A Useful Treatment For Muscular Dystrophy

Published Date: 02 Nov 2017

By Rhian Stavely 3887556

Background

The muscular dystrophies (MD’s) are a group of genetically linked diseases characterized by a progressive degradation of muscular strength and muscle fibre necrosis. While the majority of MD’s are genetically inherited recessively via the X chromosome there are instances of autosomal dominant inheritance, as well as spontaneity. A boom in modern genetic research, catalysed by the inception of the polymerase chain reaction method, has lead to the entire mapping of the human genome. Although not every aspect is fully understood, great time and effort has gone into deciphering the genetic properties that give rise to the symptoms caused by MD’s. Understanding the complexity of the genome and ways to amend errors in the nucleotide sequences appears to be the best possible option for eliminating MD’s. (Lovering RM, Porter NC & Bloch RJ, 2005)

Seeing as though there is no way possible to estimate if this is an accomplishable task, or the timeframe this may take, it is still important for treatment options to be made available to alleviate current patient’s symptoms so that they can live with a prolonged lively hood, as comfortable as possible. Few treatments affect the relentless muscle wastage as a result of MD’s with arguably the exception of corticosteroids; however side effects including increased weight gain, behavioural changes, glucose intolerance, cataracts and effects on natural maturation are concern enough to prevent its use. This has lead families resorting to unreliable nutritional supplementation such as arginine, which was later proved to cause detrimental long term effects. (Wehling-Henricks M, et al. 2010.)

For these reasons, a safe and more effective treatment to prevent muscle wastage is in much need of development. One of the most promising treatments could possibly be through the use of adenylosuccinic acid (ASA) which is thought to increase energy, physical endurance and muscular strength in patients suffering from MD. (Rybalka, E, 2007)

The Muscular Dystrophies

Contrary to the belief of many there is a range of heterogeneity in MD’s with separate genetic causatives, which are usually categorized phenotypically. The most common and most debilitating type of MD however is Duchenne's muscular dystrophy (DMD); with an incidence as high as 1 in 3600. The first clinical evidence of DMD can present itself as early as 1 year but usually between 2 and 5 years. These signs include an effected gait such as waddling and toe walking as well as pseudohypertrophy. The mean definitive age of DMD diagnosis however remains at 5 years of age. By 12 years the majority of DMD patients have severe scoliosis and/or lordosis and are wheelchair bound. Fatality is a likely event in the second decade of life due to respiratory fibrosis and cardiomyopathy leading to heart failure. (Wehling-Henricks M, et al. 2010, Davidson ZE & Truby H, 2009)

DMD is an inherited X-linked recessive genetic disorder, therefore only males are affected. There is a possibility of females also presenting DMD; however this could only occur in exceptional circumstances such as an abnormality in X chromosome presentation. This is because the other X chromosome, which is unaffected by genetic abnormalities, usually assumes the role of gene expression. The early onset of DMD decreases the likelihood of reproduction which is another main reason why females do not generally inherit DMD but rather carry the mutation unaware and asymptomatically. Approximately one third of DMD’s arise from a new sporadic mutation which accounts for DMD’s endless incidence. (Gordon ES & Hoffman EP, 2005)

The genetic variance that causes DMD is found on the Xp21 locus. This gene is known as dystrophin and subsequently codes for the protein dystrophin; this plays an important role in the structure of muscle tissue. The mutation in this genes results in the absence of dystrophin translation and protein synthesis. Becker’s muscular dystrophy (BMD) is an allelic variant to DMD which results in the abnormal quality or quantity of dystrophin. The mode of inheritance is similar but depending where the mutation has arisen a variable degree of protein functionality is retained. With BMD milder symptoms are seen with a later onset, between 5 and 15 years is expected. BMD usually does not present with scoliosis and patients can live well into their adult years and possibly a full life expectancy. (Verma S, Anziska Y & Cracco J, 2010.)

Another form of MD that is less common then DMD but can be just as symptomatic are the limb girdle muscular dystrophies (LGMD), which effect mainly the upper and lower proximal limbs. LGMD can have both an autosomal dominant and autosomal recessive mode of inheritance, with the later known to mimic DMD in severity. There are 16 identified LGMD’s with genetic differences affecting different proteins, some enzymatic rather than structural. For this reason we see a wide array of phenotype severity with LGMD.

The third most common form of MD is fascioscaplulohumeral muscular dystrophy (FSHD), an autosomal dominant disease, characterized by the weakness of facial muscles including the inability to blow and open eyes during sleep. The progression of FSHD is slow and a normal life expectancy is predicted however there is increased risk for cardiac problems to arise. There are numerous other, rarer forms of MD but the other main MD which should be noted and is the most common in adult life is myotonic muscular dystrophy. This form of MD can arise at any age with two types. One caused by a series of CTG repeats in the gene coding for the protein dystrophia myotonica protein kinase and the other CCGT repeats in the gene encoding for a zinc finger at 3q. The causative effects of these mutations remain elusive because they are situated in introns however it is thought the extra nucleotide somehow impede the splicing process. Myotonic muscular dystrophy is autosomal dominant and the series of mutation expansions increase by generation. (Lovering RM, Porter NC & Bloch RJ, 2005)

Dystrophin

Little is known about the rarer forms of MD; however a wide range of research has been conducted on the most common form DMD. Dystrophin, the gene affected by the mutation causing DMD, is the largest gene identified in any genome thus far. It is four fold the size of any other gene estimated at 2.5 million base pairs long. The sheer size of the gene gives reason as to why it is extremely susceptible to mutation. The majority of the dystrophin coding sequence largely consist of introns, only 11,000 base pairs are coding for the giant 427kD protein. Although a single nucleotide insertion or deletion in either the introns or the exons will still cause a frame shift mutation and change the whole sequence in which the amino acids are derived creating a non functional protein. This mutation is a common cause of DMD. BMD on the other hand is caused by a point mutation. The wrong nucleotide is put in place of another; however the reading frame remains intact resulting in a less functional protein then regular dystrophin, but causes largely less severe symptoms in most cases then present in DMD. (Gordon ES & Hoffman EP, 2005, Lynch GS, Schertzer JD, Ryall JG, 2007.)

To explain how dystrophin causes DMD it was thought that dystrophin was a part of the cell membrane. The absence of dystrophin caused weakening of the cell membrane and breakage upon myofibre contraction, releasing a calcium influx into the muscle fibres. Although this appears true to a degree we now know a lot more information to further and alter this concept.

Current theorists believe Dystrophin binds either directly or indirectly to proteins at the sacrolemma which is known as the dystrophin-glycoprotein complex. This connection is thought to occur at the c-terminal of dystrophin to the transmembrane protein β-Dystroglycan which is bound to α-Dystroglycan which is ligated to the extra cellular membrane as well as the sarcospans and sarcoglycans. The n-terminal of dystrophin is bound to F-actin inside the muscle fibre. This completes the molecular link between the extracellular membrane and the contractile filaments. Not only that but dystrophin is also known to bind to several other various proteins mid shaft such as syntrophin and dystrobevin with more being discovered as research progresses. (Lovering RM, Porter NC & Bloch RJ, 2005)

There are now two main hypotheses as to why dystrophin is essential to prevent muscle degeneration and remains a required protein otherwise causing MD. The first theory considers that dystrophin is a structural protein. As dystrophin connects the myofibres to the extra cellular membrane, it is thought to play a major role in stabilizing the sacrolemma. It is also thought that this creates force transduction to prevent myofibre damage. The helical structures in parts of dystrophin support this theory and may provide load bearing effect like a spring. The second theory has been conceptualized due to the several proteins that bind to dystrophin in the dystrophin-glycoprotein complex. Even though they are not affected directly, dystrophin is thought to act as scaffolding keeping signalling proteins in the correct place for proper muscle metabolism such as nueronal Nitric Oxide Synthesase (nNOS) which binds to dystrobrevin. (Judge LM, Haraguchi M & Chamberlain JS, 2006.)

Underlying problems

It has long been noted that there appears to be a disturbed energy metabolism deficiency linked with MD. Purine metabolism is greatly impaired in MD. The most effected is arguably the turnover of adenine nucleotides. (Thomson WH & Smith I. 1978)

Camiña F, et al. (1995) determined that DMD sufferers are severely deficient in ATP and ADP, their precursor ASA and adenylosuccinate synthetise.

Considering that mitochondria plays an important role in ATP synthesis, Ca+ regulation and cellular death, it has long been speculated that it may cause many of the symptoms seen in DMD. The relation between dystrophin and mitochondria is not fully understood however research from Perciva JM, et al. (2013) has concluded that the absence of dystrophin does affect the mitochondria with in the muscle and causes decreased intramuscular ATP. Dystrophin is expected to be required to maintain the subsarcolemmal pool density of mitochondria and impact its localization as well as reducing ATP synthesizing capacities leading to ATP deficiency. It is suspected this is due to the effect of impaired signalling of nitric oxide (NO) given that it is a key regulation factor for mitochondria. The impaired localization of nNOS effects NO signalling and is also thought to impair nutrient oxygen delivery to regenerating muscle fibres in patients with MD (Mungrue IN and Bredt DS, 2004)

Recent evidence by Onopiuk M, et al. (2009) also suggests a dissociated mitochondrial network is caused by MD as well as impaired energy metabolism. These results have however been shown in an mdx mouse model. Although the mdx mouse shows similar symptoms to human MD the underlying mechanism may be heterogeneous and therefore the association to human MD is questionable,

Purine cycle

The actual effect on the chemical pathway of ATP production is not understood as a direct cause in DMD. By understanding purine metabolism however the problems that do occur may be rectified. The purine cycle is a means of producing ATP, GTP and its derivatives through a series of chemical reactions usually enzymatically assisted. The adenosine cycle, which is thought to be the major purine affected in MD, produces cyclic reactions of IMP to ASA to AMP then back to IMP. Even though specific enzymes regulate these reactions an effect of concentration in one will affect the others due to their symbiotic relationship. ATP is produced by the phosphorylation of AMP. A lack of ATP may mean AMP is unable to be produced in enough quantities to compensate for ATP degradation. This gives evidence to support that a defect in the adenosine cycle is caused in DMD. In order to convert IMP to AMP, the energy produced by GTP hydrolysis powers adenylosuccinic synthesase to create ASA from IMP and aspartate. ASA is then cleaved by adenylosuccinate lyase to form AMP and fumurate which can be used to power the Krebs cycle yielding ATP production. In this instance however ATP production will be substrate limited by AMP. For conversion back to IMP, the enzyme AMP deaminase will use AMP and water as substrate to form IMP and ammonia. (H A Simmonds, 1991, Van den Berghe G, Vincent MF & Jaeken J, 1997)

Adenylosuccinic acid treatment

In order to increase intramuscular ATP, adenylosuccinic acid (ASA) has been considered to promote the concentration of AMP. The reaction between ASA and AMP is promoted by adenylosuccinate lyase. Although the concentration of this enzyme is a limiting factor itself, one way to the boost the rate of reaction may be to increase the substrate, ASA. Increased substrate could possibly increase enzymatic potential and the increased concentration could further drive the reaction forward to reach its equilibrium resulting in increased AMP product. ATP could then be replaced through unaltered pathways of phosphorylation of the AMP to theoretically boost ATP throughout the muscle fibres. A foreseeable limitation to this however remains; as the enzyme that catalyses the AMP production is limited, the rate at which ATP is produced may not be significant enough to counteract the unknown causative that decreases ATP concentration in the first place. (Carter CE & Cohen LH, 1956, Fitt PS & Parliament MB, 1982, Srivastava, H.S. 2010)

Research by Thomson WH, (1987) indicates that there is inadequate purine biosynthesis in DMD. it is also thought this indicates a possible partial defect in adenylosucchinate synthetise. The study also found that continuous infusion of ASA caused an acute and lasting increase in muscular strength. Normal heart rate and ECG were restored and a return to normal levels for serum enzymes. A noted assessment between oral allopurinol treatment and ASA infusion stated that allopurinol showed limited improvement in comparison to ASA. Thomson concluded that the disappearance of DMD symptoms appeared possible due to these findings.

This could possibly be due to the different direct targets of the purine metabolism pathway the substances affect. While ASA is aimed at increasing ASA concentration levels as a substrate to promote a reaction; the role of allopurinol is to inhibit a reaction. Alloprinol inhibits xanthine oxidase, an enzyme important for the conversion of purines into uric acid which is excreted and ultimately the end of the purine synthesis pathway. The precursor of xanthine, hypoxanthine can be recovered and synthesized into inosine which gives rise to adenine purines and potentially ATP. A major setback in the build up of these substances however is a negative feedback mechanism that halts purine production Dervieux T, et al. (2002.) Over an 18 month period no increase in adenine nucleotide production, was observed through research in a study of DMD patients treated with allopurinol by Bertorini TE, et al. (1985). Further more muscle degeneration continued with no significant reduction and no significant increase in ATP was found when compared to the placebo group. This may be attributed to the purposed negative feedback system, which also may hinder hopes of reduced symptoms due to ASA infusion treatment.

According to Bonsett CA & Rudman A (1992) however, research indicating that the use of synthetically produced ASA does have beneficial effects in patients with DMD and BMD. These include corrective trends in the parameters of creatine and calcium metabolism. Evidence is given to support an improved state of ATP production. The findings also indicate that an infusion of ASA has a possible therapeutic potential for patients with MD. Decreased creatine levels may also play a role in the symptoms seen in MD. Nabuurs, C. et al., (2013) has shown earlier this year that a creatine depletion shows similar symptoms of human MD is an mdx mouse model. Furthermore by an infusion of creatine; symptoms were able to be controlled and disappeared. This could mean that the improved energy metabolism seen in research from Bonsett CA & Rudman A (1992) maybe attributed to the indirect correction of creatine production through ASA.

The verdict

Despite promising research, the application of ASA for the treatment of MD has still not been verified to benefit MD patients. There are many aspects that need to be considered when deciding if ASA is a suitable treatment or not. Little research has been preformed using ASA as a therapeutic drug; therefore considering there is such little information made available it is irresponsible to take a yes or no stance. Additional study is definitely needed to confirm the benefits of ASA treatment for MD sufferers. Furthermore we are unaware of any possible side effects that may be caused in a full scale treatment plan. Due to the ethical issues associated with this aspect it may be beneficial to experiment in an animal model. One animal model commonly used in MD research is experimentation on mdx mice. At 2 years of age old mdx mice show similar phenotypical characteristic of human DMD (Hayes, A & Williams DA, 1998) which be useful for preliminary research in this instance. The lack of study in these areas is surprising considering the promising results yielded by the few previous studies. Considering that these studies were carried out over 20 years ago it provokes the question as to why research suddenly stopped. Perhaps there is unpublished data that has disproved ASA as a treatment in MD; this would explain why ASA has not been based in a study testing its viability as a treatment for MD since. Another possible explanation is that most MD research is based on genetic therapy. The majority of resources for muscular dystrophy research may have been moved into the field of genetics as it is seen as the only possible way to ‘cure’ the disease. Although genetic therapy holds the main hope for eliminating MD, an optimal treatment in order to give MD sufferers the best possible life is necessary. The future possibility of adenylosuccinic acid supplementation as a useful treatment for muscular dystrophy is plausible as of current knowledge. To consider application of adenylosuccinic acid as a treatment for muscular dystrophy further research must be conducted; evidence suggests however that this may be a worthwhile feat.