Myostatin, a member of the TGF-␤ family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells. muscle growth ͉ muscular dystrophy ͉ TGF-beta ͉ muscle stem cells ͉ myonuclear domain L oss of muscle mass and strength is a major clinical feature of inherited myopathies such as Duchenne muscular dystrophy (DMD) and also of more common acquired atrophies associated with disuse, aging, and cancer. This loss has fostered widespread interest in the powerful inhibitory effect of myostatin, a member of the TGF-␤ family of signaling molecules, on muscle growth (1) with specific focus on the prospect of modulating this system to counteract atrophic processes. Indeed, muscle fiber hypertrophy arising from absence or blockade of myostatin has been reported to be associated with therapeutic benefits in the mdx mouse model of DMD (2, 3). This hypertrophy has been attributed to proliferation of satellite cells (4, 5), the principal cellular source for growing and regenerating skeletal muscle (6-10), consequent upon their release from myostatin inhibition (5,11,12).Here, we have investigated the contribution of satellite cells in 2 myostatin-null mouse models, constitutive (mstn Ϫ/Ϫ ) and compact (BEH c/c ), and following myostatin blockade by AAVmediated overexpression of myostatin propeptide. These data, together with our results from in vitro studies on the effect of presence or absence of myostatin on satellite cells contradict co...

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/␤-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of ␤-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulinstimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/␤-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts. INTRODUCTIONSatellite cells are skeletal muscle adult stem cells that participate in postnatal muscle growth and regeneration. Although satellite cells are normally quiescent in adult muscle, they are responsible for muscle regeneration after injury and involved in work-or load-induced muscle fiber hypertrophy (Rosenblatt and Parry, 1992;Schultz and McCormick, 1994;De Angelis et al., 1999;Semsarian et al., 1999;Bodine et al., 2001).Ex vivo models such as myogenic cell lines were isolated from adult mouse muscle and as such are derived from adult satellite cells. At the proliferative stage, myoblasts (activated satellite cells) can be grown extensively in high serum-containing medium and express MyoD, a musclespecific transcription factor that regulates the differentiation process (Weintraub, 1993). When serum levels are lowered, myoblasts exit the cell cycle and spontaneously differentiate, giving rise to a heterogeneous population of cells. The first and major subpopulation is composed of myotubes, quiescent multinucleated cells expressing muscle-specific structural proteins. The remaining subset is composed of quiescent, mononucleated and undifferentiated cells termed reserve cells. Reserve cells retain the ability to be activated and proliferate after which they can be induced to differentiate, leading again to a new mixed population of myotubes and reserve cells. They also express at least two genes characteristic of skeletal muscle stem cells, namely, myf-5 and cd34 and from these c...

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related ␣ (Akt1) and ␤ (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKB␤ was up-regulated whereas expression of PKB␣ was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKB␤ localized in the nuclei. In a transactivation assay, PKB␤ (either wild-type or constitutively active) was more efficient than PKB␣ in activating musclespecific gene expression. Moreover, microinjection of specific antibodies to PKB␤ inhibited differentiation of muscle cells, whereas control or anti-PKB␣ antibodies did not. On the other hand, microinjection of the anti-PKB␣ antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKB␤ antibodies had no effect. Taken together, these results show that PKB␤ plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKB␣.

BackgroundIn the search for more potent and safer tuberculosis vaccines, CAF01 was identified as a remarkable formulation. Based on cationic liposomes and including a synthetic mycobacterial glycolipid as TLR-independent immunomodulator, it induces strong and protective T helper-1 and T helper-17 adult murine responses to Ag85B-ESAT-6, a major mycobacterial fusion protein. Here, we assessed whether these properties extend to early life and how CAF01 mediates its adjuvant properties in vivo.Methods/FindingsFollowing adult or neonatal murine immunization, Ag85B-ESAT-6/CAF01 similarly reduced the post-challenge bacterial growth of M. bovis BCG, whereas no protection was observed using Alum as control. This protection was mediated by the induction of similarly strong Th1 and Th17 responses in both age groups. Multifunctional Th1 cells were already elicited after a single vaccine dose and persisted at high levels for at least 6 months even after neonatal priming. Unexpectedly, this potent adjuvanticity was not mediated by a massive targeting/activation of dendritic cells: in contrast, very few DCs in the draining lymph nodes were bearing the labeled antigen/adjuvant. The increased expression of the CD40 and CD86 activation markers was restricted to the minute portion of adjuvant-bearing DCs. However, vaccine-associated activated DCs were recovered several days after immunization.ConclusionThe potent adult and neonatal adjuvanticity of CAF01 is associated in vivo with an exquisite but prolonged DC uptake and activation, fulfilling the preclinical requirements for novel tuberculosis vaccines to be used in early life.

We explored the role of CD40-CD40L (CD154) in the severe malaria elicited by Plasmodium berghei anka infection in mice. Mortality was >90% by day 8 after infection in ؉/؉ mice, but markedly decreased in CD40؊/؊ or in CD40L؊/؊ mice, as well as in ؉/؉ mice treated with anti-CD40L monoclonal antibody. Parasitemia was similar in the different conditions. Breakdown of the blood-brain barrier was evident in infected ؉/؉, but not in CD40؊/؊ mice. Thrombocytopenia was less severe in CD40؊/؊ mice than in the ؉/؉ controls. Sequestration of macrophages in brain venules and alveolar capillaries was reduced in CD40؊/؊ or in CD40L؊/؊ mice, whereas sequestration of parasitized red blood cells or polymorphonuclear leukocytes in alveolar capillaries was CD40-CD40L-independent. CD40 mRNA was increased in the brain and lung of infected mice whereas CD40L was increased in the lung. Tumor necrosis factor plasma levels were similarly increased in infected ؉/؉ or CD40؊/؊ mice. Expression of CD54 and its mRNA levels in the brain were moderately decreased in CD40-deficient mice. Thus the mortality associated with severe malaria requires CD40-CD40L interaction that contributes to the breakdown of the blood-brain barrier, macrophage sequestration, and platelet consumption.

Skeletal muscle growth and regeneration require a population of muscle stem cells, the satellite cells, located in close contact to the myofiber. These cells are specified during fetal and early postnatal development in mice from a Pax3/7 population of embryonic progenitor cells. As little is known about the genetic control of their formation and maintenance, we performed a genome-wide chronological expression profile identifying the dynamic transcriptomic changes involved in establishment of muscle stem cells through life, and acquisition of muscle stem cell properties. We have identified multiple genes and pathways associated with satellite cell formation, including set of genes specifically induced (EphA1, EphA2, EfnA1, EphB1, Zbtb4, Zbtb20) or inhibited (EphA3, EphA4, EphA7, EfnA2, EfnA3, EfnA4, EfnA5, EphB2, EphB3, EphB4, EfnBs, Zfp354c, Zcchc5, Hmga2) in adult stem cells. Ephrin receptors and ephrins ligands have been implicated in cell migration and guidance in many tissues including skeletal muscle. Here we show that Ephrin receptors and ephrins ligands are also involved in regulating the adult myogenic program. Strikingly, impairment of EPHB1 function in satellite cells leads to increased differentiation at the expense of self-renewal in isolated myofiber cultures. In addition, we identified new transcription factors, including several zinc finger proteins. ZFP354C and ZCCHC5 decreased self-renewal capacity when overexpressed, whereas ZBTB4 increased it, and ZBTB20 induced myogenic progression. The architectural and transcriptional regulator HMGA2 was involved in satellite cell activation. Together, our study shows that transcriptome profiling coupled with myofiber culture analysis, provides an efficient system to identify and validate candidate genes implicated in establishment/maintenance of muscle stem cells. Furthermore, tour de force transcriptomic profiling provides a wealth of data to inform for future stem cell-based muscle therapies.

Mice were exposed to pure oxygen for various times to explore the pulmonary platelet trapping associated with alveolar damage, its mechanism, and its role in the lesions. Platelet sequestration, evaluated by electron microscopy and by injection of radiolabeled platelets, was detectable after 72 h and reached a maximum after 96 h of exposure (i.e., shortly before death). Circulating platelets (analyzed by Facscan) showed some increase in the expression of CD11a and CD62, but little change in CD31 and CD61. Both platelet activation and lung sequestration were dependent on TNF-alpha, since antibody against TNF-alpha reduced the expression of CD11a on circulating platelets and their sequestration in the lung. Lung platelet sequestration was also decreased by anti-CD11a MoAb. Northern blot analysis of lung mRNA isolated at 96 h of oxygen exposure revealed a 7-fold increase in CD54 (intercellular adhesion molecule-1 [ICAM-1]) and a 2.5-fold increase in TNF-alpha mRNAs respectively. These results demonstrate that the platelet pulmonary trapping induced by hyperoxia is dependent upon TNF-alpha and the CD11a-CD54 adhesion molecules. However, platelet trapping does not appear to play an important pathogenic role in acute oxygen injury, since treatments that decrease trapping (anti-TNF-alpha, anti-CD11a, or antibody-induced thrombocytopenia) did not markedly attenuate the alveolar damage.

Akt1 and Akt2 are the major isoforms of Akt expressed in muscle cells and muscle tissue. We have performed siRNA silencing of Akt1 and Akt2 in C2 myoblasts to characterize their specific implication in muscle differentiation. Whereas silencing Akt2, and not Akt1, inhibited cell cycle exit and myoblast differentiation, Akt2 overexpression led to an increased proportion of differentiated myoblasts. In addition, we demonstrate that Akt2 is required for myogenic conversion induced by MyoD overexpression in fibroblasts. We show Akt2, but not Akt1, binds Prohibitin2/Repressor of Estrogen Activator, PHB2/REA, a protein recently implicated in transcriptionnal repression of myogenesis. Co-immunoprecipitation experiments on endogenous proteins showed the Akt2-REA complex does not contain Prohibitin1. We have analyzed expression and localization of PHB2/REA during proliferation and differentiation of mouse and human myoblasts. PHB2/REA shows punctated nuclear staining which partially co-localizes with Akt2 in differentiated myotubes and PHB2 levels decrease at the onset of myogenic differentiation concomitant with an increase in Akt2. There appears to be an inverse correlation between Akt2 and PHB2 protein levels where cells silenced for Akt2 expression show increased level of PHB2/REA and overexpression of Akt2 resulted in decreased Prohibitin2/REA. Taken together, these results, along with our previous observations, clearly show that Akt2 and not Akt1 plays a major and early role in cell cycle exit and myogenic differentiation and this function involves its specific interaction with PHB2/REA.