• Characterizing the role of tafazzin in allergically activated mast cells

      Maguire, Aindriu; Applied Health Sciences Program
      Introduction & Aim: Allergic inflammatory diseases are a constantly growing health concern in westernized societies. Mast cells, the driving force behind many allergic diseases, modulate various metabolic pathways to carry out their various functions following IgE-FceRI-mediated activation. Tafazzin is a cardiolipin transacylase that works to remodel cardiolipin into its mature form of tetralinoleoyl-cardiolipin. Mature cardiolipin is important for efficient energy production through oxidative phosphorylation. The aim of this project is to study the effects of a reduction in tafazzin protein content on IgE-mediated mast cell activation. Methods: Fetal liver-derived mast cells (FLMCs) were treated with 1 μg/mL doxycycline for 5 days to initiate the tafazzin knockdown. Western blotting was used to confirm a reduction in tafazzin protein content. Flow cytometry was used to ensure that the FLMCs expressed both c-kit and FceRI receptors, and that receptor expression was not impacted by the doxycycline treatment. Oxygen consumption rate was measured using a Clark-type electrode. b-hexosaminidase release assays were utilized to assess degranulation during the early phase of the allergic reaction. ELISA assays were used to examine secretion of de novo synthesized inflammatory mediators, while qPCR assays were used to examine gene transcription of these mediators. Results: The doxycycline inducible tafazzin knockdown resulted in a 99.5% reduction in tafazzin protein content. This reduction was accompanied by a 25% reduction in oxygen consumption and a 31.4% reduction in degranulation. Secretion of CCL1, CCL2, and TNF was significantly reduced, CCL9 secretion levels showed a decrease that approached significance. Secretion of CCL3 and IL-6 was not impacted. Gene expression was not impacted for any of the inflammatory mediators measured. Conclusion & Significance: These results suggest that tafazzin may play a role in mast cell oxidative phosphorylation. Functionally, the results suggest that tafazzin plays a role in degranulation and mediator secretion. However, since gene expression was not impacted it is thought that the tafazzin reduction causes these decreases due to an impairment of the exocytosis mechanism. This work adds to the growing field of immunometabolism and improves our understanding of how mast cells modulate metabolic pathways during an allergic inflammatory event.
    • The temporal relationship between cardiolipin biosynthesis and remodeling enzymes and cardiolipin content during unloading atrophy in mouse soleus

      Elkes, Mario; Applied Health Sciences Program
      Skeletal muscle is a heterogeneous tissue that consists of individual fibers that differ in contractile and metabolic properties. Skeletal muscle is also dynamic in its ability to adapt to external stimuli through changes in cell size, number, and/or fiber type composition, which are matched by mitochondrial content. Mitochondria are central to skeletal muscle adaptations and mitochondrial energetic function is highly dependent on the membrane phospholipid composition, specifically the mitochondrially exclusive cardiolipin (CL). CL biosynthesis results in nascent CL which must be remodeled by tafazzin (Taz) to form the predominant CL species in mammals, tetralinoleoyl cardiolipin (TLCL). Previous research has shown that CL content and 18:2n6 composition decreased and Taz protein content increased in tenotomy-induced atrophied mouse soleus, suggesting the upregulation of Taz may play a role in slowing this process. Thus, the purpose of this thesis was to examine the temporal relationship between enzymes of CL biosynthesis and remodelling and CL content during unload induced atrophy. Fourteen days post tenotomy resulted in reduced expression of phosphatidyl glycerol phosphate synthase (PGS1) and Taz protein, as well as a reduction in CL content. PGS1 appeared to be reduced prior to changes to CL content, while Taz was reduced following changes to CL content, suggesting that CL content is mainly dependant on PGS1, and the reduction in total CL may have reduced the need for Taz. This is the first study to show a temporal relationship between CL, CL biosynthesis and remodeling enzymes during muscle atrophy. Our identification of the CL biosynthesis proteins which are impacted during muscle atrophy resulting in reduced CL content, may pave the path for future treatment strategies to preserve the function of these enzymes during atrophy, and maintain CL content.