Relative roles of TGF‐β1 and Wnt in the systemic regulation and aging of satellite cell responses

Muscle stem (satellite) cells are relatively resistant to cell‐autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P‐Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor‐beta (TGF‐β) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age‐dependent myogenic activity of sera TGF‐β1, and its potential cross‐talk with systemic Wnt. We found that sera TGF‐β1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF‐β1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF‐β1 were inhibitory and young sera suppressed myogenesis if TGF‐β1 was activated. Our data suggest that platelet‐derived sera TGF‐β1 levels, or endocrine TGF‐β1 levels, do not explain the age‐dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF‐β neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF‐β1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF‐β receptor kinase inhibitor, which attenuated TGF‐β signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF‐β1‐dependent block on muscle regeneration, identify physiological modalities of age‐imposed changes in TGF‐β1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.     

Effects of IGF‐1 isoforms on muscle growth and sarcopenia

The decline in skeletal muscle mass and strength occurring in aging, referred as sarcopenia, is the result of many factors including an imbalance between protein synthesis and degradation, changes in metabolic/hormonal status, and in circulating levels of inflammatory mediators. Thus, factors that increase muscle mass and promote anabolic pathways might be of therapeutic benefit to counteract sarcopenia. Among these, the insulin‐like growth factor‐1 (IGF‐1) has been implicated in many anabolic pathways in skeletal muscle. IGF‐1 exists in different isoforms that might exert different role in skeletal muscle. Here we study the effects of two full propeptides IGF‐1Ea and IGF‐1Eb in skeletal muscle, with the aim to define whether and through which mechanisms their overexpression impacts muscle aging. We report that only IGF‐1Ea expression promotes a pronounced hypertrophic phenotype in young mice, which is maintained in aged mice. Nevertheless, examination of aged transgenic mice revealed that the local expression of either IGF‐1Ea or IGF‐1Eb transgenes was protective against age‐related loss of muscle mass and force. At molecular level, both isoforms activate the autophagy/lysosome system, normally altered during aging, and increase PGC1‐α expression, modulating mitochondrial function, ROS detoxification, and the basal inflammatory state occurring at old age. Moreover, morphological integrity of neuromuscular junctions was maintained and preserved in both MLC/IGF‐1Ea and MLC/IGF‐1Eb mice during aging. These data suggest that IGF‐1 is a promising therapeutic agent in staving off advancing muscle weakness.     

Age‐related loss of nitric oxide synthase in skeletal muscle causes reductions in calpain S‐nitrosylation that increase myofibril degradation and sarcopenia

Sarcopenia, the age‐related loss of muscle mass, is a highly‐debilitating consequence of aging. In this investigation, we show sarcopenia is greatly reduced by muscle‐specific overexpression of calpastatin, the endogenous inhibitor of calcium‐dependent proteases (calpains). Further, we show that calpain cleavage of specific structural and regulatory proteins in myofibrils is prevented by covalent modification of calpain by nitric oxide (NO) through S‐nitrosylation. We find that calpain in adult, non‐sarcopenic muscles is S‐nitrosylated but that aging leads to loss of S‐nitrosylation, suggesting that reduced S‐nitrosylation during aging leads to increased calpain‐mediated proteolysis of myofibrils. Further, our data show that muscle aging is accompanied by loss of neuronal nitric oxide synthase (nNOS), the primary source of muscle NO, and that expression of a muscle‐specific nNOS transgene restores calpain S‐nitrosylation in aging muscle and prevents sarcopenia. Together, the findings show that in vivo reduction of calpain S‐nitrosylation in muscle may be an important component of sarcopenia, indicating that modulation of NO can provide a therapeutic strategy to slow muscle loss during old age.     

Deficiency of ovarian ornithine decarboxylase contributes to aging‐related egg aneuploidy in mice

It has been known for more than four decades that during mammalian estrous cycles, luteinizing hormone stimulates a transitory rise in the ovaries of ornithine decarboxylase (ODC) activity and its enzymatic product putrescine, concurrent with oocyte maturation in vivo. Inhibition of this transitory ODC/putrescine rise, however, does not appear to affect oocyte maturation or ovulation. Using several mouse models and combining in vitro and in vivo approaches, we demonstrated that deficiency of ODC during oocyte maturation is correlated with increased levels of egg aneuploidies. These results suggest that the transitory ovarian ODC rise in late proestrus is important for ensuring proper chromosome segregation during oocyte maturation. Older mice (8 months of age) exhibited about 1/3 that of young mice in LH‐stimulated ovarian ODC activity and a corresponding increase in egg aneuploidies. Moreover, a combination of putrescine supplementation in mouse drinking water leading up to oocyte retrieval and in oocyte maturation medium reduced egg aneuploidies of the older mice from 12.7% to 5.3%. Therefore, ovarian ODC deficiency might be an important etiology of maternal aging‐related aneuploidies, and peri‐ovulatory putrescine supplementation might reduce the risk of aneuploid conceptions in older women.     

PGC‐1α affects aging‐related changes in muscle and motor function by modulating specific exercise‐mediated changes in old mice

The age‐related impairment in muscle function results in a drastic decline in motor coordination and mobility in elderly individuals. Regular physical activity is the only efficient intervention to prevent and treat this age‐associated degeneration. However, the mechanisms that underlie the therapeutic effect of exercise in this context remain unclear. We assessed whether endurance exercise training in old age is sufficient to affect muscle and motor function. Moreover, as muscle peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) is a key regulatory hub in endurance exercise adaptation with decreased expression in old muscle, we studied the involvement of PGC‐1α in the therapeutic effect of exercise in aging. Intriguingly, PGC‐1α muscle‐specific knockout and overexpression, respectively, precipitated and alleviated specific aspects of aging‐related deterioration of muscle function in old mice, while other muscle dysfunctions remained unchanged upon PGC‐1α modulation. Surprisingly, we discovered that muscle PGC‐1α was not only involved in improving muscle endurance and mitochondrial remodeling, but also phenocopied endurance exercise training in advanced age by contributing to maintaining balance and motor coordination in old animals. Our data therefore suggest that the benefits of exercise, even when performed at old age, extend beyond skeletal muscle and are at least in part mediated by PGC‐1α.     

Haploinsufficiency of myostatin protects against aging‐related declines in muscle function and enhances the longevity of mice

The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28–30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN^+/− and MSTN^−/− mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN^+/+ and MSTN^−/− mice, MSTN^+/− mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity.     

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.     

A tissue‐specific screen of ceramide expression in aged mice identifies ceramide synthase‐1 and ceramide synthase‐5 as potential regulators of fiber size and strength in skeletal muscle

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi‐organ investigation of age‐associated changes of ceramide species, a unique and distinctive change pattern of C_16:0 and C_18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C_16:0 and C_18:0 acyl chains, respectively, was down‐regulated in skeletal muscle of aged mice. Similarly, an age‐dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1^Δ/Δ and CerS5^Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1‐ and CerS5‐deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age‐related muscle wasting disorders in humans.     

miR‐181a regulates p62/SQSTM1, parkin,and protein DJ‐1 promoting mitochondrial dynamics in skeletal muscle aging

One of the key mechanisms underlying skeletal muscle functional deterioration during aging is disrupted mitochondrial dynamics. Regulation of mitochondrial dynamics is essential to maintain a healthy mitochondrial population and prevent the accumulation of damaged mitochondria; however, the regulatory mechanisms are poorly understood. We demonstrated loss of mitochondrial content and disrupted mitochondrial dynamics in muscle during aging concomitant with dysregulation of miR‐181a target interactions. Using functional approaches and mito‐QC assay, we have established that miR‐181a is an endogenous regulator of mitochondrial dynamics through concerted regulation of Park2, p62/SQSTM1, and DJ‐1 in vitro. Downregulation of miR‐181a with age was associated with an accumulation of autophagy‐related proteins and abnormal mitochondria. Restoring miR‐181a levels in old mice prevented accumulation of p62, DJ‐1, and PARK2, and improved mitochondrial quality and muscle function. These results provide physiological evidence for the potential of microRNA‐based interventions for age‐related muscle atrophy and of wider significance for diseases with disrupted mitochondrial dynamics.     

Tyrosinase‐catalyzed oxidation of resveratrol produces a highly reactive ortho‐quinone: Implications for melanocyte toxicity

trans‐Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, RES), a naturally occurring polyphenol, has recently attracted increased interest as a health‐beneficial agent. However, based on its p‐substituted phenol structure, RES is expected to be a substrate for tyrosinase and to produce a toxic o‐quinone metabolite. The results of this study demonstrate that the oxidation of RES by tyrosinase produces 4‐(3′,5′‐dihydroxy‐trans‐styrenyl)‐1,2‐benzoquinone (RES‐quinone), which decays rapidly to an oligomeric product (RES‐oligomer). RES‐quinone was identified after reduction to its corresponding catechol, known as piceatannol. RES‐quinone reacts with N‐acetylcysteine, a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS analyses. The production of a triadduct is not common for o‐quinones, suggesting a high reactivity of RES‐quinone. RES‐quinone also binds to bovine serum albumin through its cysteine residue. RES‐oligomer can oxidize GSH to GSSG, indicating its pro‐oxidant activity. These results suggest that RES could be cytotoxic to melanocytes due to the binding of RES‐quinone to thiol proteins.     

Weekly administration of rapamycin improves survival and biomarkers in obese male mice on high‐fat diet

Recent discoveries have revealed the key role of mTOR (target of rapamycin) in aging. Furthermore, rapamycin extends lifespan in mice, especially in female mice. Here, we treated obese male mice on high‐fat diet with rapamycin given intermittently: either weekly (once a week) or alternating bi‐weekly (three injections every other week). While only marginally reducing obesity, intermittent administration of rapamycin significantly extended lifespan. Significance was achieved for weekly treated group and for the three rapamycin‐received groups combined. In weekly treatment group, 100% mice were alive by the age of 2 years, whereas 60% of mice died in untreated group by this age. The effect of weekly treatment on survival was highly significant and cannot be fully explained by partial reduction in obesity. Alternating bi‐weekly treatments seem to be less effective than weekly treatment, although effects of additional factors (see 3 ) may not be excluded. After one year of treatment, all survived mice were sacrificed 8 days after the last administration of rapamycin to avoid its direct interference with parameters examined. Fasting levels of cardiac and hepatic p‐S6, a marker of mTORC1 activity, were lower in weekly treatment group compared with control mice. In contrast, levels of p‐Akt (S473), glucose, triglycerides and insulin were unchanged, whereas leptin and IGF‐1 tended to be lower. Thus, weekly treatment with rapamycin may slow down aging in obese male mice on high‐fat diet.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

Exercise reverses age‐related vulnerability of the retina to injury by preventing complement‐mediated synapse elimination via a BDNF‐dependent pathway

Retinal ganglion cells (RGCs) become increasingly vulnerable to injury with advancing age. We recently showed that this vulnerability can be strongly modified in mice by exercise. However, the characteristics and underlying mechanisms of retinal protection with exercise remain unknown. Hence, the aim of this study was to investigate cellular changes associated with exercise‐induced protection of aging retinal cells and the role of local and peripheral trophic signalling in mediating these effects. We focussed on two molecules that are thought to play key roles in mediating beneficial effects of exercise: brain‐derived neurotrophic factor (BDNF) and AMP‐activated protein kinase (AMPK). In middle‐aged (12 months old) C57BL/6J mice, we found that exercise protected RGCs against dysfunction and cell loss after an acute injury induced by elevation of intra‐ocular pressure. This was associated with preservation of inner retinal synapses and reduced synaptic complement deposition. Retinal expression of BDNF was not upregulated in response to exercise alone. Rather, exercise maintained BDNF levels in the retina, which were decreased postinjury in nonexercised animals. Confirming a critical role for BDNF, we found that blocking BDNF signalling during exercise by pharmacological means or genetic knock‐down suppressed the functional protection of RGCs afforded by exercise. Protection of RGCs with exercise was independent of activation of AMPK in either retina or skeletal muscle. Our data support a previously unidentified mechanism in which exercise prevents loss of BDNF in the retina after injury and preserves neuronal function and survival by preventing complement‐mediated elimination of synapses.     

Atypical antidepressants extend lifespan of Caenorhabditis elegans by activation of a non‐cell‐autonomous stress response

Oxidative stress has long been associated with aging and has recently been linked to psychiatric disorders, including psychosis and depression. We identified multiple antipsychotics and antidepressants that extend Caenorhabditis elegans lifespan and protect the animal from oxidative stress. Here, we report that atypical antidepressants activate a neuronal mechanism that regulates the response to oxidative stress throughout the animal. While the activation of the oxidative stress response by atypical antidepressants depends on synaptic transmission, the activation by reactive oxygen species does not. Lifespan extension by atypical antidepressants depends on the neuronal oxidative stress response activation mechanism. Neuronal regulation of the oxidative stress response is likely to have evolved as a survival mechanism to protect the organism from oxidative stress, upon detection of adverse or dangerous conditions by the nervous system.     

Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle‐derived nitric oxide

Muscle aging is associated with changes in myeloid cell phenotype that may influence age‐related changes in muscle structure. We tested whether preventing age‐related reductions in muscle neuronal nitric oxide synthase (nNOS) would obviate age‐related changes in myeloid cells in muscle. Our findings show that muscle aging is associated with elevations of anti‐inflammatory M2a macrophages that can increase muscle fibrosis. Expression of a muscle‐specific nNOS transgene in mice prevented age‐related increases in M2a macrophages. Transgene expression also reduced expression of collagens and decreased muscle fibrosis. The nNOS transgene prevented age‐related increases in arginase‐1 but did not influence TGFβ expression, indicating that the transgene may prevent age‐related muscle fibrosis by inhibiting the arginase‐dependent profibrotic pathway. Although aged satellite cells or fibro‐adipogenic precursor (FAPs) cells also promote fibrosis, transgene expression had no effect on the expression of key signaling molecules that regulate fibrogenic activity of those cells. Finally, we tested whether increases in M2a macrophages and the associated increase in fibrosis were attributable to aging of myeloid lineage cells. Young bone marrow cells (BMCs) were transplanted into young or old mice, and muscles were collected 8 months later. Muscles of young mice receiving young BMCs showed no effect on M2a macrophage number or collagen accumulation compared to age‐matched, nontransplanted controls. However, muscles of old mice receiving young BMCs showed fewer M2a macrophages and less accumulation of collagen. Thus, the age‐related increase in M2a macrophages in aging muscle and the associated muscle fibrosis are determined in part by the age of bone marrow cells.     

Maintenance of polygenic sex determination in a fluctuating environment: an individual‐based model

R. A. Fisher predicted that individuals should invest equally in offspring of both sexes, and that the proportion of males and females produced (the primary sex ratio) should evolve towards 1:1 when unconstrained. For many species, sex determination is dependent on sex chromosomes, creating a strong tendency for balanced sex ratios, but in other cases, multiple autosomal genes interact to determine sex. In such cases, the maintenance of multiple sex‐determining alleles at multiple loci and the consequent among‐family variability in sex ratios presents a puzzle, as theory predicts that such systems should be unstable. Theory also predicts that environmental influences on sex can complicate outcomes of genetic sex determination, and that population structure may play a role. Tigriopus californicus, a copepod that lives in splash‐pool metapopulations and exhibits polygenic and environment‐dependent sex determination, presents a test case for relevant theory. We use this species as a model for parameterizing an individual‐based simulation to investigate conditions that could maintain polygenic sex determination. We find that metapopulation structure can delay the degradation of polygenic sex determination and that periods of alternating frequency‐dependent selection, imposed by seasonal fluctuations in environmental conditions, can maintain polygenic sex determination indefinitely.     

Extracellular‐signal‐regulated kinase/mitogen‐activated protein kinase signaling as a target for cancer therapy: an updated review

Mitogen‐activated protein kinase (MAPK) signaling pathway is activated in a wide spectrum of human tumors, exhibiting cardinal oncogenic roles and sustained inhibition of this pathway is considered as a primary goal in clinic. Within this pathway, receptor tyrosine kinases such as epithelial growth factor receptor, mesenchymal–epithelial transition, and AXL act as upstream regulators of RAS/RAF/MEK/extracellular‐signal‐regulated kinase. MAPK signaling is active in both early and advanced stages of tumorigenesis, and it promotes tumor proliferation, survival, and metastasis. MAPK regulatory effects on cellular constituent of the tumor microenvironment is for immunosuppressive purposes. Cross‐talking between MAPK with oncogenic signaling pathways including WNT, cyclooxygenase‐2, transforming growth factor‐β, NOTCH and (in particular) with phosphatidylinositol 3‐kinase is contributed to the multiplication of tumor progression and drug resistance. Developing resistance (intrinsic or acquired) to MAPK‐targeted therapy also occurs due to heterogeneity of tumors along with mutations and negative feedback loop of interactions exist between various kinases causing rebound activation of this signaling. Multidrug regimen is a preferred therapeutic avenue for targeting MAPK signaling. To enhance patient tolerance and to mitigate potential adversarial effects related to the combination therapy, determination of a desired dose and drug along with pre‐evaluation of cancer‐type‐specific kinase mutation and sensitivity, especially for patients receiving triplet therapy is an urgent need.