Skeletal Muscle Fibre-Specific Knockout Of P53 Does Not Reduce Mitochondrial Content Or Enzyme Activity

Tumour protein p53 (p53) was initially characterised as a tumour suppressor protein (Matoba et al., 2006; Vousden and Prives, 2009; Muller and Vousden, 2013), serving to regulate cellular metabolism and proliferation (Zhou et al., 2003; Bensaad et al., 2006; Matoba et al., 2006). More recently, a functional role of p53 for in vivo skeletal muscle physiology has been proposed, following observations that p53 can regulate apoptosis (Saleem et al., 2009), atrophy (Fox et al., 2014), autophagy (Saleem et al., 2014), mitochondrial DNA stability (Saleem and Hood, 2013; Safdar et al., 2016), post-exercise signalling (Saleem et al., 2009, 2014), mitochondrial function (Park et al., 2009; Saleem et al., 2009, 2014; Wang et al., 2013) and endurance performance (Park et al., Prime Boosts Supplement 2009; Saleem et al., longer lasting pills 2009; Wang et al., 2013) within skeletal muscle. Whole-body knockout (KO) of p53 in mice results in a deficient skeletal muscle mitochondrial phenotype (Park et al., 2009; Saleem et al., 2009), displaying reduced mitochondrial mass, mtDNA copy number, cytochrome-c oxidase enzyme activity, and state 3 respiration (Park et al., 2009; Saleem et al., 2009). As a consequence, endurance capacity and voluntary wheel running are also reduced in p53 KO mice (Park et al., 2009; Saleem et al., 2009). In comparison, oncogenic p53 mutations found in the Li-Fraumeni syndrome increase in vivo skeletal muscle oxidative phosphorylation in humans (Wang et al., 2013), while mitochondrial respiration and content of electron transport chain proteins is increased in primary myoblasts from Li-Fraumeni carriers and in mice carrying a p53 R712H polymorphism (Wang et al., 2013). Thus, it is clear that p53 plays an important role in mitochondrial metabolism and function.



Whilst loss of p53 impairs mitochondrial function, importantly, p53 KO mice still respond to endurance exercise training (Saleem et al., 2009). Specifically, p53 KO and WT mice display similar increases in cytochrome-c oxidase activity with training, while trained p53 KO mice exhibit no difference in electron micrograph determined subsarcolemmal mitochondrial density compared to trained WT mice (Saleem et al., 2009). This suggests that p53 is not essential for endurance exercise induced mitochondrial adaptations (Saleem et al., 2009; Safdar et al., 2016), and the functional deficits of p53 KO appear to arise in the basal (i.e., non-exercised) state. Despite the wealth of evidence that p53 is important for whole-body metabolism and skeletal muscle mitochondrial function, determining the importance of p53 specifically in skeletal muscle cannot be ascertained from models of whole-body p53 deletion. In such models, it cannot be excluded that phenotypic differences in skeletal muscle physiology may arise as secondary defects due to dysfunction induced by the loss of p53 in other cell types.



Thus, to elucidate the role of p53 specifically within skeletal muscle fibres, this study determined the effect of skeletal muscle fibre-specific loss of p53 (mKO) on mitochondrial content and enzyme activity in skeletal muscle. The development and validation of the p53 mKO mouse has been described previously (Fox et al., 2014). Briefly, p53 mKO mice were generated by crossing homozygous p53 floxed mice (p53f/f; exons 2-10 of the p53 gene are flanked by LoxP restriction sites) with mice expressing cre recombinase (Cre) under the control of the muscle creatine kinase (MCK) promoter. Control mice (WT) were p53f/f littermates that lack the MCK-Cre transgene. All mice were on a C57BL/6 background. Eight-week old male mice were used for all experiments. Mice were housed in colony cages at 21°C with 12:12-h light-dark cycles and ad libitum access to standard laboratory chow (Harlan-Teklad formula 7913) and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Iowa.



Muscle was obtained from young (8-weeks old), healthy mice under basal conditions. Gastrocnemius, quadriceps and triceps muscle was rapidly dissected and rinsed to remove blood and fur before being snap-frozen in liquid nitrogen. Muscle was powdered using a Cellcrusher tissue pulverizer (Cellcrusher, Co. Cork, Ireland) on dry ice and stored at −80°C prior to analysis. Tissue was homogenised in a 10-fold mass excess of ice-cold sucrose lysis buffer (50 mM Tris, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na4P2O7-10H2O, 270 mM sucrose, 1 M Triton-X, 25 mM β-glycerophosphate, 1 μM Trichostatin A, 10 mM Nicatinamide, 1 mM 1,4-Dithiothreitol, 1% Phosphatase Inhibitor Cocktail 2; Sigma, 1% Sigma Phosphatase Inhibitor Click here Cocktail 2; Sigma, 4.8% cOmplete Mini Protease Inhibitor Cocktail; Roche) by shaking in a FastPrep 24 5G (MP Biomedicals, Santa Ana, California, USA) at 6.0 m· −1 for 80 s and centrifuging at 4°C and 8,000 g for 10 min to remove insoluble material. Protein concentrations were determined by the DC protein assay (Bio-Rad, Hercules, California, USA).