Angiotensin II Inhibits Satellite Cell Proliferation and Prevents Skeletal Muscle Regeneration

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Although patients with advanced CHF or CKD often have increased angiotensin II (Ang II) levels and cachexia and Ang II causes skeletal muscle wasting in rodents, the potential effects of Ang II on muscle regeneration are unknown. Muscle regeneration is highly dependent on the ability of a pool of muscle stem cells (satellite cells) to proliferate and to repair damaged myofibers or form new myofibers. Here we show that Ang II reduced skeletal muscle regeneration via inhibition of satellite cell (SC) proliferation. Ang II reduced the number of regenerating myofibers and decreased expression of SC proliferation/differentiation markers (MyoD, myogenin, and active-Notch) after cardiotoxin-induced muscle injury in vivo and in SCs cultured in vitro. Ang II depleted the basal pool of SCs, as detected in Myf5^nLacZ/+ mice and by FACS sorting, and this effect was inhibited by Ang II AT1 receptor (AT1R) blockade and in AT1aR-null mice. AT1R was highly expressed in SCs, and Notch activation abrogated the AT1R-mediated antiproliferative effect of Ang II in cultured SCs. In mice that developed CHF postmyocardial infarction, there was skeletal muscle wasting and reduced SC numbers that were inhibited by AT1R blockade. Ang II inhibition of skeletal muscle regeneration via AT1 receptor-dependent suppression of SC Notch and MyoD signaling and proliferation is likely to play an important role in mechanisms leading to cachexia in chronic disease states such as CHF and CKD.     

Clonal Characterization of Rat Muscle Satellite Cells: Proliferation,Metabolism and Differentiation Define an Intrinsic Heterogeneity

Satellite cells (SCs) represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB) muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC) present in major proportion (∼75%) and the high proliferative clones (HPC), present instead in minor amount (∼25%). LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (ΔΨ_m), ATP balance and Reactive Oxygen Species (ROS) generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.     

Gαi2 Signaling Is Required for Skeletal Muscle Growth,Regeneration, and Satellite Cell Proliferation and Differentiation

We have previously shown that activation of Gαi2, an α subunit of the heterotrimeric G protein complex, induces skeletal muscle hypertrophy and myoblast differentiation. To determine whether Gαi2 is required for skeletal muscle growth or regeneration, Gαi2-null mice were analyzed. Gαi2 knockout mice display decreased lean body mass, reduced muscle size, and impaired skeletal muscle regeneration after cardiotoxin-induced injury. Short hairpin RNA (shRNA)-mediated knockdown of Gαi2 in satellite cells (SCs) leads to defective satellite cell proliferation, fusion, and differentiation ex vivo. The impaired differentiation is consistent with the observation that the myogenic regulatory factors MyoD and Myf5 are downregulated upon knockdown of Gαi2. Interestingly, the expression of microRNA 1 (miR-1), miR-27b, and miR-206, three microRNAs that have been shown to regulate SC proliferation and differentiation, is increased by a constitutively active mutant of Gαi2 [Gαi2(Q205L)] and counterregulated by Gαi2 knockdown. As for the mechanism, this study demonstrates that Gαi2(Q205L) regulates satellite cell differentiation into myotubes in a protein kinase C (PKC)- and histone deacetylase (HDAC)-dependent manner.     

Association of Interleukin-6 Signalling with the Muscle Stem Cell Response Following Muscle-Lengthening Contractions in Humans

Background

The regulation of muscle stem cells in humans in response to muscle injury remains largely undefined. Recently, interleukin-6 (IL-6) has been implicated in muscle stem cell (satellite cell)-mediated muscle hypertrophy in animals; however, the role of IL-6 in the satellite cell (SC) response following muscle-lengthening contractions in humans has not been studied.

Methodology/Principal Findings

Eight subjects (age 22±1 y; 79±8 kg) performed 300 maximal unilateral lengthening contractions (3.14 rad.s⁻¹) of the knee extensors. Blood and muscle samples were collected before and at 4, 24, 72, and 120 hours post intervention. IL-6, IL-6 receptor (IL-6Rα), cyclin D1, suppressor of cytokine signling-3 (SOCS3) mRNA were measured using quantitative RT-PCR and serum IL-6 protein was measured using an ELISA kit. JAK2 and STAT3 phosphorylated and total protein was measured using western blotting techniques. Immunohistochemical analysis of muscle cross-sections was performed for the quantification of SCs (Pax7⁺ cells) as well as the expression of phosphorylated STAT3, IL-6, IL-6Rα, and PCNA across all time-points. The SC response, as defined by an amplification of Pax7⁺ cells, was rapid, increasing by 24 h and peaking 72 h following the intervention. Muscle IL-6 mRNA increased following the intervention, which correlated strongly (R² = 0.89, p<0.002) with an increase in serum IL-6 concentration. SC IL-6Rα protein was expressed on the fiber, but was also localized to the SC, and IL-6⁺ SC increased rapidly following muscle-lengthening contractions and returned to basal levels by 72 h post-intervention, demonstrating an acute temporal expression of IL-6 with SC. Phosphorylated STAT3 was evident in SCs 4 h after lengthening contraction, and the downstream genes, cyclin D1 and SOCS3 were significantly elevated 24 hours after the intervention.

Conclusions/Significance

The increased expression of STAT3 responsive genes and expression of IL-6 within SCs demonstrate that IL-6/STAT3 signaling occurred in SCs, correlating with an increase in SC proliferation, evidenced by increased Pax7⁺/PCNA⁺ cell number in the early stages of the time-course. Collectively, these data illustrate that IL-6 is an important signaling molecule associated with the SC response to acute muscle-lengthening contractions in humans.     

Changes in calpain and caspase gene expression at the mRNA level during bovine muscle satellite cell myogenesis and the correlation between the cell model and the muscle tissue

The calpain proteolytic system plays a central role in cell death and cell signaling. Caspases are a family of proteases implicated in apoptosis. The objective of this study was to explore the regulation and change trend of calpains (CAPN1 and CAPN3) and caspases (caspase-3, caspase-7, and caspase-9) expression at the mRNA level in Luxi cattle skeletal muscle satellite cells during proliferation and differentiation into myotubes. We also sought to assess whether there is a relationship between the muscle satellite cell model and skeletal muscle tissue. Satellite cells were isolated from longissimus dorsi muscle from Luxi cattle and cultured in vitro. Immunofluorescence was used to characterize satellite cells. Our study was divided into three groups: stage one, satellite cells proliferated at 50- and 80-% confluence; stage two, satellite cells differentiated at days 1, 3, 5, 7, and 15; stage three, not the satellite cells but the skeletal muscle tissue. Real-time PCR was used to quantify expression of calpains and the caspases at the mRNA level. These data demonstrated that CAPN1, CAPN3, CASP7, Myf5, and MyoG gene expression significantly increased from satellite cell proliferation to differentiation phases (P < 0.05). In contrast, CASP3 and CASP9 gene expression was significantly down-regulated during myogenesis (P < 0.05). Moreover, we put the CAPN1, CAPN3, CASP3, CASP7, CASP9, Myf5, and MyoG together to say that these genes expression had no significant correlation between the satellite cell model and the skeletal muscle tissue (P > 0.05). Here, we conclude that calpains (CAPN1 and CAPN3), caspases (caspase-3, caspase-7, and caspase-9), and Myf5 and MyoG all have important roles in satellite cell myogenesis. However, there is no relationship between the cell model and muscle tissue.     

seRNA PAM controls skeletal muscle satellite cell proliferation and aging through trans regulation of Timp2 expression synergistically with Ddx5

Muscle satellite cells (SCs) are responsible for muscle homeostasis and regeneration and lncRNAs play important roles in regulating SC activities. Here, in this study, we identify PAM (Pax7 Associated Muscle lncRNA) that is induced in activated/proliferating SCs upon injury to promote SC proliferation as myoblast cells. PAM is generated from a myoblast‐specific super‐enhancer (SE); as a seRNA it binds with a number of target genomic loci predominantly in trans. Further studies demonstrate that it interacts with Ddx5 to tether PAM SE to its inter‐chromosomal targets Timp2 and Vim to activate the gene expression. Lastly, we show that PAM expression is increased in aging SCs, which leads to enhanced inter‐chromosomal interaction and target genes upregulation. Altogether, our findings identify PAM as a previously unknown lncRNA that regulates both SC proliferation and aging through its trans gene regulatory activity.     

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca²⁺ATPases (SERCAs) are the main Ca²⁺ pumps which decrease the intracellular Ca²⁺ level by reaccumulating Ca²⁺ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca²⁺ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca²⁺-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca²⁺]i measured at the 20^th and 40^th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca²⁺-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca²⁺]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.     

Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Skeletal muscle regeneration is mediated by satellite cells (SCs). Upon injury, SCs undergo self-renewal, proliferation, and differentiation into myoblasts followed by myoblast fusion to form new myofibers. We previously showed that the heparan sulfate (HS) 6-O-endosulfatases (Sulf1 and -2) repress FGF signaling to induce SC differentiation during muscle regeneration. Here, we identify a novel role of Sulfs in myoblast fusion using a skeletal muscle-specific Sulf double null (Sulf^SK-DN) mouse. Regenerating Sulf^SK-DN muscles exhibit reduced canonical Wnt signaling and elevated non-canonical Wnt signaling. In addition, we show that Sulfs are required to repress non-canonical Wnt signaling to promote myoblast fusion. Notably, skeletal muscle-relevant non-canonical Wnt ligands lack HS binding capacity, suggesting that Sulfs indirectly repress this pathway. Mechanistically, we show that Sulfs reduce the canonical Wnt-HS binding and regulate colocalization of the co-receptor LRP5 with caveolin3. Therefore, Sulfs may increase the bioavailability of canonical Wnts for Frizzled receptor and LRP5/6 interaction in lipid raft, which may in turn antagonize non-canonical Wnt signaling. Furthermore, changes in subcellular distribution of active focal adhesion kinase (FAK) are associated with the fusion defect of Sulf-deficient myoblasts and upon non-canonical Wnt treatment. Together, our findings uncover a critical role of Sulfs in myoblast fusion by promoting antagonizing canonical Wnt signaling activities against the noncanonical Wnt pathway during skeletal muscle regeneration.     

Delineating the relationship between immune system aging and myogenesis in muscle repair

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.     

Deletion of SA β‐Gal+ cells using senolytics improves muscle regeneration in old mice

Systemic deletion of senescent cells leads to robust improvements in cognitive, cardiovascular, and whole‐body metabolism, but their role in tissue reparative processes is incompletely understood. We hypothesized that senolytic drugs would enhance regeneration in aged skeletal muscle. Young (3 months) and old (20 months) male C57Bl/6J mice were administered the senolytics dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi‐weekly for 4 months. Tibialis anterior (TA) was then injected with 1.2% BaCl₂ or PBS 7‐ or 28 days prior to euthanization. Senescence‐associated β‐Galactosidase positive (SA β‐Gal+) cell abundance was low in muscle from both young and old mice and increased similarly 7 days following injury in both age groups, with no effect of D+Q. Most SA β‐Gal+ cells were also CD11b+ in young and old mice 7‐ and 14 days following injury, suggesting they are infiltrating immune cells. By 14 days, SA β‐Gal+/CD11b+ cells from old mice expressed senescence genes, whereas those from young mice expressed higher levels of genes characteristic of anti‐inflammatory macrophages. SA β‐Gal+ cells remained elevated in old compared to young mice 28 days following injury, which were reduced by D+Q only in the old mice. In D+Q‐treated old mice, muscle regenerated following injury to a greater extent compared to vehicle‐treated old mice, having larger fiber cross‐sectional area after 28 days. Conversely, D+Q blunted regeneration in young mice. In vitro experiments suggested D+Q directly improve myogenic progenitor cell proliferation. Enhanced physical function and improved muscle regeneration demonstrate that senolytics have beneficial effects only in old mice.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

Increased Residual Force Enhancement in Older Adults Is Associated with a Maintenance of Eccentric Strength

Despite an age-related loss of voluntary isometric and concentric strength, muscle strength is well maintained during lengthening muscle actions (i.e., eccentric strength) in old age. Additionally, in younger adults during lengthening of an activated skeletal muscle, the force level observed following the stretch is greater than the isometric force at the same muscle length. This feature is termed residual force enhancement (RFE) and is believed to be a combination of active and passive components of the contractile apparatus. The purpose of this study was to provide an initial assessment of RFE in older adults and utilize aging as a muscle model to explore RFE in a system in which isometric force production is compromised, but structural mechanisms of eccentric strength are well-maintained. Therefore, we hypothesised that older adults will experience greater RFE compared with young adults. Following a reference maximal voluntary isometric contraction (MVC) of the dorsiflexors in 10 young (26.1±2.7y) and 10 old (76.0±6.5y) men, an active stretch was performed at 15°/s over a 30° ankle joint excursion ending at the same muscle length as the reference MVCs (40° of plantar flexion). Any additional torque compared with the reference MVC therefore represented RFE. In older men RFE was ∼2.5 times greater compared to young. The passive component of force enhancement contributed ∼37% and ∼20% to total force enhancement, in old and young respectively. The positive association (R ² = 0.57) between maintained eccentric strength in old age and RFE indicates age-related mechanisms responsible for the maintenance of eccentric strength likely contributed to the observed elevated RFE. Additionally, as indicated by the greater passive force enhancement, these mechanisms may be related to increased muscle series elastic stiffness in old age.     

Elevated H3K27ac in aged skeletal muscle leads to increase in extracellular matrix and fibrogenic conversion of muscle satellite cells

Epigenetic alterations occur in various cells and tissues during aging, but it is not known if such alterations are also associated with aging in skeletal muscle. Here, we examined the changes of a panel of histone modifications and found H3K27ac (an active enhancer mark) is markedly increased in aged human skeletal muscle tissues. Further analyses uncovered that the H3K27ac increase and enhancer activation are associated with the up‐regulation of extracellular matrix (ECM) genes; this may result in alteration of the niche environment for skeletal muscle stem cells, also called satellite cells (SCs), which causes decreased myogenic potential and fibrogenic conversion of SCs. In mice, treatment of aging muscles with JQ1, an inhibitor of enhancer activation, inhibited the ECM up‐regulation and fibrogenic conversion of SCs and restored their myogenic differentiation potential. Altogether, our findings not only uncovered a novel aspect of skeletal muscle aging that is associated with enhancer remodeling but also implicated JQ1 as a potential treatment approach for restoring SC function in aging muscle.     

Whey protein supplementation accelerates satellite cell proliferation during recovery from eccentric exercise

Human skeletal muscle satellite cells (SCs) are essential for muscle regeneration and remodeling processes in healthy and clinical conditions involving muscle breakdown. However, the potential influence of protein supplementation on post-exercise SC regulation in human skeletal muscle has not been well investigated. In a comparative human study, we investigated the effect of hydrolyzed whey protein supplementation following eccentric exercise on fiber type-specific SC accumulation. Twenty-four young healthy subjects received either hydrolyzed whey protein + carbohydrate (whey, n = 12) or iso-caloric carbohydrate (placebo, n = 12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to and 24, 48 and 168 h post-exercise, muscle biopsies were obtained from the exercise leg and analyzed for fiber type-specific SC content. Maximal voluntary contraction (MVC) and serum creatine kinase (CK) were evaluated as indices of recovery from muscle damage. In type II fiber-associated SCs, the whey group increased SCs/fiber from 0.05 [0.02; 0.07] to 0.09 [0.06; 0.12] (p < 0.05) and 0.11 [0.06; 0.16] (p < 0.001) at 24 and 48 h, respectively, and exhibited a difference from the placebo group (p < 0.05) at 48 h. The whey group increased SCs/myonuclei from 4?% [2; 5] to 10?% [4; 16] (p?p < 0.001) and muscle soreness and CK increased (p < 0.001), irrespective of supplementation. In conclusion, whey protein supplementation may accelerate SC proliferation as part of the regeneration or remodeling process after high-intensity eccentric exercise.     

Comparative Study of Injury Models for Studying Muscle Regeneration in Mice

Background

A longstanding goal in regenerative medicine is to reconstitute functional tissus or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised.

Methods

We have used transgenic Tg:Pax7nGFP and Flk1^GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex^®.

Results

We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl₂), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a “dead zone” devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl₂ but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.

Conclusions

Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.     

Disruption of Smad4 in Mouse Epidermis Leads to Depletion of Follicle Stem Cells

Follicle stem cells (SCs) residing in the bulge region of a hair follicle (HF) can give rise to multiple lineages during the hair cycle and wound healing. The activation and self-renewal of follicle SCs must be tightly regulated to maintain the HF and epidermal homeostasis. Here we show that, in young mice, disruption of epidermal Smad4, the common mediator of transforming growth factor-β (TGF-β) signaling, stimulated the activation of follicle SCs, leading to hyperplasia of interfollicular epidermis (IFE), HFs, and sebaceous glands (SGs). Increased proliferation of follicle SCs ultimately exhausted the SC niche, indicated by the loss of bromodeoxyuridine (BrdU) label–retaining cells (LRCs), loss of keratin 15 (K15), and CD34 expression. In addition, the colony-forming efficiency of Smad4 mutant keratinocytes was significantly decreased. Increased nuclear localization of β-catenin and increased expression of c-Myc were correlated with the overactivation and depletion of follicle SCs. We concluded that Smad4 plays a pivotal role in follicle SC maintenance.     

Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner

Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr^−/−xLcat^−/− or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr^−/−xLcat^+/+ single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr^+/+xLcat^−/− (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.     

A Disintegrin and Metalloprotease 10 (ADAM10) Is Indispensable for Maintenance of the Muscle Satellite Cell Pool

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10 (a disintegrin and metalloprotease 10), a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed a nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared with wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.     

IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage

Background

Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied.

Methodology/Principal Findings

Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s⁻¹ over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA.

Conclusions/Significant Findings

We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.