The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells.
ABSTRACT: Skeletal muscle stem cell (MuSC) function declines with age and contributes to impaired muscle regeneration in older individuals. Acting through AMPK/p27Kip1, we have identified a pathway regulating the balance between autophagy, apoptosis, and senescence in aged MuSCs. While p27Kip1 is implicated in MuSC aging, its precise role and molecular mechanism have not been elucidated. Age-related MuSC dysfunction was associated with reduced autophagy, increased apoptosis, and hypophosphorylation of AMPK and its downstream target p27Kip1. AMPK activation or ectopic expression of a phosphomimetic p27Kip1 mutant was sufficient to suppress in vitro apoptosis, increase proliferation, and improve in vivo transplantation efficiency of aged MuSCs. Moreover, activation of the AMPK/p27Kip1 pathway reduced markers of cell senescence in aged cells, which was, in part, dependent on p27Kip1 phosphorylation. Thus, the AMPK/p27Kip1 pathway likely regulates the autophagy/apoptosis balance in aged MuSCs and may be a potential target for improving muscle regeneration in older individuals.
Project description:Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPK?1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPK?1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPK?1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPK?1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPK?1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPK?1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.
Project description:The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38? and p38? mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38? and p38? in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.
Project description:Quiescent adult muscle stem cells (MuSCs) regenerate skeletal muscle upon injury throughout life. However, aged skeletal muscles fail to maintain stem cell quiescence, leading to declines in MuSC number and functionality. Although autophagy plays an important role in the maintenance of MuSC quiescence, how quiescent MuSCs and their autophagy levels are maintained throughout life is largely unknown. The current study reveals how GnRH, a hypothalamic hormone, maintains the quiescence of adult MuSCs by preventing the onset of senescence and how the decline of sex steroids in organismal ageing is implicated in MuSC ageing. Overall design: Gene expression profiling of WT and Pax7-CreER;Arf/y;Esr2–/– conditional knock out MuSCs.
Project description: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.
Project description:Age-related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age-related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age-related increase in muscle stiffness drives YAP/TAZ-mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.
Project description:Aging impairs tissue repair. This is pronounced in skeletal muscle, whose regeneration by muscle stem cells (MuSCs) is robust in young adult animals but inefficient in older organisms. Despite this functional decline, old MuSCs are amenable to rejuvenation through strategies that improve the systemic milieu, such as heterochronic parabiosis. One such strategy, exercise, has long been appreciated for its benefits on healthspan, but its effects on aged stem cell function in the context of tissue regeneration are incompletely understood. Here we show that exercise in the form of voluntary wheel running accelerates muscle repair in old animals and improves old MuSC function. Through transcriptional profiling and genetic studies, we discovered that the restoration of old MuSC activation ability hinges on restoration of Cyclin D1, whose expression declines with age in MuSCs. Pharmacologic studies revealed that Cyclin D1 maintains MuSC activation capacity by repressing TGF? signaling. Taken together, these studies demonstrate that voluntary exercise is a practicable intervention for old MuSC rejuvenation. Furthermore, this work highlights the distinct role of Cyclin D1 in stem cell quiescence.
Project description:Muscle undergoes progressive weakening and regenerative dysfunction with age due in part to the functional decline of skeletal muscle stem cells (MuSCs). MuSCs are heterogeneous but whether their gene expression changes with age and the implication of such changes are unclear. Here we show that in mice, Growth arrest-specific gene 1 (Gas1) is expressed in a small subset of young MuSCs with its expression progressively increasing in larger fractions of MuSCs later in life. Over-expression of Gas1 in young MuSCs and inactivation of Gas1 in aged MuSCs support that Gas1 reduces the quiescence and self-renewal capacity of MuSCs. Gas1 reduces Ret signaling, which is required for MuSC quiescence and self-renewal. Indeed, we show that the Ret ligand, Glial Cell-Derived Neurotrophic Factor (GDNF), can counteract Gas1 by stimulating Ret signaling and enhancing MuSC self-renewal and regeneration, thus improving muscle function. We propose that strategies aimed to target this pathway can be exploited to improve the regenerative decline of muscle stem cells.
Project description:Skeletal muscle is a regenerative tissue which can repair damaged myofibers through the activation of tissue-resident muscle stem cells (MuSCs). Many muscle diseases with impaired regeneration cause excessive adipose tissue accumulation in muscle, alter the myogenic fate of MuSCs, and deregulate the cross-talk between MuSCs and fibro/adipogenic progenitors (FAPs), a bi-potent cell population which supports myogenesis and controls intra-muscular fibrosis and adipocyte formation. In order to better characterize the interaction between adipogenesis and myogenesis, we studied muscle regeneration and MuSC function in whole body Pparg null mice generated by epiblast-specific Cre/lox deletion (Pparg?/?). We demonstrate that deletion of PPAR? completely abolishes ectopic muscle adipogenesis during regeneration and impairs MuSC expansion and myogenesis after injury. Ex vivo assays revealed that perturbed myogenesis in Pparg?/? mice does not primarily result from intrinsic defects of MuSCs or from perturbed myogenic support from FAPs. The immune transition from a pro- to anti-inflammatory MuSC niche during regeneration is perturbed in Pparg?/? mice and suggests that PPAR? signaling in macrophages can interact with ectopic adipogenesis and influence muscle regeneration. Altogether, our study demonstrates that a PPAR?-dependent adipogenic response regulates muscle fat infiltration during regeneration and that PPAR? is required for MuSC function and efficient muscle repair.
Project description:Tissue renewal and muscle regeneration largely rely on the proliferation and differentiation of muscle stem cells called muscular satellite cells (MuSCs). MuSCs are normally quiescent, but they are activated in response to various stimuli, such as inflammation. Activated MuSCs proliferate, migrate, differentiate, and fuse to form multinucleate myofibers. Meanwhile, inappropriate cues for MuSC activation induce premature differentiation and bring about stem cell loss. Recent studies revealed that stem cell regulation is disrupted in various aged tissues. We found that the expression of microRNA (miR)-155, which is an inflammation-associated miR, is upregulated in MuSCs of aged muscles, and this upregulation activates the differentiation process through suppression of C/ebp?, which is an important molecule for maintaining MuSC self-renewal. We also found that Notch1 considerably repressed miR-155 expression, and loss of Notch1 induced miR-155 overexpression. Our findings suggest that miR-155 can act as an activator of muscular differentiation and might be responsible for accelerating aging-associated premature differentiation of MuSCs.
Project description:Exercise train (ET) stimulates muscle response in pathological conditions, including aging. The molecular mechanisms by which exercise improves impaired adiponectin/adiponectin receptor 1 (AdipoR1)-related muscle actions associated with aging are poorly understood. Here we observed that in a senescence-accelerated mouse prone 10 (SAMP10) model, long-term ET modulated muscle-regenerative actions.25-week-old male SAMP10 mice were randomly assigned to the control and the ET (45?min/time, 3/week) groups for 4?months. Mice that were maintained in a sedentary condition served controls.ET ameliorated aging-related muscle changes in microstructure, mitochondria, and performance. The amounts of proteins or mRNAs for p-AMPK?, p-Akt, p-ERK1/2, p-mTOR, Bcl-XL, p-FoxO3, peroxisome proliferators-activated receptor-? coactivator, adiponectin receptor1 (adpoR1), and cytochrome c oxidase-IV, and the numbers of CD34+ /integrin-?7+ muscle stem cells (MuSCs) and proliferating cells in the muscles and bone-marrow were enhanced by ET, whereas the levels of p-GSK-3? and gp91phox proteins and apoptotic cells were reduced by ET. The ET also resulted in increased levels of plasma adiponectin and the numbers of bone-marrow (BM)-derived circulating CD34+ /integrin-?7+ MuSCs and their functions. Integrin-?7+ MuSCs of exercised mice had improved changes of those beneficial molecules. These ET-mediated aged muscle benefits were diminished by adiponectin and AdipoR1 blocking as well as AMPK inhibition. Finally, recombinant mouse adiponectin enhanced AMPK and mTOR phosphorylations in BM-derived integrin-?7+ cells.These findings suggest that ET can improve aging-related impairments of BM-derived MuSC regenerative capacity and muscle metabolic alterations via an AMPK-dependent mechanism that is mediated by an adiponectin/AdipoR1 axis in SAMP10 mice.