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In muscle cells, action potentials originating from the neuromuscular junction cause release of calcium from intracellular stores that triggers muscle contraction, a process known as excitation contraction (EC) coupling. Formation of specialized membrane structures during skeletal muscle development collectively known as the triad junction is absolutely required for the regulation and maintenance of the calcium cycling that occurs during EC coupling. The triad junction is a well-organized anatomical structure consisting of the transverse-tubule invagination of the plasma membrane and the terminal cisternae of the sarcoplasmic reticulum that stores the intracellular calcium. Many skeletal muscle diseases such as limb girdle muscular dystrophy and miyoshi myopathy present muscle weakness and fatigue phenotypes. Skeletal muscle structure defects are observed in these diseases and are postulated to be an underlying pathophysiological cause.In this project we investigated whether over-expression of Mitsugumin 29 (MG29), a protein intricately involved the biogenesis of triad structures, can improve disease phenotypes in a mouse model of limb girdle muscular dystrophy (A/J mice). We have found that adeno-associated virus (AAV) delivery of FLAG tagged MG29 (FLAG-MG29) in skeletal muscle was able to significantly improve the transverse-tubule network and triad junction structure in the A/J mice. These structural developments correlated with improvements in skeletal muscle calcium signaling as well as increased skeletal muscle contractile capacity.In addition to the critical roles in skeletal muscle development, we also tested if MG29 contributes to skeletal muscle regeneration following injury. Using cardiotoxin (CTX) induced-skeletal muscle injury, we observed that MG29 deficient mice have impaired muscle regeneration capacity compared to wild type littermates. We present evidence that functional interaction between MG29 and Bin1, another resident triad protein that promotes membrane bending and tubulation, plays a critical role in facilitating regeneration of skeletal muscle after cardiotoxin injury.MG29 belongs to the synaptophysin protein family that plays important roles in vesicle fusion and membrane recycling. Several studies have implicated single nucleotide polymorphisms in SYPL2, the gene that codes for MG29, as high risk factors for metabolic dysfunction in obesity and diabetes. We thus hypothesize that MG29 may interact with the glucose-transporter (Glut-4) machinery to modulate glucose uptake into muscle fibers. We found that the absence of MG29 leads to decreased ability of insulin-mediated uptake of blood glucose, leading to glucose-intolerance in the mg29-/- mice. AAV-mediated expression MG29 could restore the defects in glucose-handling in the mg29-/- mice. We further demonstrated that MG29 can interact with vesicle associated membrane protein 3 (VAMP3), which is found on glucose transporter 4 containing vesicles (GSVs). The MG29/VAMP3 interaction could be enhanced in skeletal muscle after insulin stimulation. Therefore, targeting MG29/VAMP3 interaction can potentially be an alternative approach to treat obesity or diabetes.The completion of these studies have progressed the MG29 research field by providing novel evidence of new interacting partners in the context of skeletal muscle and metabolic disease. Most importantly, this project establishes a proof-of-concept of the therapeutic potential of MG29 over-expression for improvement of skeletal muscle physiology.