Myc Influences Mitochondrial Dynamics Figure 1. Levels of Myc affect mitochondrial structure in rat fibroblasts. (a) Mitochondrial ultrastructure. The indicated cell lines were examined by transmission electron microscopy. Representative mitochondria are presented. Note the smaller and cristae-deficient mitochondrion of the myc2/2 cells (arrow) and the complete restoration of this defect in myc2/2wtMyc cells. (b) Mitochondrial length. Mean mitochondrial length in each cell line was determined by measuring at least 30 individual mitochondria from images of multiple cells obtained by transmission electron microscopy. The student t-test was used to calculate the p-values indicated in the figure. doi:10.1371/journal.pone.0037699.g001 monomers, which tend to predominate under conditions of low membrane potential [41]. As seen in Figure 3a, the structurally abnormal mitochondria of myc2/2 cells showed a nearly ten-fold reduction in membrane polarization relative to that of myc+/+ and myc2/2wtMyc cells, with the latter showing no evidence of hyperpolarization as has been reported to occur in activated T- cells [42]. Having established that the enforced expression of Myc restored mitochondrial membrane potential, we next asked whether basal levels of oxidative phosphorylation (OXPHOS) were affected. We have previously shown that another rat fibroblast line, Rat1a, is more reliant on glycolysis than OXPHOS for its basal energy requirements but that Myc over-expression significantly increases the rates of both [43]. We used the XF24 Extracellular Flux Analyzer to measure oxygen consumption rate (OCR), which serves as a reliable surrogate for OXPHOS [44]. Our findings in myc2/2, myc+/+ and myc2/2wtMyc cells were consistent with our previous results. As seen in Figure 3b, basal OCR, as measured between 0 and 19 min. was approximately two-fold higher in myc+/+ cells than in myc2/2 cells and an additional 2– 2.5-fold higher in myc2/2wtMyc cells; thus, OCR was largely correlated with the degree of Myc expression and/or de- regulation. The cell lines were then sequentially exposed to four metabolic inhibitors to provide a profile of bio-energetic capacity. The first inhibitor, oligomycin (Figure 3b, addition A at 19 minutes), blocks the ATP synthase activity of Complex V and led to the expected cessation of respiration in all cell lines. Cyanide p-trifluoro- methoxy-phenylhydrazone (FCCP; Figure 3b addition B at 46 minutes) was next injected to promiscuously restore proton flow. The increase in FCCP-mediated OCR provided a measure- ment of the maximal respiratory rate of cells, which was significantly higher in myc2/2wtMyc cells. The metabolic response seen between the addition of FCCP and 2-deoxy-D- glucose (2-DG), which inhibits glycolysis (addition C at 74 min- utes), was representative of the so-called ‘‘spare respiration capacity (sRC)’’ [45,46]. myc2/2 cells had the lowest sRC, displaying approximately 30% lower levels than myc+/+ cells, whereas sRC was 10.6-fold higher in myc2/2wtMyc cells. Following the addition of 2-DG, myc2/2wtMyc cells displayed a further increase in OCR that, in combination with the sRC, represents the total reserve respiratory capacity [45,46], which was 20-fold higher than the level observed in myc2/2 cells. Finally, the injection of rotenone (injection D at 102 minutes), which inhibits Complex I, resulted in the complete cessation of electron flow and oxygen consumption in all cell lines. Taken together, these data show that Myc increases both actual baseline OXPHOS as well as the potential to achieve higher levels, in the face of otherwise normal mitochondrial mass. We next investigated the relationship between OXPHOS and ATP levels in the above cell lines. We found that myc+/+ cells and myc2/2wtMyc cells each contained about three-fold higher steady-state levels of ATP than myc2/2 cells (Figure 3c). The inhibition of glycolysis by 2-DG was associated with a nearly complete loss of net ATP levels whereas the inhibition of Complex I by rotenone was associated with much more modest decreases. PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e37699