Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching

The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin-deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this, we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model, which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse, we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%), and this correlates with a graded force loss over three eccentric contractions for dKO muscles (~36% after first contraction, ~66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (~75% after first contraction, ~89% after three contractions). In dKO, protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies

Evidence for ACTN3 as a genetic modifier of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and progressive weakness. There is considerable inter-patient variability in disease onset and progression, which can confound the results of clinical trials. Here we show that a common null polymorphism (R577X) in ACTN3 results in significantly reduced muscle strength and a longer 10\u2009m walk test time in young, ambulant patients with DMD; both of which are primary outcome measures in clinical trials. We have developed a double knockout mouse model, which also shows reduced muscle strength, but is protected from stretch-induced eccentric damage with age. This suggests that \u3b1-actinin-3 deficiency reduces muscle performance at baseline, but ameliorates the progression of dystrophic pathology. Mechanistically, we show that \u3b1-actinin-3 deficiency triggers an increase in oxidative muscle metabolism through activation of calcineurin, which likely confers the protective effect. Our studies suggest that ACTN3 R577X genotype is a modifier of clinical phenotype in DMD patients

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

Six weeks of N-acetylcysteine antioxidant in drinking water decreases pathological fiber branching in MDX mouse dystrophic fast-twitch skeletal muscle

Introduction: It has been proposed that an increased susceptivity to oxidative stress caused by the absence of the protein dystrophin from the inner surface of the sarcolemma is a trigger of skeletal muscle necrosis in the destructive dystrophin deficient muscular dystrophies. Here we use the mdx mouse model of human Duchenne Muscular Dystrophy to test the hypothesis that adding the antioxidant NAC at 2% to drinking water for six weeks will treat the inflammatory phase of the dystrophic process and reduce pathological muscle fiber branching and splitting resulting in a reduction of mass in mdx fast-twitch EDL muscles. Methods: Animal weight and water intake was recorded during the six weeks when 2% NAC was added to the drinking water. Post NAC treatment animals were euthanised and the EDL muscles dissected out and placed in an organ bath where the muscle was attached to a force transducer to measure contractile properties and susceptibility to force loss from eccentric contractions. After the contractile measurements had been made the EDL muscle was blotted and weighed. In order to assess the degree of pathological fiber branching mdx EDL muscles were treated with collagenase to release single fibers. For counting and morphological analysis single EDL mdx skeletal muscle fibers were viewed under high magnification on an inverted microscope. Results: During the six-week treatment phase NAC reduced body weight gain in three- to nine-week-old mdx and littermate control mice without effecting fluid intake. NAC treatment also significantly reduced the mdx EDL muscle mass and abnormal fiber branching and splitting. Discussion: We propose chronic NAC treatment reduces the inflammatory response and degenerative cycles in the mdx dystrophic EDL muscles resulting in a reduction in the number of complexed branched fibers reported to be responsible for the dystrophic EDL muscle hypertrophy

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

A mathematical and computational review of Hartree-Fock SCF methods in Quantum Chemistry

We present here a review of the fundamental topics of Hartree-Fock theory in Quantum Chemistry. From the molecular Hamiltonian, using and discussing the Born-Oppenheimer approximation, we arrive to the Hartree and Hartree-Fock equations for the electronic problem. Special emphasis is placed in the most relevant mathematical aspects of the theoretical derivation of the final equations, as well as in the results regarding the existence and uniqueness of their solutions. All Hartree-Fock versions with different spin restrictions are systematically extracted from the general case, thus providing a unifying framework. Then, the discretization of the one-electron orbitals space is reviewed and the Roothaan-Hall formalism introduced. This leads to a exposition of the basic underlying concepts related to the construction and selection of Gaussian basis sets, focusing in algorithmic efficiency issues. Finally, we close the review with a section in which the most relevant modern developments (specially those related to the design of linear-scaling methods) are commented and linked to the issues discussed. The whole work is intentionally introductory and rather self-contained, so that it may be useful for non experts that aim to use quantum chemical methods in interdisciplinary applications. Moreover, much material that is found scattered in the literature has been put together here to facilitate comprehension and to serve as a handy reference.Comment: 64 pages, 3 figures, tMPH2e.cls style file, doublesp, mathbbol and subeqn package

Age- and Gender-Related Changes in Contractile Properties of Non-Atrophied EDL Muscle

Background: In humans, ageing causes skeletal muscles to become atrophied, weak, and easily fatigued. In rodent studies, ageing has been associated with significant muscle atrophy and changes in the contractile properties of the muscles. However, it is not entirely clear whether these changes in contractile properties can occur before there is significant atrophy, and whether males and females are affected differently. Methods and Results: We investigated various contractile properties of whole isolated fast-twitch EDL muscles from adult (2–6 months-old) and aged (12–22 months-old) male and female mice. Atrophy was not present in the aged mice. Compared with adult mice, EDL muscles of aged mice had significantly lower specific force, longer tetanus relaxation times, and lower fatiguability. In the properties of absolute force and muscle relaxation times, females were affected by ageing to a greater extent than males. Additionally, EDL muscles from a separate group of male mice were subjected to eccentric contractions of 15 % strain, and larger force deficits were found in aged than in adult mice. Conclusion: Our findings provide further insight into the muscle atrophy, weakness and fatiguability experienced by the elderly. We have shown that even in the absence of muscle atrophy, there are definite alterations in the physiological properties of whole fast-twitch muscle from ageing mice, and for some of these properties the alterations are mor

Expected Performance of the ATLAS Experiment - Detector, Trigger and Physics

A detailed study is presented of the expected performance of the ATLAS detector. The reconstruction of tracks, leptons, photons, missing energy and jets is investigated, together with the performance of b-tagging and the trigger. The physics potential for a variety of interesting physics processes, within the Standard Model and beyond, is examined. The study comprises a series of notes based on simulations of the detector and physics processes, with particular emphasis given to the data expected from the first years of operation of the LHC at CERN