Skeletal muscle disease is a source of pain and debilitation for many thousands of people worldwide (2). These diseases can result from a range of extrinsic causes (3) or genetic mutations involving structural (4, 5), metabolic (6) or signaling components of the muscle (7). The goal of this project will be to understand the function of the recently identified disease-causing gene, STAC3 (8, 9). Global ablation of STAC3 in mice causes complete fetal paralysis and perinatal death due to lack of muscle contraction (9). The lethality of STAC3 deletion at birth demonstrates the importance of this protein for normal muscle development and function;however, perinatal lethality precludes analysis of STAC3 function in juvenile and adult mice, the life stages most relevant for understanding human disease. Because defects in calcium handling cause disease in juvenile and adult humans, the first aim of this study will be to ablate STAC3 in postnatal mice using an inducible cre-lox model. This strategy will eliminate most but not all STAC3 and is expected to accurately model mutations that affect protein expression or stability. Characterization of these mutants will provide deeper insight into the role of STAC3 in postnatal muscle function and will provide a rational basis for understanding human phenotypes resulting from STAC3 mutations.
The second aim of the proposed project will be to identify interaction partners of STAC3. It is likely that STAC3 interacts with complexes associated with similar phenotypes, namely the dihydropyridine receptor complex or the ryanodine receptor, so components of these complexes will be systematically evaluated for interaction using protein pull-down from cultured muscle cells. In addition to individual candidate analysis, mass spectrometry will be used to cross validate positively interacting candidates and to provide an unbiased method to discover unexpected interactions.
The third aim of this project will be to understand which residues in the STAC3 protein are required for its function. To accomplish this aim, a knockout C2C12 myoblast cell line will be generated using Transcription Activator-Like Effector Nucleases (widely known as TALENs). These cells can then be easily manipulated to express mutant constructs using retroviral transduction followed by assessment of calcium release in response to excitation. This strategy will provide a low cost, reproducible system for rapidly characterizing mutant constructs and will reduce the number of animals needed to complete this study by eliminating the need for primary knockout myoblasts. Mutants that are unable to rescue calcium release will then be assessed for loss of interaction with proteins identified in aim 2. Completion of these aims will improve understanding of STAC3 function and excitation-contraction coupling, which may serve as a basis for new treatment strategies for a variety of muscle maladies.
Skeletal muscle is vitally important to a normal, healthy lifestyle and to maintenance of metabolic homeostasis (1). Resulting from genetic and other causes, diseases of skeletal muscle are a major burden for millions of people worldwide. Through this study, we will explore how molecular defects in the gene STAC3 affect the ability of muscle to function normally.