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The niche is a conserved regulator of stem cell quiescence and function. During ageing, stem cell function declines. To what extent and by what means age-related changes within the niche contribute to this phenomenon are unknown. Here we demonstrate that t

Optimal regeneration of skeletal muscle in response to injury requires the contribution of tissue resident stem cells termed satellite cells. Normally residing at the interface between the muscle fiber and overlying basal lamina it is generally understood with age the satellite cell pool exhibits decline both in number and function. Over the past decade mechanisms that contribute to these declines have begun to emerge. Implicit in aged-related satellite cell dysfunction and decline is the involvement of signals from the environment. Many of the signals that become deregulated with age have conserved functions during distinct stages of muscle fiber formation both in early development and regeneration. In particular, modulations in Wnt, TGFβ, Notch and FGF emanating from aged skeletal muscle fibers or the systemic milieu have emerged as age-related alterations that significantly impact both the maintenance of the satellite cell pool and skeletal muscle regenerative efficacy. In this review we will summarize how the aforementioned pathways contribute to skeletal muscle development and regeneration. We will then discuss deregulation of these cascades with age and how they contribute to satellite cell depletion and dysfunction. The review will also summarize some of the challenges we face in trying to draw parallels between murine and human satellite cell aging. Finally, we will highlight the few examples whereby FDA approved drugs may be exploited to modulate specific signaling cascades in effort to preserve skeletal muscle regenerative function with age.

Embryonic skeletal muscle growth is contingent upon a population of somite derived satellite cells, however, the contribution of these cells to early postnatal skeletal muscle growth remains relatively high. As prepubertal postnatal development proceeds, the activity and contribution of satellite cells to skeletal muscle growth diminishes. Eventually, at around puberty, a population of satellite cells escapes terminal commitment, continues to express the paired box transcription factor Pax7, and reside in a quiescent state orbiting the myofiber periphery adjacent to the basal lamina. After adolescence, some satellite cell contributions to muscle maintenance and adaptation occur, however, their necessity is reduced relative to embryonic, early postnatal, and prepubertal growth., (Copyright © 2024. Published by Elsevier Inc.)

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway., Competing Interests: Declaration of interests K.A. is a member of the Advisory Board of Molecular Cell., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mesenchymal progenitors of the lateral plate mesoderm give rise to various cell fates within limbs, including a heterogeneous group of muscle-resident mesenchymal cells. Often described as fibro-adipogenic progenitors, these cells are key players in muscle development, disease, and regeneration. To further define this cell population(s), we perform lineage/reporter analysis, flow cytometry, single-cell RNA sequencing, immunofluorescent staining, and differentiation assays on normal and injured murine muscles. Here we identify six distinct Pdgfra + non-myogenic muscle-resident mesenchymal cell populations that fit within a bipartite differentiation trajectory from a common progenitor. One branch of the trajectory gives rise to two populations of immune-responsive mesenchymal cells with strong adipogenic potential and the capability to respond to acute and chronic muscle injury, whereas the alternative branch contains two cell populations with limited adipogenic capacity and inherent mineralizing capabilities; one of the populations displays a unique neuromuscular junction association and an ability to respond to nerve injury., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

During development, resident stem cell populations contribute to the growth and maturation of tissue and organs. In skeletal muscle, muscle stem cells, or satellite cells (SCs), are responsible for the maturation of postnatal myofibers. However, the role SCs play in later stages of postnatal growth, and thus, when they enter a mature quiescent state is controversial. Here, we discuss the current literature regarding the role SCs play in all stages of postnatal growth, from birth to puberty onset to young adulthood. We additionally highlight the implications of SC loss or dysfunction during developmental stages, both in the context of experimental paradigms and disease settings., (© 2021 Federation of European Biochemical Societies.)

Background: Radiotherapy is commonly used to treat childhood cancers and can have adverse effects on muscle function, but the underlying mechanisms have yet to be fully elucidated. We hypothesized that endurance exercise following radiation treatment would improve skeletal muscle function., Methods: We utilized the Small Animal Radiation Research Platform (SARRP) to irradiate juvenile male mice with a clinically relevant fractionated dose of 3× (every other day over 5 days) 8.2 Gy X-ray irradiation locally from the knee to footpad region of the right hindlimb. Mice were then singly housed for 1 month in cages equipped with either locked or free-spinning voluntary running wheels. Ex vivo muscle contractile function, RT-qPCR analyses, resting cytosolic and sarcoplasmic reticulum (SR) store Ca 2+ levels, mitochondrial reactive oxygen species levels (MitoSOX), and immunohistochemical and biochemical analyses of muscle samples were conducted to assess the muscle pathology and the relative therapeutic impact of voluntary wheel running (VWR)., Results: Irradiation reduced fast-twitch extensor digitorum longus (EDL) muscle-specific force by 27% compared to that of non-irradiated mice, while VWR post-irradiation improved muscle-specific force by 37%. Radiation treatment similarly reduced slow-twitch soleus muscle-specific force by 14% compared to that of non-irradiated mice, while VWR post-irradiation improved specific force by 18%. We assessed intracellular Ca 2+ regulation, oxidative stress, and mitochondrial homeostasis as potential mechanisms of radiation-induced pathology and exercise-mediated rescue. We found a significant reduction in resting cytosolic Ca 2+ concentration following irradiation in sedentary mice. Intriguingly, however, SR Ca 2+ store content was increased in myofibers from irradiated mice post-VWR compared to mice that remained sedentary. We observed a 73% elevation in the overall protein oxidization in muscle post-irradiation, while VWR reduced protein nitrosylation by 35% and mitochondrial reactive oxygen species (ROS) production by 50%. Finally, we found that VWR significantly increased the expression of PGC1α at both the transcript and protein levels, consistent with an exercise-dependent increase in mitochondrial biogenesis., Conclusions: Juvenile irradiation stunted muscle development, disrupted proper Ca 2+ handling, damaged mitochondria, and increased oxidative and nitrosative stress, paralleling significant deficits in muscle force production. Exercise mitigated aberrant Ca 2+ handling, mitochondrial homeostasis, and increased oxidative and nitrosative stress in a manner that correlated with improved skeletal muscle function after radiation., (© 2022. The Author(s).)

Background: As paediatric cancer survivors are living into adulthood, they suffer from the age-related, accelerated decline of functional skeletal muscle tissue, termed sarcopenia. With ionizing radiation (radiotherapy) at the core of paediatric cancer therapies, its direct and indirect effects can have lifelong negative impacts on paediatric growth and maintenance of skeletal muscle. Utilizing our recently developed preclinical rhabdomyosarcoma mouse model, we investigated the late effects of paediatric radiation treatment on skeletal muscles from late adolescent (8 weeks old) and middle-aged (16 months old) mice., Methods: Paediatric C57BL/6J male mice (3 weeks old) were injected with rhabdomyosarcoma cells into their right hindlimbs, and then fractionated irradiation (3 × 8.2 Gy) was administered to those limbs at 4 weeks old to eliminate the tumours. Radiation-alone and tumour-irradiated mice were assessed at either 8 weeks (3 weeks post-irradiation) or 16 months (14 months post-irradiation) of age for muscle physiology, myofibre characteristics, cell loss, histopathology, fibrosis, inflammatory gene expression, and fibrotic gene expression., Results: Mice that received only paediatric radiation demonstrated reduced muscle mass (-17%, P < 0.001), muscle physiological function (-25%, P < 0.01), muscle contractile kinetics (-25%, P < 0.05), satellite cell number (-45%, P < 0.05), myofibre cross-sectional area (-30%, P < 0.0001), and myonuclear number (-17%, P < 0.001). Paediatric radiation increased inflammatory gene expression, increased fibrotic gene expression, and induced extracellular matrix protein deposition (fibrosis) with tumour elimination exacerbating some phenotypes. Paediatric tumour-eliminated mice demonstrated exacerbated deficits to function (-20%, P < 0.05) and myofibre size (-17%, P < 0.001) in some muscles as well as further increases to inflammatory and fibrotic gene expression. Examining the age-related effects of paediatric radiotherapy in middle-aged mice, we found persistent myofibre atrophy (-20%, P < 0.01), myonuclear loss (-18%, P < 0.001), up-regulated inflammatory and fibrotic signalling, and lifelong fibrosis., Conclusions: The results from this paediatric radiotherapy model are consistent and recapitulate the clinical and molecular features of accelerated sarcopenia, musculoskeletal frailty, and radiation-induced fibrosis experienced by paediatric cancer survivors. We believe that this preclinical mouse model is well poised for future mechanistic insights and therapeutic interventions that improve the quality of life for paediatric cancer survivors., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1 -/- ), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1 -/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors., Competing Interests: JL, MM, JC, PU, PF, KS, SP, SK, JC, WL, RH, LB, JM, KD, BL, SM, GV, JC, YJ, SB, CA No competing interests declared, (© 2021, Larouche et al.)

Background: Skeletal muscle wasting (SMW) in cancer patients is associated with increased morbidity, mortality, treatment intolerance and discontinuation, and poor quality of life. This is particularly true for patients with pancreatic ductal adenocarcinoma (PDAC), as over 85% experience SMW, which is responsible for ~30% of patient deaths. While the established paradigm to explain SMW posits that muscle catabolism from systemic inflammation and nutritional deficiencies, the cause of death, and the cellular and molecular mechanisms responsible remain to be elucidated. To address this, we investigated the relationship between tumour burden and survival in the KCKO murine PDAC model., Methods: Female C57BL/6J mice 6-8 weeks of age underwent orthotopic injection with KCKO-luc tumour cells. Solid tumour was verified on Day 5, post-tumour inoculation. In vivo, longitudinal lean mass and tumour burden were assessed via dual-energy X-ray absorptiometry and IVIS imaging, respectively, and total body weight was assessed, weekly. Animals were sacrificed at a designated end point of 'failure to thrive'. After sacrifice, lower limb hind muscles were harvested for histology and RNA extraction., Results: We found a strong correlation between primary tumour size and survival (r 2  = 0.83, P < 0.0001). A significant decrease in lower limb lean mass was first detected at Day 38 post-implantation vs. no tumour controls (NTCs) (P < 0.0001). SMW was confirmed by histology, which demonstrated a 38%, 32.7%, and 39.9% decrease in fibre size of extensor digitorum longus, soleus, and tibialis anterior muscles, respectively, in PDAC mice vs. NTC (P < 0.002). Histology also revealed a 67.6% increase in haematopoietic cells within the muscle of PDAC mice when compared with NTC. Bulk RNAseq on muscles from PDAC mice vs. NTC revealed significant increases in c/ebpβ/Δ, il-1, il-6, and tnf gene expression. Pathway analyses to identify potential upstream factors revealed increased adipogenic gene expression, including a four-fold increase in igfbp-3. Histomorphometry of Oil Red-O staining for fat content in tibialis anterior muscles demonstrated a 95.5% increase in positively stained fibres from PDAC mice vs. NTC., Conclusions: Together, these findings support a novel model of PDAC-associated SMW and mortality in which systemic inflammation leads to inflammatory cell infiltration into skeletal muscle with up-regulated myocellular lipids., (© 2021 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

During prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained. Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.

During aging, muscle mass decreases, leading to sarcopenia, associated with low-level chronic inflammation (inflammaging), which induces sarcopenia by promoting proteolysis of muscle fibers and inhibiting their regeneration. Patients with a variety of pathologic conditions associated with sarcopenia, including rheumatoid arthritis (RA), have systemically elevated TNFα serum levels, and transgenic mice with TNFα overexpression (TNF-Tg mice, a model of RA) develop sarcopenia between adolescence and adulthood before they age. However, if and how TNFα contributes to the pathogenesis of sarcopenia during the normal aging process and in RA remains largely unknown. We report that TNFα levels are increased in skeletal muscles of aged WT mice, associated with muscle atrophy and decreased numbers of satellite cells and Type IIA myofibers, a phenotype that we also observed in adult TNF-Tg mice. Aged WT mice also have increased numbers of myeloid lineage cells in their skeletal muscles, including macrophages and granulocytes. These cells have increased TNFα expression, which impairs myogenic cell differentiation. Expression levels of TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, which mediates signaling by some TNF receptor (TNFR) family members, are elevated in skeletal muscles of both aged WT mice and adult TNF-Tg mice. TRAF6 binds to TNFR2 in C2C12 myoblasts and mediates TNFα-induced muscle atrophy through NF-κB-induced transcription of the muscle-specific E3 ligases, Atrogen1 and Murf1, which promote myosin heavy-chain degradation. Haplo-deficiency of TRAF6 prevents muscle atrophy and the decrease in numbers of satellite cells, Type IIA myofibers, and myogenic regeneration in TRAF6 +/- ;TNF-Tg mice. Our findings suggest that pharmacologic inhibition of TRAF6 signaling in skeletal muscles during aging could treat/prevent age- and RA-related sarcopenia by preventing TNFα-induced proteolysis and inhibition of muscle fiber regeneration. © 2020 American Society for Bone and Mineral Research., (© 2020 American Society for Bone and Mineral Research.)

Most prostate cancers depend on androgens for growth, and therefore, the mainstay treatment for advanced, recurrent, or metastatic prostate cancer is androgen deprivation therapy (ADT). A prominent side effect in patients receiving ADT is an obese frailty syndrome that includes fat gain and sarcopenia, defined as the loss of muscle function accompanied by reduced muscle mass or quality. Mice bearing Pten-deficient prostate cancers were examined to gain mechanistic insight into ADT-induced sarcopenic obesity. Castration induced fat gain as well as skeletal muscle mass and strength loss. Catabolic TGF-β family myokine protein levels were increased immediately prior to strength loss, and pan-myokine blockade using a soluble receptor (ActRIIB-Fc) completely reversed the castration-induced sarcopenia. The onset of castration-induced strength and muscle mass loss, as well as the increase in catabolic TGF-β family myokine protein levels, were coordinately accelerated in tumor-bearing mice relative to tumor-free mice. Notably, growth differentiation factor 11 (GDF11) increased in muscle after castration only in tumor-bearing mice, but not in tumor‑free mice. An early surge of GDF11 in prostate tumor tissue and in the circulation suggests that endocrine GDF11 signaling from tumor to muscle is a major driver of the accelerated ADT-induced sarcopenic phenotype. In tumor-bearing mice, GDF11 blockade largely prevented castration-induced strength loss but did not preserve muscle mass, which confirms a primary role for GDF11 in muscle function and suggests an additional role for the other catabolic myokines.

Objective: To examine and quantify the sexual dimorphism in pathologic features manifested in the musculoskeletal and cardiopulmonary systems and incidence of mortality in the tumor necrosis factor-transgenic (TNF-Tg; Tg3647 strain) mouse model of inflammatory erosive arthritis., Methods: Kaplan-Meier survival estimates were determined in male and female Tg3647 mice and sex-matched wild-type (WT) littermate mice. Longitudinal and cross-sectional pathologic outcomes in the musculoskeletal and cardiopulmonary systems were assessed via ultrasound, micro-computed tomography, grip strength measurements, histologic and serologic analyses, flow cytometry, and skeletal muscle physiologic measures., Results: Compared to male Tg3647 mice (n = 30), female Tg3647 mice (n = 34) had significantly shorter lifespans (P < 0.001) and exhibited the following pathologic features (n = 4-6 per group; P < 0.05 versus male Tg3647 littermates): gross deficits in body mass and muscle weight, early-onset inflammatory arthritis with severity of end-stage arthritis that was as severe as that seen in male transgenic mice, and early onset and increased severity of inflammatory interstitial lung disease (ILD). Histologically, the ILD observed in Tg3647 mice was characterized by inflammatory cell accumulation and pulmonary arteriole thickening, which was concomitant with the presence of right ventricular hypertrophy, a feature that was also more severe in the female compared to male Tg3647 mice (P < 0.05). No sexual dimorphisms in TNF-induced deficient grip strength, axial skeletal growth, or bone loss were found. Globally, the extent of the pathologic changes observed in female Tg3647 mice was greater than that observed in male Tg3647 mice when each group was compared to their sex-matched WT littermates., Conclusion: These findings indicate that TNF selectively drives the early onset of arthritis and progression of pathologic changes in the cardiopulmonary system in female Tg3647 mice. These results in the Tg3647 mouse identify it as a suitable model to better understand the mechanisms underlying sexual dimorphism and cardiopulmonary disease in the setting of inflammatory arthritis and other connective tissue diseases., (© 2019, American College of Rheumatology.)

The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7 CreERT2/+ ; Rosa26 nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7 CreERT2/+ ; Rosa26 DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The neuromuscular junction (NMJ) is the specialized site that connects the terminal of a motor neuron axon to skeletal muscle. As a synapse NMJ integrity is essential for transducing motor neuron signals that initiate skeletal muscle contraction. Many diseases and skeletal muscle aging are linked to impaired NMJ function and the associated muscle wasting. In this chapter we review the components of an NMJ and, the processes of NMJ development, maturation, and regeneration. Also, we briefly discuss the cellular and molecular mechanisms of NMJ decline in the context of disease and aging., (© 2018 Elsevier Inc. All rights reserved.)

Background: Sarcopenia, the age-related loss of skeletal muscle, is a side effect of androgen deprivation therapy (ADT) for prostate cancer patients. Resident stem cells of skeletal muscle, satellite cells (SCs), are an essential source of progenitors for the growth and regeneration of skeletal muscle. Decreased androgen signaling and deficits in the number and function of SCs are features of aging. Although androgen signaling is known to regulate skeletal muscle, the cellular basis for ADT-induced exacerbation of sarcopenia is unknown. Furthermore, the consequences of androgen deprivation on SC fate in adult skeletal muscle remain largely unexplored., Methods: We examined SC fate in an androgen-deprived environment using immunofluorescence and fluorescence-activated cell sorting (FACS) with SC-specific markers in young castrated mice. To study the effects of androgen deprivation on SC function and skeletal muscle regenerative capacity, young castrated mice were subjected to experimental regenerative paradigms. SC-derived-cell contributions to skeletal muscle maintenance were examined in castrated Pax7 CreER/+ ; ROSA26 mTmG/+ mice. SCs were depleted in Pax7 CreER/+ ; ROSA26 DTA/+ mice to ascertain the consequences of SC ablation in sham and castrated skeletal muscles. Confocal immunofluorescence analysis of neuromuscular junctions (NMJs), and assessment of skeletal muscle physiology, contractile properties, and integrity were conducted., Results: Castration led to SC activation, however this did not result in a decline in SC function or skeletal muscle regenerative capacity. Surprisingly, castration induced SC-dependent maintenance of young skeletal muscle. The functional dependence of skeletal muscles on SCs in young castrated mice was demonstrated by an increase in SC-derived-cell fusion within skeletal muscle fibers. SC depletion was associated with further atrophy and functional decline, as well as the induction of partial innervation and the loss of NMJ-associated myonuclei in skeletal muscles from castrated mice., Conclusion: The maintenance of skeletal muscles in young castrated mice relies on the cellular contributions of SCs. Considering the well-described age-related decline in SCs, the results in this study highlight the need to devise strategies that promote SC maintenance and activity to attenuate or reverse the progression of sarcopenia in elderly androgen-deprived individuals., Competing Interests: CONFLICT OF INTEREST STATEMENT AK, WL, NP, SF, JJK, KLN, and JVC, declare no conflict of interest.

Purpose of Review: Cancer cachexia remains understudied and there are no standard treatments available despite the publication of an international consensus definition and the completion of several large phase III intervention trials in the past 6 years. In September 2015, The University of Rochester Cancer Center NCORP Research Base led a Symposium on Cancer Cachexia and Sarcopenia with goals of reviewing the state of the science, identifying knowledge gaps, and formulating research priorities in cancer cachexia through active discussion and consensus., Recent Findings: Research priorities that emerged from the discussion included the implementation of morphometrics into clinical decision making, establishing specific diagnostic criteria for the stages of cachexia, expanding patient selection in intervention trials, identifying clinically meaningful trial endpoints, and the investigation of exercise as an intervention for cancer cachexia., Summary: Standardizing how we define and measure cancer cachexia, targeting its complex biologic mechanisms, enrolling patients early in their disease course, and evaluating exercise, either alone or in combination, were proposed as initiatives that may ultimately result in the improved design of cancer cachexia therapeutic trials.

Neuromuscular junction degeneration is a prominent aspect of sarcopenia, the age-associated loss of skeletal muscle integrity. Previously, we showed that muscle stem cells activate and contribute to mouse neuromuscular junction regeneration in response to denervation (Liu et al., 2015). Here, we examined gene expression profiles and neuromuscular junction integrity in aged mouse muscles, and unexpectedly found limited denervation despite a high level of degenerated neuromuscular junctions. Instead, degenerated neuromuscular junctions were associated with reduced contribution from muscle stem cells. Indeed, muscle stem cell depletion was sufficient to induce neuromuscular junction degeneration at a younger age. Conversely, prevention of muscle stem cell and derived myonuclei loss was associated with attenuation of age-related neuromuscular junction degeneration, muscle atrophy, and the promotion of aged muscle force generation. Our observations demonstrate that deficiencies in muscle stem cell fate and post-synaptic myogenesis provide a cellular basis for age-related neuromuscular junction degeneration and associated skeletal muscle decline.

Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFβ superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4 , the downstream cofactor for canonical TGFβ superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration. Specific deletion of Smad4 in adult mouse SCs led to increased propensity for terminal myogenic commitment connected to impaired proliferative potential. Furthermore, SC-specific Smad4 disruption compromised adult skeletal muscle regeneration. Finally, loss of Smad4 in aged SCs did not promote aged skeletal muscle regeneration. Therefore, SC-specific reduction of Smad4 is a feature of aged regenerating skeletal muscle and Smad4 is a critical regulator of SC and MP amplification during skeletal muscle regeneration., Competing Interests: The authors declare that no competing interests exist.

First line treatment for recurrent and metastatic prostate cancer is androgen deprivation therapy (ADT). Use of ADT has been increasing in frequency and duration, such that side effects increasingly impact patient quality of life. One of the most significant side effects of ADT is sarcopenia, which leads to a loss of skeletal muscle mass and function, resulting in a clinical disability syndrome known as obese frailty. Using aged mice, we developed a mouse model of ADT-induced sarcopenia that closely resembles the phenotype seen in patients, including loss of skeletal muscle strength, reduced lean muscle mass, and increased adipose tissue. Sarcopenia onset occurred about 6 weeks after castration and was blocked by a soluble receptor (ActRIIB-Fc) that binds multiple TGFβ superfamily members, including myostatin, growth differentiation factor 11, activin A, activin B, and activin AB. Analysis of ligand expression in both gastrocnemius and triceps brachii muscles demonstrates that each of these proteins is induced in response to ADT, in 1 of 3 temporal patterns. Specifically, activin A and activin AB levels increase and decline before onset of strength loss at 6 weeks after castration, and myostatin levels increase coincident with the onset of strength loss and then decline. In contrast, activin B and growth differentiation factor 11 levels increase after the onset of strength loss, 8-10 weeks after castration. The observed patterns of ligand induction may represent differential contributions to the development and/or maintenance of sarcopenia. We hypothesize that some or all of these ligands are targets for therapy to ameliorate ADT-induced sarcopenia in prostate cancer patients.

Skeletal muscle maintenance depends on motor innervation at neuromuscular junctions (NMJs). Multiple mechanisms contribute to NMJ repair and maintenance; however muscle stem cells (satellite cells, SCs), are deemed to have little impact on these processes. Therefore, the applicability of SC studies to attenuate muscle loss due to NMJ deterioration as observed in neuromuscular diseases and aging is ambiguous. We employed mice with an inducible Cre, and conditionally expressed DTA to deplete or GFP to track SCs. We found SC depletion exacerbated muscle atrophy and type transitions connected to neuromuscular disruption. Also, elevated fibrosis and further declines in force generation were specific to SC depletion and neuromuscular disruption. Fate analysis revealed SC activity near regenerating NMJs. Moreover, SC depletion aggravated deficits in reinnervation and post-synaptic morphology at regenerating NMJs. Therefore, our results propose a mechanism whereby further NMJ and skeletal muscle decline ensues upon SC depletion and neuromuscular disruption.

Across different niches, subsets of highly functional stem cells are maintained in a relatively dormant rather than proliferative state. Our understanding of proliferative dynamics in tissue-specific stem cells during conditions of increased tissue turnover remains limited. Using a TetO-H2B-GFP reporter of proliferative history, we identify skeletal muscle stem cell, or satellite cells, that retain (LRC) or lose (nonLRC) the H2B-GFP label. We show in mice that LRCs and nonLRCs are formed at birth and persist during postnatal growth and adult muscle repair. Functionally, LRCs and nonLRCs are born equivalent and transition during postnatal maturation into distinct and hierarchically organized subsets. Adult LRCs give rise to LRCs and nonLRCs; the former are able to self-renew, whereas the latter are restricted to differentiation. Expression analysis revealed the CIP/KIP family members p21(cip1) (Cdkn1a) and p27(kip1) (Cdkn1b) to be expressed at higher levels in LRCs. In accordance with a crucial role in LRC fate, loss of p27(kip1) promoted proliferation and differentiation of LRCs in vitro and impaired satellite cell self-renewal after muscle injury. By contrast, loss of p21(cip1) only affected nonLRCs, in which myogenic commitment was inhibited. Our results provide evidence that restriction of self-renewal potential to LRCs is established early in life and is maintained during increased tissue turnover through the cell cycle inhibitor p27(kip1). They also reveal the differential role of CIP/KIP family members at discrete steps within the stem cell hierarchy.

The niche is a conserved regulator of stem cell quiescence and function. During ageing, stem cell function declines. To what extent and by what means age-related changes within the niche contribute to this phenomenon are unknown. Here we demonstrate that the aged muscle stem cell niche, the muscle fibre, expresses Fgf2 under homeostatic conditions, driving a subset of satellite cells to break quiescence and lose their self-renewing capacity. We show in mice that relatively dormant aged satellite cells robustly express sprouty 1 (Spry1), an inhibitor of fibroblast growth factor (FGF) signalling. Increasing FGF signalling in aged satellite cells under homeostatic conditions by removing Spry1 results in the loss of quiescence, satellite cell depletion and diminished regenerative capacity. Conversely, reducing niche-derived FGF activity through inhibition of Fgfr1 signalling or overexpression of Spry1 in satellite cells prevents their depletion. These experiments identify an age-dependent change in the stem cell niche that directly influences stem cell quiescence and function.

Mammalian limb and trunk skeletal muscles are composed of muscle fibers that differ in contractile and molecular properties. They are commonly divided into four categories according to the myosin heavy chain that they express: I, IIA, IIX and IIB, ranging from slowest to fastest. Individual motor axons innervate tens of muscle fibers, nearly all of which are of the same type. The mechanisms accounting for this striking specificity, termed motor unit homogeneity, remain incompletely understood, in part because there have been no markers for motoneuron types. Here we show in mice that the synaptic vesicle protein SV2A is selectively localized in motor nerve terminals on slow (type I and small type IIA) muscle fibers; its close relatives, SV2B and SV2C, are present in all motor nerve terminals. SV2A is broadly expressed at birth; fast motoneurons downregulate its expression during the first postnatal week. An inducible transgene incorporating regulatory elements from the Sv2a gene permits selective labeling of slow motor units and reveals their composition. Overexpression of the transcriptional co-regulator PGC1α in muscle fibers, which converts them to a slow phenotype, leads to an increased frequency of SV2A-positive motor nerve terminals, indicating a fiber type-specific retrograde influence of muscle fibers on their innervation. This retrograde influence must be integrated with known anterograde influences in order to understand how motor units become homogeneous.

A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.

We examined the role of post-transcriptional mechanisms in controlling utrophin A mRNA expression in slow versus fast skeletal muscles. First, we determined that the half-life of utrophin A mRNA is significantly shorter in the presence of proteins isolated from fast muscles. Direct plasmid injection experiments using reporter constructs containing the full-length or truncated variants of the utrophin 3'UTR into slow soleus and fast extensor digitorum longus muscles revealed that a region of 265 nucleotides is sufficient to confer lower levels of reporter mRNA in fast muscles. Further analysis of this region uncovered a conserved AU-rich element (ARE) that suppresses expression of reporter mRNAs in cultured muscle cells. Moreover, stability of reporter mRNAs fused to the utrophin full-length 3'UTR was lower in the presence of fast muscle protein extracts. This destabilization effect seen in vivo was lost upon deletion of the conserved ARE. Finally, we observed that calcineurin signaling affects utrophin A mRNA stability through the conserved ARE. These results indicate that ARE-mediated mRNA decay is a key mechanism that regulates expression of utrophin A mRNA in slow muscle fibers. This is the first demonstration of ARE-mediated mRNA decay regulating the expression of a gene associated with the slow myogenic program.

Ca2+ signalling plays an important role in excitation-contraction coupling and the resultant force output of skeletal muscle. It is also known to play a crucial role in modulating both short- and long-term muscle cellular phenotypic adaptations associated with these events. Ca2+ signalling via the Ca2+/calmodulin (CaM)-dependent phosphatase calcineurin (CnA) and via Ca2+/CaM-dependent kinases, such as CaMKI and CaMKII, is known to regulate hypertrophic growth in response to overload, to direct slow versus fast fibre gene expression, and to contribute to mitochondrial biogenesis. The CnA- and CaMK-dependent regulation of the downstream transcription factors nuclear factor of activated T cells (NFAT) and myocyte-specific enhancer factor 2 are known to activate muscle-specific genes associated with a slower, more oxidative fibre phenotype. We have also recently shown the expression of utrophin A, a cytoskeletal protein that accumulates at the neuromuscular junction and plays a role in maturation of the postsynaptic apparatus, to be regulated by CnA-NFAT and Ca2+/CaM signalling. This regulation is fibre-type specific and potentiated by interactions with the transcriptional regulators and coactivators GA binding protein (also known as nuclear respiratory factor 2) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Another downstream target of CnA signalling may be myostatin, a transforming growth factor-beta family member that is a negative regulator of muscle growth. While the list of the downstream targets of CnA/NFAT- and Ca2+/CaM-dependent signalling is emerging, the precise interaction of these pathways with the Ca2+-independent pathways p38 mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2, phosphoinositide-3 kinase, and protein kinase B (Akt/PKB) must also be considered when deciphering fibre responses and plasticity to altered contractile load.

In this study, we crossbred mdx mice with transgenic mice expressing a small peptide inhibitor for calmodulin (CaM), known as the CaM-binding protein (CaMBP), driven by the slow fiber-specific troponin I slow promoter. This strategy allowed us to determine the impact of interfering with Ca(2+)/CaM-based signaling in dystrophin-deficient slow myofibers. Consistent with impairments in the Ca(2+)/CaM-regulated enzymes calcineurin and Ca(2+)/CaM-dependent kinase, the nuclear accumulation of nuclear factor of activated T-cell c1 and myocyte enhancer factor 2C was reduced in slow fibers from mdx/CaMBP mice. We also detected significant reductions in the levels of peroxisome proliferator gamma co-activator 1alpha and GA-binding protein alpha mRNAs in slow fiber-rich soleus muscles of mdx/CaMBP mice. In parallel, we observed significantly lower expression of myosin heavy chain I mRNA in mdx/CaMBP soleus muscles. This correlated with fiber-type shifts towards a faster phenotype. Examination of mdx/CaMBP slow muscle fibers revealed significant reductions in A-utrophin, a therapeutically relevant protein that can compensate for the lack of dystrophin in skeletal muscle. In accordance with lower levels of A-utrophin, we noted a clear exacerbation of the dystrophic phenotype in mdx/CaMBP slow fibers as exemplified by several pathological indices. These results firmly establish Ca(2+)/CaM-based signaling as key to regulating expression of A-utrophin in muscle. Furthermore, this study illustrates the therapeutic potential of using targets of Ca(2+)/CaM-based signaling as a strategy for treating Duchenne muscular dystrophy (DMD). Finally, our results further support the concept that strategies aimed at promoting the slow oxidative myofiber program in muscle may be effective in altering the relentless progression of DMD.

We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1alpha alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1alpha with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1alpha.

Utrophin up-regulation in muscle fibers of Duchenne muscular dystrophy patients represents a potential therapeutic strategy. It is thus important to delineate the regulatory events presiding over utrophin in muscle in attempts to develop pharmacological interventions aimed at increasing utrophin expression. A number of studies have now shown that under several experimental conditions, the abundance of utrophin is increased without a corresponding elevation in its mRNA. Here, we examine whether utrophin expression is regulated at the translational level in regenerating muscle fibers. Treatment of mouse tibialis anterior muscles with cardiotoxin to induce muscle degeneration/regeneration led to a large (approximately 14-fold) increase in the levels of utrophin A with a modest change in expression of its transcript (40%). Isolation of the mouse utrophin A 5'-untranslated region (UTR) revealed that it is relatively long with a predicted high degree of secondary structure. In control muscles, the 5'-UTR of utrophin A caused an inhibition upon translation of a reporter protein. Strikingly, this inhibition was removed during regeneration, indicating that expression of utrophin A in regenerating muscles is translationally regulated via its 5'-UTR. Using bicistronic reporter vectors, we observed that this translational effect involves an internal ribosome entry site in the utrophin A 5'-UTR. Thus, internal ribosome entry site-mediated translation of utrophin A can, at least partially, account for the discordant expression of utrophin A protein and transcript in regenerating muscle. These findings provide a novel target for up-regulating levels of utrophin A in Duchenne muscular dystrophy muscle fibers via pharmacological interventions.

Although the molecular defect causing Duchenne/Becker muscular dystrophy (DMD/BMD) was identified nearly 20 years ago, the development of effective therapeutic strategies has nonetheless remained a daunting challenge. Over the years, a variety of different approaches have been explored in an effort to compensate for the lack of the DMD gene product called dystrophin. This review not only presents some of the most promising molecular, cellular, and pharmacological strategies but also highlights some issues that need to be addressed before considering their implementation. Specifically, we describe current strategies being developed to exogenously deliver healthy copies of the dystrophin gene to dystrophic muscles. We present the findings of several studies that have focused on repairing the mutant dystrophin gene using various approaches. We include a discussion of cell-based therapies that capitalize on the use of myoblast or stem cell transfer. Finally, we summarize the results of several studies that may eventually lead to the development of appropriate drug-based therapies. In this context, we review our current knowledge of the mechanisms regulating expression of utrophin, the autosomal homologue of dystrophin. Given the complexity associated with the dystrophic phenotype, it appears likely that a combinatorial approach involving different therapeutic strategies will be necessary for the appropriate management and eventual treatment of this devastating neuromuscular disease.

Upregulation of utrophin in muscle is currently being examined as a potential therapy for Duchenne muscular dystrophy patients. In this context, we generated transgenic mice harboring a 1.3 kb human utrophin A promoter fragment driving expression of the lacZ gene. Characterization of reporter expression during postnatal muscle development revealed that the levels and localization of beta-galactosidase parallel expression of utrophin A transcripts. Moreover, we noted that the utrophin A promoter is more active in slow soleus muscles. Additionally, expression of the reporter gene was regulated during muscle regeneration in a manner similar to utrophin A transcripts. Together, these results show that the utrophin A promoter-lacZ construct mirrors expression of utrophin A mRNAs indicating that this utrophin A promoter fragment confers temporal and spatial patterns of expression in skeletal muscle. This transgenic mouse will be valuable as an in vivo model for developing and testing molecules aimed at increasing utrophin A expression.

Duchenne muscular dystrophy (DMD) is a progressive and ultimately fatal skeletal muscle disease. Currently, the most effective therapy is the administration of a subclass of glucocorticoids, most notably deflazacort. Although deflazacort treatment can attenuate DMD progression, extend ambulation, and maintain muscle strength, the mechanism of its action remains unknown. Prior observations have shown that activation of a JNK1-mediated signal transduction cascade contributes to the progression of the DMD phenotype, in part by phosphorylation and inhibition of a calcineurin sensitive NF-ATc1 transcription factor. Here, we observed that deflazacort treatment restored myocyte viability in muscle cells with constitutive activation of JNK1 and in dystrophic mdx mice. However, deflazacort treatment did not alter JNK1 activity itself, but rather led to an increase in the activity of the calcineurin phosphatase and an up-regulation of NF-ATc1-dependent gene expression. The prophylactic effect of deflazacort treatment was associated with increased expression of NF-ATc1 target genes such as the dystrophin homologue utrophin. Moreover, the muscle sparing effects of deflazacort were completely abolished when used in conjunction with the calcineurin inhibitor cyclosporine. Collectively, these results show that deflazacort attenuates loss of dystrophic myofiber integrity by up-regulating the activity of the phosphatase calcineurin, which in turn negates JNK1 inhibition of NF-ATc1-mediated phosphorylation and nuclear exclusion of NF-ATc1.

Utrophin has been studied extensively in recent years in an effort to find a cure for Duchenne muscular dystrophy. In this context, we previously showed that mice expressing enhanced muscle calcineurin activity (CnA*) displayed elevated levels of utrophin around their sarcolemma. In the present study, we therefore crossed CnA* mice with mdx mice to determine the suitability of elevating calcineurin activity in preventing the dystrophic pathology. Muscles from mdx/CnA* displayed increased nuclear localization of NFATc1 and a fiber type shift towards a slower phenotype. Measurements of utrophin levels in mdx/CnA* muscles revealed an approximately 2-fold induction in utrophin expression. Consistent with this induction, we also observed that members of the dystrophin-associated protein (DAP) complex were present at the sarcolemma of mdx/CnA* mouse muscle. This restoration of the utrophin-DAP complex was accompanied by significant reductions in the extent of central nucleation and fiber size variability. Importantly, assessment of myofiber sarcolemmal damage, as monitored by the intracellular presence of IgM and albumin as well as by Evans blue uptake in vivo, revealed a net amelioration of membrane integrity. Finally, immunofluorescence experiments using Mac-1 antibodies showed a reduction in the number of infiltrating immune cells in muscles from mdx/CnA* mice. These results show that elevated calcineurin activity attenuates the dystrophic pathology and thus provides an effective target for pharmacological intervention.

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.

The neuromuscular junction has been used for several decades as an excellent model system to examine the cellular and molecular events involved in the formation and maintenance of a differentiated chemical synapse. In this context, several laboratories have focused their efforts over the last 15 years on the important contribution of transcriptional mechanisms to the regulation of the development and plasticity of the postsynaptic apparatus in muscle fibers. Converging lines of evidence now indicate that post-transcriptional events, operating at the level of mRNA stability and targeting, are likely to also play key roles at the neuromuscular junction. Here, we present the recent findings highlighting the role of these additional molecular events and extend our review to include data showing that post-transcriptional events are also important in the control of the expression of genes encoding synaptic proteins in muscle cells placed under different conditions. Finally, we discuss the possibility that mis-regulation of post-transcriptional events can occur in certain neuromuscular diseases and cause abnormalities of the neuromuscular junction., (Copyright 2002 Wiley Periodicals, Inc.)

Duchenne muscular dystrophy (DMD) is the most prevalent inherited muscle disease and results from mutations/deletions in the X-linked dystrophin gene. Although several approaches have been envisaged to counteract the effects of this progressive disease, there is currently no cure available. One strategy consists in utilizing a protein normally expressed in DMD muscle which, once expressed at appropriate levels and at the correct subcellular location, could compensate for the lack of dystrophin. A candidate for such a role is the dystrophin-related protein now referred to as utrophin. In contrast to dystrophin, which is expressed along the length of healthy muscle fibers, utrophin accumulates at the neuromuscular junction in both normal and DMD fibers. Several years ago, we began a series of experiments to determine the mechanisms responsible for the expression of utrophin at the neuromuscular synapse. Initially, we showed that utrophin transcripts accumulate preferentially within the postsynaptic sarcoplasm. To determine whether selective transcription of the utrophin gene accounts for this synaptic accumulation of utrophin mRNAs, we injected several utrophin promoter-reporter constructs directly into mouse muscle and demonstrated the preferential synaptic expression of the reporter gene. These results suggested that local transcriptional activation of the utrophin gene is responsible for the accumulation of utrophin mRNAs at the neuromuscular junction. In these studies, we also demonstrated that an N-box motif contained within the utrophin promoter plays a critical role in directing the synapse-specific expression of the utrophin gene. Additionally, our studies have shown that the ets-factors GABP alpha and beta are part of a protein complex that can bind to the N-box motif to transactivate the gene in muscle cells in culture and in vivo. In these experiments, we also noted that the nerve-derived trophic factors agrin and ARIA/heregulin regulate expression of utrophin via the activation of GABP alpha and beta which in turn, transactivate the utrophin gene via the N-box motif. Although these studies demonstrate that transcriptional activation can regulate utrophin mRNA levels, it is possible that additional mechanisms are also involved. In particular, the association of mRNAs with cytoskeletal elements and RNA-binding proteins may contribute to the accumulation of utrophin transcripts within the postsynaptic sarcoplasm. In recent studies, we have begun to examine this and we have now identified specific regions within the 3' untranslated region that are necessary for targeting and stabilizing utrophin mRNAs in skeletal muscle cells. A series of in vivo studies have also led us to conclude that post-transcriptional mechanisms are indeed important in regulating the abundance of utrophin transcripts in muscle. Together, these studies should lead to the identification of cis- and trans-acting elements regulating transcription of the utrophin gene as well as the stability and targeting of its mRNA in muscle cells. The results should therefore, identify specific targets that may become important in designing specific pharmacological interventions directed at increasing the expression of utrophin into extrasynaptic regions of DMD muscle fibers. In addition, these findings will contribute to our basic understanding of the cellular and molecular events involved in the formation, maintenance and plasticity of the neuromuscular synapse.

In addition to showing differences in the levels of contractile proteins and metabolic enzymes, fast and slow muscles also differ in their expression profile of structural and synaptic proteins. Because utrophin is a structural protein expressed at the neuromuscular junction, we hypothesize that its expression may be different between fast and slow muscles. Western blots showed that, compared with fast extensor digitorum longus (EDL) muscles, slow soleus muscles contain significantly more utrophin. Quantitative RT-PCR revealed that this difference is accompanied by a parallel increase in the expression of utrophin transcripts. Interestingly, the higher levels of utrophin and its mRNA appear to occur in extrasynaptic regions of muscle fibers as shown by immunofluorescence and in situ hybridization experiments. Furthermore, nuclear run-on assays showed that the rate of transcription of the utrophin gene was nearly identical between EDL and soleus muscles, indicating that increased mRNA stability accounts for the higher levels of utrophin in slow muscles. Direct plasmid injections of reporter gene constructs showed that cis-acting elements contained within the utrophin 3'-untranslated region (3'-UTR) confer greater stability to chimeric LacZ transcripts in soleus muscles. Finally, we observed a clear difference between EDL and soleus muscles in the abundance of RNA-binding proteins interacting with the utrophin 3'-UTR. Together, these findings highlight the contribution of posttranscriptional events in regulating the expression of utrophin in muscle.