The effect of training on the expression of mitochondrial biogenesis- and apoptosis-related proteins in skeletal muscle of patients with mtDNA defects

Mitochondrial myopathy patients (MMPs) have impaired oxidative phosphorylation and exercise intolerance. Endurance training of MMPs improves exercise tolerance, but also increases mutational load. To assess the regulation of mitochondrial content in MMPs, we measured proteins involved in 1) biogenesis, 2) oxidative stress, and 3) apoptosis in MMPs and healthy controls (HCs) both before and after endurance training. Before training, MMPs had a greater mitochondrial content, along with a 1.4-fold (P < 0.05) higher expression of the biogenesis regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha). The DNA repair enzyme 8-oxoguanine DNA glycolase-1 (OGG-1), the antioxidant manganese superoxide dismutase (MnSOD), and the apoptotic proteins AIF and Bcl-2 were higher in MMPs compared with HCs. Aconitase, an enzyme sensitive to oxidative stress, was 52% lower (P < 0.05) in MMPs when calculated based on an estimate of mitochondrial volume and oxidative stress-induced protein modifications tended to be higher in MMPs compared with HCs. Endurance training (ET) induced increases in mitochondrial content in both HC subjects and MMPs, but there was no effect of training on the regulatory proteins Tfam or PGC-1alpha. In MMPs, training induced a selective reduction of OGG-1, an increase in MnSOD, and a reduction in aconitase activity. Thus, before training, MMPs exhibited an adaptive response of nuclear proteins indicative of a compensatory increase in mitochondrial content. Following training, several parallel adaptations occurred in MMPs and HCs, which may contribute to previously observed functional improvements of exercise in MMPs. However, our results indicate that muscle from MMPs may be exposed to greater levels of oxidative stress during the course of training. Further investigation is required to evaluate the long-term benefits of endurance training as a therapeutic intervention for mitochondrial myopathy patients.     

Signaling of mitochondrial biogenesis following oxidant injury

Mitochondrial dysfunction is a common consequence of ischemia-reperfusion and drug injuries. For example, sublethal injury of renal proximal tubular cells (RPTCs) with the model oxidant tert-butylhydroperoxide (TBHP) causes mitochondrial injury that recovers over the course of six days. Although regeneration of mitochondrial function is integral to cell repair and function, the signaling pathway of mitochondrial biogenesis following oxidant injury has not been examined. A 10-fold overexpression of the mitochondrial biogenesis regulator PPAR-gamma cofactor-1alpha (PGC-1alpha) in control RPTCs resulted in a 52% increase in mitochondrial number, a 27% increase in respiratory capacity, and a 30% increase in mitochondrial protein markers, demonstrating that PGC-1alpha mediates mitochondrial biogenesis in RPTCs. RPTCs sublethally injured with TBHP exhibited a 50% decrease in mitochondrial function and increased mitochondrial autophagy. Compared with the controls, PGC-1alpha levels increased 12-fold on days 1, 2, and 3 post-injury and returned to base line on day 4 as mitochondrial function returned. Inhibition p38 MAPK blocked the up-regulation of PGC-1alpha following oxidant injury, whereas inhibition of calcium-calmodulin-dependent protein kinase, calcineurin A, nitric-oxide synthase, and phosphoinositol 3-kinase had no effect. The epidermal growth factor receptor (EGFR) was activated following TBHP exposure, and the EGFR inhibitor AG1478 blocked the up-regulation of PGC-1alpha. Additional inhibitor studies revealed that the sequential activation of Src, p38 MAPK, EGFR, and p38 MAPK regulate the expression of PGC-1alpha following oxidant injury. In contrast, although Akt was activated following oxidant injury, it did not play a role in PGC-1alpha expression. We suggest that mitochondrial biogenesis following oxidant injury is mediated by p38 and EGFR activation of PGC-1alpha.     

Sirtuin 1 (SIRT1) deacetylase activity is not required for mitochondrial biogenesis or peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) deacetylation following endurance exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.     

A metabolic shift induced by a PPAR panagonist markedly reduces the effects of pathogenic mitochondrial tRNA mutations

Mutations in mitochondrial DNA-encoded tRNA genes are associated with many human diseases. Activation of peroxisome proliferator-activated receptors (PPARs) by synthetic agonists stimulates oxidative metabolism, induces an increase in mitochondrial mass and partially compensates for oxidative phosphorylation system (OXPHOS) defects caused by single OXPHOS enzyme deficiencies in vitro and in vivo. Here, we analysed whether treatment with the PPAR panagonist bezafibrate in cybrids homoplasmic for different mitochondrial tRNA mutations could ameliorate the OXPHOS defect. We found that bezafibrate treatment increased mitochondrial mass, mitochondrial tRNA steady state levels and enhanced mitochondrial protein synthesis. This improvement resulted in increased OXPHOS activity and finally in enhanced mitochondrial ATP generating capacity. PPAR panagonists are known to increase the expression of PPAR gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Accordingly, we found that clones of a line harbouring a mutated mitochondrial tRNA gene mutation selected for the ability to grow in a medium selective for OXPHOS function had a 3-fold increase in PGC-1α expression, an increase that was similar to the one observed after bezafibrate treatment. These findings show that increasing mitochondrial mass and thereby boosting residual OXPHOS capacity can be beneficial to an important class of mitochondrial defects reinforcing the potential therapeutic use of approaches stimulating mitochondrial proliferation for mitochondrial disorders.     

Pyruvate induces mitochondrial biogenesis by a PGC-1 alpha-independent mechanism

Oxidative cells increase mitochondrial mass in response to stimuli such as changes in energy demand or cellular differentiation. This plasticity enables the cell to adapt dynamically to achieve the necessary oxidative capacity. However, the pathways involved in triggering mitochondrial biogenesis are poorly defined. The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells. C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 h. Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate, and maximal oxidative capacity. The increase in mitochondrial biogenesis was independent of increases in peroxisomal proliferator activator receptor- coactivator-1 (PGC-1) and PGC-1 mRNA expression. To further assess whether PGC-1 expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1 before treatment with pyruvate. Despite a 70% reduction in PGC-1 mRNA, the effect of pyruvate was preserved. Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1 null mice. These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity. oxidative metabolism; peroxisomal proliferator activator receptor- coactivator-1, mitochondria; muscle     

PGC-1alpha over-expression promotes recovery from mitochondrial dysfunction and cell injury

Cell death from mitochondrial dysfunction and compromised bioenergetics is common after ischemia-reperfusion injury and toxicant exposure. Thus, promoting mitochondrial biogenesis is therapeutically attractive for sustaining oxidative phosphorylation and maintaining ATP-dependent cellular functions. Here, we evaluated increased mitochondrial biogenesis prior to or after oxidant exposure in primary cultures of renal proximal tubular cells (RPTC). Over-expression of the mitochondrial biogenesis regulator PPAR-gamma cofactor-1 alpha (PGC-1alpha) in control RTPC increased basal and uncoupled cellular respiration, ATP, and mitochondria. Increasing mitochondrial number/function prior to oxidant exposure did not preserve mitochondrial function, but potentiated dysfunction and cell death. However, increased mitochondrial biogenesis after oxidant injury accelerated recovery of mitochondrial function. In oxidant treated RPTC, mitochondrial protein expression was reduced by 50%. Also, ATP and cellular respiration decreased 48 h after oxidant exposure, whereas mitochondrial function in injured RPTC over-expressing PGC-1alpha returned to control values. Thus, up-regulation of mitochondrial biogenesis after oxidant exposure accelerates recovery of mitochondrial and cellular functions.     

Statin-Induced Increases in Atrophy Gene Expression Occur Independently of Changes in PGC1α Protein and Mitochondrial Content

One serious side effect of statin drugs is skeletal muscle myopathy. Although the mechanism(s) responsible for statin myopathy remains to be fully determined, an increase in muscle atrophy gene expression and changes in mitochondrial content and/or function have been proposed to play a role. In this study, we examined the relationship between statin-induced expression of muscle atrophy genes, regulators of mitochondrial biogenesis, and markers of mitochondrial content in slow- (ST) and fast-twitch (FT) rat skeletal muscles. Male Sprague Dawley rats were treated with simvastatin (60 or 80 mg·kg^-1·day^-1) or vehicle control via oral gavage for 14 days. In the absence of overt muscle damage, simvastatin treatment induced an increase in atrogin-1, MuRF1 and myostatin mRNA expression; however, these were not associated with changes in peroxisome proliferator gamma co-activator 1 alpha (PGC-1α) protein or markers of mitochondrial content. Simvastatin did, however, increase neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS) and AMPK α-subunit protein expression, and tended to increase total NOS activity, in FT but not ST muscles. Furthermore, simvastatin induced a decrease in β-hydroxyacyl CoA dehydrogenase (β-HAD) activity only in FT muscles. These findings suggest that the statin-induced activation of muscle atrophy genes occurs independent of changes in PGC-1α protein and mitochondrial content. Moreover, muscle-specific increases in NOS expression and possibly NO production, and decreases in fatty acid oxidation, could contribute to the previously reported development of overt statin-induced muscle damage in FT muscles.     

Insulin-resistant muscle is exercise resistant: evidence for reduced response of nuclear-encoded mitochondrial genes to exercise

Mitochondrial dysfunction, associated with insulin resistance, is characterized by low expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and nuclear-encoded mitochondrial genes. This deficit could be due to decreased physical activity or a decreased response of gene expression to exercise. The objective of this study was to investigate whether a bout of exercise induces the same increase in nuclear-encoded mitochondrial gene expression in insulin-sensitive and insulin-resistant subjects matched for exercise capacity. Seven lean and nine obese subjects took part. Insulin sensitivity was assessed by an 80 mU.m(-2).min(-1) euglycemic clamp. Subjects were matched for aerobic capacity and underwent a single bout of exercise at 70 and 90% of maximum heart rate with muscle biopsies at 30 and 300 min postexercise. Quantitative RT-PCR and immunoblot analyses were used to determine the effect of exercise on gene expression and protein abundance and phosphorylation. In the postexercise period, lean subjects immediately increased PGC-1alpha mRNA level (reaching an eightfold increase by 300 min postexercise) and protein abundance and AMP-dependent protein kinase phosphorylation. Activation of PGC-1alpha was followed by increase of nuclear respiratory factor-1 and cytochrome c oxidase (subunit VIc). However, in insulin-resistant subjects, there was a delayed and reduced response in PGC-1alpha mRNA and protein, and phosphorylation of AMP-dependent protein kinase was transient. None of the genes downstream of PGC-1alpha was increased after exercise in insulin resistance. Insulin-resistant subjects have a reduced response of nuclear-encoded mitochondrial genes to exercise, and this could contribute to the origin and maintenance of mitochondrial dysfunction.