Increase In Mitochondrial Content Research Articles

Impact of chronic muscle use and disuse on innate immune signaling in skeletal muscle

Mitochondria exhibit high levels of adaptability to various stimuli such as chronic muscle use (i.e. exercise) or disuse, such as immobilization or denervation. These changes in the mitochondrial network ultimately affect the health and functioning of skeletal muscle, which contributes to whole-body metabolism, locomotion, and postural stability. It is now acknowledged that mitochondrial dysfunction can activate inflammatory pathways through the recognition of damage-associated molecular patterns (DAMPs). The objective of this study was to examine how changes in mitochondria brought about by either chronic muscle inactivity or chronic exercise training affect innate immune system activation. We hypothesized that an inverse relationship between mitochondrial content and innate immune signaling would be evident in skeletal muscle. To investigate this, skeletal muscle from the hindlimbs of mice was collected after 7 days of sciatic muscle denervation, or after 4.5 weeks of endurance swim training involving 240 minutes of daily exercise. We analyzed the expression of the NLRP3 inflammasome, innate immune system signaling proteins, mitochondrial markers, and mitochondrial DNA (mtDNA) content using western blotting, cytochrome C oxidase (COX) activity, ELISA and qPCR. Following 7 days of denervation, there was a significant decrease in both COX-IV protein and COX activity, indicating a reduction in mitochondrial content. This was accompanied by an increase (p<0.05) in NLRP3, procaspase-1, gasdermin-D (GSDMD) and its active N-terminal fragment, GSDMD-N, as well as a notable increase in IL-1β. In addition, other possible converging pathways were also upregulated including cGAS-STING, and gasdermin-E. In comparison, following exercise training, there was a significant increase in mitochondrial content, apparent through increases in COX-IV protein, COX activity and mitochondrial RNA content. This was associated with decreases in procaspase-1 and caspase-1 protein expression alongside reduced STING activation. These findings highlight an inverse relationship between mitochondrial content and the activation of several inflammatory pathways that possibly converge upon NLRP3 inflammasome activation and pyroptotic cell death, leading to detriments in skeletal muscle health. This work aims to further the understanding of innate immune signaling pathways within muscle which can potentially highlight therapeutic targets to regulate its activation under divergent metabolic conditions. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Priyanka Khemraj is the holder of an Ontario Graduate Scholarship. David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spexin ameliorated obesity-related metabolic disorders through promoting white adipose browning mediated by JAK2-STAT3 pathway

BackgroundSpexin, a 14 amino acid peptide, has been reported to regulate obesity and its associated complications. However, little is known about the underlying molecular mechanism. Therefore, this study aimed to investigate the effects of spexin on obesity and explore the detailed molecular mechanisms in vivo and in vitro.MethodsMale C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity, and mice fed a standard fat diet were used as controls. Then, these mice were treated with SPX or Vehicle by intraperitoneal injection for an additional 12 weeks, respectively. The metabolic profile, fat-browning specific markers and mitochondrial contents were detected. In vitro, 3T3-L1 cells were used to investigate the molecular mechanisms.ResultsAfter 12 weeks of treatment, SPX significantly decreased body weight, serum lipid levels, and improved insulin sensitivity in HFD-induced obese mice. Moreover, SPX was found to promote oxygen consumption in HFD mice, and it increased mitochondrial content as well as the expression of brown-specific markers in white adipose tissue (WAT) of HFD mice. These results were consistent with the increase in mitochondrial content and the expression of brown-specific markers in 3T3-L1 mature adipocytes. Of note, the spexin-mediated beneficial pro-browning actions were abolished by the JAK2/STAT3 pathway antagonists in mature 3T3-L1 cells.ConclusionsThese data indicate that spexin ameliorates obesity-induced metabolic disorders by improving WAT browning via activation of the JAK2/STAT3 signaling pathway. Therefore, SPX may serve as a new therapeutic candidate for treating obesity.

Camphene as a Protective Agent in Myocardial Ischemia/Reperfusion Injury.

Myocardial ischemia/reperfusion injury (I/R) and the resulting heart failure is one of the main causes of mortality and morbidity worldwide. Camphene has been shown to have anti-inflammatory and hypolipidemic properties; however, its role in the protection of the heart from ischemia and reperfusion has not been investigated. The cardioprotective role of camphene and the mechanism that mediates its action against I/R injury was evaluated in the present study. A single dose of camphene was administered in adult rats prior to ex vivo I/R induction. Infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining and cardiomyocyte injury was assessed by determining the release of the enzyme lactate dehydrogenase (LDH). Camphene pretreatment provided significant protection reducing myocardial infarct size and cell death after I/R. The effect was correlated with the reduction in oxidative stress as evidenced by the determination of protein carbonylation, GSH/GSSG ratio, the increase in mitochondrial content as determined by CS activity, and the modulation of antioxidant defense mechanisms (expression of Nrf2 and target genes and activities of CAT, MnSOD, and GR). Furthermore, ferroptosis was decreased, as demonstrated by downregulation of GPx4 expression and reduction in lipid peroxidation. The results suggest that camphene can protect the heart against I/R injury by maintaining redox homeostasis and can hold therapeutic potential for mitigating the detrimental effects of I/R in the heart.

The role of GPR81–cAMP–PKA pathway in endurance training-induced intramuscular triglyceride accumulation and mitochondrial content changes in rats

The athlete's paradox phenomenon involves the accumulation of intramuscular triglycerides (IMTG) in both insulin-resistant and insulin-sensitive endurance athletes. Nevertheless, a complete understanding of this phenomenon is yet to be achieved. Recent research indicates that lactate, a common byproduct of physical activity, may increase the accumulation of IMTG in skeletal muscle. This is achieved through the activation of G protein-coupled receptor 81 (GPR81) leads to the suppression of the cyclic adenosine monophosphate–protein kinase A (cAMP–PKA) pathway. The mechanism accountable for the increase in mitochondrial content in skeletal muscle triggered by lactate remains incomprehensible. Based on current research, our objective is to explore the role of the GPR81-inhibited cAMP–PKA pathway in the aggregation of IMTG and the increase in mitochondrial content as a result of prolonged exercise. The GPR81–cAMP–PKA-signaling pathway regulates the buildup of IMTG caused by extended periods of endurance training (ET). This is likely due to a decrease in proteins related to fat breakdown and an increase in proteins responsible for fat production. It is possible that the GPR81–cAMP–PKA pathway does not contribute to the long-term increase in mitochondrial biogenesis and content, which is induced by chronic ET. Additional investigation is required to explore the possible hindrance of the mitochondrial biogenesis and content process during physical activity by the GPR81–cAMP–PKA signal.

Significance of quantitative analyses of the impact of heterogeneity in mitochondrial content and shape on cell differentiation.

Mitochondria, classically known as the powerhouse of cells, are unique double membrane-bound multifaceted organelles carrying a genome. Mitochondrial content varies between cell types and precisely doubles within cells during each proliferating cycle. Mitochondrial content also increases to a variable degree during cell differentiation triggered after exit from the proliferating cycle. The mitochondrial content is primarily maintained by the regulation of mitochondrial biogenesis, while damaged mitochondria are eliminated from the cells by mitophagy. In any cell with a given mitochondrial content, the steady-state mitochondrial number and shape are determined by a balance between mitochondrial fission and fusion processes. The increase in mitochondrial content and alteration in mitochondrial fission and fusion are causatively linked with the process of differentiation. Here, we critically review the quantitative aspects in the detection methods of mitochondrial content and shape. Thereafter, we quantitatively link these mitochondrial properties in differentiating cells and highlight the implications of such quantitative link on stem cell functionality. Finally, we discuss an example of cell size regulation predicted from quantitative analysis of mitochondrial shape and content. To highlight the significance of quantitative analyses of these mitochondrial properties, we propose three independent rationale based hypotheses and the relevant experimental designs to test them.

The role of TFE3 in mediating skeletal muscle mitochondrial adaptations to exercise training.

Transcription factor E3 (TFE3) is a transcription factor that activates the expression of lysosomal genes involved in the clearance of dysfunctional mitochondria, termed mitophagy. With exercise, TFE3 is presumed to optimize the mitochondrial pool through the removal of organelles via lysosomes. However, the molecular mechanisms of the involved pathways remain unknown. Wild-type (WT) and TFE3 knockout (KO) mice were subjected to 6 wk of voluntary wheel running as an endurance training regimen. This was followed by a 45-min bout of in situ stimulation of the sciatic nerve innervating hindlimb muscles to evaluate muscle fatigue and contractile properties. A subset of animals was treated with colchicine to measure autophagy and mitophagy flux. Fatigability during stimulation was reduced with training in WT animals, as seen by a 13% increase in the percentage of maximum force at 5 min of stimulation, and a 30% increase at 30 minutes. Permeabilized fiber oxygen consumption was also improved with training. Concurrent with improved muscle and mitochondrial function, cytochrome c oxidase (COX) activity and COX I protein expression were increased in trained WT animals compared to untrained animals, signifying an increase in mitochondrial content. These training adaptations were abolished with the loss of TFE3. Surprisingly, the absence of TFE3 did not affect lysosomal content nor did it blunt the induction of mitophagy flux with contractile activity compared to WT mice. Our results suggest that the loss of TFE3 compromises beneficial training adaptations that lead to improved muscle endurance and mitochondrial function.NEW & NOTEWORTHY Our understanding of the role of transcription factor E3 (TFE3) in skeletal muscle is very limited. This research shows that TFE3 plays a direct role in skeletal muscle mitochondrial enhancement with exercise training, thereby introducing a paradigm shift in our perception of the function of TFE3 in mitochondrial maintenance, beyond mitophagy. This research serves to introduce TFE3 as a protein that holds promise as a future therapeutic target for metabolic diseases and skeletal muscle dysfunction.

Iguratimod Regulates CD4+ T-Cell Homeostasis and Function By Restoring PINK1/Parkin-Mediated Mitophagy in Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction and suppressed platelet production, resulting in isolated thrombocytopenia. Subpopulation imbalance and dysfunction of CD4+ T cells are involved in the pathogenesis of ITP. However, the exact mechanism of CD4+ T-cell abnormalities is not fully understood. Mitophagy, a process that eliminates damaged and dysfunctional mitochondria, has conventionally been considered the primary mechanism responsible for mitochondrial quality control. Mitophagy deficiency has been implicated in a series of inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). However, whether it plays a role in ITP immune abnormalities and its specific mechanism remain unclear. Iguratimod is a small molecule compound that is widely used as a novel antirheumatic drug in the treatment of rheumatoid arthritis. Iguratimod inhibits the production of several inflammatory cytokines, including IL-1, IL-6, IL-8, and tumor necrosis factor (TNF), by enhancing mitophagy. Our previous clinical study demonstrated the efficacy of iguratimod in the treatment of ITP. This study aimed to explore whether iguratimod regulates CD4+ T-cell homeostasis and function by enhancing mitophagy in ITP. Methods BM CD4+ T cells were isolated from ITP patients and healthy donors. The subgroup of CD4+ T cells was analyzed by flow cytometry. ELISA was used to evaluate cytokine and chemokine levels in bone marrow plasma. Mitochondrial mass, morphology, quality and mitophagy were evaluated by flow cytometry, confocal microscopy, electron microscopy and western blotting. RNA sequencing and metabolomics were performed. Epigenetic state alteration was assessed by a transposase-accessible chromatin assay with high throughput sequencing (ATAC-seq). Iguratimod-treated BM-derived ITP-CD4+ T cells and an ITP active mouse model were used to explore its regulation of mitophagy and effects on T-cell homeostasis and function. Results Th1/Th2 imbalance and Treg reduction were observed in the bone marrow of ITP patients, consistent with a previous report. Flow cytometry and confocal microscopy revealed an increase in mitochondrial content and mtROS in CD4+ T cells but a decrease in mitochondrial membrane potential. Furthermore, electron microscopy revealed structural damage to mitochondria in ITP-CD4+ T cells. These data confirmed an accumulation of damaged mitochondria in ITP-CD4+ T cells. The expression levels of LC3II/LC3I, PINK1 and parkin decreased in CD4+ T cells, and the colocalization of mitochondria and lysosomes decreased, which suggested compromised PINK1/parkin-mediated mitophagy in ITP-CD4+ T cells. RNA-seq revealed transcriptional changes associated with mitochondrial function, mitophagy and metabolic pathways in ITP-CD4+ T cells. Moreover, metabolomics showed alterations in TCA cycle metabolites. Since epigenetic programs are regulated by metabolites, ATAC-seq was performed to obtain the epigenetic state. We found that ITP-CD4+ T cells altered chromatin accessibility in regions with T cell differentiation, transcription factors and T-cell function. After in vitro treatment with iguratimod, the Th1/Th2 imbalance was corrected, and the number of Tregs was restored. The content, morphology and membrane potential of mitochondria in ITP-CD4+ T cells were restored after iguratimod intervention. The expression level of PINK1 and parkin and the colocalization of mitochondria and lysosomes were increased, suggesting an improvement in mitophagy. In vivo, iguratimod accelerated platelet recovery in an ITP active mouse model. After treatment, the Th1/Th2 imbalance and the Treg number were restored, and the mitophagy of CD4+ T cells was corrected in the bone marrow of the ITP mouse model. Conclusion PINK1/Parkin-mediated mitophagy deficiency contributed to mitochondrial dysfunction and altered the metabolism of ITP-CD4+ T cells, which affected their differentiation and function by regulating the epigenetic state of immune response genes. Iguratimod could restore CD4+ T-cell subpopulations and treat ITP by enhancing mitophagy.

Ultrastructure of noise-induced cochlear synaptopathy

Acoustic overexposure can eliminate synapses between inner hair cells (IHCs) and auditory nerve fibers (ANFs), even if hair-cell function recovers. This synaptopathy has been extensively studied by confocal microscopy, however, understanding the nature and sequence of damage requires ultrastructural analysis. Here, we used focused ion-beam scanning electron microscopy to mill, image, segment and reconstruct ANF terminals in mice, 1 day and 1 week after synaptopathic exposure (8–16 kHz, 98 dB SPL). At both survivals, ANF terminals were normal in number, but 62% and 53%, respectively, lacked normal synaptic specializations. Most non-synapsing fibers (57% and 48% at 1 day and 1 week) remained in contact with an IHC and contained healthy-looking organelles. ANFs showed a transient increase in mitochondrial content (51%) and efferent innervation (34%) at 1 day. Fibers maintaining synaptic connections showed hypertrophy of pre-synaptic ribbons at both 1 day and 1 week. Non-synaptic fibers were lower in mitochondrial content and typically on the modiolar side of the IHC, where ANFs with high-thresholds and low spontaneous rates are normally found. Even 1 week post-exposure, many ANF terminals remained in IHC contact despite loss of synaptic specializations, thus, regeneration efforts at early post-exposure times should concentrate on synaptogenesis rather than neurite extension.

Remodelling of the Mitochondrial Bioenergetic Pathways in Human Cultured Fibroblasts with Carbohydrates.

Mitochondrial oxidative phosphorylation defects underlie many neurological and neuromuscular diseases. Patients' primary dermal fibroblasts are one of the most commonly used in vitro models to study mitochondrial pathologies. However, fibroblasts tend to rely more on glycolysis than oxidative phosphorylation for their energy when cultivated in standard high-glucose medium, rendering it difficult to expose mitochondrial dysfunctions. This study aimed to systematically investigate to which extent the use of galactose- or fructose-based medium switches the fibroblasts' energy metabolism to a more oxidative state. Highly proliferative cells depend more on glycolysis than less proliferative cells. Therefore, we investigated two primary dermal fibroblast cultures from healthy subjects: a highly proliferative neonatal culture and a slower-growing adult culture. Cells were cultured with 25 mM glucose, galactose or fructose, and 4 mM glutamine as carbon sources. Compared to glucose, both galactose and fructose reduce the cellular proliferation rate, but the galactose-induced drop in proliferation is much more profound than the one observed in cells cultivated in fructose. Both galactose and fructose result in a modest increase in mitochondrial content, including mitochondrial DNA, and a disproportionate increase in protein levels, assembly, and activity of the oxidative phosphorylation enzyme complexes. Galactose- and fructose-based media induce a switch of the prevalent biochemical pathway in cultured fibroblasts, enhancing aerobic metabolism when compared to glucose-based medium. While both galactose and fructose stimulate oxidative phosphorylation to a comparable degree, galactose decreases the cellular proliferation rate more than fructose, suggesting that a fructose-based medium is a better choice when studying partial oxidative phosphorylation defects in patients' fibroblasts.

Aberrant LETM1 elevation dysregulates mitochondrial functions and energy metabolism and promotes lung metastasis in osteosarcoma

Osteosarcoma is a differentiation-deficient disease, and despite the unique advantages and great potential of differentiation therapy, there are only a few known differentiation inducers, and little research has been done on their targets. Cell differentiation is associated with an increase in mitochondrial content and activity. The metabolism of some tumor cells is characterized by impaired oxidative phosphorylation, as well as up-regulation of aerobic glycolysis and pentose phosphate pathways. Leucine-containing zipper and EF-hand transmembrane protein 1 (LETM1) is involved in the maintenance of mitochondrial morphology and is closely associated with tumorigenesis and progression, as well as cancer cell stemness. We found that MG63 and 143B osteosarcoma cells overexpress LETM1 and exhibit abnormalities in mitochondrial structure and function. Knockdown of LETM1 partially restored the mitochondrial structure and function, inhibited the pentose phosphate pathway, promoted oxidative phosphorylation, and led to osteogenic differentiation. It also inhibited spheroid cell formation, proliferation, migration, and invasion in an in vitro model. When LETM1 was knocked down in vivo, there was reduced tumor formation and lung metastasis. These data suggest that mitochondria are aberrant in LETM1-overexpressing osteosarcoma cells, and knockdown of LETM1 partially restores the mitochondrial structure and function, inhibits the pentose phosphate pathway, promotes oxidative phosphorylation, and increases osteogenic differentiation, thereby reducing malignant biological behavior of the cells.

High fat diet feeding induces brain mitochondrial dysfunction in female Ossabaw swine

Study objective: Emerging evidence now suggest reduced mitochondrial function as a key initiator in Alzheimer’s disease (AD)-like pathology increasing the need for models of AD that display mitochondrial dysfunction. Dyslipidemia is a common aliment from overconsumption of a high fat diet (HFD) that increases the risk of AD and may contribute to mitochondrial dysfunction. Rodent studies utilize HFDs to induce brain pathology resembling that of AD. Unsurprisingly, rodent studies are rarely recapitulated in humans, which leads to a very low clinical success rate and highlights the need for more translatable models to study AD-like pathology. Swine seem to share many characteristics of humans and might offer a more translatable model to study HFD induced pathology. Thus, the objective of this study was to determine if HFD feeding in swine could offer a more translatable model to study mitochondrial dysfunction during neurodegeneration. Hypothesis: 6-months of HFD feeding will decrease indices of mitochondrial function and biogenesis in female Ossabaw swine. Methods: To characterize the effects of a HFD on brain mitochondrial health in a highly translatable swine model, high resolution respirometry measurements were taken from isolated brain mitochondria from in female Ossabaw swine fed a HFD for 6-months. Additionally, indices of mitochondrial biogenesis in whole tissue was measured (transcriptional markers of biogenesis and citrate synthase activity). Results: HFD feeding significantly increased body weight (p<0.05) with trending increases in blood glucose (p<0.10). Maximal uncoupled mitochondrial respiration was significantly decreased (p<0.05) with state 3-complex I and state 3 complex I & II trending down in the dorsal hippocampus (DH) after HFD diet feeding (p<0.10). Additionally, state 2 respiration was significantly decreased in the prefrontal cortex (PFC) after HFD feeding (p<0.05). HFD feeding increased transcriptional markers of mitochondrial biogenesis in the DH and PFC (p<0.05), without altering whole homogenate citrate synthase activity in either brain region. Conclusions: These data indicate HFD feeding in Ossabaw pigs could offer a potential model to study pathologies relating to mitochondrial dysfunction during neurodegenerative process. Additionally, the DH was found to be more vulnerable to HFD-induced perturbations, evident by the exacerbated deficits in mitochondrial respiration in this brain region. Finally, these data suggest HFD-induced increase in transcriptional markers of mitochondrial biogenesis may be a compensatory response to HFD-induced mitochondrial respiration deficits. However, citrate synthase activity was not different between groups indicating this upregulation of transcriptional markers was unable to translate to an increase in mitochondrial content in the DH and PFC. The project was supported by the MU Office of Postdoctoral Education Research Grant Program. Additional funding was also generously provided Scott Rector. Images in figure 2 adapted and modified from “Krebs cycle template”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates . This work was also supported with resources and the use of facilities at the University of Missouri and Harry S. Truman Memorial Veterans Hospital in Columbia, Missouri. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.

The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.

Human skeletal muscle mitochondrial responses to single-leg intermittent or continuous cycle exercise training matched for absolute intensity and total work.

There is renewed interest in the potential for interval (INT) training to increase skeletal muscle mitochondrial content including whether the response differs from continuous (CONT) training. Comparisons of INT and CONT exercise are impacted by the manner in which protocols are "matched", particularly with respect to exercise intensity, as well as inter-individual differences in training responses. We employed single-leg cycling to facilitate a within-participant design and test the hypothesis that short-term INT training would elicit a greater increase in mitochondrial content than work- and intensity-matched CONT training. Ten young healthy adults (five males and five females) completed 12 training sessions over 4 weeks with each leg. Legs were randomly assigned to complete either 30 min of CONT exercise at a challenging sustainable workload (~50% single-leg peak power output; Wpeak) or INT exercise that involved 10 × 3-min bouts at the same absolute workload. INT bouts were interspersed with 1min of recovery at 10% Wpeak and each CONT session ended with 10min at 10% Wpeak. Absolute and mean intensity, total training time, and volume were thus matched between legs but the pattern of exercise differed. Contrary to our hypothesis, biomarkers of mitochondrial content including citrate synthase maximal activity, mitochondrial protein content and subsarcolemmal mitochondrial volume increased after CONT (p < 0.05) but not INT training. Both training modes increased single-leg Wpeak (p < 0.01) and time to exhaustion at 70% of single-leg Wpeak (p < 0.01). In a work- and intensity-matched comparison, short-term CONT training increased skeletal muscle mitochondrial content whereas INT training did not.

The receptor for advanced glycation end products (RAGE) is involved in mitochondrial function and cigarette smoke-induced oxidative stress.

The mechanisms underlying muscle dysfunction with Chronic Obstructive Pulmonary Disease (COPD) are poorly understood. Indirect evidence has recently suggested a role of Advanced Glycation End Products (AGEs) and their receptor (RAGE) in the pathophysiology of COPD. Accordingly, this study aimed to examine the redox balance and mitochondrial alterations in the skeletal muscle of a mouse model deficient in the receptor for AGE (RAGE-KO) and wild-type C57BL/6 exposed to cigarette smoke for 8-months using immunoblotting, spectrophotometry, and high-resolution respirometry. Cigarette smoke exposure increased by two-fold 4-HNE levels (P < 0.001), a marker of oxidative stress, and markedly downregulated contractile proteins, mitochondrial respiratory complexes, and uncoupling proteins levels (P < 0.001). Functional alterations with cigarette smoke exposure included a greater reliance on complex-I supported respiration (P < 0.01) and lower relative respiratory capacity for fatty acid (P < 0.05). RAGE knockout resulted in 47% lower 4-HNE protein levels than the corresponding WT control mice exposed to cigarette smoke (P < 0.05), which was partly attributed to increased Complex III protein levels. Independent of cigarette smoke exposure, RAGE KO decreased mitochondrial specific maximal respiration (P < 0.05), resulting in a compensatory increase in mitochondrial content measured by citrate synthase activity (P < 0.001) such that muscle respiratory capacity remained unaltered. Together, these findings suggest that knockout of RAGE protected the skeletal muscle against oxidative damage induced by 8 months of cigarette smoke exposure. In addition, this study supports a role for RAGE in regulating mitochondrial content and function and can thus serve as a potential therapeutic target.

Ammonium chloride administration prevents training-induced improvements in mitochondrial respiratory function in the soleus muscle of male rats.

Exercise training can increase both mitochondrial content and mitochondrial respiration. Despite its popularity, high-intensity exercise can be accompanied by mild acidosis (also present in certain pathological states), which may limit exercise-induced adaptations to skeletal muscle mitochondria. The aim of this study was to determine if administration of ammonium chloride (0.05 g/kg) to Wistar rats before each individual exercise session (5 high-intensity exercise sessions/wk for 8 wk) reduced training-induced increases in mitochondrial content (measured by citrate synthase activity and protein content of electron transport system complexes) and respiration (measured in permeabilized muscle fibers). In the soleus muscle, the exercise-training-induced increase in mitochondrial respiration was reduced in rats administered ammonium chloride compared to control animals, but mitochondrial content was not altered. These effects were not present in the white gastrocnemius muscle. In conclusion, ammonium chloride administration before each exercise session over 8 wk reduced improvements in mitochondrial respiration in the soleus muscle but did not alter mitochondrial content. This suggests that mild acidosis may affect training-induced improvements in the respiration of mitochondria in some muscles.

IL-6 deletion decreased REV-ERBα protein and influenced autophagy and mitochondrial markers in the skeletal muscle after acute exercise

Interleukin 6 (IL-6) acts as a pro and anti-inflammatory cytokine, has an intense correlation with exercise intensity, and activates various pathways such as autophagy and mitochondrial unfolded protein response. Also, IL-6 is interconnected to circadian clock-related inflammation and can be suppressed by the nuclear receptor subfamily 1, group D, member 1 (Nr1d1, protein product REV-ERBα). Since IL-6 is linked to physical exercise-modulated metabolic pathways such as autophagy and mitochondrial metabolism, we investigated the relationship of IL-6 with REV-ERBα in the adaptations of these molecular pathways in response to acute intense physical exercise in skeletal muscle. The present study was divided into three experiments. In the first one, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were divided into three groups: Basal time (Basal; sacrificed before the acute exercise), 1 hour (1hr post-Ex; sacrificed 1 hour after the acute exercise), and 3 hours (3hr post-Ex; sacrificed 3 hours after the acute exercise). In the second experiment, C2C12 cells received IL-6 physiological concentrations or REV-ERBα agonist, SR9009. In the last experiment, WT mice received SR9009 injections. After the protocols, the gastrocnemius muscle or the cells were collected for reverse transcription-quantitative polymerase chain reaction (RTq-PCR) and immunoblotting techniques. In summary, the downregulation of REV-ERBα, autophagic flux, and most mitochondrial genes was verified in the IL-6 KO mice independent of exercise. The WT and IL-6 KO treated with SR9009 showed an upregulation of autophagic genes. C2C12 cells receiving IL-6 did not modulate the Nr1d1 mRNA levels but upregulated the expression of some mitochondrial genes. However, when treated with SR9009, IL-6 and mitochondrial gene expression were upregulated in C2C12 cells. The autophagic flux in C2C12 suggest the participation of REV-ERBα protein in the IL-6-induced autophagy. In conclusion, the present study verified that the adaptations required through physical exercise (increases in mitochondrial content and improvement of autophagy machinery) might be intermediated by an interaction between IL-6 and REVERBα.

Axonal response of mitochondria to demyelination and complex IV activity within demyelinated axons in experimental models of multiple sclerosis.

Axonal injury in multiple sclerosis (MS) and experimental models is most frequently detected in acutely demyelinating lesions. We recently reported a compensatory neuronal response, where mitochondria move to the acutely demyelinated axon and increase the mitochondrial content following lysolecithin-induced demyelination. We termed this homeostatic phenomenon, which is also evident in MS, the axonal response of mitochondria to demyelination (ARMD). The aim of this study is to determine whether ARMD is consistently evident in experimental demyelination and how its perturbation relates to axonal injury. In the present study, we assessed axonal mitochondrial content as well as axonal mitochondrial respiratory chain complex IV activity (cytochrome c oxidase or COX) of axons and related these to axonal injury in nine different experimental disease models. We used immunofluorescent histochemistry as well as sequential COX histochemistry followed by immunofluorescent labelling of mitochondria and axons. We found ARMD a consistent and robust phenomenon in all experimental disease models. The increase in mitochondrial content within demyelinated axons, however, was not always accompanied by a proportionate increase in complex IV activity, particularly in highly inflammatory models such as experimental autoimmune encephalomyelitis (EAE). Axonal complex IV activity inversely correlated with the extent of axonal injury in experimental disease models. Our findings indicate that ARMD is a consistent and prominent feature and emphasise the importance of complex IV activity in the context of ARMD, especially in autoimmune inflammatory demyelination, paving the way for the development of novel neuroprotective therapies.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

Physical exercise improves mitochondrial function in ovariectomized rats.

Estrogen deficiency causes metabolic disorders in humans and rodents, including in part due to changes in energy expenditure. We have shown previously that skeletal muscle mitochondrial function is compromised in ovariectomized (Ovx) rats. Since physical exercise is a powerful strategy to improve skeletal muscle mitochondrial content and function, we hypothesize that exercise training would counteract the deficiency-induced skeletal muscle mitochondrial dysfunction in Ovx rats. We report that exercised Ovx rats, at 60-65% of maximal exercise capacity for 8 weeks, exhibited less fat accumulation and body weight gain compared with sedentary controls. Treadmill exercise training decreased muscle lactate production, indicating a shift to mitochondrial oxidative metabolism. Furthermore, reduced soleus muscle mitochondrial oxygen consumption confirmed that estrogen deficiency is detrimental to mitochondrial function. However, exercise restored mitochondrial oxygen consumption in Ovx rats, achieving similar levels as in exercised control rats. Exercise-induced skeletal muscle peroxisome proliferator-activated receptor-γ coactivator-1α expression was similar in both groups. Therefore, the mechanisms by which exercise improves mitochondrial oxygen consumption appears to be different in Ovx-exercised and sham-exercised rats. While there was an increase in mitochondrial content in sham-exercised rats, demonstrated by a greater citrate synthase activity, no induction was observed in Ovx-exercised rats. Normalizing mitochondrial respiratory capacity by citrate synthase activity indicates a better oxidative phosphorylation efficiency in the Ovx-exercised group. In conclusion, physical exercise sustains mitochondrial function in ovarian hormone-deficient rats through a non-conventional mitochondrial content-independent manner.

Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila.

The effects of aging on the brain are widespread and can have dramatic implications on the overall health of an organism. Mitochondrial dysfunction is a hallmark of brain aging, but, the interplay between mitochondrial quality control, neuronal aging, and organismal health is not well understood. Here, we show that aging leads to a decline in mitochondrial autophagy (mitophagy) in the Drosophila brain with a concomitant increase in mitochondrial content. We find that induction of BCL2-interacting protein 3 (BNIP3), a mitochondrial outer membrane protein, in the adult nervous system induces mitophagy and prevents the accumulation of dysfunctional mitochondria in the aged brain. Importantly, neuronal induction of BNIP3-mediated mitophagy increases organismal longevity and healthspan. Furthermore, BNIP3-mediated mitophagy in the nervous system improves muscle and intestinal homeostasis in aged flies, indicating cell non-autonomous effects. Our findings identify BNIP3 as a therapeutic target to counteract brain aging and prolong overall organismal health with age.