Our Scientists

Muscular dystrophies, a group of genetic diseases that primarily affect skeletal muscle, are characterized by progressive muscle weakness. Duchenne muscular dystrophy is caused by mutations in the dystrophin gene that lead to the complete absence of dystrophin in skeletal muscle. Research in my laboratory on the function of dystrophin led to the discovery of the skeletal muscle dystrophin-glycoprotein complex (DGC), which spans the muscle cell membrane and links the sub-sarcolemma actin cytoskeleton to the surrounding basement membrane. Defects in genes that encode either components of the complex itself or mediators of its requisite post-translational modifications lead to distinct forms of muscular dystrophy. My current and future research focuses on four related areas: (1) the molecular pathogenesis of DGC disorders, (2) the mechanistic basis of maintaining muscle membrane integrity, (3) the molecular pathogenesis of disorders arising from defects in dystroglycan glycosylation, and (4) the structural basis of dystroglycan function as a basement membrane receptor.

The Molecular Pathogenesis of Disorders of the Dystrophin-Glycoprotein Complex
Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response. This form of fatigue fails to correlate with either central or peripheral fatigue and is disproportionate to activity level—and is thus a major determinant of disability. We have investigated the molecular basis of the exercise-induced fatigue response, using an interdisciplinary approach involving an integrated in vivo activity assay, genetically defined mouse models, laser Doppler imaging, magnetic resonance imaging, and patient biopsy analysis.

We have demonstrated that loss of neuronal nitric oxide synthase (nNOS), a DGC component, from the skeletal muscle sarcolemma exacerbates the fatigue experienced after mild exercise. This loss leads to a deficiency in the normal contraction-induced cGMP-dependent attenuation of local vasoconstriction, resulting in postexercise narrowing of the muscle vasculature. This decrease in blood flow manifests as an exaggerated fatigue response to mild activity and, in dystrophic muscle, causes exercise-induced muscle edema. We also showed that sarcolemmal nNOS levels are reduced in patient biopsies representing numerous distinct myopathies. This suggests a common mechanism of fatigue. In mouse models with mislocalized nNOS, exercise-induced fatigue and muscle edema can be relieved pharmacologically by enhancing nitric oxide–cGMP signaling from active muscle. These findings reveal a novel therapeutic strategy for addressing fatigue symptoms in neuromuscular disease patients in whom nNOS is mislocalized or reduced.

Although the muscular dystrophies affect primarily skeletal muscle, the protein components of the DGC are expressed in many tissues and fat infiltration is seen in many muscular dystrophies. To understand the possible nonmuscle function of the DGC, we studied white adipocytes, which share a common precursor with myocytes. White adiopcytes express a cell-specific sarcoglycan complex that contains β-, δ-, and ε-sarcoglycan. In addition, the adipose sarcoglycan complex associates with sarcospan and the laminin-binding form of dystroglycan. In studying multiple sarcoglycan-null mouse models, we discovered that loss of α-sarcoglycan has no consequences for expression of the adipocyte sarcoglycan complex but that, in adipocytes, the loss of either β- or δ-sarcoglycan leads to a concomitant loss of the sarcoglycan complex and sarcospan, as well as to a dramatic reduction in dystroglycan. Furthermore, in collaboration with Jeffrey Pessin (Albert Einstein College of Medicine), we have demonstrated that mice lacking the sarcoglycan complex in both adipose tissue and skeletal muscle (β-sarcoglycan-null mice) are glucose intolerant and exhibit whole-body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles.

The Mechanistic Basis of Maintaining Muscle Membrane Integrity
Compromised integrity of the muscle sarcolemma has been proposed to initiate muscle fiber pathology in muscular dystrophy, yet the molecular basis for this compromised integrity has never been clearly established. To study the possible role of dystroglycan in maintaining sarcolemmal integrity, we developed a novel in situ laser damage assay and measured lengthening contraction-induced muscle damage. To directly test the function of the dystroglycan-mediated link between the basal lamina and the sarcolemma, we examined the sarcolemmal integrity of the muscle fibers from the Large^myd mouse, an animal model for dystroglycan hypoglycosylation that lacks only the laminin globular domain-binding O-glycan that is present in wild-type counterparts. We found that despite maintaining an intact DGC, Large^myd muscle fibers have reduced sarcolemmal integrity and exhibit detachment from the basal lamina. This detachment makes Large^myd muscles highly susceptible to lengthening contraction-induced injury. Furthermore, we demonstrated that recombinant glycosylated α-dystroglycan can restore sarcolemma integrity of the Large^myd muscle fibers. Therefore, dystroglycan-dependent tight physical attachment of the basal lamina to the sarcolemma is important for transmission of the basal lamina's structural strength to the sarcolemma, providing resistance to mechanical stress. Our findings establish—for the first time—a mechanism that accounts for the increased susceptibility of patients with hypoglycosylated dystroglycan to contraction-induced muscle injury, and highlight the importance of this protective basic cellular mechanism in the context of mechanical damage.

We are also interested in understanding the role of dystroglycan in maintaining sarcolemmal integrity in cardiac muscle, since several forms of muscular dystrophy linked to dystroglycan hypoglycosylation are associated with the development of cardiomyopathy. We have shown that gene-targeted loss of dystroglycan function in ventricular cardiac myocytes is sufficient to induce a progressive cardiomyopathy (characterized by focal cardiac fibrosis, cardiac hypertrophy, and dilation) that ultimately leads to heart failure. Our findings suggest that, in cardiac myocytes, the ability of dystroglycan to function as an extracellular matrix receptor is crucial for limiting the spread of myocardial membrane damage to neighboring cardiac myocytes, and that loss of dystroglycan matrix receptor function in cardiac muscle cells is central to the development of cardiomyopathy in glycosylation-deficient muscular dystrophies.

Muscle cells also utilize dystroglycan-independent mechanisms to maintain sarcolemmal integrity. One example is plasma membrane repair, which involves the patching of new membrane to the surface membrane at or near the site of membrane disruption. Our previous data demonstrated that the novel membrane protein dysferlin (a gene mutated in two forms of dystrophy: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy) plays an important role in the Ca₂₊-dependent membrane repair of skeletal muscle. To study the role of membrane repair in muscular dystrophy, we generated mice deficient for both dystrophin and dysferlin. These mice exhibited not only more severe muscular dystrophy but also an accelerated progression of cardiomyopathy.

Since a number of inherited forms of dilated cardiomyopathy arise from mutations in genes encoding proteins that link the cytoskeleton to the extracellular matrix, we studied the possible role of dysferlin-dependent membrane repair in the heart. Using a modified membrane repair assay, we demonstrated that cardiac muscle possesses a Ca²⁺-regulated membrane-resealing mechanism that repairs membrane damage. Dysferlin plays an essential role in this process. In our study, the disruption of dysferlin-mediated membrane repair rendered the heart highly susceptible to stress-induced ventricular injury. In particular, stress exercise in dysferlin-null mice led to dysfunction of the left ventricle and to increased Evans blue dye uptake in dysferlin-deficient cardiomyocytes.Our results suggest that dysferlin-mediated membrane repair is necessary for maintaining the integrity of the cardiomyocyte membrane, particularly under conditions of mechanical stress. In addition, they provide a plausible explanation for the association of cardiomyopathy with most muscular dystrophy–associated mutations: contraction-induced injury causes myocyte necrosis because cumulative membrane damage cannot be effectively repaired.

The Molecular Pathogenesis of Disorders in Dystroglycan Glycosylation
Previous work in my laboratory revealed that dystroglycan glycosylation plays a role in the pathogenesis of Fukuyama congenital muscular dystrophy, muscle-eye-brain disease, and Walker-Warburg syndrome, each of which is a congenital muscular dystrophy with a range of associated developmental brain and eye defects. Genetic data showed that mutations in proteins with homology to glycosyltransferases (POMT1, POMT2, POMGnT1, FKTN, FKRP, and LARGE) are linked to these congenital muscular dystrophies (also called dystroglycanopathies). Biochemical analysis of muscle biopsies revealed a convergent role for these proteins in the glycosylation of α-dystroglycan, a process required for functional activity. We have confirmed these findings and extended our work on the dystroglycanopathies to milder forms of limb-girdle muscular dystrophy with or without brain involvement and confirmed that the more severe cases are characterized by a nearly complete lack of functional glycosylation; those that are milder show only reduced glycosylation. (This work has been facilitated by the Iowa Wellstone Muscular Dystrophy Cooperative Research Center, which is under my direction.)

We were also interested in determining if the loss of dystroglycan could result in the full spectrum of congenital brain and eye malformations observed in the dystroglycanopathies. Attempts to investigate the mechanism(s) underlying the brain and eye pathology have been technically challenging, since eliminating either dystroglycan or a glycosyltransferase involved in its post-translational modification causes early embryonic lethality in mice. We showed that mice with epiblast-specific loss of dystroglycan develop brain and eye defects that broadly resemble the clinical spectrum of the human disease. Specifically, we established that (1) dystroglycan is the key substrate affected by loss of glycosyltransferase activity, and loss of its function is responsible for the full spectrum of brain and eye malformations observed in the dystroglycanopathies; and (2) dystroglycan stabilizes basement membrane structures in both the brain and eye, and disruption of the basement membrane structure is a common mechanism that underlies developmental defects in the dystroglycan-deficient brain and eye. These findings demonstrate that dystroglycan plays a central role in the dystroglycanopathies and suggest that novel defects in post-translational processing or mutations of the dystroglycan gene itself may underlie disease cases in which no causative mutation has been found.

To further our understanding of the biology of dystroglycan glycosylation, we have also investigated the hypoglycosylation of dystroglycan in epithelial cancer cells. The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is expressed in epithelial cells and mediates interactions between the cell membrane and basement membranes in various epithelia. In earlier studies in collaboration with Mina Bissel (Lawrence Berkeley National Laboratory), we had shown that in many epithelium-derived cancers, α-dystroglycan is not detected, although the cotranslated β-dystroglycan is. Our more recent study revealed that in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers, α-dystroglycan is correctly expressed and trafficked to the cell membrane but fails to bind laminin because of the silencing of the LARGE gene. Exogenous expression of LARGE in these cancer cells restored the normal glycosylation and laminin binding of α-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings suggest that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells.

The Structural Basis of Dystroglycan Function as a Basement Membrane Receptor
We are also studying post-translational processing of dystroglycan by the novel glycosyltransferase LARGE. My laboratory found that the amino-terminal domain of α-dystroglycan is essential for recognition by LARGE. This enzyme-substrate recognition motif is critical for the post-translational modification of dystroglycan and is necessary for the functional maturation of the protein into a receptor for extracellular matrix components. We revealed the significance of this pathway in vivo, as gene transfer of LARGE into muscle-specific gene-knockout mice rescued domain-specific functions of dystroglycan in the whole animal. Thus, molecular recognition of dystroglycan by LARGE in the biosynthetic pathway is essential to the production of functional dystroglycan.

We have explored the potential of LARGE overexpression as a treatment for glycosyltransferase-deficient muscular dystrophies. We showed that the enzyme LARGE modifies the sugar moieties of α-dystroglycan and prevents muscular dystrophy in myd mice. We also demonstrated that high levels of LARGE restore the function of α-dystroglycan and modulate its glycosylation in myoblasts and fibroblasts from patients afflicted with Fukuyama congenital muscular dystrophy, muscle-eye-brain disease, and Walker-Warburg syndrome. We also discovered that LARGE-dependent glycosylation of α-dystroglycan is required for dystroglycan function and that induction of LARGE restores the function of α-dystroglycan, regardless of the type of glycosyltransferase that is mutated in patients. This work indicates that LARGE has broad therapeutic potential.

This work was supported in part by grants from the National Institutes of Health and the Muscular Dystrophy Association.

As of September 02, 2010