Duchenne Muscular Dystrophy Cell Bio


The term muscular dystrophy (MD) describes a group of approximately 40 inherited disorders characterized by progressive muscle weakness and wasting, with the most common and severe form being Duchenne muscular dystrophy (DMD).1 DMD is an inherited X-linked recessive disorder with an incidence of 1 in 3500 live male births.1 2 3 4 5 Onset of symptoms usually occurs in the preschool years between the ages of 2 and 5,4 with definitive diagnosis between 54 and 6 years of age.3 DMD is characterized by progressive symmetric muscle weakness, gait disturbances and progressive muscle degeneration and substitution with fat and connective tissue, stemming from progressive loss of contractile function.1 2 3 4 This loss of contractile function is due to the absence of the cellular protein dystrophin.1 3 4 If dystrophin remains present, but in an altered form, it will result in Becker muscular dystrophy (BMD), a less severe form of MD.6 Knowing this, current research in DMD is aimed at restoring the production of dystrophin, resulting in a clinical presentation similar to the milder BMD.6 Presently, there is no treatment for DMD, but current management of the disease has increased life expectancy into the late 20s and 30s.4


Figure 2. Normal Dystrophin Function


Normal Function

Dystrophin is a relatively large protein (427 kDa) found in the cytoskeleton of various cells, mostly skeletal, cardiac, and smooth muscle cells.7 8 Research has shown dystrophin is located on the cytoplasmic side of the cell membrane, linking the cytoskeleton to a dystrophin protein complex (DPC) on the surface membrane of muscle fibers, which then interact with the extracellular matrix. The attachment point of dystrophin to the muscle extracellular matrix is thought to be located at the merosin (laminin M) component. 7 8 Although the exact mechanisms of dystrophin have been elusive, numerous suggestions have linked dystrophin to maintenance of cell membrane integrity, prevention of cell membrane damage caused by muscle contraction or stretch, regulation of normal cell membrane permeability, assistance with calcium homeostasis, and maintenance of cell membrane stiffness.7 8 9 10

Genetic research has identified the primary defect which is responsible for the amount of dystrophin deficiency seen in DMD. It has been localized to a mutation of the dystrophin gene, found at the Xp21 band on the short arm of the X chromosome, meaning males are primarily afflicted with DMD.1 2 3 4 7 8 9 10 DMD results when the mutation on the X chromosome disrupts the translational reading frame.6

Abnormal Function

Individuals expressing the genetic mutation at the dystrophin gene exhibit significantly diminished amounts of dystrophin in skeletal muscle, which has been shown to be absent or greatly deficient (less than 3% of normal amounts) in patients with DMD.7 Dystrophic muscles also display greatly reduced amounts of other DPC glycoproteins associated with the cell membrane.11 Research has illustrated that this deficiency leads to widespread consequences affecting multiple cellular processes ultimately leading to muscle cell membrane breakdown, apoptosis and necrosis.


DMD is a fatal neuromuscular disease affecting boys that unfortunately has a 100% mortality rate.12 13 As with many diseases, animal models have played an important role in trying to improve the quality of life and possibly find a definitive cure for individuals with DMD.12 Fortunately for scientists, in contrast to most neuromuscular pathologies, several animal models are available for use in DMD research.12 A lot has been discovered about the underlying cellular mechanisms of DMD from the animal model studies.12 When looking for appropriate animal models, researchers look for models that allow a reliable prediction of the response to a given therapeutic intervention in humans.12 In the case of DMD, the animals also need to have similar underlying genetic defects comparable to patients with DMD.12 Homologues of DMD have been identified in several animals including mice, dogs, cats, fish, and invertebrates.12 13 14 These models have been used to explore therapeutic approaches such as cell replacement and cell transplantation strategies.13

mdx Mouse Model

One of three naturally occuring mammalian animal models is the C57BL/10ScSn mdx mouse, commonly referred to as the mdx mouse.15 16 The mdx mouse is a genetic and biochemical homologue of DMD, carrying a point mutation on exon 23 of the mouse dystrophin gene introducing a premature stop codon, leading to the absence of full-length dystrophin.12 13 14 Despite the absence of dystrophin expression in the muscles, the mdx mice display a relatively mild phenotype compared to DMD patients.14 The first expression of muscle pathology occurs between 2-4 weeks of age with a marked degeneration and regeneration of muscle fibers.17 Fibrosis in most mdx mouse muscle is less pronounced than in DMD patients with the exception of the diaphragm muscle.18 Respiratory pathology is evident in older mice, indicating that respiratory capacity decreases with age.19 Abnormal cardiac function is also seen in the mdx mouse sharing several characteristics of DMD-associated cardiomyopathy.20 Mdx mice have been used to explore gene therapies, somatic gene transfer techniques, and oligonucleotide therapies.13 However, due to the small size of the mdx mice, they cannot be used to address certain issues associated with DMD, such as performing gene or cell therapy on large volumes of muscle. In addition to the mdx mouse, researchers have also characterized several additional strains that have different mutations and differential expression of dystrophin isoforms and similar pathological phenotype.14 However, the mdx mouse still remains the most valuable animal model of the mice.14

Canine Model

The second naturally occuring mammalian animal model is the canine model (cxmd).21 Spontaneous mutations of the dystrophin gene resulting in X-linked muscular dystrophy have been identified in several breeds of the domestic dog: the Golden Retriever, the Rottweiler, the German short-haired pointer, and a beagle model.13 22 23 24 Canine cxmd results from a point mutation at the 3’ consensus splice site of intron 6 of the dystrophin gene, which leads to skipping of exon 7, a disruption in the open reading frame, and premature termination of translation.21 Of the canine cxmd models, the pathogenesis of the Golden Retriever dystrophic (GRMD) dog is the most similar to that of human patients with DMD, characterized by progressive muscle wasting, degeneration and fibrosis, and a shortened life span.14 GRMD dogs suffer very rapidly from the disease, much like it is seen in humans.12 Incomplete muscle repair of lesions start to develop in utero and between the ages of 6-9 weeks affected dogs begin to show symptoms including progressive weakness and gait abnormalities leading to pronounced muscle atrophy, fibrosis, and contractures by the age of 6 months.25 The dogs quickly become less active and typically lose the ability to walk by 12 months of age.25 The dogs typically die from cardiac or respiratory complications within days, months, or 2 to 4 years after birth, depending on the severity of the disease.25 Research on the GRMD models has focused on myoblast transfer, gene transfer, and oligonucleotide therapy.13 Beagle dogs have been mated with affected Golden Retrievers to obtain a breed of a smaller size which is an advantage for some therapeutical approaches. Although this model has not been widely used, it seems to be an attractive alternative to the golden retriever because it has an improved survival rate and are ideal candidates for drug therapies.12 Even though the GRMD models seem to be the most appropriate animal model in DMD research, they are not an ideal laboratory animal.13 14 Colonies are expensive to maintain, require extra daily care, and there is a high degree of variability in disease severity between breeds and among littermates although they all carry the same genetic mutation.12 14

Feline Model

The final naturally-occuring dystrophin deficient mammalian animal model is the feline model, known as the hypertrophic feline muscular dystrophy (hfmd) model.12 This model lacks approximately 200 kb of the dystrophin gene.26 The skeletal muscle of the hfmd cat undergoes cycles of degeneration and regeneration but, unlike humans with DMD, it does not develop the debilitating fibrosis that is characteristic of DMD.13 The cats eventually die due to compression of the esophagus by the hypertrophied diaphragm or because of impaired water intake caused by glossal hypertrophy, which then leads to renal failure.27 It is not widely used as a model due to the limited pathological similarity to DMD.13

Non-Mammalian Models

Two non-mammalian models of disease have been used in DMD-related gene analysis and drug studies.28 29 Zebrafish30 and the nematode C. elegans29 both express a dystrophin homologue and are useful models due to the fact that they can be maintained in large numbers, are readily genetically manipulable, and they exemplify physiological simplicity. However, these are not ideal models due to the fact that they are non-mammalian models which have different musculature and pathology from human patients with DMD.13

When deciding what animal model is the best option in DMD research many criteria need to be considered. These conditions include: the genetic basis of the disease should be the same for both humans and the animal model; the model should demonstrate key hallmarks of the disease, DMD; the animals should be readily available, easy to handle and maintainable in standard laboratory conditions; disease progression should be well characterized and documented; and the phenotype should be reproducible.12 Based on these conditions, the feline and non-mammalian models are not yet characterized or well documented and the pathology differs to a large degree from humans.12 Therefore, the mdx mouse and GRMD dog are currently the best option for animal models in DMD research. Although the GRMD model is the closest model of the human disease, it is an expensive model to maintain. The mdx mouse has the advantages of being small, reproducible, commercially available, inexpensive, and well researched.


Figure 4. Pathophysiology of Muscular Dystrophy



People with DMD experience chronic inflammation and impaired regeneration in addition to dystrophin mutations.31 32 The inflammatory cascade begins shortly after birth with increased presence of inflammatory gene clusters found prior to symptom onset as early as 8-10 months after birth.33 It has been proposed that in people with DMD, the cell membrane has increased permeability due to a plasma membrane defect in the muscle fibers.34 The exact mechanism for leakage is unknown, however it is hypothesized that there is a disruption in the lipid bilayer. This membrane permeability is increased following exercise suggesting damage may have a mechanical component (ie: exercise stress induces tears or clefts allowing for leakage.)35 36 An alternative hypothesis suggests macrophage cytotoxicity is responsible for membrane lysis rather than mechanical stress.37

Regardless of the mechanism, increased permeability of the cell membrane allows efflux of intracellular creatine kinase (CK) into the serum along with an influx of extra-cellular calcium.34 36 Persistent inflammation in combination with poor regeneration causes the progressive muscular degeneration characteristic of DMD. The inflammatory process in DMD is not fully understood as there are a number of possible locations for abnormal cellular communication or function.

Following the inflammatory cascade, it is hypothesized that profibrotic cytokine transforming growth factor (TGF-B1) is induced leading to muscle wasting due to failed regeneration.33 37 The ability for muscle cells to regenerate is lost in DMD, this is assumed to be due to an exhaustion of satellite cells during ongoing degeneration and regeneration cycles.31

The following is a list of some of the cellular agents present in the complex inflammatory cascade of DMD.

Creatine Kinase (CK)

In a typical cell, CK catalyzes the creation of creatine and adenosine triphosphate (ATP) from phosphocreatine and ADP.34 In DMD, physical activity leads to a massive release of CK and a respondent inflammatory response36 CK levels are significantly increased from 200 units/L in healthy individuals to 5,000-15,000 units/L in people with DMD.36 Patients with DMD who have been active exhibit increased levels of serum CK levels which are notably decreased with bedrest.35


Figure 5. Calcium’s Role in Inflammation


Due to membrane permeability, it is hypothesized that a large amount of calcium is allowed into the cell. This increase in intracellular calcium may stimulate the calcium activated neutral protease (m-CANP) and other proteases which debase the muscle proteins by promoting myofibrillar protein degeneration.33 38

Toll-like receptors (TLRs)

These proteins are pattern recognition receptors. They recognize pathogen-associated molecular patterns. TLRs are one of the earliest recognizable markers of inflammation. TLRs orchestrate the inflammatory response by a cascade of cellular communication. In DMD, TRLs (specifically TRL-7) are strongly activated. One theory is that the up-regulation of TLRs creates a downstream event resulting in increased activity of the NF-kB pathway.33

Nuclear Factor Kappa B (NF-kB)

NF-kB is a downstream inflammatory-response transcription factor found in the cytosol. It is initially bound to IkB proteins.33 39 In response to inflammatory cytokines, NF-kB is released from IkB protein bondage and translocates to the nucleus to regulate gene expression. Specifically, in DMD, NF-kB promotes expression of HLA-A, HLA-B, and HLA-C target genes as well as cytokines and chemokines.33 HLA genes recognize molecules as non-self and initiate an infiltration of macrophages or induce apoptosis of cells that have been identified as pathogenic.32 40

The NF-kB pathway is chronically active in dystrophinopathies including DMD. The classic chronic inflammation associated with DMD is decreased with pharmacological inhibition of the NF-kB pathway. It is possible that the NF-kB pathway not only promotes inflammation and degeneration, but may simultaneously limit the activation of progenitor populations interfering with their ability to repair damaged muscle tissue.39


Figure 6. Inflammatory Immune Response in a Simplified Diagram

Tumor Necrosis Factor-alpha (TNF-alpha)

TNF-alpha is a pro-inflammatory cytokine that is increased in blood serum approximately 1000 times in DMD.31 The exact influence of TNF-alpha on DMD has not been determined. TNF-alpha accumulates quickly in damaged myofibers and may have a direct role in regenerating muscle, however it is also hypothesized that increased TNF-alpha may directly inhibit muscle function and increase muscle breakdown and fibrosis.31 33 37 TNF-alpha also induces NF-kB activity.39


In patients with DMD, the rennin-angiotensin system increases the fibrotic cytokine TGF-B and upregulates TNF-alpha which further perpetuates the inflammatory process. Fibrosis occurs as matrix metalloproteinase (MMP) inhibitors such as TIMP1 and 2 are upregulated.32

Free Radicals

Before the discovery of the deficient proteins associated with the pathophysiology of muscular dystrophy, it has been postulated that free radicals play a significant role in the disease process. Several mechanisms of free radical involvement have been proposed by multiple researchers, including disruption of signaling pathways, alterations in tissue response to the presence of disease, and behavioral changes in the affected individual that may trigger free radical production.9 Other researched hypotheses that support the role of free radicals in the pathophysiology of DMD incorporate the inclusion of mitochondrial function into reactive oxygen species (ROS) formation, a form of free radical.8 10

The exact role that free radicals may have in the pathophysiology of DMD is unknown, as well as the significance to which free radicals contribute to the disease process. In regards to free radicals, it is uncertain as to whether oxidative stress is an early, initial event that triggers the pathologic event of dystrophic muscle cell necrosis or whether it’s a consequence of other cellular events that occur in the pathophysiology of DMD. Research has shown that muscles of DMD patients exhibit elevated products of lipid peroxidation, elevated levels of enzymatic markers of oxidative stress, and increased concentrations of antioxidants Vitamin E and coenzyme Q in the surface muscle membranes. These findings support the presence of oxidative stress at the sarcolemma membrane.9 Even after these results, however, and following observations made by previous researchers, Tidball and colleagues9 postulated that oxidative stress has the potential to induce DMD pathology, but is not sufficient enough to solely cause DMD without the contribution from other disruptions in homeostasis.9

Reduction in nNOS/NO

One enzyme that has been found to play a significant role in the link between dystrophin-deficient muscle cells and free radicals is neuronal nitric oxide synthase (nNOS). This enzyme is a member of the dystrophin-glycoprotein complex located on the cytoplasmic surface of the sarcolemma. In dystrophin-deficient muscle cells, this enzyme is reduced to less than 20% of levels that are found in normal muscle, and the mRNA to produce this enzyme is also significantly decreased.41 With a reduction in this enzyme to catalyze reactions, the production of the free radical nitric oxide (NO) is reduced. NO is a free radical that protects the muscle against oxidative injury by potentially functioning as an antioxidant, and mediates the signal pathways between dystrophin and other proteins.42 43

One consequence that has been hypothesized to result from a reduced production of NO is disruptions in the synaptic structure of dystrophin-deficient muscle cells in mdx mice. The neuromuscular junctions (NMJs) located within mdx mice muscle cells demonstrate abnormal structure, a decreased number of acetylcholine receptors (AchRs), and an altered distribution of AchRs and other post-synaptic membrane proteins, all of which impair synaptic transmission. The alterations in the NMJ in mdx mice may be coincidentally caused by the same ligands that are responsible for localizing nNOS at the NMJ. Therefore, defects in the nNOS localization (and therefore the production of NO) at the NMJ may contribute to changes in the structure and function of NMJs in mdx mice muscles.44

Increased Levels of SOD-1

Reduced muscle use, as seen in individuals with DMD who progress through the disease process, can alter free radical production and associated enzyme activity. In particular, a free radical called superoxide dismutase 1 (SOD-1) is dramatically increased in activity-reduced DMD muscle, and this increase in SOD-1 leads to increases in lipid peroxidation. A function of SOD-1 is to convert superoxide into hydrogen peroxide, which in turn, can cause oxidative damage to the lipids that form the cell membrane (lipid peroxidation).45 In addition, nNOS activity and expression have both been found to be significantly reduced in disease-free muscle after reduced use.9

Intracellular Calcium and the Production of ROS

According to Whitehead et al.,8 strong evidence supports the finding that total calcium levels in muscle samples from patients with DMD were greater than those without DMD. Similarly, Williams and Allen46 reported increased ROS levels in mdx mice were associated with abnormal intracellular calcium. Therefore, a hypothesis can be made that calcium homeostasis is disrupted in the disease process of DMD. Two plausible hypotheses have been proposed to identify this increase in muscle calcium levels. One hypothesis is that dystrophin plays a structural role in keeping the cell intact during stretch contractions (ie. eccentric contractions). Without dystrophin, the stretch contraction mechanically pulls on the sarcolemma causing membrane tears. Calcium is then thought to enter the intracellular matrix through these membrane tears. Several research studies, however, have disregarded this mechanism of calcium influx, and have stated that the intrinsic sarcolemma strength is upheld by the lipid bi-layer, which is not affected by the lack of dystrophin.8 A second hypothesis is that dystrophin plays a significant role in collecting ion channels in the sarcolemma, and in dystrophin-deficient muscle cells, the malfunction of a “calcium leak channel” may cause an increase in intracellular calcium.7 47 Based on these hypotheses, muscle contractions that result in membrane tears and local entrances of calcium into the cell stimulates the establishment of calcium leak channels in the sarcolemma. Elevated levels of intracellular calcium also trigger certain proteases, such as calpain, which in turn activate calcium leak channels as well as degrade skeletal muscle proteins. This pathway thereby continues to feed itself, and an elevated amount of intracellular calcium results.8 In addition to the calcium leak channels, another channel, called stretch-activated channel (SAC), has been found to be increased in muscle cells of mdx mice. These mechanosensitive channels are thought to be the primary passageway for calcium to enter the cell, and through SACs, the membrane permeability to calcium increases, thereby creating this large cascade of calcium into the cell.8 For a brief time period, excess intracellular calcium can be effectively managed by the mitochondria and sarcoplasmic reticulum before being overwhelmed. Supporting this theory, calcium concentration in the sarcoplasmic reticulum has been found to greater in mdx myotubules when compared to myotubules in normal muscle tissue.48 49

So one might ask, what is the significance of excess calcium inside the cell? One finding is that abnormal uptake of calcium by mitochondria can create an increased production of free radicals, and in mdx mice, this mitochondrial oxidative phosphorylation has been found to be impaired.50

Mitochondria and ROS Production

Many studies have illustrated the fact that the majority of ROS production seen in cells occurs via the mitochondria as a toxic by-product of oxidative phosphorylation, which is the central means of ATP and heat production for our bodies.8 10 51 Initially, ROS are encased within the mitochondria and are unable to harm other intracellular structures. However, if the amount of ROS within the mitochondria becomes too high, they may begin to damage proteins, lipids, and acids inside the mitochondria, decrease oxidative phosphorylation output, and increase ROS production even further.51 In this event, the cell becomes significantly impaired and must be removed by activation of the mitochondrial permeability transition pore (mtPTP). This structure is hypothesized to be comprised of various proteins which sense mitochondrial ROS and calcium levels. When these levels become unstable the mtPTP is activated which opens a channel, causing proteins and ROS to be expulsed into the cytosol and eventually leading to cell death.51 The expanding role of increased levels of ROS in DMD has caused this mechanism to gain traction as another source of excessive ROS through a cycle of amplified ROS formation causing increased mtPTP opening and vice versa.10

Aside from ROS production due to oxidative phosphorylation, monoamine oxidases A and B (MAO-A; MAO-B) have displayed the ability to cause oxidative deamination of certain neurotransmitters and dietary amines in the outer mitochondrial membrane. This process creates aldehydes, ammonia, and hydrogen peroxide, which are all considered ROS. Various studies involving the mitochondria have exhibited reduced ROS levels in muscle cells in mdx mice animal models using MAO-inhibition10 or PTP inhibition through ablation of cyclophilin D, a mitochondrial protein which promotes PTP activity.52 53

Inflammatory Cells and ROS

Free radicals may also interact with inflammatory cells to cause muscle damage in the disease process of DMD. Reactive oxygen species have been found to trigger the activation of the NF-ĸB pathway. NF-ĸB, a transcription factor, controls the presence of pro-inflammatory cells including TNFα and IL-1β. Increased levels of these pro-inflammatory cells have been found in mdx mice before muscle death even occurs. Besides promoting inflammation, another role of TNFα is the production of mitochondrial free radicals. This system, therefore, is self-perpetuating in that increased ROS activates the expression of NF-ĸB, which in turn activates TNFα, causing a production of more mitochondrial ROS.54 Furthermore, other inflammatory cells such as neutrophils and macrophages have the ability to produce ROS. In fact, macrophage-driven free radicals have been hypothesized to cause a significant amount of the muscle damage seen in mdx mice.9 The presence of NO can inhibit these free radicals. However, a reduction in nNOS present in dystrophin-deficient muscle does not produce sufficient amounts of NO to combat these free radicals, and therefore, cell damage by ROS occurs.55


Apoptosis is a specific form of cell death in which apoptotic cells shrink and are then rapidly phagocytized by neighboring cells.56 This genetically controlled cell suicide takes place through one of two possible mechanistic pathways.56 One potential pathway is via an extrinsic system Fas and FasL involving transmembrane receptors of the death receptor family.31 FasL ligand induces apoptosis through cognate interactions with its receptor Fas.31 Fas is not typically expressed in normal muscle tissue, but has been found in diseased muscle fibers.57 The second pathway is via an intrinsic, endogenous system such as the mitochondrial Bax/Bcl-2.31 Bcl-2 is a major and well-characterized anti-apoptotic protein that prevents the release of pro-apoptotic proteins through the outer mitochondrial membrane.57 Bax, on the other hand, is a protein that promotes cell death in response to apoptotic stimuli.31 A common end-point for both of these pathways is the activation of a series of cysteine proteases known as caspases.56 These caspases can be broken down into two families: effectors and initiators.56 Effectors, such as caspase 3, are responsible for proteolytic cleavage that leads to cell disassembly.56

Apoptosis, along with other mechanisms, has been proposed to cause pathogenesis in several neuromuscular diseases.58 59 Dystrophin deficiency associated with DMD results in chronic inflammation and severe skeletal muscle degeneration, where the extent of muscle fibrosis contributes to disease severity.31 Normally dystrophin interacts with several members of the dystrophin glycoprotein complex, which forms a mechanical as well as signaling link from the extracellular matrix to the cytoskeleton.31 Mutations in dystrophin result in membrane damage, allowing massive infiltration of immune cells, chronic inflammation, apoptosis, necrosis, and severe muscle degeneration.31 Normal muscles have the capacity to regenerate in response to injury. This ability is absent in DMD, resulting in severe skeletal muscle degeneration and triggering of apoptosis.31 Not long ago, it was believed that, in people with DMD, necrosis, a process associated with cell membrane damage, osmotic imbalances, ion fluxes, cell swelling, and death, was the only mechanism that resulted in muscle degeneration.37 60 It is now accepted that the onset of death in dystrophin deficient muscles is an apoptotic process and that later stages of the disease involve both apoptosis and necrosis.61

Researchers first found evidence of apoptotic muscle loss in dystrophin-deficient muscle in a study conducted on mdx mice in which apoptotic events were reported to precede muscle fiber necrosis.59 These findings supported the results already found in patients with DMD displaying apoptosis-associated features such as DNA fragmentation and the upregulation of Bcl-2, Bax, and caspases.62 Another study looked at the muscles of individuals with DMD to examine the role of apoptosis.63 Apoptotic nuclei, which are very rare in normal muscles (less than 0.1%), were detected in the dystrophic muscles of these individuals as well as mosaic patterns of Bcl-2 and Bax which typically characterize dystrophic muscles.63 This study was the first to show an apoptotic process in muscle fibers of patients with DMD.63 There has been a correlation found between apoptosis and caspase 3, in which caspase 3 activity was only found in dystrophin-deficient muscle fibers.56 Another difference between healthy muscle and muscles in a person with DMD is that normal muscles are always positive for Bcl-2 and negative for Fas and Bax.56 57 In patients with DMD, all muscles were found to have some fibers expressing Fas, Bax expression was significantly higher, and Bcl-2 expression was significantly lower.31 56 57

It is clear that apoptosis plays a major role in the degeneration of muscle fibers in individuals with DMD. Scientists are now trying to use this knowledge to create anti-apoptosis therapies to slow down or stop the cellular death taking place in the dystrophin-deficient muscle fibers.58 However, since some apoptotic activity is normal in typical development, scientists are still trying to determine if anti-apoptosis therapies will globally cause more harm than good.62


Musculoskeletal System


Figure 7. Normal Skeletal Muscle


Figure 8. DMD Skeletal Muscle

Mechanical Damage

Based on the location and overall structure of dystrophin, authors have proposed that the protein provides stability to the muscle membrane during stresses involved with normal muscle contraction.64 In various studies involving older mdx mice, muscle fibers have been shown to have abnormal splitting and branching,65 suggesting that the surface membrane of dystrophic muscle fibers is more vulnerable to forces placed upon it during contractions and/or stretches, especially those which are eccentric in nature, leading to further damage.65 66 67 68 Supporting this theory, serum creatine kinase (CK) has found to be grossly elevated in mdx mice, indicating chronic damage and breakdown in the muscle fiber membrane.69 Muscle membrane damage and degeneration is not limited to skeletal muscles of the limbs. Stedman, et al.18 found diaphragm degeneration after 25 days of age in mdx mice, leading to loss of tissue compliance/elasticity, loss of regenerative capacity, fibrosis, increased collagen density, and reduced maximal force production compared to control mice.18

Force Production and Fatigue Level

Impaired myofibrillar function as a result of mechanical damage will undoubtedly lead to decreases in muscle force production and fatigue resistance. Oxidative stress and cell death have also been linked with these deficits seen in dystrophic muscle.10 Indeed, increased propensity to muscle fatigue is a major functional impairment associated with DMD. Boys with DMD between 8-10 years of age maintained a contraction of the biceps brachii at 60-70% of their maximum volitional contraction (MVC) for 10 seconds, compared to healthy boys who could maintain 60-70% MVC for 45 seconds.70 One mechanism proposed to contribute to these impairments is the reduction in activity of several enzymes, such as GAPDH, involved in glycolysis in DMD muscle. These enzymes have been found to be susceptible to oxidative stress in vivo and their inhibition due to oxidative stress in DMD can slow ATP production significantly in skeletal muscle.71 72

Membrane Permeability

Past theories regarding membrane permeability in DMD centered on the proposal that dystrophin-deficiency leads to defective muscle surface membrane function resulting in instability, increased permeability, and chronic myofiber leakage.73 74 This leakage can potentially allow an influx of ROS, calcium, and/or other harmful molecules into the muscle cell, ultimately resulting in the damage seen in DMD. The rise in permeability has been suggested to be caused by transient muscle membrane tears caused by stretch and contractions,8 as noted in above sections, which would also explain the elevation of CK levels in the bloodstream.74 Chronic, major membrane leakage would most definitely result in cell death. If this theory would be true it would seem as though involved cells’ ionic composition would only be affected to a minor extent, therefore allowing the cell to survive a certain amount of increased permeability.7 McArdle et al.7 conclude that DMD muscle cells have no innate increased permeability to external calcium or ROS, but after degeneration occurs via other mechanisms, the cell experiences an influx of calcium as well as a release of proteins, propagating myofibrillar damage.7

Excess Intra/Extracellular Calcium

In addition to CK levels, elevated amounts of extracellular calcium are noted in mdx mice, which is indicative of chronic degenerative changes of the muscle fiber membrane.69 Similarly, Turner et al.47 have shown increased activity of a calcium leak channel in muscles of mdx mice, which may provide another avenue of calcium entry into the muscle fiber itself.47 75 A rise in the amount of calcium uptake into the cell can disrupt calcium homeostasis and lead to damage and eventually necrosis of the cell. A significant correlation has been found between dystrophin-deficiency and decreased Troponin I activity (a calcium regulating protein)76 as well as increased calpain activity. Calpain is a calcium-activated cysteine protease which has been shown to degrade a range of cytoskeletal and membrane proteins during periods of both muscle degeneration and regeneration.77 78 79 Authors have illustrated greater calpain activity in mdx mice compared to control mice80 and have found less cumulative muscle damage in mdx transgenic mice developed to inhibit calpain processes.81

nNOS Deficiency

As stated before, dystrophin deficiency affects other proteins of the DPC as well as dystrophin itself. One DPC protein that is lost in dystrophin absence is neuronal nitric oxide synthase (nNOS), reduced to less than 20% of levels seen in normal muscle. Multiple studies have linked nNOS with beneficial oxidative effects in normal muscle, and low levels have been found in DMD affected muscles which lead to oxidative damage via free radicals.7 8 9 10 In other studies, dystrophin deficient muscles are more susceptible to oxidative damage and stress in vitro.9 Exact mechanisms of free radicals leading to DMD characteristics are explained in earlier sections. Loss of nNOS is also thought to reduce the ability of dystrophic muscle to grow, repair, and regenerate through low values of nitric oxide (NO). Release of NO accelerates the cell cycle via the secretion of hepatocyte growth factor, which potentially assists muscle growth.9 Experimental administration of NOS inhibitors to mice experiencing muscle crush injuries reduced the total amount of muscle repair seen in one study.82

Two-Hit Hypothesis

Researchers, such as Rando,83 have proposed an interpretation of the multiple cellular breakdowns seen with DMD called the two-hit hypothesis. The basis of this argument relies on the fact that while oxidative damage has the potential to promote the disease, it cannot sufficiently cause DMD in isolation of other contributing factors which also disturb homeostasis. Rando suggested that muscle ischemia due to the loss of NO in DMD, which helps regulate vasodilation, leads to a recurrent ischemia-reperfusion injury in the sarcolemma. This type of ischemic injury, when followed by a second disturbing factor caused by DPC defects, leads to the muscle necrosis often seen in DMD.83 His hypothesis has received experimental support in one study whose results found that total muscle damage was greater in mdx mice after mechanical stress was placed on the sarcolemma following a period of ischemia-reperfusion than the sum of the damage caused by mechanical damage or ischemia-reperfusion in separate mdx mice.84 Other studies have agreed that dystrophin-deficiency leading to DPC loss alone is not sufficient to cause muscle breakdown in the mdx mouse model, supporting the two-hit hypothesis.7


People with DMD have been found to have low bone mineral density (BMD) even in the absence of steroid therapy.85 Some proposed mechanisms for decreased BMD in this population are low muscle mass, reduced mechanical loading, narrow bones, short stature as well as glucocorticoid treatment.2 In DMD, it is proposed that the bones are not subject to the same weight bearing and mechanical loading as in the typical population.2 85 Mechanical stress on the bone is an important determinant of BMD and bone strength. Osteocytes respond to changes in mechanical loading and bone metabolism.86 In healthy individuals, mechanical loading of the bone induces osteocyte signaling through the gap junction, initiating osteoblastic bone formation and/or inhibiting osteoclastic resorption.86 In people with DMD, especially those who become non-ambulatory, mechanical stresses on the bone differ from that of healthy individuals.

In DMD, BMD is found to be affected primarily in the lumbar spine and the proximal femur, with decreased BMD being found in the proximal femur prior to loss of ambulation.85 Additionally, between 75-90% of non-ambulatory people with DMD develop scoliosis which has been found to be associated with increased pain and cardio-respiratory compromise.87

Cardiovascular System


Figure 9. Normal Heart vs. Dilated Cardiomyopathic Heart

Cardiomyopathy in DMD

Patients with DMD can develop preclinical cardiac involvement before turning six years old and often these cardiac abnormalities remain unnoticed until the patient is in his midteens due to the masking of symptoms by decreased exercise tolerance.88 89 By the third decade, patients with DMD have nearly a 100% incidence of cardiomyopathy, and an estimated 20% of deaths are attributed to cardiomyopathy.89 90 As respiratory complications decrease due to advances in non-invasive ventilation, cardiomyopathy related deaths are expected to rise.90 Dilated cardiomyopathy is characterized by enlarged heart chambers and reduced cardiac wall thickness.89 With advanced fibrosis in the cardiac tissue, arrhythmias including atrial fibrillation, atrioventricular block, ventricular tachycardia, and ventricular fibrillation can occur.91

The pathogenesis of cardiomyopathy in DMD is believed to be similar to that of the skeletal muscle.89 The absence of dystrophin in the heart muscle causes a dysfunction in the stretch-activated membrane ion channels.89 During ventricular filling the cardiomyocytes stretch, but the stretch-activated channels do not open correctly and this induces an increased influx of calcium.89 The increase in calcium induces calpains which degrade troponin I and interfere with the contraction of the cardiomyocyte. The membrane destruction allows more calcium into the cell and eventually the calcium overload leads to cardiomyocyte death.89 While this process is detrimental to skeletal muscle, cardiac muscle contracts at least 86,400 times a day, accelerating the process.89 In normal cardiomyocytes, small amounts of calcium enter the cell and induce larger amounts of intracellular calcium to be released into the cytoplasm.89 This process is escalated in DMD and the high calcium concentration activates the cascade of protein degredation and death.89

As the myocardiocytes begin to die, they initiate the inflammatory process.89 Macrophages migrate to clear the damaged cells and fibroblasts invade to form scar tissue or fibrosis.89 The fibrotic tissue is inflexible and restricts the efficiency of myocardial contraction.89 Fibrosis typically begins in the left ventricular wall beginning in the epicardium and spreading to the endocardium.89 This fibrotic region will eventually stretch, become thin, lose contractility, and result in dilated cardiomyopathy.89 The dilation of the heart increases left ventricular volume, decreases systolic function, and can lead to mitral valve regurgitation.89 These effects result in decreased cardiac output and hemodynamic decompensation.89

The lack of dystrophin in cardiomyocytes may also have other consequences such as increased oxidative stress and pronounced reductions in nitric oxide signaling due to an impaired activity of neuronal nitric oxide synthase (nNOS). These abnormalities lead to an increased susceptibility to opening of the permeability transition pore during periods of stress and a greater release of cytocrome c.92 In failing hearts, mitochondrial membranes demonstrate increased permeability leading to the release of proapoptotic factors such as cytochrome c and activation of cleavage of procaspases 3 and 9 leading to mitochondrial death followed by cardiomyocyte death.92

Since there is already leaking of creatine kinase from the skeletal muscles, the typical cardiac specific protein CK-MB cannot be used to diagnose heart problems for DMD.88 89 Instead, levels of cardiac troponin I are used as well as brain natriuretic peptide, a cardiac hormone.89 BNP is released by the atrial cells following left ventricular overloading and increased wall stress.89

Treatment of Cardiomyopathy

A decreased cardiac output activates the renin-angiotensin-aldosterone system (RAAS).89 The enzyme renin is activated and cleaves angiotensinogen to form angiotensin I.89 The endothelial cells produce angiotensin-converting enzyme (ACE) to transform angiotensin I to angiotensin II which stimulates the adrenal cortex to secrete aldosterone, promoting fluid and sodium retention.89 Angiotensin II and aldosterone facilitate the formation of fibrosis and overgrowth of connective tissue in the heart.89 Specifically, angiotensin II enhances the activity of TFG-B1, a fibrogenic cytokine.89 Therefore, ACE-inhibitors, angiotensin receptor blockers, and aldosterone antagonists are used to decrease the hyperactivity of the RAAS system from the decreased cardiac output.89 The use of ACE inhibitors in young patients with DMD could delay the onset and progression of left ventricular dysfunction.93 Beta blockers can also improve systolic function and help with arrhythmias.93

A non-ionic, synthetic surfactant, poloxamer 188, is now being researched for its ability to help cardiomyocytes withstand a greater stretch without collapsing.88 Poloxamer 188 has a hydrophobic center that would insert into the membrane defects or tears and hydrophilic arms that would anchor it to the surface membrane.88

For treatment of arrhythmias, there should be consideration of an implantable cardioverter-defibrillator.88 However, by the time many patients are considering this treatment due to decreased ejection fraction, severe kyphoscoliosis can complicate the device implantation.88

Peripheral Vascular Effects of DMD

The reduction of neuronal NOS in the sarcolemma plays a role in abnormal blood flow during exercise in individuals with DMD. In a study performed by Sander et al.,94 the regulation of muscle blood flow during exercise was investigated in both normal skeletal muscle and dystrophin-deficient skeletal muscle in humans. Results of this study reveal that a lack of dystrophin (as in DMD) impairs the regulation of the vasoconstrictor response.94 In normal skeletal muscle, the production of NO by nNOS is activated via muscle contraction. NO then diffuses to local arterioles where it signals norepinephrine to bind to the α-adrenergic receptor, which provokes vasoconstriction. The presence of NO inhibits α-adrenergic vasoconstriction by an unknown mechanism.95 A possible mechanism involved in this inadequate vasoregulation is the reduced levels of nNOS at the dystrophin-glycoprotein complex (DGC) located on the sarcolemma. As stated above in the free radical section, nNOS is reduced in dystrophin-deficient muscle, and therefore, the production of nitric oxide (NO) is also reduced. In the absence of dystrophin at the sarcolemma, nNOS is not properly anchored correctly, and instead, is re-directed into the cytosol.41 96 Therefore, without nNOS, a lack of NO results and is unavailable to regulate the vasoconstrictor response. Without regulation, as seen in mdx mice, there is no inhibition of the α-adrenergic vasoconstriction pathway. Therefore, prolonged vasoconstriction occurs, and local muscle ischemia results due to an inadequate blood supply to the exercising muscle. This prolonged local muscle ischemia may directly contribute to muscle cell necrosis in patients with DMD.43 95

A recent study performed by Percival et al.97 in 2008 investigated the functional effect of nNOS deficiency in nNOS knockout mice using an in situ method where the muscle remains in its normal physiological environment. Results showed that in male mice only, nNOS deficiency caused a reduction in muscle bulk and maximum tetanic force production. In both male and female mice, nNOS deficiency resulted in an increased risk of developing contraction-related muscle fatigue. Therefore, it can by postulated that nNOS deficiency has an effect on the maintenance of muscle bulk, generation of force production, and induced fatigue in addition to its role in muscle cell necrosis in patients with dystrophinopathies.97

Respiratory System

In DMD, the lower extremity and proximal musculature are impaired more than distal upper extremity musculature. The diaphragm is an imperative respiratory muscle that experiences more muscular damage than other skeletal muscles.38 There are conflicting theories regarding the mechanism of injury of the diaphragm. The diaphragm may experience injury as a result of an increased workload in comparison to skeletal muscle. An alternative theory suggests damage accumulates due to contraction-induced injury.98 The cellular mechanism of degradation is proposed to be similar to that of the musculoskeletal system. Regardless of the mechanism, a decrease in type 2 muscle fibers are found within the diaphragm with type 1 fiber-sparing until later in the disease process. One possible explanation of this phenomenon is increased stress on the type 2 fibers due to larger fiber size.98

As a result of diaphragm weakness and fibrosis, people with DMD experience decrease ventilation, chronic respiratory insufficiency with decreased vital capacity, tidal volume and minute ventilation, as well as an ineffective cough.38 99 100 Expiratory lung strength begins to decline at age 7 and continues to worsen with age.38 Vital capacity (VC) also decreases at a rate of 8-12% per year and when VC reaches less than 1L, circulating oxygen desaturation leads to death within one year.99 Forced vital capacity is one of the best indicators of clinical condition.101 Mechanical ventilation becomes necessary in many patients with DMD during the later stages of disease, however the appropriate time to initiate ventilator assistance is variable and there is currently no standard system for determining appropriateness for mechanical ventilation.99 100

Immune System

Evidence suggests that a significant portion of the pathophysiology associated with DMD may be due to the immune cells that invade in response to injury in the mechanically injured muscle fibers.102 The signals that lead to successful muscle repair in healthy muscle may promote muscle wasting and fibrosis in dystrophic muscle.38 As neutrophils, macrophages, and lymphocytes accumulate at the site of injury, they produce TNF-alpha which initiates the NF-kB pathway, causing cell death. Macrophages play a large role in disease progression as M1 macrophages kill myocytes in vitro and are characterized by cytotoxic activity.32 Abnormal intracellular signaling cascades that regulate the immune system contribute substantially to the degenerative process in DMD as evidenced by the presence of immune cell infiltrates in dystrophic muscles in early disease stages.103 Animal studies on the mdx mouse have proven the association between increased influx of neutrophils, M1 macrophages, T cells and mast cells on DMD progression.32

Role of the Immune System in DMD

Normal Functioning of the Cells Involved in the Immune System

Cellular Component Response found in Individuals with DMD
Cytokines Elevated levels of various inflammatory cytokines (TNF-alpha, TGF-f3) are noted in dystrophic skeletal muscles.38 TGF-b has been found to be overexpressed in people with DMD after the age of 6 months and may be implicated in decreased muscle function secondary to fibrosis.103
TNF-alpha Elevated levels of TNF-alpha are present in dystrophic skeletal muscle and are believed to increase catabolism in mature muscle fibers, inducing muscle wasting.38
Macrophages A persistent inflammatory response is observed in human dystrophic skeletal muscle that leads to an altered extracellular environment, including an increased presence of macrophages.38 In the mdx mouse model, macrophage infiltration is the most prominent immune feature, and elevated macrophage levels in the muscle can be seen as early as 2 weeks of age.103
Mast cells People with DMD have 10 times the amount of mast cells present in their skeletal muscle when compared to healthy controls, and these immune cells are associated with the initiation and progression of skeletal muscle lesions in DMD.103
Genes Major Histocompatibility Complex (MHC) class I proteins can be detected on the muscle fibers of people with DMD as early as 6 months of age, and MHC class II proteins as early as 1 year of age.103 Typical skeletal muscle fibers have little to no MHC class I proteins on their surface, but in people with DMD these proteins are present at 3 times the normal level.103
T-cells T-cells are present in people with DMD as early as 6 to 12 months of age and may contribute to the course of the disease by targeting unknown antigens on the surface of the dystrophic muscle fibers.103
Lymphocytes One study that looked at immunodepressed mdx mice that were lacking B and T lymphocytes showed a decrease in muscle fibrosis and a reduction of TGF-beta1, suggesting that these lymphocytes play a role in necrosis in this population.104
CD4+,8+ Elevated concentrations of activated CD8+ and helper CD4+ cells have been found in dystrophic muscles of mdx mice, and they are significantly elevated in their skeletal muscle in comparison to controls.103 Depletion of CD4+ and/or CD8+ cells in mdx mice resulted in reduction in muscle pathology.103 104
Dendritic cells An increased number of dendritic cells are found in the myofibrils in dystrophic muscle. In the presence of TNF-alpha, dendritic cells upregulate adhesion and co-stimulatory molecules to increase T-cell response.105
Interleukins (IL) IL-1beta is a key contributor to inflammatory myopathies and its expression is increased in dystrophic muscles.103 Increased IL-1beta expression prior to initial onset of the disease can trigger pro-inflammatory immune response in dystrophic muscles.103



The mechanism of action of corticosteroids is not completely clear in DMD.36 106 107 108 Glucocorticoids typically suppress inflammation by binding to glucocorticoid receptors. By binding to these receptors they enhance the transcription of anti-inflammatory genes and inhibit pro-inflammatory factors such as NF-kB and activator protein-1.107 However, corticosteroids cannot be effective solely because they are immunosuppressants. Studies with other immunosuppressant drugs such as azathioprine do not show beneficial effects in DMD as prednisone and deflazacort have.106 109 A few studies have shown that prednisolone can increase utrophin, another protein that prevents eccentric contraction-induced injuries, and dystrophin protein expression in DMD muscle cultures and mdx mouse muscle cells while other studies have failed to reproduce these effects.107 Steroids are postulated to reduce muscle necrosis and inflammation that usually results in fibrosis, inhibit muscle proteolysis, stimulate myoblast profliferation and thereby increase muscle regeneration and growth, stabilize muscle fiber membranes, reduce cytosolic calcium concentrations, and up regulate utrophin.108 109 110 There have been studies that show a lower amount of T-cells in patients with DMD treated with prednisone, however other inflammatory cells such as B-cells, macrophages, natural killer cells, and necrotic fibers were no different.109

Corticosteroids delay the disease progression but do not reverse the course of the disease.107 The recommended dosage of prednisone for optimal increase of strength is 0.75 mg/kg/day.90 107 Beneficial effects of corticosteroid use have included increased muscle strength, prolonged walking, delayed decline of pulmonary function, reduced incidence of cardiomyopathy, and a reduced need for scoliosis surgery.36 90 108 While prednisone is the most widely used corticosteroid, deflazacort has been shown to have less side effects and similar benefits, however it is not approved in the US.90 111
The most frequent side effect of steroid use is weight gain.109 Other side effects of corticosteroids include acne, personality changes, hirsutism, growth retardation, vertebral fractures, and reduced bone density with potential osteoporosis.36 109 Long term use of steroids can result in adrenal suppression, increased susceptibility to infection, hypertension, impaired glucose tolerance, gastrointestinal irritation, and skin fragility.109

Along with characteristics of the disease process itself, corticosteroid therapy is implicated in low BMD in people with DMD.2 Corticosteroids have been shown to decrease bone density by affecting osteoblastic bone formation and osteoclastic remodeling.112 Bone loss from steroid therapy is induced by osteocyte and osteoblast apoptosis.113 The mechanism can involve changes in gene transcription in that it binds to the glucocorticoid receptor, resulting in conformational changes and nuclear translocation of the ligand-bound receptor, followed by cis or trans interactions with DNA which affects gene transcription.113 Glucocorticoids can also act on bone without effecting gene transcription through modulation of extracellular signal-related kinases (ERKs) and c-Jun N-terminal kinase (JNK).113 They can act on prolinerich tyrosine kinase 2 (Pyk2), which causes cytoskeltal reorganization, cell detachment and apoptosis.113 Corticosteroids also suppress intestinal calcium absorption and decrease sex hormone synthesis, which negatively impacts BMD.114

Gene Therapy

Over the last decade, many gene therapy studies have focused on adding a new gene through viral or non-viral delivery of dystrophin to the dysfunctional muscle tissue.115 There have been challenges, however, with this type of gene therapy. The extensive size of the dystrophin coding region (11kb) and the long-term expression of gene constructs into skeletal muscle and delivery efficiency have limited clinical applicability.115 As a result, current research studies focus on correcting the dysfunctional gene instead of gene addition.115


Figure 10. Exon Skipping in Muscular Dystrophy

Exon Skipping

DMD occurs when there is a mutation of the dystrophin gene that disrupts the reading frame and leads to a premature abortion of dystrophin synthesis. The mutation is usually a result of a deletion of an exon.116 The exon is the piece of the DNA that is responsible for encoding the information for the amino acid sequence of the protein.117 The ultimate goal of gene therapy is to deliver a gene copy that is functional or repair the gene that is damaged.118 The exon skipping approach is specific to individual mutations because different mutations require skipping of different exons.119 The identification of revertant fibers in the dystrophic muscle can be done through exon skipping, resulting in dystrophin protein expression in order to guide the use of antisense oligonucleotides (AONs).116 In order to restore the damaged reading frame, AONs can be used to induce skipping of the exon that contains a premature stop codon from the pre-mRNA by binding to the specific splice sites.118 The remaining protein gene will not be as long after the splicing, but what remains will be functional.120 The severe DMD phenotype is converted into a milder phenotype known as Becker muscular dystrophy (BMD).121 A series of AONs have been identified through research to induce the skipping of 20 different exons.122 When considering all patients with DMD, greater than 75% would see benefits from skipping of these exons.121 Exon skipping has the ability to correct for the dysfunctional dystrophin which can target the actual cause of muscular dystrophy.120 The process of exon skipping is a well studied area of science with promising research studies in the mdx mouse models,123 canine models,13 and even in human cell culture models.124

Aartsma-Rus et al.124 conducted a study that included six patients with DMD who were affected by different mutations on different exons. Each of the six DMD patients had myotube cultures that were transfected with the AON specific for skipping the mutated exon. All six of the patient’s culture showed exon skipping and resulted in shorter remaining proteins. Dystrophin was detected, as soon as 16 hours after the transfection, in 75%-80% of the cultures and the reading frames were restored in the treated cells through exon skipping and resulted in dystrophins similar to BMD.124 These proteins are most likely more functional. The formation of the dystrophin-glycoprotein complex was also re-established following the restoration of dystrophin synthesis.124 This study demonstrates a successful therapeutic approach to minimize the severity of DMD in over 75% of the mutations reported in DMD by taking into account different deletions and mutations. The following year, a similar study121 was conducted to determine the effectiveness of inducing skipping of multiple exons to create deletions. The researchers found the approach to be very challenging from a technical standpoint as there is great variability and the levels of intended multiexon skips are usually low.121 It is also important to look at the condition of the muscle tissue following an AON treatment. It has been shown that dystrophin transcripts are targeted in the exon skipping approach, but the transcripts are only expressed in muscle tissue. As DMD progresses, fibrotic and adipose tissue replace the muscle so AON treatment should begin in the earliest of stages to maximize the clinical outcome.125 Exon skipping using AONs has proven to be successful in animal and human models, but further research is warranted to determine any negative side effects associated with the gene therapy and continue the scientific search for the DMD cure.

Adeno-Associated Virus-Mediated Gene Therapy

Adeno-associated virus (AAV) is a small virus that infects humans causing a mild immune response. Its properties have made it successful in gene therapy as the delivering agent into the cells without eliciting infectious diseases in humans.126 The AAV vector can package the dystrophin in smaller forms while preserving the signaling and structural support. The mdx mouse model has been successful in reversing the dystrophic phenotype127 but further research is warranted to determine the effects this therapy has in humans. Human have multiple muscle groups affected by MD making the vector delivery significantly more challenging. Advances in this type of gene therapy are underway with the possibility of systemic delivery in humans in the future.126 It is very important to consider the body’s immune system response to AAV. Mendell et al.,128 found that in human muscle, transgene expression was successful after viral vector mediated gene delivery and lasted for up to 3 months.128 In this study of three people with MD, only one experienced a cytotoxic T-cell response at a minimal level that did not prevent the resulting gene expression.128 Further research needs to be completed to ensure eradication of a T-cell mediated response with adeno-associated virus-mediated gene therapy.

Immunity to Dystrophin

Mendell et al.129 has determined that an immune reaction occurs to dystrophin in patients with DMD that leads to issues with strengthening of the affected muscles. Research has been conducted over the last decade in an attempt to discover a virus that can deliver a correct copy of the DMD gene to the affected muscles to enhance the strengthening properties of that muscle.128 129 Wang et al.127 found that the mdx mouse obtained strength gains in the affected muscles through adeno-associated virus-mediated gene therapy. In order to translate the animal model into a clinical model, Mendell et al.129 injected a viral vector into the biceps muscle of one arm in six different males with DMD. The viral vector packaged the corrected gene for injection into the involved muscle, similar to the mdx mouse model. However, after three months, there was no evidence of dystrophin production.129 T cells are lymphocytes that are involved with cell-mediated immunity. If a foreign cell is recognized, the T cells will destroy it to protect the body from infection. Delivering the correct copy of the DMD gene into the skeletal muscle may have resulted in an immune response where the T-cells destroyed it. The body may have seen the new gene as foreign since these patients do not have dystrophin in skeletal muscles to begin with.129 T cell immunity was present in two of the six patients upon investigation.129 T cell immunity to dystrophin was also discovered in a patient before the injection of the correct gene was given. The small numbers of T cells were able to recognize dystrophin in muscle cells that are responsible for self correction of the dysfunctional gene. When the new gene was injected, the immune system response was amplified.129 As a result of this study, it is now known that the small amount of dystrophin produced in DMD muscle for self correction can trigger autoimmunity through the T cells and the corrected gene injected to treat the disease may not be effective.129 Future research will focus on discovering a method to turn off the T cell response so the corrected dystrophin gene can be effective in slowing muscle loss and the detrimental effects on patients with DMD.129


Duchenne muscular dystrophy (DMD) is an inherited X-linked recessive disorder affecting male births resulting in progressive muscle weakness and wasting.1 Dystrophin, a cellular protein, is absent in the disease that leads to loss of contractile function of skeletal muscle.1 2 3 4 There have been many studies on human and animal models trying to determine which cellular mechanism to alter in the hopes of finding a treatment. It is evident that there are many cellular mechanisms involved in the disease process of DMD. Inflammation sets off a cascade leading to the production of free radicals and eventually apoptosis. The cellular cascade affects many body systems including the musculoskeletal system, cardiovascular system, respiratory system, and immune system. Current medical management includes the use of corticosteroids in attempt to slow the progression of the disease and gene therapy with the hope of altering the disease to a milder form. Unfortunately, despite the ongoing research related to DMD, there is no known treatment or cure. More research needs to be conducted to determine a definitive cure for this devastating disorder.

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