Biology of autophagy in skeletal muscles

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Autophagy is a protein recycling pathway whereby cellular contents can be delivered to the lysosome to yield recyclable nutrient components. Recent evidence suggests that autophagy plays a very important role in skeletal muscle autophagy. We intend to establish a research program at McGill University aimed at studying the basic aspects of autophagy in skeletal muscles and at training graduate students in basic cell biology research. The proposal consists of 3 sections. Section 1: We will evaluate the roles of mitochondrial reactive oxygen species (ROS) and permeability transition pore (PTP) as signal transducers triggering the induction of skeletal muscle autophagy in response to catabolic stimuli. To this end, we will use mice over-expressing peroxiredoxin 3 (Trx3+/+)(mitochondrial antioxidant enzyme) and mice deficient in cyclophilin D (Ppif-/-, essential component of mitochondrial PTP). Autophagy in Trx3+/+ and Ppif-/- mice and their littermate wild type mice will be examined along with proteasomal degradation pathways and mitochondrial respiration. Section 2: We will define morphological and biochemical characteristics of autophagy membranes in cultured skeletal muscle cells by using a fluorscent marker for autophagosomes. We will identify autophagy formation using confocal and electron microscopy as well proteomics approaches. Section 3: We will identify the transcriptional factors that regulate autophagy-related genes in skeletal muscles by using bioinformatics tools to analyze promoters of autophagy genes. Activation of predicted transcription factors will be verified in muscle cells using specialized technqiues and their functional importance in autophagy regulation will be verified with siRNA approach.