On the Pathogenesis of Duchenne Muscular Dystrophy

Dcvelop. Med. Child Yeurol. 1975, 17, 527-533

Review Article Gerald M. Fenichel

On the Pathogenesis of Duchenne Muscular Dystrophy 'b

Introduction There are a t present three active h y -

potheses concerning the pathogenesis of Duchenne muscular dystrophy. The I'NS-

culur hypothesis has sought a defect in the micro-circulation and has led to some interesting observations on biogenic amine metabolism. The neurogenir hypothesis has considered the myopathy to be the result of an abnormal neurotrophism from 'sick' motor neurons. The niyogrnic hypothesis seeks the primary fault in the muscle fiber and includes ( a ) structural defects, ( h ) disturbances in enzyme systems and ( c ) over-production of connective tissue.

In order t o evaluate the relative merits of the three hypotheses. it is imperative that we limit our analysis to the information concerned strictly with Duchenne dystrophy. There is no evidence to indicate that other human myopathies o r any animal dystrophy is pathophysiologically identical to Duchenne muscular dystrophy (DMD). This simple fact is frequently ig- nored when the case is presented for a specific hypothesis.

The Vascular/Biogenic Amine Hypothesis The notion that an ischemic mechanism

might induce the muscle necrosis of Du- chenne dystrophy was first suggested by

the pattern of the myopathology and then reinforced by some report> of biogenic amine abnormalities. One of the earliest and more consistent histological features in the muscles of patients with Duchenne dystrophy is a clustering of the fibers which are undergoing necrosis (Pearson 1962, Engel 1967). The fibers within each cluster are at approximately the same stage of degeneration and contrast sharply with the surrounding normal cells. A similar focal necrosis has been produced in rabbits by micro-embolization of the muscle, using small dextran particles injected into the femoral artery (Hathaway rt cr l . 1970). These investigators themselves pointed out the important reservations in comparing this experiniental niyopathy with DMD; firstly there is the species difference, secondly there is the consideration that muscle tissue has on ly a limited number of responses to injury, and finally there is the fact that similarities in histopathology d o not necessarily indicate a common pathogenetic mechanism. Nevertheless, this experimental myopathy was thought to share similarities with DMD and to be different from the pathology identified as large vessel occlusion and exemplitied by juvenile dermatomyositis. A recent study has re-evaluated the dktribution of ne- crotic muscle fibers in biopies from DMI)

Professor and Chairman, Department of Neurology, School of Medicine, Vanderbilt University, Nash-

* Second Annual Presidential Address to the Child Neurology Society, Madison, Wixonsin, Octoher ville, Tennessee 37232. Director, Jerry Lewis Neuroinuscular Disease Research Center.

1974.

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DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1975, 17

and found them to be randomly distributed (O’Brien e t a / . 1974). If this is confirmed by others, then the animal model would be negated.

The morphology of the micro-circulation and its blood flow have been examined directly in patients with DMD (Jerusalem et a/. 1974a, Koehler 1974). The mean capillary size is significantly increased, but the mean fiber area served per capillary is unchanged. The only consistent morphological change is a replication of the basal lamina of the capillary wall. This is recorded with equal frequency in the muscles of patients with vasculitis and to a lesser extent in patients with spinal muscular atrophies and other myopathies.

Blood flow at rest and during the hy- peremia which follows exercise is normal and capillary diffusion capacity is unim- paired, as measured by the local xenon injection method (Paulson et a/. 1974). Thus, at present there is no evidence of a structural or functional defect in the micro-circulation of patients with DMD to support an ischemic etiology.

The interest in the micro-circulation did, however, intensify the research effort to identify an abnormality of biogenic amine metabolism in this disease.

Early accounts of an increased concen- tration of serotonin in the cerebrospinal fluid, and elevations in the urinary excre- tion of epinephrine and norepinephrine in patients with DMD (Stern et a/. 1956, Misra et al. 1965) have not been confirmed by careful measurements using refined techniques (Mendell et al. 1972). However, a significant defect in the initial uptake of serotonin by platelets has been observed in patients with DMD, but not in other myopathies (Murphy et a/. 1973). The experimental injection of serotonin does not produce histological lesions in skeletal muscle, but when it is combined either with aortic ligation (Mendell et a/. 1971) or pretreatment with imipramine

(Mendell and Parker 1974), a focal muscle necrosis is observed. The imipramine experiment is of particular interest, since this drug blocks the uptake of biogenic amines into platelets and thereby simulates the disturbance reported in patients with DMD. It is also an inhibitor of phospho- diesterase and thereby elevates levels of

I shall return later to some considera- tions of cyclic nucleotide abnormalities in regard to the biogenic amine hypothesis and as a bridge to the neurogenic hypothesis, but first I should mention some interesting work utilizing the fluorescent histochemical technique for biogenic amines in DMD. In muscles taken at biopsy from patients with DMD, a lime-green fluorescence is observed in groups of fibers which appear otherwise normal on serial section (Wright et a/. 1973). Muscles from patients with other neuromuscular dis- orders do not exhibit this fluorescence, nor do the muscles of rats treated with imipramine and serotonin (Olson and Mendell 1974). These histological and histochemical observations can be reproduced in rats by injection of pargyline, a monoamine oxidase inhibitor (Yu et a/. 1974). The source and identity of this fluorescent material in the muscle cells of boys with DMD and in rats treated with pargyline is still undetermined.

Returning now to the cyclic nucleotides, the observation has been reported that while the basal enzyme activity of muscle adenyl cyclase is normal in children with DMD, its response to stimulation by epinephrine is only 30 per cent of normal (Mawatari e t a / . 1974).

This decreased reactivity of the enzyme may be part of a non-specific defect in the plasma membrane which results in an abnormality of the catecholamine receptor, or it may be a significant step in the pathogenetic mechanism. Unfortunately, this same question can be posed for all the

Cyclic AMP.

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GERALD M. FENICHEL

H 529

reported defects in biogenic amine uptake and metabolism. While we ponder that question, we must also keep in mind that the cyclic nucleotides and catecholamines play an important r6le in neuromuscular transmission on both the presynaptic and postsynaptic side of the neuromuscular junction (Goldberg and Singer 1969, Lentz 1972, McAfee and Greengard 1972, Takamori et a/. 1973).

The Neurogenic Hypothesis The neurogenic hypothesis originated

with studies on the dystrophic mouse (Conrad and Glaser 1962, Glaser and Seashore 1967), and subsequent observations have continued to substantiate the theory of a neurogenic basis of murine dystrophy (McComas and Mrozek 1967, Duchen and Stenafi 1971, Harris and Wilson 1971, Salafsky 1971, Hironaka et a/. 1972, Kumpel and Dubowitz 1974, Peterson 1974).

These reports in the dystrophic mouse, coupled with the presence of fibrillation potentials in patients with DMD (Norris and Chatfield 1955), suggested that neurogenic factors might also play a r61e in human disease. McComas and his group were the first to make the case for a neurogenic etiology of human dystrophy (McComas et a/. 1970, 1971a, 1974). Using a new method for estimating the number of motor units in the muscle and then correlating this result with the iso- metric twitch tension of the same muscle, they have been able to demonstrate a progressive reduction in the number of functioning motor units, without a reduction in their size. They also reported a slowing of the nerve impulse conduction velocity in the distal but not the proximal portion of the axon. The theory was pos- tulated that muscular dystrophy is a chronic dysfunction of motor neurons, eventually leading to their physiological failure (McComas et a/. 19716).

The motor neurons were referred to as “sick’, a state characterized by “difficulty in maintaining satisfactory synaptic con- nections with muscle fibers.” This difficulty is manifested by impaired neuromuscular transmission during maximal effort or repetitive nerve stimulation and by an inability to acquire previously denervated muscle fibers.

This intriguing theory has been examined by others in a variety of ways. One group, using a technique for counting the number of limb motor neurons in the lumbrosacral spinal cord, found a normal number in patients dying from DMD (Tomlinson et al. 1973, 1974; Irving ef a/ . 1974). The same technique has also demonstrated no reduction in the number of motor neurons in murine dystrophy (Papapetropolous and Bradley 1972). While it might be anti- cipated that ‘sick’ motor neurons would eventually die and their numbers be decreased, this observation alone is not con- clusive evidence against a neurogenic hypothesis. Abnormalities in function may not always be reflected in morphological abnormalities which can be observed at the light-microscopy level.

The distal portion of the nerve and the neuromuscular junction have been studied as intensively as the motorneuron cell. The intramuscular nerves and axon terminals are morphologically normal (Jer- usalem et al. 19746). and collateral innervation cannot be demonstrated by calcu- lations of the terminal innervation ratio (Coers et al. 1973), despite some electrophysiological data to the contrary (Des- medt and Borenstein 1973). A consistent finding in one study (Jerusalem et a/. 19746) is a focal atrophy of the postsynaptic reg- ion of the neuromuscular junction. These postsynaptic alterations were not believed to have been preceded by a presynaptic disturbance, since the nerve terminals overlying the atrophic postsynaptic folds are normal. This abnormality can be


experimentally produced in rats by chronic treatment with neostigmine (Engel et al. 1973). Chronic and irreversible cholinesterase inhibition produces not only end- plate atrophy but also the histological features of myopathy (Fenichel et al. 1972).

Recent reports have challenged the original work of McComas and colleagues by claiming that the techniques used by his group were not capable of recording motor-unit action potentials of small amplitude (Scarpalezos and Panayiotopo- lous 1973, Brown 1974, Panayiotopolous et al. 1974). Using additional methods and equipment, the motor-unit potentials were found to be normal in number but of small size, in keeping with the traditional EMG evidence of a primary myopathy.

Despite this challenge to the most con- vincing evidence supporting a neurogenic hypothesis for human dystrophy, neurogenic factors cannot be discounted. The motor neuron has a trophic influence on skeletal muscle which extends beyond the phenomenon of contraction to include also the maintenance of structure and the quality of function. The symbiosis between these two cells is such that no event can occur in one without some effect upon the other. Let us then consider what is known about the muscle cell in Duchenne muscular dystrophy.

The Myogenic Hypothesis The search for a basic abnormality

within the muscle cell has been extensive. Many disturbances of structure and function have been identified, but there is a continuing difficulty in discriminating primary pathogenetic factors from the secondary effects of cellular necrosis. Genetically determined defects ultimately are expressed as an abnormality in the synthesis of cell proteins. It would there- fore be convenient to review the muscle studies in terms of the three protein

components: (a) structural proteins, (b) enzymes and (c) repressor proteins (Perry 1971).

Most structural proteins of the muscle cell serve its contractile function. The myofibrillary proteins, (myosin, actin, troponin and tropomyosin) have been examined and not noted to have any definite abnormality (Furakawa and Peter 1972, Penn et al. 1972, Samaha 1972, 1973). The membrane system is also part of the contractile apparatus and consists of (a) an external portion (the sarcolemma) which infolds itself deep into the interior of the fiber to form the transverse tubular system, and (6) the sarcoplasmic reticulum, an internal network of tubules and sacs. Depolarization of the end-plate produces the propagation of a current in the transverse tubular system which causes the release of calcium ions from the adjacent sacs of the sarcoplasmic reticulum. The released calcium activates myosin ATrase and initiates contraction. Rebinding of calcium by the sarcoplasmic reticulum is required for relaxation.

The sarcoplasmic reticulum isolated from muscle of patients with Duchenne dystrophy is reported to be impaired in its ability to bind calcium (Samaha and Gergely 1969, Takagai et al. 1973). The protein and lipid composition of the sarcoplasmic reticulum are normal, but the phosphatidylcholine content is decreased. It has been suggested that the abnormal phospholipid content, as well as the deficiency in calcium binding, may be due to contamination of the membrane fractions by microsomes from the adipose and connective tissues, which are greatly increased in this disease (Takagai et a/. 1973).

A variety of enzyme changes have been described in DMD but as yet none have been found to be specific. A decrease in the activity of some enzymes is observed in many severe myopathies (Kar and Pearson

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GEKALU M . FENlCHEL

1972), while others are increased in activity as a reflection of the active lipogenesis and collagen synthesis which characterizes the disease (Kar and Pearson 1973).

With no significant defect identifiable from studies on the major structural proteins and enzymes, attention has recently been directed to protein synthesis. increased protein synthesis has been identified, as measured by the rate of aminoacid incorporation as well as a change in the distribution of muscle polyribosomes (Monckton and Nihei 1969). This observation has been confirmed and extended to demonstrate that the heavy polyribosomes from dystrophic muscle produce four to five times more collagen than control fractions (lonasescu rt a/. I971 ). An increase in the rate of collagen synthesis has also been identified in a significant percentage of carriers (lonasescu et a/. 1973).

The concept that DMD may be a disorder of connective tissue is not a new one (Bourne and Golarz 1959). If this were the primary fault, the genetic mechanism could be through a defective repressor gene which fails to turn off collagen synthesis. An alternative explanation is, of course, that the over-production of collagen is not a primary fault, but rather a nonspecific cellular response to injury.

These interesting alternatives are worthy areas for further exploration.

Conclusions What sense can be made from these

apparently conflicting notions and observations? Alfred North Whitehead stated: “The aim of science is to seek the simplest explanation of complex facts”. I have no simple explanation for the complex facts of DMD, but it does seem to me that the one place where all the hypotheses collide is the neuroniuscular junction. The motor neuron has a trophic effect on skeletal muscle, mediated by a transmitted sub- stance or substances. I t directs protein synthesis and influences the metabolic identity of the cell. The myocyte is not the silent servant of the motor neuron: it actively feeds back information to the nerve which alters neurotransmission to suit changes which have occurred pri- marily in the muscle cell. The motor unit is in turn part of a larger living system which needs and has a multiplicity of devices to alter the events at the synapse. Among these devices are hormones which, acting through cyclic nucleotides at the cellular level, delicately and precisely tune events on both sides of the neuromuscular junction.

SUMMARY The relative merits of the three presently most active hypotheses (vascular, neurogenic,

and myogenic) concerning the pathogenesis of Duchenne muscular dystrophy are analysed and discussed and the literature is comprehensively reviewed.

ZUSAMMENFASSUNG Zur Pathogenese der Duclienne’schen Muskeldvstropliie

Die entsprechenden Hauptpunkte der drei gegenwartig bedeutensten Hypothesen (vaskular, neurogen und myogen) zur Pathogenese der Duchenne’schen Muskeldystrophie werden analysiert und diskutiert und es wird ein umfassender Literaturuberblick gegeben.

53 1


RESUMEN Sobre la patogenia de la distrojia muscular de Duchenne

Se analizan y discuten 10s mkritos relativos de las tres hip6tesis actualmente m9s activas (vascular, neurogdnica y miogknica) sobre la patogenia de la distrofia muscular de Duche- nne. Se revisa la literatura ampliamente.

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On the Pathogenesis of Duchenne Muscular Dystrophy