcriptional activation and repression during myogenesis. CONCLUSIONS: These results uncover p38α association and function on chromatin at novel classes of target genes during skeletal muscle cell differentiation. This is consistent with this MAPK isoform being a transcriptional regulator.

The Chromatin Remodeling Complex Chd4/NuRD Controls Striated Muscle Identity and Metabolic Homeostasis.

Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life.

Epigenetic control of adult skeletal muscle stem cell functions.

Skeletal muscle regeneration in the adult (de novo myogenesis) depends on a resident population of muscle stem cells (satellite cells) that are normally quiescent. In response to injury or stress, satellite cells are activated and expand as myoblast cells that differentiate and fuse to form new muscle fibers or return to quiescence to maintain the stem cell pool (self-renewal). Satellite cell-dependent myogenesis is a well-characterized multi-step process orchestrated by muscle-specific transcription factors, such as Pax3/Pax7 and members of the MyoD family of muscle regulatory factors, and epigenetically controlled by mechanisms such as DNA methylation, covalent modification of histones and non-coding RNAs. Recent results from next-generation genome-wide sequencing have increased our understanding about the highly intricate layers of epigenetic regulation involved in satellite cell maintenance, activation, differentiation and self-renewal, and their cross-talk with the muscle-specific transcriptional machinery.