Sarcoplasmic reticulum calcium handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wild-type littermates.

NEW FINDINGS: What is the central question of this study? What are the early effects of dystrophin deficiency on sarcoplasmic reticulum Ca2+ handling in the mdx mouse? What is the main finding and its importance? In the mdx mouse, Ca2+ handling by the sarcoplasmic reticulum is little affected by the absence of dystrophin when looking at fibres without branches that have recently regenerated after massive myonecrosis. This has important implications for our understanding of Ca2+ pathology in the mdx mouse. ABSTRACT: There is a variety of results in the literature regarding the effects of dystrophin deficiency on the Ca2+ handling properties of the sarcoplasmic reticulum (SR) in the mdx mouse, an animal model of Duchenne muscular dystrophy. One possible source of variation is the presence of branched fibres. Fibre branching, a consequence of degenerative-regenerative processes such as muscular dystrophy, has in itself a significant influence on the function of the SR. In this study, we attempted to detect early effects of dystrophin deficiency on SR Ca2+ handling by using unbranched fibres from the immediate post-necrotic stage in mdx mice (recently regenerated after massive necrosis). Using kinetically corrected fura-2 fluorescence signals measured during twitch and tetanus, we analysed the amplitude, rise time and decay time of Δ[Ca2+ ]i in unfatigued and fatigued fibres. Decay was also resolved into SR pump and SR leak components. Fibres from mdx mice were similar in all respects to fibres from wild-type littermates apart from: (1) a smaller amplitude of the initial spike of Δ[Ca2+ ]i during a tetanus; and (2) a mitigation of the fall in Δ[Ca2+ ]i amplitude during the course of fatigue. Our findings suggest that the early effects of a loss of dystrophin on SR Ca2+ handling in mdx mice are subtle, and we emphasize the importance of distinguishing between Ca2+ pathology that is attributable to lack of dystrophin and Ca2+ pathology that is attributable to muscle degeneration.

Dystrophin-negative slow-twitch soleus muscles are not susceptible to eccentric contraction induced injury over the lifespan of the mdx mouse.

Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease in humans and is characterized by the absence of a functional copy of the protein dystrophin from skeletal muscle. In dystrophin-negative humans and rodents, regenerated skeletal muscle fibers show abnormal branching. The number of fibers with branches and the complexity of branching increases with each cycle of degeneration/regeneration. Previously, using the mdx mouse model of DMD, we have proposed that once the number and complexity of branched fibers present in dystrophic fast-twitch EDL muscle surpasses a stable level, we term the "tipping point," the branches, in and of themselves, mechanically weaken the muscle by rupturing when subjected to high forces during eccentric contractions. Here, we use the slow-twitch soleus muscle from the dystrophic mdx mouse to study prediseased "periambulatory" dystrophy at 2-3 wk, the peak regenerative "adult" phase at 6-9 wk, and "old" at 58-112 wk. Using isolated mdx soleus muscles, we examined contractile function and response to eccentric contraction correlated with the amount and complexity of regenerated branched fibers. The intact muscle was enzymatically dispersed into individual fibers in order to count fiber branching and some muscles were optically cleared to allow laser scanning confocal microscopy. We demonstrate throughout the lifespan of the mdx mouse that dystrophic slow-twitch soleus muscle is no more susceptible to eccentric contraction-induced injury than age-matched littermate controls and that this is correlated with a reduction in the number and complexity of branched fibers compared with fast-twitch dystrophic EDL muscles.

Branched fibers from old fast-twitch dystrophic muscles are the sites of terminal damage in muscular dystrophy.

A striking pathological feature of dystrophinopathies is the presence of morphologically abnormal branched skeletal muscle fibers. The deterioration of muscle contractile function in Duchenne muscular dystrophy is accompanied by both an increase in number and complexity of these branched fibers. We propose that when number and complexity of branched fibers reaches a critical threshold, or "tipping point," the branches in and of themselves are the site of contraction-induced rupture. In the present study, we use the dystrophic mdx mouse and littermate controls to study the prediseased dystrophic fast-twitch extensor digitorum longus (EDL) muscle at 2-3 wk, the peak myonecrotic phase at 6-9 wk, and finally, "old," at 58-112 wk. Using a combination of isolated muscle function contractile measurements coupled with single-fiber imaging and confocal microscope imaging of cleared whole muscles, we identified a distinct pathophysiology, acute fiber rupture at branch nodes, which occurs in "old" fast-twitch EDL muscle approaching the end stage of the dystrophinopathy muscle disease, where the EDL muscles are entirely composed of complexed branched fibers. This evidence supports our concept of "tipping point" where the number and extent of fiber branching reach a level where the branching itself terminally compromises muscle function, irrespective of the absence of dystrophin.

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.

Lifespan Analysis of Dystrophic mdx Fast-Twitch Muscle Morphology and Its Impact on Contractile Function.

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient mdx mouse model, we studied the morphological and contractile chronology of dystrophic skeletal muscle pathology in fast-twitch Extensor Digitorum Longus muscles from animals 4-22 months of age containing 100% regenerated muscle fibers. Catastrophically, the older age groups lost ∼80% of their maximum force after one eccentric contraction (EC) of 20% strain with the greatest loss of ∼92% recorded in senescent 22-month-old mdx mice. In old age groups, there was minimal force recovery ∼24% after 120 min, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-phase model where a "tipping point" is reached when branched fibers rupture irrevocably on EC. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (>24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 °C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at >24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Protein kinase CK2 regulates metal toxicity in neuronal cells.

Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 µM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and ß) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

Lysozyme depolymerization of photo-activated chitosan adhesive films.

Effective tissue bioadhesion of rose bengal-chitosan films can be achieved by photoactivation using a green laser. In this study, lysozyme was incorporated in these films to enhance the rate of depolymerization and assess the laser impact on lysozyme. The lysozyme loaded films exhibited a 21% mass loss after 4 weeks implantation in rats while control films (without lysozyme) had only 7% mass loss. Capillary electrophoresis-mass spectroscopy showed that chitosan degraded into monomers and oligomers of glucosamine and N-acetyl-glucosamine. Irradiation with laser did not affect the depolymerization of adhesive by lysozyme suggesting that the inclusion of lysozyme in the bioadhesive is a viable technique for tailoring the depolymerization.