Upregulated IL-1ß in dysferlin-deficient muscle attenuates regeneration by blunting the response to pro-inflammatory macrophages.

BACKGROUND: Loss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. Dysferlin-deficient muscle is characterized by inflammatory foci and macrophage infiltration with subsequent decline in muscle function. Whereas macrophages function to remove necrotic tissue in acute injury, their prevalence in chronic myopathy is thought to inhibit resolution of muscle regeneration. Two major classes of macrophages, classical (M1) and alternative (M2a), play distinct roles during the acute injury process. However, their individual roles in chronic myopathy remain unclear and were explored in this study. METHODS: To test the roles of the two macrophage phenotypes on regeneration in dysferlin-deficient muscle, we developed an in vitro co-culture model of macrophages and muscle cells. We assayed the co-cultures using ELISA and cytokine arrays to identify secreted factors and performed transcriptome analysis of molecular networks induced in the myoblasts. RESULTS: Dysferlin-deficient muscle contained an excess of M1 macrophage markers, compared with WT, and regenerated poorly in response to toxin injury. Co-culturing macrophages with muscle cells showed that M1 macrophages inhibit muscle regeneration whereas M2a macrophages promote it, especially in dysferlin-deficient muscle cells. Examination of soluble factors released in the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1ß and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1ß, which inhibited it. Importantly, blockade of IL-1ß signaling significantly improved differentiation of dysferlin-deficient cells. CONCLUSIONS: We propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1ß signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle.

Defective skeletal muscle growth in lamin A/C-deficient mice is rescued by loss of Lap2α.

Mutations in lamin A/C result in a range of tissue-specific disorders collectively called laminopathies. Of these, Emery-Dreifuss and Limb-Girdle muscular dystrophy 1B mainly affect striated muscle. A useful model for understanding both laminopathies and lamin A/C function is the Lmna(-/-) mouse. We found that skeletal muscle growth and muscle satellite (stem) cell proliferation were both reduced in Lmna(-/-) mice. Lamins A and C associate with lamina-associated polypeptide 2 alpha (Lap2α) and the retinoblastoma gene product, pRb, to regulate cell cycle exit. We found Lap2α to be upregulated in Lmna(-/-) myoblasts (MBs). To specifically test the contribution of elevated Lap2α to the phenotype of Lmna(-/-) mice, we generated Lmna(-/-)Lap2α(-/-) mice. Lifespan and body mass were increased in Lmna(-/-)Lap2α(-/-) mice compared with Lmna(-/-). Importantly, the satellite cell proliferation defect was rescued, resulting in improved myogenesis. Lmna(-/-) MBs also exhibited increased levels of Smad2/3, which were abnormally distributed in the cell and failed to respond to TGFß1 stimulation as in control cells. However, using SIS3 to inhibit signaling via Smad3 reduced cell death and augmented MB fusion. Together, our results show that perturbed Lap2α/pRb and Smad2/3 signaling are important regulatory pathways mediating defective muscle growth in Lmna(-/-) mice, and that inhibition of either pathway alone or in combination can ameliorate this deleterious phenotype.

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice.

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kß-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

Uncoordinated transcription and compromised muscle function in the lmna-null mouse model of Emery- Emery-Dreyfuss muscular dystrophy.

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting.

Novel LMNA mutations in patients with Emery-Dreifuss muscular dystrophy and functional characterization of four LMNA mutations.

Mutations in LMNA cause a variety of diseases affecting striated muscle including autosomal Emery-Dreifuss muscular dystrophy (EDMD), LMNA-associated congenital muscular dystrophy (L-CMD), and limb-girdle muscular dystrophy type 1B (LGMD1B). Here, we describe novel and recurrent LMNA mutations identified in 50 patients from the United States and Canada, which is the first report of the distribution of LMNA mutations from a large cohort outside Europe. This augments the number of LMNA mutations known to cause EDMD by 16.5%, equating to an increase of 5.9% in the total known LMNA mutations. Eight patients presented with either p.R249W/Q or p.E358K mutations and an early onset EDMD phenotype: two mutations recently associated with L-CMD. Importantly, 15 mutations are novel and include eight missense mutations (p.R189P, p.F206L, p.S268P, p.S295P, p.E361K, p.G449D, p.L454P, and p.W467R), three splice site mutations (c.IVS4 + 1G>A, c.IVS6 - 2A>G, and c.IVS8 + 1G>A), one duplication/in frame insertion (p.R190dup), one deletion (p.Q355del), and two silent mutations (p.R119R and p.K270K). Analysis of 4 of our lamin A mutations showed that some caused nuclear deformations and lamin B redistribution in a mutation specific manner. Together, this study significantly augments the number of EDMD patients on the database and describes 15 novel mutations that underlie EDMD, which will contribute to establishing genotype-phenotype correlations.

Dynamics of muscle fibre growth during postnatal mouse development.

BACKGROUND: Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle. RESULTS: Here, we report on the dynamics of postnatal myofibre growth in the mouse extensor digitorum longus (EDL) muscle, which is essentially composed of fast type II fibres in adult. We found that there was no net gain in myofibre number in the EDL between P7 and P56 (adulthood). However, myofibre cross-sectional area increased by 7.6-fold, and length by 1.9-fold between these ages, resulting in an increase in total myofibre volume of 14.1-fold: showing the extent of myofibre hypertrophy during the postnatal period. To determine how the number of myonuclei changes during this period of intense muscle fibre hypertrophy, we used two complementary mouse models: 3F-nlacZ-E mice express nlacZ only in myonuclei, while Myf5nlacZ/+ mice have beta-galactosidase activity in satellite cells. There was a approximately 5-fold increase in myonuclear number per myofibre between P3 and P21. Thus myofibre hypertrophy is initially accompanied by a significant addition of myonuclei. Despite this, the estimated myonuclear domain still doubled between P7 and P21 to 9.2 x 103 microm3. There was no further addition of myonuclei from P21, but myofibre volume continued to increase, resulting in an estimated approximately 3-fold expansion of the myonuclear domain to 26.5 x 103 microm3 by P56. We also used our two mouse models to determine the number of satellite cells per myofibre during postnatal growth. Satellite cell number in EDL was initially approximately 14 satellite cells per myofibre at P7, but then fell to reach the adult level of approximately 5 by P21. CONCLUSIONS: Postnatal fast muscle fibre type growth is divided into distinct phases in mouse EDL: myofibre hypertrophy is initially supported by a rapid increase in the number of myonuclei, but nuclear addition stops around P21. Since the significant myofibre hypertrophy from P21 to adulthood occurs without the net addition of new myonuclei, a considerable expansion of the myonuclear domain results. Satellite cell numbers are initially stable, but then decrease to reach the adult level by P21. Thus the adult number of both myonuclei and satellite cells is already established by three weeks of postnatal growth in mouse.

Genotype-phenotype correlations in laminopathies: how does fate translate?

A-type laminopathies are a group of diseases resulting from mutations in the intermediate filament proteins lamin A and C (both encoded by the LMNA gene), but for which the pathogenic mechanisms are little understood. In some laminopathies, there is a good correlation between the presence of a specific LMNA mutation and the disease diagnosed. In others however, many different mutations can give rise to the same clinical condition, even though the mutations may be distributed throughout one, or more, of the three functionally distinct protein domains of lamin A/C. Conversely, certain mutations can cause multiple laminopathies, with related patients carrying an identical mutation even having separate diseases, often affecting different tissues. Therefore clarifying genotype-phenotype links may provide important insights into both disease penetrance and mechanism. In the present paper, we review recent developments in genotype-phenotype correlations in laminopathies and discuss the factors that could influence pathology.

Presenilin-1 acts via Id1 to regulate the function of muscle satellite cells in a gamma-secretase-independent manner.

Muscle satellite cells are the resident stem cells of adult skeletal muscle. Here, we have examined the role of the multifunctional protein presenilin-1 (PS1) in satellite cell function. PS1 acts as a crucial component of the gamma-secretase complex, which is required to cleave single-pass transmembrane proteins such as Notch and amyloid-beta precursor protein. PS1, however, also functions through gamma-secretase-independent pathways. Activation of satellite cells was accompanied by induction of PS1, with PS1 knockdown enhancing their myogenic differentiation, but reducing their self-renewal. Transfection with siRNA against PS1 led to accelerated myogenic differentiation during muscle regeneration in vivo. Conversely, constitutive expression of PS1 resulted in the suppression of myogenic differentiation and promotion of the self-renewal phenotype. Importantly, we found that PS1 also acts independently of its role in gamma-secretase activity in controlling myogenesis, which is mediated in part by Id1 (inhibitor of DNA binding 1), a negative regulator of the myogenic regulatory factor MyoD. PS1 can control Id1, which affects satellite cell fate by regulating the transcriptional activity of MyoD. Taken together, our observations show that PS1 is a key player in the choice of satellite cell fate, acting through both gamma-secretase-dependent and gamma-secretase-independent mechanisms.

Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells.

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin alpha7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells.

Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

Does satellite cell dysfunction contribute to disease progression in Emery-Dreifuss muscular dystrophy?

Muscular dystrophies comprise at least 34 conditions, characterized by progressive skeletal muscle weakness and degeneration. The loci affected include mutations in both muscle-specific genes and genes that are more widely expressed such as LMNA and EMD, responsible for EDMD (Emery-Dreifuss muscular dystrophy). LMNA encodes A-type lamins, whereas EMD encodes emerin, both located in the nuclear envelope. Mutation or loss of A-type lamins or emerin in the terminally differentiated myonuclei of muscle fibres results in muscle damage. Importantly, since LMNA and EMD are also expressed by the resident skeletal muscle stem cells, the satellite cells, the mutations that cause muscle damage may also directly compromise the regenerative response. Thus EDMD is different from dystrophic conditions such as Duchenne muscular dystrophy, where the mutated gene is only expressed in the muscle fibres. In this brief review, we examine the evidence that myoblasts carrying EDMD-causing mutations are compromised, and discuss the possibility that such dysfunction results in reduced efficiency of muscle regeneration, so actively contributes to disease progression.

beta-Catenin promotes self-renewal of skeletal-muscle satellite cells.

Satellite cells are the resident stem cells of adult skeletal muscle. As with all stem cells, how the choice between self-renewal or differentiation is controlled is central to understanding their function. Here, we have explored the role of beta-catenin in determining the fate of myogenic satellite cells. Satellite cells express beta-catenin, and expression is maintained as they activate and undergo proliferation. Constitutive retroviral-driven expression of wild-type or stabilised beta-catenin results in more satellite cells expressing Pax7 without any MyoD -- therefore, adopting the self-renewal pathway, with fewer cells undergoing myogenic differentiation. Similarly, preventing the degradation of endogenous beta-catenin by inhibiting GSK3beta activity also results in more Pax7-positive-MyoD-negative (Pax7(+)MyoD(-)) satellite-cell progeny. Consistent with these observations, downregulation of beta-catenin using small interfering RNA (siRNA) reduced the proportion of satellite cells that express Pax7 and augmented myogenic differentiation after mitogen withdrawal. Since a dominant-negative version of beta-catenin had the same effect as silencing beta-catenin using specific siRNA, beta-catenin promotes self-renewal via transcriptional control of target genes. Thus, beta-catenin signalling in proliferating satellite cells directs these cells towards the self-renewal pathway and, so, contributes to the maintenance of this stem-cell pool in adult skeletal muscle.

Diacylglycerol kinase-alpha mediates hepatocyte growth factor-induced epithelial cell scatter by regulating Rac activation and membrane ruffling.

Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.

Ghrelin and des-acyl ghrelin promote differentiation and fusion of C2C12 skeletal muscle cells.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.