INTRODUCTION

Muscular dystrophies are a group of genetic diseases that primarily affect skeletal muscle and are characterized by progressive muscle weakness. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that lead to the complete absence of dystrophin in skeletal and cardiac muscle. Research in my laboratory on the function of dystrophin led to the identification and purification of the dystrophin-glycoprotein complex (DGC) from skeletal muscle, which provides an essential structural link between the actin cytoskeleton and the extracellular matrix. Defects in genes encoding a number of components of this complex lead to distinct forms of muscular dystrophy. Current projects in my laboratory are aimed at determining the function of the DGC to understand the molecular pathogenesis of muscular dystrophy and associated cardiomyopathy and to develop therapeutic approaches to treat muscular dystrophy.

Dystrophin-Glycoprotein Complex

The DGC is a large oligomeric complex of membrane proteins in the sarcolemma of skeletal muscle. Biochemical and structural characterization of the DGC indicates that it consists of dystrophin, a large, rod-shaped cytoskeletal protein that binds F-actin; a- and b-dystroglycan, which bind the G domain of laminin-2 and the cysteine-rich region of dystrophin, respectively; the syntrophins, intracellular proteins that bind the carboxyl terminus of dystrophin; and the sarcoglycan-sarcospan (SG-SSPN) complex. Based on interactions of the DGC with the extracellular matrix and the cytoskeleton, and the consequences of loss of function in genes encoding DGC componenets, we have proposed that at least one function of the DGC is to provide mechanical reinforcement of the sarcolemma and to maintain membrane integrity during cycles of contraction and relaxation. The absence of dystrophin would disrupt these interactions, rendering the sarcolemma susceptible to damage from muscle contraction and thus leading to muscle cell necrosis in patients with DMD.

Dystroglycan and Basement Membrane Assembly

A major emphasis of my laboratory is determining the cellular function of dystroglycan, a-Dystroglycan binds the extracellular matrix component laminin-2 with high affinity, while b-dystroglycan anchors dystrophin to the sarcolemma membrane. Thus, through its interactions with laminin and dystrophin, dystroglycan acts as a transmembrane link between the extracellular matrix and the cystoskeleton. Dystroglycan expression is not restricted to muscle but is widely expressed in many cell types, particularly those associated with basement membranes. Targeted inactivation of the dystroglycan gene in the mouse demonstrated that dystroglycan is required for embryonic development and suggested that dystroglycan mediates the assembly and/or maintenance of the basement membrane.

Based on genetic analysis of embryonic stem (ES) cells, we have recently shown that dystroglycan is required for the formation of basement membranes, shedding light on the process of basement membrane formation in vivo. Specifically, we have defined a dystroglycan-dependent interaction with laminin as a requisite, initial step in the formation of basement membranes. Moreover, we have shown thatt dystroglycan mediates the formation of laminin clusters on the surface of ES cells. Our work indidates that laminin clustering may be a general mechanism related to basement membrane assembly. We are generating mice with a skeletal muscle-specific disruption of the dystroglycan gene to examine directly dystroglycan's function in skeletal muscle.

Molecular Pathogenesis of Limb-Girdle Muscular Dystrophy

Limb-girdle muscular dystrophy (LGMD) is genetically and clinically heterogeneous; it may be inherited in an autosomal-dominant or -recessive manner, and it has different rates of progression and severity. Our studies of the structure and funcion of the DGC suggested that a primary deficiency in a dystrophin-associated protein could be responsible for an autosomal-recessive LGMD. Several years ago we showed that a-sarcoglycan (adhalin) is deficient in skeletal muscle of patients with severe childhood autosomal-recessive muscular dystrophy. Subsequent work showed that missense mutations in a-sarcoglycan cause a form of LGMD.

The involvement of the sarcoglycan complex in the pathoenesis of LGMD has become increasingly clear. Work from many laboratories, including my own, shows that mutations in the skeletal muscle sarcoglycan genes lead to four forms of LGMD. LGMD families harbor mnay different mutations, which cause diesase ranging in severity from mild impairment with slow progression to severe disabilitily and rapid deterioration.

The availibility of accurate animal models of LGMD will greatly facilitate the investigation of the molecular pathogenesis of this disease. We are studyin a-, b- and d-sarcoglycan-null mice. Observations of these mutatnt mice indicate that they display complete deficiency of the SG-SSPN complex at the sarcolemma surrounding muscle fibers and that they develop progressive muscular dystrophy. In addition, we have shown that the sarcoglycan complex is responsible for the anchoring of a-dystroglycan to the sarcolemma membrane.

Gene Transfer for LGMD

As with any hereditary disorder, there is considerable interest in developing genetic therapy for LGMD. Exploration of potential treatments has been facilitated by the use of animal models of muscle dystrophy, such as the BIO 14.6 hamster, which has a deletion in the d-sarcoglycan gene, and our sarcoglycan-deficient mice. We have found that the skeletal muscle phenotype in the BIO 14.6 hamster can be corrected via direct intramuscular injection of an adenovirus that contains the normal d-sarcoglycan cDNA. High levels of expression and the renewed expression of all sarcoglycan proteins in the sarcolemma can be generated from a single injection. Importantly, the expression of the sarcoglycan complex results in the stabilization of dystroglycan. This restoration of the DGC results in a dramatic reduction in membrane permeability and reduces the level of central nucleation found in the infected fibers, a hallmark of progressive muscle degeneration.

The most remarkable finding of this study was that persistent expression from the d-sarcoglycan adenovirus can be detected for as long as 12 months. Our goal is to demonstrate the feasibility of viral-mediated sarcoglycan gene transfer into sarcoglycan-null mice and its therapeutic potential. (A grant from the Muscular Dystrophy Association provided support for this project.)

Sarcoglycan-Defient Cardiomyopathy

Cardiomyopathy is a multifactorial disease, and recent experiments have implicated a role for the DGC in the pathogenesis of both hereditary and acquired forms of cardiomyopathy. To investigate mechanisms in the pathogenesis of cardiomyopathy associated with mutations of the DGC, we analyzed genetically engineered mice deficient for either a-sarcoglycan (Sgca) or d-sarcoglycan(Sgcd). We found that only Sgcd-null mice develop cardiomyopathy, with focal areas of necrosis as the histological hallmark in cardiac and skeletal muscle. Absence of the SG-SSPN complex in skeletal and cardiac membranes was observed in both animal models. Loss of vascular smooth muscle SG-SSPN complex was only detected in Sgcd-null mice and associated with irregularities of the coronary vasculature. Administration of a vascular smooth muscle relaxant prevents onset of myocardial necrosis.

Our data indicate that disruption of the SG-SSPN complex in vascular smooth muscle perturbs vascular function, which initiates cardiomyopathy and exacerbates muscular dystrophy. Our results present a novel pathogenetic mechanism for muscular dystrophy and cardiomyopathy and reveal new insights into the involvement of the Sg-SSPN complex in vascular smooth muscle function.