Understanding basic regulation of mitochondrial bioenergetics and adaptations to exercise

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Within muscle, mitochondria influence several processes, including metabolic homeostasis, apoptosis and redox balance. However, the regulation of mitochondria remains poorly elucidated. Therefore, a better understanding of these dynamic structures could provide insight into many cellular processes. By incorporating molecular, biochemical and physiological approaches my long-term goals are to determine a) mechanisms regulating mitochondrial bioenergetics, b) mitochondrial biogenesis, and c) understand how these effect substrate utilization in skeletal muscle. My specific hypotheses and short-term plans are: 1. Regulation of mitochondrial FAT/CD36 translocation: I have previously shown that exercise redistributes FAT/CD36 to mitochondrial membranes, however, the molecular basis for this subcellular trafficking remains unknown. I hypothesize that the C-terminal YCACR motif of FAT/CD36 and AMP activated protein kinase (AMPK) signaling are both required for this cellular process. I will transfect wild type and C-terminal mutants (C-terminal amino acids deleted) into the skeletal muscle of FAT/CD36 knock out mice. Thereafter I will provide metabolic challenges (eg. sciatic nerve stimulated contraction) and determine the ability of FAT/CD36 to accumulate on mitochondrial membranes and the functional consequence on rates of fatty acid oxidation/respiration. Similar experiments in wild type and AMPK kinase dead mice will determine the necessity of AMPK signaling in mediating mitochondrial FAT/CD36 translocation. 2. Regulation of carnitine palmitoyltransferase-I (CPTI) malonyl-CoA (M-CoA) kinetics: I have previously shown that exercise augments the ability of M-CoA to inhibit CPTI, but the molecular basis for this remains unknown. To ascertain direct regulation of CPTI, substrate kinetics (PCoA and M-CoA) will be performed and CPTI will be immunoprecipitated and examined for redox modifications and acetylation status before and after exercise in humans. In addition, CPTI substrate kinetics will be determined various ways in the presence and absence of the cytoskeletal network (specifically ß-tubulin) to determine the ability of the cytoskeletal network to regulate CPTI. 3. The metabolic role of the mitochondrial reticulum: I have previously shown that over-expressing mitofusin-2 (MFN2) independently does not influence mitochondrial bioenergetics. I therefore hypothesize that the primary metabolic role of MFN2 is to anchor mitochondria to the sarcoplasmic reticulum, creating an efficient micro domain for ADP transport from SERCA to mitochondria. Therefore, I will over-and-under express MFN2 in rat skeletal muscle and subsequently determine a) muscle fatigue rates, b) intracellular calcium levels during contraction, and c) calcium stimulated mitochondrial respiration kinetics. 4. Regulation of mitochondrial biogenesis: Scarce information exists regarding post-transcriptional mechanisms that influence mitochondrial biogenesis. In eukaryotic cells a variety of RNA binding proteins (RBPs) have been identified which regulate mRNA stability. The role of these RBPs in mammalian muscle remains unknown. Therefore, I will over-express HuR (stabilizer), CUB-BP1 and AUF1 (both destabilizers) alone and in combination in rat muscle to determine the effect on mRNA profiles, whole muscle metabolism, mitochondrial content/respiratory capacity, and responses to acute and chronic muscle contraction. SIGNIFICANCE; All required methodologies have been established in my laboratory. The proposed studies will provide fundamental knowledge on diverse cellular processes, and therefore, HQP within my laboratory will be comprehensively trained.