Inhibition of Atrogin-1/MAFbx Mediated MyoD Proteolysis Prevents Skeletal Muscle Atrophy In Vivo

Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed

Identification of essential sequences for cellular localization in the muscle-specific ubiquitin E3 ligase MAFbx/Atrogin 1

Contact: [email protected] audienceIn skeletal muscle atrophy, upregulation and nuclear accumulation of the Ubiquitin E3 ligase MAFbx is essential for accelerated muscle protein loss, but the nuclear/cytoplasmic shuttling of MAFbx is undefined. Here we found that MAFbx contains two functional nuclear localization signals (NLS). Mutation or deletion of only one NLS induced cytoplasmic localization of MAFbx. We identified a non-classical NES located in the leucine charged domain (LCD) of MAFbx, which is leptomycin B insensitive. We demonstrated that mutation (L169Q) in LLXXL motif of LCD suppressed cytoplasmic retention of MAFbx. Nucleocytoplasmic shuttling of MAFbx represents a novel mechanism for targeting its substrates and its cytosolic partners in muscle atrophy

Beyond mice: Emerging and transdisciplinary models for the study of early-onset myopathies

International audienceThe use of the adapted models to decipher patho-physiological mechanisms of human diseases is always a great challenge. This is of particular importance for early-onset myopathies, in which pathological mutations often impact not only on muscle structure and function but also on developmental processes. Mice are currently the main animal model used to study neuromuscular disorders including the early-onset myopathies. However strategies based on simple animal models and on transdisciplinary approaches exploring mechanical muscle cell properties emerge as attractive, non-exclusive alternatives. These new ways provide valuable opportunities to improve our knowledge on how mechanical, biochemical, and genetic/epigenetic cues modulate the formation, organization and function of muscle tissues. Here we provide an overview of how single cell and micro-tissue engineering in parallel to non-mammalian, Drosophila and zebrafish models could contribute to filling gaps in our understanding of pathogenic mechanisms underlying early-onset myopathies. We also discuss their potential impact on designing new diagnostic and therapeutic strategies

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient

International audienceSpecific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A

MyoD undergoes a distinct G2/M-specific regulation in muscle cells.

Noyaux et cycle cellulaire (resp. Danièle Hernandez-Verdun)International audienceThe transcription factors MyoD and Myf5 present distinct patterns of expression during cell cycle progression and development. In contrast to the mitosis-specific disappearance of Myf5, which requires a D-box-like motif overlapping the basic domain, here we describe a stable and inactive mitotic form of MyoD phosphorylated on its serine 5 and serine 200 residues by cyclin B-cdc2. In mitosis, these modifications are required for releasing MyoD from condensed chromosomes and inhibiting its DNA-binding and transcriptional activation ability. Then, nuclear MyoD regains instability in the beginning of G1 phase due to rapid dephosphorylation events. Moreover, a non-phosphorylable MyoD S5A/S200A is not excluded from condensed chromatin and alters mitotic progression with apparent abnormalities. Thus, the drop of MyoD below a threshold level and its displacement from the mitotic chromatin could present another window in the cell cycle for resetting the myogenic transcriptional program and to maintain the myogenic determination of the proliferating cells

Modulation of cell signaling pathways related to the cytoskeleton reduces desmin aggregation.

<p>(A) C2C12 cells were co-transfected with a GFP-tagged desmin mutant D399Y and constructs coding for either wild type (WT) or dominant-negative mutant (DN) kinases or kinase-modulating proteins [i.e., Rac1, p21-activated protein kinase (PAK1), Rho kinase (ROCK), mammalian Diaphanous (mDia), protein kinase C (PKC), p38-regulated/activated protein kinase (PRAK) and transforming growth factor β activated kinase 1 (TAK1)]. At 20 h after transfection, cells were fixed and the total number of cells (n = 1500) and the number of cells with aggregates were counted. Experiments were performed 4 times. The percentage of cells with aggregates is displayed on a box plot graph (Tukey's diagram). Asterisk indicates a result statistically different from the control co-transfected with the desmin mutant and the empty vector pcDNA3 (p < 0.05 calculated with a non-parametric test). The black horizontal bar represents the median value; limits of the rectangle represent the first (25% lower values) and the third (75% lower values) quartiles, respectively. Error bars, Tukey's adjacent values. (B) Same treatment as for (A) except that cells were fixed 48 h after transfection.</p

PKC WT, PAK1 WT, and Rac1 DN induce autophagy in C2C12 myoblasts.

<p>(A) LC3 levels in C2C12 cells expressing several constructs. Cells were transfected for 16 h with TAK1 WT, PKC WT, RhoK WT (ROCK), Rac1 DN, PAK1 WT or pcDNA3 (CNTL), together with a GFP-LC3 construct to measure autophagy in co-transfected cells. Cells were then lysed and GFP-LC3-II levels quantified in Western blots to estimate autophagy intensity. A representative result is shown in panel A. Actin was used as loading control. Levels of activation of autophagy were obtained by quantification of the LC3-II (anti-GFP antibody) band normalized to actin (anti-actin antibody). The control value (CNTL) was assigned to 1.0. (B) Statistical analysis of activation of autophagy. Experiments shown in (A) were repeated 3 times and displayed as dot plots. Asterisk indicates a result statistically different from the control co-transfected with the empty vector pcDNA3 (p < 0.05 calculated with a non-parametric test).</p

Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy

<div><p>Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the <i>desmin</i> gene (<i>DES</i>), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant <i>DES</i>. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.</p></div

Stimulation of autophagy with PP242 reduces desmin aggregation in myoblasts.

<p>(A) C2C12 cells were transiently transfected for 4 h with GFP-Desmin D399Y mutant, washed, and treated for 16 h with PP242 (10 μM). They were fixed and several fields photographed. A typical field is displayed. Green dots are cell aggregates and blue dots are cell nuclei visualized with DAPI. Cells from the control panel (CNTL) were treated with DMSO. Scale bar, 30 μm. (B) Quantification of 3 independent experiments (n = 1200 total cells for each condition in each experiment). The left panel displays results obtained with the GFP-Desmin Q389P construct, and in the right panel, from GFP-Desmin D399Y transfections. (C) Same as for (B), except that myc-Desmin Q389P (left panel) and myc-Desmin D399Y (right panel) constructs were used. Cells were transiently transfected for 4 h with these constructs, washed, and treated 16 h with PP242 (10 μM) or DMSO as solvent (CNTL). They were fixed and then processed for immunocytochemistry with an anti-myc antibody. Numbers of cells with aggregates per field and number of myc-positive cells without aggregates but displaying a normal desmin network were counted. The percentage of cells with aggregates among all myc-positive cells was calculated, taking into account 5 fields per experimental condition (n = 1400) in 3 independent experiments. Transfection efficiency was 25%. Significant differences from the control are indicated with asterisk (p<0.05 calculated with a non-parametric test).</p