Genetic and Functional Analysis of Cell Adhesion in Muscle

University dissertation from Stockholm : Stockholm University

Abstract: Skeletal muscle is one of the most abundant tissues in the body, and its main function is to generate the force for movement. The mature muscle cell is a giant, elongated, multinucleated cell surrounded by a specialized, extracellular matrix (ECM), the basement membrane (BM). The BM in muscle, as in other tissues, is composed of laminin, type IV collagen, entactin/nidogen and heparan sulphate proteoglycan. One major component of the BM in muscle is laminin-2, which is composed of a heavy chain laminina2 and two light chains,b1 and lamining1. Laminin-2 is predominantly expressed in skeletal muscle and peripheral nerve but is also found in other tissues.Cell adhesion to the basement membrane is mediated by cell surface receptors, which thereby link the BM to the cytoskeleton. This linkage is thought to be important for generating the force required for movement. Mutations in adhesion molecules in muscle cause muscular dystrophy, proving the importance of cell adhesion in muscle.In order to analyze the molecular mechanisms of cell adhesion in muscle, we have analyzed laminin-2 and two other muscle adhesion proteins, laminina-sarcoglycan and tetranectin, in muscle development and regeneration. Most importantly, we have developed in vitro and in vivo models for laminin-2 deficient muscular dystrophy.We generated several lines of mutant embryonic stem (ES) cell with disruption of the laminin- laminina2 chain gene. We found that homozygous null mutant ES cells differentiate normally in vitro, giving rise to cardiomyocytes, myotubes, and smooth muscle cells in addition to many other cell types. However, the myotubes that are formed are unstable. They detach, collapse, and degenerate, a process which is initiated at the appearance of the mature, contractile phenotype of the cells. We propose that the detachment and death of contracting myotubes in vitro has its counterpart in vivo, and that contraction-induced myofiber damage, along with the lack of survival cues provided by laminin-2/merosin, is a significant contribution to muscle degeneration in merosin-deficient muscular dystrophy.We used laminin laminina2 mutant mice to study the expression of laminin-2 in development and regeneration using the lacZ gene as a reporter for the lama2 gene. We found that the lacZ/lama2 gene is highly expressed in the early stages of myogenesis and is down regulated when myogenesis is completed. Most importantly, the gene is up-regulated early in muscle regeneration, suggesting that laminin-2 plays an important role in this process. Despite the prominent expression of lama2 in normal development, laminina2 null mutant mice have no obvious developmental defect. Instead, they develop muscular dystrophy two weeks after birth. We found extensive apoptosis in null mutant mice, and this cell death is dramatically reduced in mice in which laminin-2 expression is restored in skeletal muscle by expression of a wild type LAMA2 transgene. Most of the apoptotic cells in null mutant mice are newly formed myofibers, suggesting that laminin-2 is needed for maturation and survival of regenerated myotubes. The apparent abortive muscle regeneration in laminin-2 deficiency suggests that the severe disease of MCMD is caused by insufficient regeneration after muscle damage.We have expressed a human LAMA2 transgene under the regulation of a muscle-specific creatine kinase promoter in mice with complete or partial deficiency of merosin. The transgene restored the synthesis and localization of laminin-2 in skeletal muscle, and greatly improved muscle morphology and integrity and the health and longevity of the mice. However, the transgenic mice share with the non-transgenic dystrophic mice a progressive lameness of hind legs, suggesting a nerve defect. These results indicate that the absence of merosin in tissues other than the muscle, such as nervous tissue, is a critical component of MCMD.We have cloned and characterized, a-sarcoglycan/adhalin, a member of the dystrophin associated sarcoglycan complex in muscle. We showed that a-sarcoglycan is expressed very late in myogenic differentiation both in vitro and in vivo. In fact, the expression is associated with the capacity of muscle cells to contract. The sarcoglycans may therefore have a role in muscle contraction. We also analyzed an ECM-associated molecule, tetranectin. We showed that expression of tetranectin is closely associated with skeletal muscle development and regeneration, and with muscle cell differentiation in vitro.In summary, our studies show the importance of laminin-2 in skeletal muscle. We have provided new information on three markers for different stages of myogenic differentiation, laminin laminina2, laminina-sarcoglycan, and tetranectin. In addition, our studies contribute to a better understanding of the mechanism of human disease caused by laminin-2 deficiency.

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