Local muscle cooling does not impact expression of mitochondrial-related genes

Recovery that takes place in a cold environment after endurance exercise elevates PGC-1α mRNA whereas ERRα and NRF2 mRNA expression are inhibited. However, the effect of local skeletal muscle cooling on mitochondrial-related gene expression is unknown.PurposeTo determine the impact of local skeletal muscle cooling during recovery from an acute bout of exercise on mitochondrial-related gene expression.MethodsRecreationally-trained male cyclists (n=8, age 25±3 y, height 181±6 cm, weight 79±8 kg, 12.8±3.6% body fat, VO_2peak 4.52±0.88 L·min⁻¹ protocol) completed a 90-min variable intensity cycling protocol followed by 4 h of recovery. During recovery, ice was applied intermittently to one leg (ICE) while the other leg served as a control (CON). Intramuscular temperature was recorded continuously. Muscle biopsies were taken from each vastus lateralis at 4 h post-exercise for the analysis of mitochondrial-related gene expression.ResultsIntramuscular temperature was colder in ICE (26.7±1.1 °C) than CON (35.5±0.1 °C) throughout the 4 h recovery period (p<0.001). There were no differences in expression of PGC-1α, TFAM, NRF1, NRF2, or ERRα mRNA between ICE and CON after the 4 h recovery period.ConclusionLocal muscle cooling after exercise does not impact the expression of mitochondrial biogenesis-related genes compared to recovery from exercise in control conditions. When these data are considered with previous research, the stimuli for cold-induced gene expression alterations may be related to factors other than local muscle temperature. Additionally, different intramuscular temperatures should be examined to determine dose-response of mitochondrial-related gene expression.     

Promotion of mitochondrial biogenesis via the regulation of PARIS and PGC-1α by parkin as a mechanism of neuroprotection by carnosic acid

BackgroundImpairment of mitochondrial biogenesis is associated with the pathological progression of Parkinson's disease (PD). Parkin-interacting substrate (PARIS) can be ubiquitinated by parkin and prevents the repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α).PurposeThis study investigated whether the neuroprotective mechanism of carnosic acid (CA) from rosemary is mediated via the regulation of PARIS and PGC-1α by parkin.MethodsThe Western blotting and RT-PCR were used to determine protein and mRNA, respectively. To investigate the protein-protein interaction of between PARIS and ubiquitin, the immunoprecipitation assay (IP assay) was utilized. Silencing of endogenous parkin or PGC-1α was performed by using transient transfection of small interfering RNA (siRNA).ResultsSH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) increased PARIS protein, decreased PGC-1α protein, and reduced protein and mRNA of mitochondrial biogenesis-related genes. CA pretreatment reversed the effects of 6-OHDA. By IP assay, the interaction of PARIS with ubiquitin protein caused by CA was stronger than that caused by 6-OHDA. Moreover, knockdown of parkin attenuated the ability of CA to reverse the 6-OHDA-induced increase in PARIS and decrease in PGC-1α expression. PGC-1α siRNA was used to investigate how CA influenced the effect of 6-OHDA on the modulation of mitochondrial biogenesis and apoptosis. In the presence of PGC-1α siRNA, CA could no longer significantly reverse the reduction of mitochondrial biogenesis or the induction of cleavage of apoptotic-related proteins by 6-OHDA.ConclusionThe cytoprotective of CA is related to the enhancement of mitochondrial biogenesis by inhibiting PARIS and inducing PGC-1α by parkin. The activation of PGC-1α-mediated mitochondrial biogenesis by CA prevents the degeneration of dopaminergic neurons, CA may have therapeutic application in PD.     

Effects of low-intensity prolonged exercise on PGC-1 mRNA expression in rat epitrochlearis muscle

We previously reported that the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) mRNA in rat epitrochlearis muscle was increased after swimming exercise training. In the present study, we demonstrated further that PGC-1 mRNA expression in the epitrochlearis muscle of 4-5-week-old male Sprague-Dawley rats was increased after a 6-h acute bout of low-intensity swimming exercise. With this increase, the expression level was approximately 8-fold of control and immersion group rats that stayed for 6-h in warm water, maintained at the identical temperature of the swimming barrel (35 degrees C) (p<0.01). Second, PGC-1 mRNA expression in the muscle was found to have increased 6-h after 30 10-s tetani contractions were induced by in vitro electrical stimulation. Finally, PGC-1 mRNA expression in the muscle incubated for 18-h with 0.5mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR: a 5' AMP-activated protein kinase (AMPK) activator) was elevated to approximately 3-fold of the control muscle (n=6, p<0.001). AMPK activity in epitrochlearis muscle after the swimming was also found to be elevated to approximately 4-fold of the pre-exercise value (p<0.001). These results may suggest that an acute bout of low-intensity prolonged swimming exercise directly enhances the PGC-1 mRNA expression in the activated muscle during exercise, possibly through, at least in part, an AMPK-related mechanism.     

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.     

Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest

The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (T_c) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while T_c were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.     

Effect of combined aerobic and strength exercises on the regulation of mitochondrial biogenesis and protein synthesis and degradation in human skeletal muscle

We tested the hypothesis that strength exercise after intermittent aerobic exercise might activate signaling pathways that regulate mitochondrial biogenesis (activation of the AMPK and p38 pathways; the expression of PGC-1α, NT-PGC-1α, TFAM, and VEGFA mRNA), protein synthesis (phosphorylation level of p70S6K1^Thr389 and eEF2^Thr56; the expression IGF-1Ea, IGF-1Ec (MGF), and REDD1 mRNA) and proteolysis (phosphorylation level of FOXO1^Ser256; the expression of MURF1, MAFbx, and Myostatin mRNA) in trained skeletal muscles. Nine amateur endurance-trained athletes performed an intermittent aerobic cycling (70 min), followed by one-leg strength exercise (ES: four sets of knee extensions till exhaustion), while the other leg was resting (E). Gene expression and protein level were evaluated in samples from m. vastus lateralis taken before the exercise, 40 min, 5 and 22 h after the aerobic exercise. The phosphorylation level of the АСС^Ser79/222 (an endogenous marker of AMPK activity) and the expression of PGC-1α-related gene TFAM (a marker of mitochondrial biogenesis) were increased after E exercise and did not changed after ES exercise. The expression of PGC-1α and truncated isoform NT-PGC-1α was increased in both legs as well. Insulin concentration in blood was decreased significantly (7.5-fold) after aerobic exercise; the phosphorylation level of FOXO^Ser256 (a regulator of ubiquitin-related proteolysis) was decreased in both legs, which means that it was activated in both types of exercises; at the same time, the expression of the E3-ubiquitin ligase gene MURF1, its target, was only increased after E exercise. Neither aerobic or combined exercise had a significant effect on the regulation of protein synthesis: there were no changes in either expression of IGF-1Ea and IGF-1Ec(MGF) mRNA isoforms or the phosphorylation levels of markers of protein synthesis p70S6K1^Thr389 and eEF2^Thr56. Thus, the performance of strength exercise immediately after aerobic one prevented the activation of mitochondrial biogenesis in endurance-trained muscles: activation of AMPK pathway and the expression of TFAM are decreased, while protein synthesis regulation is not affected. At the same time, the strength exercise inhibited the expression of MURF1 gene (a marker of ubiquitin proteasome system), which was induced by aerobic exercise. We suggest that strength exercise performed immediately after intense intermittent aerobic exercise may have a negative effect on aerobic performance if used chronically.     

An acute bout of high-intensity interval training increases the nuclear abundance of PGC-1α and activates mitochondrial biogenesis in human skeletal muscle

Low-volume, high-intensity interval training (HIT) increases skeletal muscle mitochondrial capacity, yet little is known regarding potential mechanisms promoting this adaptive response. Our purpose was to examine molecular processes involved in mitochondrial biogenesis in human skeletal muscle in response to an acute bout of HIT. Eight healthy men performed 4 × 30-s bursts of all-out maximal intensity cycling interspersed with 4 min of rest. Muscle biopsy samples (vastus lateralis) were obtained immediately before and after exercise, and after 3 and 24 h of recovery. At rest, the majority of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis, was detected in cytosolic fractions. Exercise activated p38 MAPK and AMPK in the cytosol. Nuclear PGC-1α protein increased 3 h into recovery from exercise, a time point that coincided with increased mRNA expression of mitochondrial genes. This was followed by an increase in mitochondrial protein content and enzyme activity after 24 h of recovery. These findings support the hypothesis that an acute bout of low-volume HIT activates mitochondrial biogenesis through a mechanism involving increased nuclear abundance of PGC-1α.     

Dynamic mobilization of PGC-1α mediates mitochondrial biogenesis for the protection of RGC-5 cells by resveratrol during serum deprivation

Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1α; instead, resveratrol facilitated PGC-1α translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1α subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.     

Cardiac mitochondrial biogenesis in endotoxemia is not accompanied by mitochondrial function recovery

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0–24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6 h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24 h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.