Minocycline treatment reduces mass and force output from fast-twitch mouse muscles and inhibits myosin production in C2C12 myotubes

Minocycline, a tetracycline-class of antibiotic, has been tested with mixed effectiveness on neuromuscular disorders such as amyotrophic lateral sclerosis, autoimmune neuritis and muscular dystrophy. The independent effect of minocycline on skeletal muscle force production and signalling remain poorly understood. Our aim here is to investigate the effects of minocycline on muscle mass, force production, myosin heavy chain abundance and protein synthesis. Mice were injected with minocycline (40 mg/kg i.p.) daily for 5 days and sacrificed at day six. Fast-twitch EDL, TA muscles and slow-twitch soleus muscles were dissected out, the TA muscle was snap-frozen and the remaining muscles were attached to force transducer whilst maintained in an organ bath. In C2C12 myotubes, minocycline was applied to the media at a final concentration of 10 µg/mL for 48 h. In minocycline treated mice absolute maximal force was lower in fast-twitch EDL while in slow-twitch soleus there was an increase in the time to peak and relaxation of the twitch. There was no effect of minocycline treatment on the other contractile parameters measured in isolated fast- and slow-twitch muscles. In C2C12 cultured cells, minocycline treatment significantly reduced both myosin heavy chain content and protein synthesis without visible changes to myotube morphology. In the TA muscle there was no significant changes in myosin heavy chain content. These results indicate that high dose minocycline treatment can cause a reduction in maximal isometric force production and mass in fast-twitch EDL and impair protein synthesis during myogenesis in C2C12 cultured cells. These findings have important implications for future studies investigating the efficacy of minocycline treatment in neuromuscular or other muscle-atrophy inducing conditions

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

Background: A common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world’s population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0 + 20% stretch did not cause eccentric damage. In contrast, L0 + 30% stretch produced a significant ~ 40% deficit in maximum force; here, we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. Methods: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA. Results: We show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar branching. Conclusions: The absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0 + 30%, but not L0 + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3-deficient humans have been reported to have a faster decline in muscle function with increasing age, that is, as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibre breakages

The role of branched muscle fibres and ACTN3 polymorphism as a genetic disease modifier in Duchenne nuscular dystrophy

Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease in humans, with an incidence of 1 in 3300 live male births. DMD is characterized by progressive cycles of skeletal muscle necrosis/regeneration triggered by the absence of the protein dystrophin from the inner surface of the sarcolemma. In DMD and dystrophin-negative mdx mice, regenerated skeletal muscle fibres are branched and deterioration of muscle contractile function with age is correlated with an increase in both the number and complexity of branched fibres. In this thesis, I present four papers in support of my hypothesis, that when the number and complexity of branched fibres in dystrophin-negative muscles reaches a critical threshold, termed ‘tipping point’, the branches in and of themselves, mechanically weaken the muscle and are susceptible to rupturing when subjected to high forces such as those experienced during eccentric/lengthening contractions. Methodologically, the papers utilise a combination of isolated muscle function contractile measurements coupled with single fibre imaging and confocal microscopy of cleared whole muscles. All experiments use intact muscles isolated from the dystrophic mdx mouse, double knockout (dk)Actn3KO/mdx (dKO) mouse and littermate controls. In conclusion, I propose a two-phase model to explain the aetiology of DMD. Phase-one involves the absence of dystrophin triggering a pathological increase in [Ca2+]in resulting in skeletal muscle fibre necrosis followed immediately by regeneration. The process proceeds cyclically, increasing the number of abnormally branched regenerated dystrophin-deficient muscle fibres. Once the number and complexity of branched fibres passes a level I term ‘tipping point’, phase-two occurs; now eccentric contractions cause force deficits as a consequence of branches rupturing. In the final stage, phase-two will tend to have a positive feedback component, as breaking branches will no longer support the eccentrically contracting muscle, placing additional stress on the remaining branches during the contraction. It is important to note that depending on the complexity of branching and forces experienced by the muscle, phase-one and phase-two are not mutually exclusive and will occur simultaneously

Neurodegeneration Risk Factor TREM2 R47H Mutation Causes Distinct Sex- and Age- Dependent Musculoskeletal Phenotype

Indiana University-Purdue University Indianapolis (IUPUI)Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), a receptor expressed in myeloid cells including microglia in brain and osteoclasts in bone has been proposed as a link between brain and bone disease. Previous studies identified an AD-associated mutation (R47H) which is known to confer an increased risk for developing AD. In these studies, we used a heterozygous model of the TREM2 R47H variant (TREM2R47H/+), which does not exhibit cognitive defects, as a translational model of genetic risk factors that contribute to AD, and investigated whether alterations to TREM2 signaling could also contribute to bone and skeletal muscle loss, independently of central nervous system defects. Our study found that female TREM2R47H/+ animals experience bone loss in the femoral mid-diaphysis between 4 and 13 months of age as measured by microCT, which stalls out by 20 months of age. Female TREM2R47H/+ animals also experience significant decreases in the mechanical and material properties of the femur measured by three-point bending at 13 months of age, but not at 4 or 20 months. Interestingly, male TREM2R47H/+ animals do not demonstrate any discernable differences in bone geometry or strength until 20 months of age, where we observed slight changes in the bone volume and material properties of male TREM2R47H/+ bones. Ex vivo osteoclast differentiation assays demonstrate that only male TREM2R47H/+ osteoclasts differentiate more after 7 days with osteoclast differentiation factors compared to WT, but qPCR follow-up showed sexdependent differences in intracellular signaling. However, bone is not the only musculoskeletal tissue affected by the TREM2 R47H variant. Skeletal muscle strength measured by both in vivo plantar flexion and ex vivo contractility of the soleus is increased and body composition is altered in female TREM2R47H/+ mice compared to WT, and this is not likely due to bone-muscle crosstalk. These studies suggests that TREM2 R47H expression in the bone and skeletal muscle are likely impacting each tissue independently. These data demonstrate that AD-associated variants in TREM2 can alter bone and skeletal muscle strength in a sex-dimorphic manner independent of the presence of central neuropathology

Investigating the regenerative effects of adipose-­derived mesenchymal stem cell conditioned media on sarcopenic and progeric skeletal muscle

Ageing, defined as the progressive deterioration of molecular, cellular, tissue and whole organism function, is a primary risk factor for numerous diseases, such as cardiovasculature disease, neurodegeneration and cancer. In recent years, advances in our knowledge of key determinant mechanisms that underpin ageing decline, drives the notion that these features can be attenuated and targeted therapeutically, enabling elderly individuals to experience an enhanced quality of life into advanced old age. Sarcopenia comprises the age-­‐related loss of muscle mass, quality and function and contributes to overall frailty, immobility and a greater risk of falls. The use of stem cell-­‐ derived conditioned media (CM) holds great clinical potential and recent studies have reported many beneficial effects in a number of tissue models of injury and disease. We want to develop a novel anti-­‐ageing therapy for the treatment of age-­‐associated declines in sarcopenia. First, we characterise the skeletal muscle profile in a novel use of the Ercc1d/-­‐ murine model of progeria and compare it to the naturally-­‐aged phenotype. We examine the effects of CM, generated from adipose-­‐derived mesenchymal stem cells (ADMSCs), on skeletal muscle composition, function and satellite cell (SC) activity in sarcopenia and progeria. We show that CM has beneficial regulatory effects on mechanisms underpinning the declines associated with the Hallmarks of Ageing, for example, enhancing mitochondrial function and reducing oxidative stress. Importantly, we also demonstrate that CM harbours pro-­‐angiogenic effects, which we hypothesise is unlikely to impact on skeletal muscle alone. Remarkably, we report the Ercc1d/-­‐ mice appear to launch a survival programme and delay the progression of age-­‐related deterioration. A further feature associated with ageing skeletal muscle is the impaired regenerative function and myofibre turnover following injury and daily use. Key factors attributed to this decline in repair involve compromised SC activity as well as the depletion of the stem cell pool, known to occur with age. We want to first, examine the influence of the myofibre microenvironment on SC behaviour. Second, we investigate the use of non-­‐muscle cell types as a source to generate muscle cells. We show that three stem cell types, ADMSC, dental pulp stem cells (DP) and amniotic fluid stem cells (AFS) and one non-­‐stem cell line, MDA-­‐MB-­‐231 (MDA) breast cancer cells, adopted amoeboid-­‐based migration (blebbing) once seeded onto myofibres. We also show that the regulation of the migratory mechanisms, known to be controlled by the Rac and Rho signalling pathways, is conserved in each of these cell types. Remarkably, we also demonstrate that a rapidly growing non-­‐muscle stem cell (AFS), as well as a non-­‐stem cell (MDA) initiate expression of MyoD and furthermore, the AFS cells were directed, through exposure to the myofibre microenvironment, to fuse and form myotube structures that express myosin heavy chain (MHC+)

Etude de la physiopathologie de la dystrophie musculaire tibiale et de la dystrophie des ceintures 2J et stratégies thérapeutiques

La titine est une protéine géante exprimée dans les muscles squelettiques et cardiaque. Un certain nombre de mutations pathogéniques ont été identifiées dans son dernier exon codant. La mutation la plus fréquente, FINmaj, conduit au remplacement de 4 acides aminés et est retrouvée chez de nombreux patients en Finlande. Cette mutation cause une dystrophiemusculaire tibiale (TMD) à l état hétérozygote et une dystrophie des ceintures de type 2J(LGMD2J) à l état homozygote.Pour obtenir une modèle d étude de la physiopathologie de ces maladies et évaluer des stratégiesthérapeutiques, nous avons introduit la mutation FINmaj dans le génome murin par une stratégiede Knock-In par recombinaison homologue. Ce modèle a été caractérisé et a permis de montrer qu il reproduit en grande partie les symptômes de la TMD et de la LGMD2J aux niveaux histologique et moléculaire. L étude de ce modèle murin a permis une meilleure compréhension de la physiopathologie de ces deux maladies et nous amené à étudier plus attentivement les interactions de la titine en C-ter avec ses partenaires afin de mieux comprendre l implication de la ligne M dans la vie du sarcomère. L étude de la physiopathologie de la TMD et de la LGM2J a permis de montrer que la calpaïne 3 (une protéase à l origine d une autre dystrophie des ceintures), jouait un rôle majeur dans laTMD. Cette constatation nous a permis d envisager une approche thérapeutique pour cette dernière visant à diminuer les symptômes en régulant négativement la calpaïne 3. Une approche de thérapie génique a aussi été testée dans le but de traiter ces deux pathologies: le trans-épissage des derniers exons de la titine. En effet, étant donné la grande taille de l'ADNc de la titine (~100 kb), des stratégies classiques de transfert de gène n étaient pas envisageables. Pour s'affranchir de ce problème, nous avons testé une approche de trans-épissage de l ARN pour remplacer le ou les derniers exons du messager de la titine. Nous avons pu ainsi démontrer la faisabilité de la correction de la titine in vitro.Titin is a giant protein expressed in both skeletal muscles and heart. Several pathogenic mutations were identified in its last coding exon. The most frequent mutation commonly referred to as FINmaj, results in the replacement of 4 amino acids and affects a subset of patients in Finland. The mutation causes a Tibial Muscular Dystrophy (TMD) when present on one allele and a Limb Girdle Muscular Dystrophy phenotype 2J (LGMD2J) when present on both alleles.To obtain a model for studying the physiopathology of these diseases and evaluating therapeutic strategies, we introduced the FINmaj mutation in the murine genome by a knock-in strategy by homologous recombination. This model was characterized and showed that it reproduces mainly the symptoms of both the human TMD and LGMD2J at histological and molecular levels.The study of this mouse model has allowed a better understanding of the pathophysiology of these two diseases and we have to study more carefully the interactions beyond titin C-ter with its partners to better understand the involvement of the M line in life of the sarcomere.The study of the pathophysiology of TMD and LGM2J showed that calpain 3 (a protease thatlind to an other limb-girdle muscular dystrophy), played a major role in TMD. This finding allowed us to consider a treatment approach for TMD to reduce symptoms by regulating negatively calpain 3. A gene therapy approach was also tested: the trans-splicing of the last exon of titin.Indeed, given the large size of the cDNA of titin (~ 100 kb), conventional strategies of genetransfer were not envisaged. To overcome this problem, we tested an approach to exchange the last exon or the last exons of the titin messenger. We were able to demonstrate the correction of titin in vitro.EVRY-Bib. électronique (912289901) / SudocSudocFranceF