Duchenne muscular dystrophy – What causes the increased membrane permeability in skeletal muscle?

doi:10.1016/j.biocel.2010.11.005

The International Journal of Biochemistry & Cell Biology

Volume 43, Issue 3, March 2011, Pages 290-294

https://doi.org/10.1016/j.biocel.2010.11.005 Get rights and content

Abstract

Duchenne muscular dystrophy is a severe muscle wasting disease caused by a mutation in the gene for dystrophin – a cytoskeletal protein connecting the contractile machinery to a group of proteins in the cell membrane. At the end stage of the disease there is profound muscle weakness and atrophy. However, the early stage of the disease is characterised by increased membrane permeability which allows soluble enzymes such as creatine kinase to leak out of the cell and ions such as calcium to enter the cell. The most widely accepted theory to explain the increased membrane permeability is that the absence of dystrophin makes the membrane more fragile so that the stress of contraction causes membrane tears which provide the increase in membrane permeability. However other possibilities are that increases in intracellular calcium caused by altered regulation of channels activate enzymes, such as phospholipase A₂, which cause increased membrane permeability. Increases in reactive oxygen species (ROS) are also present in the early stages of the disease and may contribute both to membrane damage by peroxidation and to the channel opening. Understanding the earliest phases of the pathology are critical to therapies directed at minimizing the muscle damage.

Introduction

Duchenne muscular dystrophy (DMD) is a severe degenerative disease of muscle which affects boys who have a mutation in the dystrophin gene leading to absence of the dystrophin protein in muscle. Dystrophin is a cytoskeletal protein which links intracellular γ-actin of the cytoskeleton to a group of proteins in the cell membrane, the dystrophin-associated protein complex (DAPC). The DAPC is further linked to the extracellular matrix through laminin (Fig. 1A). In DMD not only is dystrophin absent, but the proteins of the DAPC are also greatly reduced (Ervasti and Campbell, 1991) while several other proteins normally associated with the DAPC show increased expression (Gervasio et al., 2008) (Fig. 1B). While the primary cause of the disease is the absence of dystrophin, the complex pathways which link the absence of dystrophin to the profound muscle wasting, inflammation and fibrosis observed at the end stage of the disease are unclear.

A cardinal feature of the disease, present from birth and before physical symptoms, is a very large elevation of plasma creatine kinase suggesting that there is increased permeability of the muscle surface membrane allowing soluble muscle enzymes to leak out of the cell. Early electron microscopy studies on DMD described focal disruptions of the surface membrane and noted contracture of the neighbouring myofibrils (Mokri and Engel, 1975). This first led to the hypothesis that damage to the surface membrane was an early feature of the disease and the suggestion that Ca²⁺ influx through a membrane defect might contribute to the disease. Experimentally the increased membrane permeability has been repeatedly confirmed by studies in which markers which are normally membrane impermeant, such as albumin and Evans Blue dye, can be found inside muscle fibres.

In order to understand the earliest phase of the disease, a key question is the mechanism whereby the absence of dystrophin exacerbates the increase in membrane permeability membrane. A popular view is that contraction can cause mechanical injury (membrane tears) and that, in the absence of dystrophin, the sarcolemma is more fragile and therefore predisposed to membrane tears (Petrof et al., 1993, Davies and Nowak, 2006). The purpose of this article is to review the evidence for the hypothesis that membrane tears are the cause of the increased membrane permeability. We believe the evidence for this hypothesis is weak and discuss alternative mechanisms for the increased membrane permeability.

Section snippets

Evidence for membrane tears

Muscles are subjected to stress and strain during normal contractions and these are exacerbated when the muscle is stretched by a large external force during a contraction (eccentric contraction). It has been known for many years that eccentric contractions in normal people lead to a mild form of muscle damage, characterised by weakness and delayed onset of swelling, stiffness and soreness. It is also known that leakage of creatine kinase from the muscle occurs during this delayed damage so

Alternative explanations for increased membrane permeability after stretched contractions

As noted above, if the cause of increased permeability were membrane tears one would predict increases in permeability starting immediately after the stretched contraction and persisting only for a minute or so. Instead there is a slow increase in intracellular Na⁺ ([Na⁺]_i) and [Ca²⁺]_i starting after the contractions and reaching a maximum after 10–20 min (for review see Allen et al., 2010). Furthermore the increase in ion levels is eliminated by drugs which block stretch-activated channels

Role of absence of dystrophin

On the interpretation described above key events in the development of increased membrane permeability are (i) the elevation of [Ca²⁺]_i and (ii) the production of ROS. The evidence discussed above suggests that the elevated [Ca²⁺]_i arises through activation of a Ca²⁺ permeable channel, probably a stretch-activated channel. A series of electrophysiological studies have demonstrated increased channel activity in mdx muscles but the identity of the channel remains unclear (for review see Allen et

Conclusions

Currently there is no treatment for DMD which prevents or reverses the inevitable progression of the disease. There are high hopes that gene or stem cell therapy in one of its many forms will eventually replace the missing dystrophin and lead to a definitive treatment. The most advanced clinical approach is anti-sense oligonucleotide treatment which allows synthesis of a shortened dystrophin with the mutated exon deleted (Kinali et al., 2009). However the best expectation for this treatment is

Acknowledgements

Supported by the National Health and Medical Research Council of Australia.

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The fragility of muscle cell membrane increases the influx of Ca2+ ions and efflux of creatine kinase. Increased Ca2+ contributes to damage of muscle membrane and ROS (Reactive oxygen species) generation (Allen and Whitehead, 2011). To repair muscle damage, body stimulates inflammatory cytokines and immune cells such as T-lymphocytes (CD4+, CD8+) and monocytes followed by increases the severity of muscle pathology.
Muscular dystrophy is a well-known genetically heterogeneous group of rare muscle disorders. This progressive disease causes the breakdown of skeletal muscles over time and leads to grave weakness. This breakdown is caused by a diverse pattern of mutations in dystrophin and dystrophin associated protein complex. These mutations lead to the production of altered proteins in response to which, the body stimulates production of various cytokines and immune cells, particularly reactive oxygen species and NFκB. Immune cells display/exhibit a dual role by inducing muscle damage and muscle repair. Various anti-oxidants, anti-inflammatory and glucocorticoid drugs serve as potent therapeutics for muscular dystrophy. Along with the above mentioned therapeutics, induced pluripotent stem cells also serve as a novel approach paving a way for personalized treatment. These pluripotent stem cells allow regeneration of large numbers of regenerative myogenic progenitors that can be administered in muscular dystrophy patients which assist in the recovery of lost muscle fibers. In this review, we have summarized gene-editing, immunological and induced pluripotent stem cell based therapeutics for muscular dystrophy treatment.
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Further, both primary and immortalized DMD lines displayed stunted contractile apparatus protein levels on day 4 under 2D differentiation conditions when compared to the healthy lines. Since dystrophin links the cytoskeleton of muscle fibers to the extracellular matrix (ECM) [1], and serves to facilitate the transmission of forces generated by the actino-myosin sarcomeric machinery to the sarcolemma and surrounding ECM [12,13], we sought to evaluate DMD-associated phenotypes in the context of a 3D ECM culture environment, and focused our attention on a panel of immortalized DMD myoblast lines (Supplementary Table 1). We leveraged our previously described MyoTACTIC culture device [17,43,51], which supports the self-organization of aligned myotubes across two flexible micro-posts within days of seeding the cell/ECM mixture into the oval-shaped culture reservoir.
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Medicine in focusDuchenne muscular dystrophy – What causes the increased membrane permeability in skeletal muscle?

Abstract

Introduction

Section snippets

Evidence for membrane tears

Alternative explanations for increased membrane permeability after stretched contractions

Role of absence of dystrophin

Conclusions

Acknowledgements

Prog Biophys Mol Biol

J Mol Cell Cardiol

Cell

J Biol Chem

Lancet Neurol

J Biol Chem

Neurosci Lett

Neuromuscular Disorders

Calcium and the damage pathways in muscular dystrophy

Can J Physiol Pharmacol

Defective membrane repair in dysferlin-deficient muscular dystrophy

Nature

Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers

J Cell Sci

Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle

J Cell Sci

Molecular mechanisms of muscular dystrophies: old and new players

Nat Rev Mol Cell Biol

Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle

J Cell Sci

Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

J Physiol

Caveolinopathies: from the biology of caveolin-3 to human diseases

Eur J Hum Genet

TRPC1 binds to caveolin-3 and is regulated by Src kinase: role in Duchenne muscular dystrophy

J Cell Sci

Revisiting TRPC1 and TRPC6 mechanosensitivity

Pflugers Arch

Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death

J Gen Physiol

Medicine in focus
Duchenne muscular dystrophy – What causes the increased membrane permeability in skeletal muscle?

Ca²⁺-independent phospholipase A₂ enhances store-operated Ca²⁺ entry in dystrophic skeletal muscle fibers