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.
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 ΔCa2+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 ΔCa2+i during a tetanus; and (2) a mitigation of the fall in ΔCa2+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.
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.
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.
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.
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.
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
...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
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.
Duchenne muscular dystrophy is characterized by progressive wasting and cycles of regeneration in skeletal muscle. Our laboratory work suggests, branched fibres, could be responsible for the terminal ...phase of muscle damage in old (58–112 weeks) dystrophic mice. A recent study in 12 week old dystrophic mice reported that the majority of force loss produced by a series of eccentric contractions (EC) in extensor digitorum longus (EDL) muscles recovers (65%) within 120 minutes, concluding this is incompatible with the assumption that EC force loss is due to mechanical damage.
The aim of this project was to assess the recovery post EC damage in adult (~16 weeks) and old (~88 weeks) dystrophic muscles. Male mdx mice and littermate controls were euthanized, EDL muscles dissected from the hind limbs and maintained in Krebs solution at room temperature. The muscles were maximally stimulated, and a series of 6× EC performed to assess force loss. Muscles were left to rest for up to 120 minutes whilst measuring recovery. Single muscle fibres were isolated enzymatically to assess the degree of fibre branching.
Our findings replicated force recovery in young mdx mice with simple fibre branches. However, minimal recovery (~23%) in muscles of 88 week old mdx mice at 120 minutes post EC was observed. This data supports our “tipping point” hypothesis and shows a distinct pathophysiology in aged mdx mice with EC force loss due to acute fibre rupture at branch nodes that occurs in “old” dystrophic EDL muscles with >70% complex branched fibres. These findings have important implications for pre‐clinical drug studies that use protection from EC damage in young mdx mice as a marker for drug efficacy.
A better understanding of the pathology of muscle damage in the mdx mice will improve our ability to test and interpret pre‐clinical drug studies using this model.
Olthoff,
J. T.,
Lindsay,
A.,
Abo-Zahrah,
R.,
Baltgalvis,
K. A.,
Patrinostro,
X.,
Belanto,
J. J., . . .
Ervasti,
J. M. (
2018).
Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.
Nat Commun,
9(
1),
5104. doi:
10.1038/s41467-018-07639-3
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
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
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
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
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.
•Lysozyme was loaded in chitosan boiadhesive to enhance rate of depolymerization.•The impact of the laser used for adhesive photoactivation on lysozyme was assessed.•Loading lysozyme in the ...bioadhesive enhanced the depolymerization rate.•Irradiation with laser did not affect the depolymerization of adhesive by lysozyme.
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.
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.
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