LONDON - Almost since the discovery of the dystrophin gene in 1989, it was assumed that the many forms of muscular dystrophy were caused by simple structural defects due to absent proteins. These proteins, which form a complex at the sarcolemma, include not only dystrophin (whose absence causes Duchenne and Becker muscular dystrophies), but also the dystroglycans and the sarcoglycans, implicated in other types of muscular dystrophies.
However, according to Francesco Muntoni, MD, of the Imperial School of Medicine in London, this view has begun to change in the past several years, in light of a growing understanding of the interactions among the many proteins of the sarcolemmal complex. Dr. Muntoni shared an evolving perspective on the pathophysiology of muscular dystrophy at the 17th World Congress of Neurology here in London in June.
DYSTROPHIN: NOT JUST A STRUCTURAL MOLECULE
It is clear dystrophin has multiple roles. It is a structural molecule that is involved in force transduction, Dr. Muntoni said. However, more recently it has become clear that dystrophin and proteins of the complex have a very clear role in signaling.
The evidence that dystrophin is a structural molecule is strong, Dr. Muntoni said. Dystrophin binds filamentous actin(F actin) at its N terminus within the muscle fiber, and has homology- that is, similar critical attributes - with several other well-recognized structural proteins of muscle. One of the earliest recognized functions of dystrophin was the binding of cortical actin to the extracellular matrix via dystroglycan. It's got the right credentials to be a structural molecule, Dr. Muntoni said.
How does the loss of the structural properties of dystrophin lead to muscle degeneration? Much speculation has centered on a disruption of calcium homeostasis, Dr. Muntoni said. It is thought that calcium enters the dystrophin-deficient muscle as a consequence of the structural abnormality of the sarcolemma, the plasma membrane of a muscle fiber. However, while it is well documented that calcium is increased in Duchenne muscle, the mechanism for this increase is still uncertain.
Dr. Muntoni noted that the absence of dystrophin also causes loss of other structural proteins. The significance of dystophin's interaction with actin is highlighted in some patients whose dystrophin gene has a mutation in the actin binding domain.Despite producing a lot of dystrophin, he said,these patients have a very severe Duchenne muscular dystrophy phenotype.
Although the structural pedigree of dystrophin is unimpeachable, it's not nearly the whole story. The truth is, though you have a single gene defect, it leads to deficiency not of a single protein function, but of many different functions, Dr. Muntoni said.
Consider the interaction of dystrophin with F actin. This form of actin is a major molecule in the trafficking of GLUT-4, the glucose transporter in muscle fiber. If you don't have well arranged cortical actin cytoskeleton, you will have a deficiency of GLUT4 at the sarcolemma, and this has been demonstrated biochemically. I am not suggesting this has a major role in the pathophysiology of DMD, Dr. Muntoni said, but it indicates how naïve we have been in thinking there is only one function of dystrophin.
Dr. Muntoni suggested that more central to the pathology of the muscular dystrophies is the role of the entire protein complex in signaling, especially through its function in stabilizing neuronal nitric oxide synthase, or nNOS. This enzyme creates nitric oxide, a molecule known to be an important mediator of smooth muscle activity, as well as other functions. The enzyme binds to dystrobrevin, another protein in the complex. Dystrobrevin deficiency causes muscular dystrophy without causing any structural abnormalities of the complex, Dr. Muntoni said. When one or more components of the complex are absent, the level of nNOS goes down, and nitric oxide signaling is disrupted.
Signaling by nNOS in the muscle appears to be at least partly responsible for modulation of blood flow. The nNOS mediates sympathetic vasoconstriction in the contracting muscle, Dr. Muntoni said. If there is no nNOS, there is an abnormal blood flow response to exercise. This has been demonstrated in children with DMD and in animal models.
COMPLEX INTERACTIONS
The roles of other proteins of the complex in the pathogenesis of muscular dystrophies are also coming into clearer focus. The sarcoglycans are a group of proteins at the sarcolemmal membrane that interact with dystrophin and with alpha- and beta-dystroglycans. Dr. Muntoni noted that patients with beta sarcoglycan mutations have a demonstrated calcium dysfunction, and that work in mice has shown that long-term treatment with verapamil, a calcium channel blocker, can suppress the development of cardiomyopathy in this model. This may be important for clinical application, Dr. Muntoni said.
The role of the dystroglycans is somewhat less clear. Missense mutations in the dystroglycan-binding site of dystrophin will cause Duchenne muscular dystrophy. That's a powerful indication that, together with the actin cytoskeleton, you need dystroglycan, or else you will develop muscular dystrophy, Muntoni said. But why do you need it?
The dystroglycan gene codes for two proteins, the alpha and beta forms, which have fundamental roles in assembly of the basement membrane, an amorphous extracellular layer of epithelium, muscle, fat, and Schwann cells.
Dr. Muntoni predicted that patients completely lacking dystroglycan would never be found, based on knockout experiments showing the nonviablity of the genotype. Patients with reduced dystroglycans have been identified, and chimeric mice that express dystroglycan in the rest of body but lack it in skeletal muscle develop muscular dystrophy.
Secondary deficiency of dystroglycan also appear in Duchenne muscular dystrophy, and in one form of congenital muscular dystrophy. A paper published in June in Nature Genetics shows that a mutation in a gene that adds sugars to alpha-dystroglycan causes a form of muscular dystrophy in mice (2001; 28:151-154). In humans, there is an as-yet uncharacterized form of congenital muscular dystrophy in which dystroglycan is down-regulated. It is likely that an increasing number of conditions will be associated with secondary abnormalities of dystroglycan, Dr. Muntoni predicted.
Dr. Muntoni suggested that the lesson from these discoveries is to stay open to new ideas. Don't marry one single hypothesis, he said, because the true picture is likely to be much more complex than the simple models suggest.
Sharon Hesterlee, PhD, who is Director of Research Development for the Muscular Dystrophy Association, commented that this deeper understanding of the multiple roles of dystrophin is a welcome development. As we've learned more about the proteins at the sarcolemma, we've begun to see that signaling is potentially a very important function of the proteins of the complex. Especially with the involvement of nitric oxide synthase, this may give us some new therapeutic directions to explore.
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