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.     

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.     

Peroxisome proliferator‐activated receptor γ coactivator 1α regulates mitochondrial calcium homeostasis,sarcoplasmic reticulum stress,and cell death to mitigate skeletal muscle aging

Age‐related impairment of muscle function severely affects the health of an increasing elderly population. While causality and the underlying mechanisms remain poorly understood, exercise is an efficient intervention to blunt these aging effects. We thus investigated the role of the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a potent regulator of mitochondrial function and exercise adaptation, in skeletal muscle during aging. We demonstrate that PGC‐1α overexpression improves mitochondrial dynamics and calcium buffering in an estrogen‐related receptor α‐dependent manner. Moreover, we show that sarcoplasmic reticulum stress is attenuated by PGC‐1α. As a result, PGC‐1α prevents tubular aggregate formation and cell death pathway activation in old muscle. Similarly, the pro‐apoptotic effects of ceramide and thapsigargin were blunted by PGC‐1α in muscle cells. Accordingly, mice with muscle‐specific gain‐of‐function and loss‐of‐function of PGC‐1α exhibit a delayed and premature aging phenotype, respectively. Together, our data reveal a key protective effect of PGC‐1α on muscle function and overall health span in aging.     

Mitochondrial function in skeletal myofibers is controlled by a TRF2‐SIRT3 axis over lifetime

Telomere shortening follows a developmentally regulated process that leads to replicative senescence of dividing cells. However, whether telomere changes are involved in postmitotic cell function and aging remains elusive. In this study, we discovered that the level of the TRF2 protein, a key telomere‐capping protein, declines in human skeletal muscle over lifetime. In cultured human myotubes, TRF2 downregulation did not trigger telomere dysfunction, but suppressed expression of the mitochondrial Sirtuin 3 gene (SIRT3) leading to mitochondrial respiration dysfunction and increased levels of reactive oxygen species. Importantly, restoring the Sirt3 level in TRF2‐compromised myotubes fully rescued mitochondrial functions. Finally, targeted ablation of the Terf2 gene in mouse skeletal muscle leads to mitochondrial dysfunction and sirt3 downregulation similarly to those of TRF2‐compromised human myotubes. Altogether, these results reveal a TRF2‐SIRT3 axis controlling muscle mitochondrial function. We propose that this axis connects developmentally regulated telomere changes to muscle redox metabolism.     

The evolution of skeletal muscle performance: gene duplication and divergence of human sarcomeric α‐actinins

In humans, there are two skeletal muscle α‐actinins, encoded by ACTN2 and ACTN3, and the ACTN3 genotype is associated with human athletic performance. Remarkably, approximately 1 billion people worldwide are deficient in α‐actinin‐3 due to the common ACTN3 R577X polymorphism. The α‐actinins are an ancient family of actin‐binding proteins with structural, signalling and metabolic functions. The skeletal muscle α‐actinins diverged ～250–300 million years ago, and ACTN3 has since developed restricted expression in fast muscle fibres. Despite ACTN2 and ACTN3 retaining considerable sequence similarity, it is likely that following duplication there was a divergence in function explaining why α‐actinin‐2 cannot completely compensate for the absence of α‐actinin‐3. This paper focuses on the role of skeletal muscle α‐actinins, and how possible changes in functions between these duplicates fit in the context of gene duplication paradigms.     

Lysyl oxidase‐like 2 inhibitor rescues D‐galactose‐induced skeletal muscle fibrosis

Aging‐related sarcopenia is currently the most common sarcopenia. The main manifestations are skeletal muscle atrophy, replacement of muscle fibers with fat and fibrous tissue. Excessive fibrosis can impair muscle regeneration and function. Lysyl oxidase‐like 2 (LOXL2) has previously been reported to be involved in the development of various tissue fibrosis. Here, we investigated the effects of LOXL2 inhibitor on D‐galactose (D‐gal)‐induced skeletal muscle fibroblast cells and mice. Our molecular and physiological studies show that treatment with LOXL2 inhibitor can alleviate senescence, fibrosis, and increased production of reactive oxygen species in fibroblasts caused by D‐gal. These effects are related to the inhibition of the TGF‐β1/p38 MAPK pathway. Furthermore, in vivo, mice treatment with LOXL2 inhibitor reduced D‐gal‐induced skeletal muscle fibrosis, partially enhanced skeletal muscle mass and strength and reduced redox balance disorder. Taken together, these data indicate the possibility of using LOXL2 inhibitors to prevent aging‐related sarcopenia, especially with significant fibrosis.     

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.     

SIRT1‐mediated ERβ suppression in the endothelium contributes to vascular aging

SIRT1 has many important molecular functions in aging, and the estrogen receptors (ERs) have a vasculoprotective effect, although the detailed mechanism for the roles of SIRT1 and ERs in vascular aging remains unclear. We found that ERβ expression in the endothelium was reduced in aging mice, and the expression of ERα and SIRT1 did not change, while SIRT1 activity declined. Further investigation showed that the ERβ expression was regulated by SIRT1 through complexes of SIRT1‐PPARγ/RXR‐p300 that bind to a PPRE (PPAR response element) site on the ERβ promoter, and the declined SIRT1 function in aging mice was due to compromised phosphorylation at S154. A single‐mutant SIRT1‐C152(D) restored the reduced ERβ expression in the endothelium with minimized reactive oxygen species generation and DNA damage and increased mitochondrial function and fatty acid metabolism. In high‐fat diet aging mice, the endothelium‐specific delivery of ERβ or SIRT1‐C152(D) on the vascular wall reduced the circulating lipids with ameliorated vascular damage, including the restored vessel tension and blood pressure. We conclude that SIRT1‐mediated ERβ suppression in the endothelium contributes to vascular aging, and the modulation of SIRT1 phosphorylation through a single‐mutant SIRT1‐C152(D) restores this effect.     

Mitochondrial‐targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial‐targeted peptide SS‐31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5 month old) and old (27 month old) mice were injected intraperitoneally with either saline or 3 mg kg^?1 of SS‐31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and ³¹P magnetic resonance spectroscopy. Age‐related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS‐31 treatment, while SS‐31 had no observable effect on young muscle. These effects of SS‐31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H₂O₂ emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS‐31 treatment, and eight days of SS‐31 treatment led to increased whole‐animal endurance capacity. These data demonstrate that SS‐31 represents a new strategy for reversing age‐related deficits in skeletal muscle with potential for translation into human use.     

AMPKα1‐LDH pathway regulates muscle stem cell self‐renewal by controlling metabolic homeostasis

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. AMPKα1^?/? MuSCs displayed a high self‐renewal rate, which impairs muscle regeneration. AMPKα1^?/? MuSCs showed a Warburg‐like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non‐limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1^?/? phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1^?/? MuSC self‐renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.     

Conditional TGF‐β1 treatment increases stem cell‐like cell population in myoblasts

The limitation in successfully acquiring large populations of stem cell has impeded their application. A new method based on the dedifferentiation of adult somatic cells to generate induced multipotent stem cells would allow us to obtain a large amount of autologous stem cells for regenerative medicine. The current work was proposed to induce a sub‐population of cells with characteristics of muscle stem cells from myoblasts through conditional treatment of transforming growth factor (TGF)‐β₁. Our results show that a lower concentration of TGF‐β₁ is able to promote C2C12 myoblasts to express stem cell markers as well as to repress myogenic proteins, which involves a mechanism of dedifferentiation. Moreover, TGF‐β₁ treatment promoted the proliferation‐arrested C2C12 myoblasts to re‐enter the S‐phase. We also investigated the multi‐differentiation potentials of the dedifferentiated cells. TGF‐β₁ pre‐treated C2C12 myoblasts were implanted into mice to repair dystrophic skeletal muscle or injured bone. In addition to the C2C12 myoblasts, similar effects of TGF‐β₁ were also observed in the primary myoblasts of mice. Our results suggest that TGF‐β₁ is effective as a molecular trigger for the dedifferentiation of skeletal muscle myoblasts and could be used to generate a large pool of progenitor cells that collectively behave as multipotent stem cell‐like cells for regenerative medicine applications.     

Age modifies respiratory complex I and protein homeostasis in a muscle type‐specific manner

Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow‐twitch soleus (SOL) and fast‐twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age‐induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I‐derived H₂O₂ production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial‐targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue‐specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.     

The role of TGF‐β1 during skeletal muscle regeneration

The injury of adult skeletal muscle initiates series of well‐coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF‐β family members is TGF‐β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF‐β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.     

TNF‐α‐mediated reduction in PGC‐1α may impair skeletal muscle function after cigarette smoke exposure

Skeletal muscle dysfunction contributes to exercise limitation in COPD. In this study cigarette smoke exposure was hypothesized to increase expression of the inflammatory cytokine, TNF‐α, thereby suppressing PGC‐1α, and hence affecting down stream molecules that regulate oxygen transport and muscle function. Furthermore, we hypothesized that highly vascularized oxidative skeletal muscle would be more susceptible to cigarette smoke than less well‐vascularized glycolytic muscle. To test these hypotheses, mice were exposed to cigarette smoke daily for 8 or 16 weeks, resulting in 157% (8 weeks) and 174% (16 weeks) increases in serum TNF‐α. Separately, TNF‐α administered to C2C12 myoblasts was found to dose‐dependently reduce PGC‐1α mRNA. In the smoke‐exposed mice, PGC‐1α mRNA was decreased, by 48% in soleus and 23% in EDL. The vascular PGC‐1α target molecule, VEGF, was also down‐regulated, but only in the soleus, which exhibited capillary regression and an oxidative to glycolytic fiber type transition. The apoptosis PGC‐1α target genes, atrogin‐1 and MuRF1, were up‐regulated, and to a greater extent in the soleus than EDL. Citrate synthase (soleus—19%, EDL—17%) and β‐hydroxyacyl CoA dehydrogenase (β‐HAD) (soleus—22%, EDL—19%) decreased similarly in both muscle types. There was loss of body and gastrocnemius complex mass, with rapid soleus but not EDL fatigue and diminished exercise endurance. These data suggest that in response to smoke exposure, TNF‐α‐mediated down‐regulation of PGC‐1α may be a key step leading to vascular and myocyte dysfunction, effects that are more evident in oxidative than glycolytic skeletal muscles. J. Cell. Physiol. 222: 320–327, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Neurotrophic regulation of mouse muscle β-amyloid protein precursor and α1-antichymotrypsin as revealed by axotomy

Kunitz-inhibitor containing forms of the β-amyloid precursor protein (βAPP), known also as protease nexin II (PNII), and α₁-antichymotrypsin (α₁-ACT), a serpin, are important components of the serine protease and inhibitor balance in many tissues. In the nervous system, this balance may have trophic or growth factor activity at different stages of development, after injury and in disease states. In the current study, using immunocytochemistry and Western blotting with antibodies against the human homologues, we analyzed whether denervation affected the localization of βAPP and α₁-ACT in adult mouse muscle following axotomy. In mouse muscle, antitive band and anti-human βAPP antibody a band at 92 kD in both normal and denervated extracts. βAPP was present in normal mouse muscle at both neuromuscular junctions and within intramuscular nerves. α₁-ACT was also detected at neuromuscular junctions, on the perineruim and endothelial cell surfaces. Following axotomy, both βAPP and α₁-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction, α₁-ACT decreased more rapidly with βAPP lingering before disappearing. Since both α₁-ACT as well as βAPP are present within senile plaques in Alzheimer's disease brains such experiments with the nicotinic, cholinergic neuromuscular synapse in denervated muscle may help to focus experiments on the mechanism of synapse loss as well as plaque deposition in this disease. © 1994 John Wiley & Sons, Inc.     

Isosteviol sodium protects the cardiomyocyte response associated with the SIRT1/PGC‐1α pathway

Cardiomyocyte dysfunction is attributed to excess oxidative damage, but the molecular pathways involved in this process have not been completely elucidated. Evidence indicates that isosteviol sodium (STVNa) has cardioprotective effects. We therefore aimed to identify the effect of STVNa on cardiomyocytes, as well as the potential mechanisms involved in this process. We established two myocardial hypertrophy models by treating H9c2 cells with high glucose (HG) and isoprenaline (ISO). Our results showed that STVNa reduced H9c2 mitochondrial damage by attenuating oxidative damage and altering the morphology of mitochondria. The results also indicated that STVNa had a positive effect on HG‐ and ISO‐induced damages via mitochondrial biogenesis. The protective effects of STVNa on cardiomyocytes were associated with the regulation of the SIRT1/PGC‐1α signalling pathway. Importantly, the effects of STVNa involved different methods of regulation in the two models, which was confirmed by experiments using an inhibitor and activator of SIRT1. Together, the results provide the basis for using STVNa as a therapy for the prevention of cardiomyocyte dysfunctions.