Rationale: Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health. Objective: The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity. Methods and Results: Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis.As p53 Key Words: aerobic Ⅲ exercise Ⅲ mitochondrial DNA Ⅲ p53 Ⅲ TFAM A cross populations aerobic exercise capacity inversely correlates with cardiovascular disease and all-cause mortality. 1-3 Our report of a marked reduction in the maximal exercise capacity of p53 homozygous knockout (p53 Ϫ/Ϫ ) mice and subsequent confirmation by others provided physiological evidence for p53 as an important mediator of aerobic metabolism. 4,5 We previously showed that p53 promotes mitochondrial respiration in human and murine cells by regulating the transcription of Synthesis of Cytochrome c Oxidase 2 (SCO2), a gene essential for oxidative phosphorylation. 4,6 A concurrent report demonstrating that p53 directly suppresses glycolysis through TIGAR, a p53-dependent regulator of glycolysis and apoptosis, suggested that p53 can coordinate aerobic and glycolytic metabolism. 7 Multiple factors contribute to aerobic exercise capacity, but one major determinant is the mitochondrial content of skeletal muscle as demonstrated by a genetic selection experiment. 8 A recent study showed decreased mitochondrial density in the skeletal muscle of p53-deficient mice, 5 but the genetic mechanism orchestrating this change has remained unclear. A number of other studies have also associated p53 with exercise response and mitochondrial function. For example, p53 levels are increased after acute exercise, and the transition from glycolysis to oxidative metabolism during development is dependent on p53. 9,10 p53 can transactivate ribonucleotide reductase p53R2 (RRM2B) which is important for maintaining mtDNA in skeletal muscle. 11 Additionally, 2 recent studies have shown asso-

We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+ in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metallation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu+ pools in SCO1 and SCO2 patient fibroblasts are largely unaltered relative to wildtype controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.