Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage

Noise exposure causes noise-induced hearing loss (NIHL). NIHL exhibits loss of inner ear sensory hair cells and is often irreparable. Although oxidative stress is involved in hearing loss, the complex mechanisms involved in NIHL are unclear. Hypoxia-inducible factor 1α (HIF-1α) has been suggested to be essential for protecting sensory hair cells. Additionally, it has been shown that ROS is involved in modulating the stability of HIF-1α. To investigate the NIHL pathogenesis, we established a tert-butyl hydroperoxide (t-BHP)-induced oxidative stress damage model in hair-like HEI-OC1 cells and an NIHL model in C57BL/6 mice. Protein and mRNA expression were determined, and biochemical parameters including reactive oxygen species (ROS) accumulation, glucose uptake, adenosine triphosphat (ATP) production, and mitochondrial content were evaluated. In HEI-OC1 cells, t-BHP induced ROS accumulation and reduced mitochondrial content and oxygen consumption, but the ATP level was unaffected. Additionally, there was increased glucose uptake and lactate release along with elevated expression of HIF-1α, glucose transporter 1, and several glycolytic enzymes. Consistently, noise trauma induced oxidative stress and the expression of HIF-1α and glycolytic enzymes in mice. Thus, we concluded that ROS induced HIF-1α expression, which promoted glycolysis, suggesting a metabolic shift maintained the ATP level to attenuate hair cell damage in NIHL.

Mitochondria-targeted antioxidant supplementation augments acute exercise-induced increases in muscle PGC1α mRNA and improves training-induced increases in peak power independent of mitochondrial content and function in untrained middle-aged men

ABSTRACTThe role of mitochondrial ROS production and signalling in muscle adaptations to exercise training has not been explored in detail. Here we investigated the effect of supplementation with the mitochondria-targeted antioxidant MitoQ on a) the skeletal muscle mitochondrial and antioxidant gene transcriptional response to acute high-intensity exercise and b) skeletal muscle mitochondrial content and function following exercise training. In a randomised, double-blind, placebo-controlled, parallel design study, 23 untrained men (age: 44 ± 7 years, VO2peak: 39.6 ± 7.9 ml/kg/min) were randomised to receive either MitoQ (20 mg/d) or a placebo for 10 days before completing a bout of high-intensity interval exercise (cycle ergometer, 10 × 60 s at VO2peak workload with 75 s rest). Blood samples and vastus lateralis muscle biopsies were collected before exercise and immediately and 3 hours after exercise. Participants then completed high-intensity interval training (HIIT; 3 sessions per week for 3 weeks) and another blood sample and muscle biopsy were collected. MitoQ supplementation augmented acute exercise-induced increases in skeletal muscle mRNA expression of the major regulator of proteins involved in mitochondrial biogenesis peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α). Despite this, training-induced increases in skeletal muscle mitochondrial content were unaffected by MitoQ supplementation. HIIT-induced increases in VO2peak and 20 km time trial performance were also unaffected by MitoQ while MitoQ augmented training-induced increases in peak power achieved during the VO2peak test. These data suggest that MitoQ supplementation enhances the effect of training on peak power, which may be related to the augmentation of skeletal muscle PGC1α expression following acute exercise. However, this effect does not appear to be related to an effect of MitoQ supplementation on HIIT-induced mitochondrial biogenesis in skeletal muscle and may therefore be the result of other adaptations mediated by PGC1α.

High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our findings suggest enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training-induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous findings.

The Influence of Age, Sex and Exercise on Autophagy, Mitophagy and Lysosome Biogenesis in Skeletal Muscle

BackgroundAging decreases skeletal muscle mass and quality. Maintenance of healthy muscle is regulated by a balance between protein and organellar synthesis and their degradation. The autophagy lysosome system is responsible for the selective degradation of protein aggregates and organelles, such as mitochondria (i.e., mitophagy). Little data exist on the independent and combined influence of age, biological sex and exercise on the autophagy system and lysosome biogenesis. The purpose of this study was to characterize sex differences in autophagy and lysosome biogenesis in young and aged muscle, and to determine if acute exercise influences these processes.MethodsYoung (4-6 months) and aged (22-24 months) male and female mice, were assigned to a sedentary, or an acute exercise group. Mitochondrial content, the autophagy-lysosome system and mitophagy were measured via protein analysis. A Tfeb-promoter-construct was utilized to examine Tfeb transcription, and nuclear-cytosolic fractions allowed us to examine Tfeb localization in sedentary and exercised muscle with age and sex.ResultsOur results indicate that female mice, both young and old, had more mitochondrial protein than age-matched males, and mitochondrial content was only reduced with age in the male cohort. Although young female mice had a greater abundance of autophagy, mitophagy and lysosome proteins than young males, we measured increases with age irrespective of sex. Interestingly, young sedentary female mice had indices of greater autophagosomal turnover than male counterparts. Exhaustive exercise was able to stimulate autophagic clearance in young male mice, but not in the other groups. Similarly, nuclear Tfeb protein was enhanced to a greater extent in young male than in young female mice following exercise, but no changes were observed in aged mice. Finally, Tfeb-promoter activity was upregulated following exercise in both young and aged muscle.ConclusionsThe present study demonstrates that biological sex influences mitochondrial homeostasis, the autophagy-lysosome system and mitophagy in skeletal muscle with age. Further, our data suggest that young male mice have a more profound ability to activate these processes with exercise than in the other groups. Ultimately, this may contribute to a greater remodeling of muscle in response to exercise training in males.

Blocked O-GlcNAc cycling alters mitochondrial morphology, function, and mass

O-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

Revisiting the contribution of mitochondrial biology to the pathophysiology of skeletal muscle insulin resistance

While the etiology of type 2 diabetes is multifaceted, the induction of insulin resistance in skeletal muscle is a key phenomenon, and impairments in insulin signaling in this tissue directly contribute to hyperglycemia. Despite the lack of clarity regarding the specific mechanisms whereby insulin signaling is impaired, the key role of a high lipid environment within skeletal muscle has been recognized for decades. Many of the proposed mechanisms leading to the attenuation of insulin signaling — namely the accumulation of reactive lipids and the pathological production of reactive oxygen species (ROS), appear to rely on this high lipid environment. Mitochondrial biology is a central component to these processes, as these organelles are almost exclusively responsible for the oxidation and metabolism of lipids within skeletal muscle and are a primary source of ROS production. Classic studies have suggested that reductions in skeletal muscle mitochondrial content and/or function contribute to lipid-induced insulin resistance; however, in recent years the role of mitochondria in the pathophysiology of insulin resistance has been gradually re-evaluated to consider the biological effects of alterations in mitochondrial content. In this respect, while reductions in mitochondrial content are not required for the induction of insulin resistance, mechanisms that increase mitochondrial content are thought to enhance mitochondrial substrate sensitivity and submaximal adenosine diphosphate (ADP) kinetics. Thus, this review will describe the central role of a high lipid environment in the pathophysiology of insulin resistance, and present both classic and contemporary views of how mitochondrial biology contributes to insulin resistance in skeletal muscle.

Venetoclax Reverses Metabolic Reprogramming Induced By S1P Modulator FTY720, Suppresses Oxidative Phosphorylation and Synergistically Targets Multiple Myeloma

Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic modalities that bypass these resistance mechanisms. FTY720, also known as fingolimod, is an S1P modulator approved by the FDA to treat the relapsing form of multiple sclerosis. Previously we reported that FTY720 exhibits potent anti-myeloma effect in vitro and in vivo in disseminated xenograft model of MM (Beider et al., Clin Cancer Res 2017). Cytotoxic activity of FTY720 was associated with down-regulation of anti-apoptotic protein MCL-1 while not affecting BCL-2 levels. It is therefore conceivable that BCL-2 inhibition using BH3-mimetic venetoclax may improve responses to FTY720. Incubation of human MM cell lines (n=8) and primary MM cells (n=3) with venetoclax and FTY720 combination synergistically potentiated cell death (CI<0.02), regardless of the MM cells t (11; 14) status. The robust apoptosis induced by venetoclax /FTY720 treatment was accompanied by cytochrome C release, activation of caspase-3 and extensive DNA damage, demonstrated by increased TUNEL staining and elevated levels of phosphorylated histone H2AX, respectively . These effects were associated with down-regulation of BCL-2 protein, stabilization of pro-apoptotic Bak protein, loss of mitochondrial membrane potential, ER stress induction, and inhibition of the AKT/mTOR signaling pathway. Furthermore, the venetoclax /FTY720 combination markedly induced mitochondrial calcium flux and mitochondrial ROS generation. Corresponding with mitochondrial destabilization, venetoclax/FTY720 combination promoted the release of apoptosis-inducing factor (AIF) from the mitochondria to the cytosol and subsequently increased AIF nuclear localization, suggesting its functional role in the execution phase of the apoptosis in response to the dual treatment. AIF is a mitochondrial oxidoreductase that contributes to cell death programs and participates in the assembly of the respiratory chain. Of note, single-agent treatment with FTY720 profoundly up-regulated mitochondrial AIF levels. Given the regulative role of AIF in mitochondrial bioenergetics, we could suggest that increased mitochondrial levels of AIF upon FTY720 exposure may support adaptive responses and promote MM survival upon mitochondrial stress. We thus investigated a possible effect of venetoclax and FTY720 separately or in combination on the metabolic activity of MM cells, observing distinct metabolic profiles of single versus combined exposures. FTY720 significantly suppressed glycolysis, down-regulating the transcript levels of the glycolytic enzymes HK2, PDK1, and LDHA. Glycolytic suppression may result in upregulation of mitochondrial content, which maintains cell survival. In accordance, increased mitochondrial activity was detected in FTY720-treated MM cells, detected by high uptake of MitoSpy Red, a dye that stains mitochondria in a membrane potential-dependent manner. To determine if the changes in the mitochondrial content also altered mitochondrial function, bioenergetic analysis was undertaken. FTY720-treated MM cells demonstrated increased levels of NDUFB8 and UQCRC2 (subunits of mitochondrial respiratory complexes I and III, respectively). Furthermore, FTY720 exposure up-regulated ATP production, suggesting an increase in tumor-protective oxidative phosphorylation (OXPHOS). In agreement, inhibition of mitochondrial electron transport chain using rotenone sensitized MM cells to FTY720, synergistically promoting cell death. Notably, co-treatment with venetoclax effectively reversed the metabolic changes mediated by FTY720, reducing mitochondrial mass, suppressing mitochondrial activity and strongly down-regulating the pathways related to OXPHOS. Furthermore, venetoclax/FTY720 combination significantly reduced glutathione (GSH) levels, therefore suppressing antioxidative cell responses. To conclude, we unveil venetoclax role in the metabolic regulation in MM cells. Venetoclax reverses metabolic reprogramming induced by FTY720, suppresses mitochondrial respiration, induces vigorous mitochondrial damage and preferentially targets MM cells in combination with FTY720. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Figure 1 Figure 1. Disclosures Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding.

Human studies of mitochondrial biology demonstrate an overall lack of binary sex differences: A multivariate meta-analysis

Mitochondria are maternally inherited organelles that play critical tissue-specific roles, including hormone synthesis and energy production, that influence development, health, and aging. However, whether mitochondria from women and men exhibit consistent biological differences remains unclear, representing a major gap in biomedical knowledge. This meta-analysis systematically examined 4 domains and 6 subdomains of mitochondrial biology (total 39 measures), including mitochondrial content, respiratory capacity, reactive oxygen species (ROS) production, morphometry, and mitochondrial DNA copy number. Standardized effect sizes (Hedges g) of sex differences were computed for each measure using data in 2,258 participants (51.5% women) from 50 studies. Only two measures demonstrated aggregate binary sex differences: higher mitochondrial content in women (g = 0.20, chi2 p = 0.01), and higher ROS production in skeletal muscle in men (g = 0.49, chi2 p < 0.0001). Sex differences showed weak to no correlation with age or BMI. Studies with small sample sizes tended to overestimate effect sizes (r = -0.17, p < 0.001), and sex differences varied by tissue examined. Our findings point to a wide variability of findings in the literature concerning possible binary sex differences in mitochondrial biology. Studies specifically designed to capture sex- and gender-related differences in mitochondrial biology are needed, including detailed considerations of physical activity and sex hormones.