Changes In Mitochondrial Content Research Articles

A Scoping Review Investigating the "Gene-Dosage Theory" of Mitochondrial DNA in the Healthy Skeletal Muscle.

This review provides an overview of the evidence regarding mtDNA and valid biomarkers for assessing mitochondrial adaptions. Mitochondria are small organelles that exist in almost all cells throughout the human body. As the only organelle, mitochondria contain their own DNA, mitochondrial DNA (mtDNA). mtDNA-encoded polypeptides are subunits of the enzyme complexes in the electron transport chain (ETC) that are responsible for production of ATP to the cells. mtDNA is frequently used as a biomarker for mitochondrial content, since changes in mitochondrial volume are thought to induce similar changes in mtDNA. However, some exercise studies have challenged this "gene-dosage theory", and have indicated that changes in mitochondrial content can adapt without changes in mtDNA. Thus, the aim of this scoping review was to summarize the studies that used mtDNA as a biomarker for mitochondrial adaptions and address the question as to whether changes in mitochondrial content, induce changes in mtDNA in response to aerobic exercise in the healthy skeletal muscle. The literature was searched in PubMed and Embase. Eligibility criteria included: interventional study design, aerobic exercise, mtDNA measurements reported pre- and postintervention for the healthy skeletal muscle and English language. Overall, 1585 studies were identified. Nine studies were included for analysis. Eight out of the nine studies showed proof of increased oxidative capacity, six found improvements in mitochondrial volume, content and/or improved mitochondrial enzyme activity and seven studies did not find evidence of change in mtDNA copy number. In conclusion, the findings imply that mitochondrial adaptions, as a response to aerobic exercise, can occur without a change in mtDNA copy number.

Mitochondrial adaptations to calorie restriction and bariatric surgery in human skeletal muscle: a systematic review with meta-analysis

ObjectiveWe performed a meta-analysis to determine the changes induced by calorie restriction (CR) and bariatric surgery on human skeletal muscle mitochondria. MethodsA systematic search of Medline and Web of Science was conducted. Controlled trials exploring CR (≥14 days) and mitochondrial function and/or content assessment were included. Moreover, studies analyzing weight loss following gastric surgery were included for comparison purposes. Human muscle data from 28 studies assessing CR (520 muscle samples) and from 10 studies assessing bariatric surgery (155 muscle samples) were analyzed in a random effect meta-analysis with three a priori chosen covariates. Main resultsWe report a decrease (p < 0.05) (mean (95 % CI)) in maximal mitochondrial state 3 respiration in response to CR (−0.44 (−0.85, −0.03)) but not in response to surgery (−0.33 (−1.18, 0.52)). No changes in mitochondrial content were reported after CR (−0.05 (−0.12, 0.13)) or in response to surgery (0.23 (−0.05, 0.52)). Moreover, data from CR subjects showed a reduction in complex IV (CIV) activity (−0.29 (−0.56, −0.03)) but not in CIV content (−0.21 (−0.63, 0.22)). Similar results were obtained when the length of the protocol, the initial body mass index, and the estimated energy deficit were included in the model as covariates. ConclusionThe observation of reduced maximal mitochondrial state 3, uncoupled respiration, and CIV activity without altering mitochondrial content suggests that, in human skeletal muscle, CR mainly modulates intrinsic mitochondrial function.

CD38 Is a Key Regulator of Tumor Growth By Modulating the Metabolic Signature of Malignant Plasma Cells

Introduction:Multiple myeloma (MM) is a disease of malignant plasma cells, characterized by high CD38 expression. Although the CD38-targeting monoclonal antibodies are highly effective, resistance invariably arises. Tumor CD38 levels decrease after anti-CD38 therapy, but the expression is rarely permanently silenced. This suggests that CD38 expression may offer a tumor cell survival advantage, but the direct impact of CD38 loss on tumor dynamics has not been extensively characterized.Methods:CD38 knockout (KO) cell lines were generated by CRISPR-Cas9. Immunocompetent Balb/c and immunodeficient NSG mice were injected subcutaneously with either non-targeting (NT) or CD38 KO J558 cells. Stromal adhesion was compared using labeled NT and KO cells, with OP-9 murine stroma cells. Cellular NAD content was quantified using the Promega Glo Assay. Mitochondria were isolated with the Mitochondria Isolation Kit (Thermo Scientific). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were quantified using the Seahorse Assay. Response to hypoxia was evaluated using a modular hypoxic chamber. Cell cycle was quantified using propidium iodine staining.Results:To examine the role of CD38 in murine models, we utilized the CD38-expressing, murine plasmacytoma cell line J558. Strikingly, CD38 KO cells injected into Balb/c mice demonstrated significantly decreased tumor volume compared to NT (113 mm 3 (KO) vs. 1293 mm 3 (NT) at day 25, p <0.001). In contrast, in vitro cell proliferation and colony formation between KO and NT J558 cells were nearly identical, suggesting that the effects of CD38-loss were highly context dependent. Since tumoral CD38 expression may negatively modulate the immune response, we next compared CD38 KO and NT cells injected into immunodeficient NSG mice. CD38 KOs demonstrated an approximately 2.2-fold decreased tumor volume compared to the NT (708 mm 3 (KO) vs. 1592 mm 3 (NT), p=0.07). Further examination of the role of CD38 on the immune microenvironment are ongoing. Considering that some tumor growth impairment was maintained in immunodeficient mice, we next interrogated the effect of CD38 loss on other aspects of cell proliferation using J558 as well as human MM cell lines RPMI-8226 and NCI-H929. Daratumumab induced CD38 internalization has been shown to reduce stromal adhesion of MM cells. Similarly, CD38 KO cells demonstrated reduced stromal adhesion (2.5-fold decrease for J558, p<0.005 and 2-fold decrease for H929, p<0.005). Although stroma is a known promoter of cell survival and proliferation, we further questioned whether the NAD-metabolizing activity of CD38 modulates tumor growth. CD38 overexpression can drive down intracellular NAD and impair mitochondrial biogenesis. Accordingly, we found significantly higher NAD levels in the KO J558 tumor cells compared to NT (2-fold change, p <0.05). Additionally, CD38 KO cells demonstrated significantly higher levels of mitochondrial protein compared with the NTs (5-fold in J558 and 2-fold in H929). CD38 KO cell lines also showed markedly increased metabolic activity, with nearly 2-fold increase in basal OCR and ECAR, as well as in spare respiratory and glycolytic capacity. Given the contrast between in vivo and in vitro growth capacity, we questioned whether changes in mitochondrial content and metabolic function could confer an advantage for CD38-expressing cells under conditions of hypoxia, which is an important characteristic of the tumor microenvironment. Strikingly, under hypoxia, but not normoxia, CD38 KO MM cells demonstrated significantly more cell cycle arrest, defined by G0/G1 blockage (p=0.003 for H929 and p=0.004 for RPMI).Conclusion:We have shown that CD38 KO cells demonstrate decreased tumor growth in vivo but not in vitro. While the immune modulatory potential of CD38 is recognized, some of the growth impairment we observed may be explained by non-immune mediated mechanisms such as reduced stroma adherence as well as changes in cell metabolism. Loss of CD38 was associated with increased mitochondrial respiration, but also elevated ECAR and glycolytic rate. Higher reliance on mitochondrial respiration could explain impaired CD38 KO proliferation rates under hypoxia, possibly as a result of increased generation of reactive oxygen species. DisclosuresGhiaur: Menarini Richerche: Research Funding; Syros Pharmaceuticals: Consultancy.

Mitochondria-cytokine crosstalk following skeletal muscle injury and disuse: a mini-review.

Skeletal muscle mitochondria are highly adaptable, highly dynamic organelles that maintain the functional integrity of the muscle fiber by providing ATP for contraction and cellular homeostasis (e.g., Na+/K+ ATPase). Emerging as early modulators of inflammation, mitochondria sense and respond to cellular stress. Mitochondria communicate with the environment, in part, by release of physical signals called mitochondrial-derived damage-associated molecular patterns (mito-DAMPs) and deviation from routine function (e.g., reduced ATP production, Ca2+ overload). When skeletal muscle is compromised, mitochondria contribute to an acute inflammatory response necessary for myofibril regeneration; however, exhaustive signaling associated with altered or reduced mitochondrial function can be detrimental to muscle outcomes. Here, we describe changes in mitochondrial content, structure, and function following skeletal muscle injury and disuse and highlight the influence of mitochondria-cytokine crosstalk on muscle regeneration and recovery. Although the appropriate therapeutic modulation following muscle stressors remains unknown, retrospective gene expression analysis reveals that interleukin-6 (IL-6), interleukin-1β (IL-1β), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1) are significantly upregulated following three unique muscle injuries. These cytokines modulate mitochondrial function and execute bona fide pleiotropic roles that can aid functional recovery of muscle, however, when aberrant, chronically disrupt healing partly by exacerbating mitochondrial dysfunction. Multidisciplinary efforts to delineate the opposing regulatory roles of inflammatory cytokines in the muscle mitochondrial environment are required to modulate regenerative behavior following skeletal muscle injury or disuse. Future therapeutic directions to consider include quenching or limited release of mito-DAMPs and cytokines present in cytosol or circulation.

Aglycemic growth enhances carbohydrate metabolism and induces sensitivity to menadione in cultured tumor-derived cells

BackgroundHepatocellular carcinoma (HCC) is the most prevalent form of liver malignancy and carries poor prognoses due to late presentation of symptoms. Treatment of late-stage HCC relies heavily on chemotherapeutics, many of which target cellular energy metabolism. A key platform for testing candidate chemotherapeutic compounds is the intrahepatic orthotopic xenograft (IOX) model in rodents. Translational efficacy from the IOX model to clinical use is limited (in part) by variation in the metabolic phenotypes of the tumor-derived cells that can be induced by selective adaptation to subculture conditions.MethodsIn this study, a detailed multilevel systems approach combining microscopy, respirometry, potentiometry, and extracellular flux analysis (EFA) was utilized to examine metabolic adaptations that occur under aglycemic growth media conditions in HCC-derived (HEPG2) cells. We hypothesized that aglycemic growth would result in adaptive “aerobic poise” characterized by enhanced capacity for oxidative phosphorylation over a range of physiological energetic demand states.ResultsAglycemic growth did not invoke adaptive changes in mitochondrial content, network complexity, or intrinsic functional capacity/efficiency. In intact cells, aglycemic growth markedly enhanced fermentative glycolytic substrate-level phosphorylation during glucose refeeding and enhanced responsiveness of both fermentation and oxidative phosphorylation to stimulated energy demand. Additionally, aglycemic growth induced sensitivity of HEPG2 cells to the provitamin menadione at a 25-fold lower dose compared to control cells.ConclusionsThese findings indicate that growth media conditions have substantial effects on the energy metabolism of subcultured tumor-derived cells, which may have significant implications for chemotherapeutic sensitivity during incorporation in IOX testing panels. Additionally, the metabolic phenotyping approach used in this study provides a practical workflow that can be incorporated with IOX screening practices to aid in deciphering the metabolic underpinnings of chemotherapeutic drug sensitivity.

Nanoparticle-encapsulated antioxidant improves placental mitochondrial function in a sexually dimorphic manner in a rat model of prenatal hypoxia.

Pregnancy complications associated with prenatal hypoxia lead to increased placental oxidative stress. Previous studies suggest that prenatal hypoxia can reduce mitochondrial respiratory capacity and mitochondrial fusion, which could lead to placental dysfunction and impaired fetal development. We developed a placenta-targeted treatment strategy using a mitochondrial antioxidant, MitoQ, encapsulated into nanoparticles (nMitoQ) to reduce placental oxidative stress and (indirectly) improve fetal outcomes. We hypothesized that, in a rat model of prenatal hypoxia, nMitoQ improves placental mitochondrial function and promotes mitochondrial fusion in both male and female placentae. Pregnant rats were treated with saline or nMitoQ on gestational day (GD) 15 and exposed to normoxia (21% O2 ) or hypoxia (11% O2 ) from GD15-21. On GD21, male and female placental labyrinth zones were collected for mitochondrial respirometry assessments, mitochondrial content, and markers of mitochondrial biogenesis, fusion and fission. Prenatal hypoxia reduced complex IV activity and fusion in male placentae, while nMitoQ improved complex IV activity in hypoxic male placentae. In female placentae, prenatal hypoxia decreased respiration through the S-pathway (complex II) and increased N-pathway (complex I) respiration, while nMitoQ increased fusion in hypoxic female placentae. No changes in mitochondrial content, biogenesis or fission were found. In conclusion, nMitoQ improved placental mitochondrial function in male and female placentae from fetuses exposed to prenatal hypoxia, which may contribute to improved placental function. However, the mechanisms (ie, changes in mitochondrial respiratory capacity and mitochondrial fusion) were distinct between the sexes. Treatment strategies targeted against placental oxidative stress could improve placental mitochondrial function in complicated pregnancies.

Brain Transcriptome Analysis of a Protein-Truncating Mutation in Sortilin-Related Receptor 1 Associated With Early-Onset Familial Alzheimer's Disease Indicates Early Effects on Mitochondrial and Ribosome Function.

The early cellular stresses leading to Alzheimer's disease (AD) remain poorly understood because we cannot access living, asymptomatic human AD brains for detailed molecular analyses. Sortilin-related receptor 1 (SORL1) encodes a multi-domain receptor protein genetically associated with both rare, early-onset familial AD (EOfAD) and common, sporadic, late-onset AD (LOAD). SORL1 protein has been shown to act in the trafficking of the amyloid β A4 precursor protein (AβPP) that is proteolysed to form one of the pathological hallmarks of AD, amyloid-β (Aβ) peptide. However, other functions of SORL1 in AD are less well understood. To investigate the effects of heterozygosity for an EOfAD-like mutation in SORL1 on the brain transcriptome of young-adult mutation carriers using zebrafish as a model organism. We performed targeted mutagenesis to generate an EOfAD-like mutation in the zebrafish orthologue of SORL1 and performed RNA-sequencing on mRNA isolated from the young adult brains of siblings in a family of fish either wild type (non-mutant) or heterozygous for the EOfAD-like mutation. We identified subtle differences in gene expression indicating changes in mitochondrial and ribosomal function in the mutant fish. These changes appear to be independent of changes in mitochondrial content or the expression of AβPP-related proteins in zebrafish. These findings provided evidence supporting that EOfAD mutations in SORL1 affect mitochondrial and ribosomal function and provide the basis for future investigation elucidating the nature of these effects.

Are Alterations in Skeletal Muscle Mitochondria a Cause or Consequence of Insulin Resistance?

As a major site of glucose uptake following a meal, skeletal muscle has an important role in whole-body glucose metabolism. Evidence in humans and animal models of insulin resistance and type 2 diabetes suggests that alterations in mitochondrial characteristics accompany the development of skeletal muscle insulin resistance. However, it is unclear whether changes in mitochondrial content, respiratory function, or substrate oxidation are central to the development of insulin resistance or occur in response to insulin resistance. Thus, this review will aim to evaluate the apparent conflicting information placing mitochondria as a key organelle in the development of insulin resistance in skeletal muscle.

Effects of voluntary wheel running on mitochondrial content and dynamics in rat skeletal muscle.

This study reports that in rat skeletal muscle the proteins specifically responsible for mitochondrial dynamics, mitofusin-2 (MFN2) and mitochondrial dynamics protein 49 (MiD49), are higher (p < 0.05) in oxidative soleus (SOL) muscle compared with predominantly glycolytic extensor digitorum longus (EDL) muscle, but not seen for optic atrophy 1 (OPA1; p = 0.06). Markers of mitochondrial content, complex I component, NADH:Ubiquinone oxidoreductase subunit A9 (NDUFA9) and complex IV protein, cytochrome C oxidase subunit IV (COXIV; p < 0.05) were also higher in SOL compared with EDL muscle; however, there was no difference in mitochondrial content between muscles, as measured using a citrate synthase assay (p > 0.05). SOL and EDL muscles were compared between age-matched sedentary rats that were housed individually with (RUN) or without (SED) free-access to a running wheel for 12 weeks and showed no change in mitochondrial content, as examined by the abundances of NDUFA9 and COXIV proteins, as well as citrate synthase activity, in either muscle (p > 0.05). Compared to SED animals, MiD49 and OPA1 were not different in either EDL or SOL muscles, and MFN2 was higher in SOL muscles from RUN rats (p < 0.05). Overall, these findings reveal that voluntary wheel running is an insufficient stimulus to result in a significantly higher abundance of most markers of mitochondrial content or dynamics, and it is likely that a greater stimulus, such as either adding resistance to the wheel or an increase in running volume by using a treadmill, is required for mitochondrial adaptation in rat skeletal muscle.

Chronic Kidney Disease Causes Skeletal Mitochondrial Myopathy Through Disruption of the Electron Transport System

IntroductionChronic kidney disease (CKD) is associated with debilitating myopathic symptoms including muscle wasting, weakness, and fatigue. Mitochondria have been implicated as a mediator of skeletal muscle dysfunction in CKD, however, the underlying mechanisms of mitochondrial dysfunction in CKD are unknown.PurposeThe purpose of this study was to define the impact of CKD on muscle mitochondrial energy transduction, metabolomic profiles, and muscle contractile function.Methods8‐week old C57BL/6J mice (N=6–10/group/sex) were fed a casein control or adenine supplemented‐diet for 10‐weeks. CKD was confirmed through measurement of glomerular filtration rate (GFR), blood urea nitrogen, and kidney histology. A comprehensive mitochondrial phenotyping platform included respiratory function, H2O2 emission, matrix dehydrogenase and OXPHOS enzyme activity, and ATP synthase activity was employed. Muscle function, size, metabolomic profiles were also assessed.ResultsCKD resulted in a 30–40% decrease in OXPHOS sensitivity supported by pyruvate/malate, glutamate/malate, succinate/rotenone, and octanoylcarnitine/malate (all P&lt;0.05) using a creatine kinase clamp to control the extracellular energy demand for mitochondria. CKD decreased maximal ADP‐stimulated respiratory capacity (4139.1 ± 564.4 vs 3448.7 ± 782.2 pmols/s/mg, P&lt;0.05), despite no change in mitochondrial content (citrate synthase activity and western blotting). Significant correlations between GFR and skeletal muscle mitochondrial respiratory function were observed in all substrate conditions (all P&lt;0.03). Enzymatic assays demonstrated significant decreases (~35%) in the activity of pyruvate and alpha‐ketoglutarate dehydrogenases (P&lt;0.05), and non‐significant decreases in glutamate dehydrogenase, malic enzyme, and isocitrate dehydrogenase (12–36%). Mitochondrial H2O2 production was modestly increased with pyruvate/malate (P=0.043) but not different with octanoylcarnitine/malate or glutamate/malate (P=0.18 and 0.23 respectively). With both sexes combined, muscle force production was not different, however, when separated male CKD mice had lower force production (293.1 ± 20.8 vs 236.8 ± 25.5, P&lt;0.05). Muscle weights and myofiber areas confirmed atrophy in male CKD mice. Metabolomics analyses indicated alterations in amino acid catabolism including arginine, tryptophan, phenylalanine/tyrosine, increased allantoin, and accumulation of uremic metabolites previously found to negatively impact mitochondrial energetics.ConclusionTo our knowledge, this is the first study to define the exact mechanisms by which CKD alters skeletal muscle mitochondrial function. Using mitochondrial phenotyping, CKD was found to disrupt OXPHOS enzymes and matrix dehydrogenases resulting in impaired oxidative phosphorylation and elevated H2O2 production. These discoveries provide the foundation for targeted mitochondrial therapies aimed to treat skeletal myopathy in CKD patients.Support or Funding InformationSupported by R01 HL148597 (STS and TER).

Ultrastructural characterization of mitochondrial damage in experimental autoimmune neuritis

Data are sparse about mitochondrial damage in GBS and in its most frequently employed animal model, experimental autoimmune neuritis (EAN).We here characterized changes in mitochondrial content and morphology at different time points during EAN by use of ultrastructural imaging and immunofluorescent labelling.Histological examination revealed that demyelinated axons and their adjacent Schwann cells showed reduced mitochondrial content and remaining mitochondria appeared swollen with greater diameter in Schwann cells and unmyelinated axons.Our findings indicate that in EAN, particularly mitochondria in Schwann cells are damaged. Further studies are warranted to address whether these changes are amenable to novel, mitoprotective treatments.

In vitro comparison of hydroxocobalamin (B12a) and the mitochondrial directed therapy by a succinate prodrug in a cellular model of cyanide poisoning

The objective of this study was to compare the use of hydroxocobalamin (B12a) and a succinate prodrug to evaluate for improvement in mitochondrial function in an in vitro model of cyanide poisoning. Peripheral blood mononuclear cells (PBMC) and human aortic smooth muscle cells (HASMC) incubated with 50 mM of sodium cyanide (CN) for five minutes serving as the CN group compared to controls. We investigated the following: (1) Mitochondrial respiration; (2) Superoxide and mitochondrial membrane potential with microscopy; (3) Citrate synthase protein expression. All experiments were performed with a cell concentration of 2−3 × 106 cells/ml for both PBMC and HASMC. There were four conditions: (1) Control (no exposure); (2) Cyanide (exposure only); (3) B12a (cyanide exposure followed by B12a treatment); (4) NV118 (cyanide followed by NV118 treatment). In this study the key findings include: (1) Improvement in key mitochondrial respiratory states with the succinate prodrug (NV118) but not B12a; (2) Attenuation of superoxide production with treatment of NV118 but not with B12a treatment; (3) The changes in respiration were not secondary to increased mitochondrial content as measured by citrate synthase; (4) The use of easily accessible human blood cells showed similar mitochondrial response to both cyanide and treatment to HASMC. The use of a succinate prodrug to circumvent partial CIV inhibition by cyanide with clear reversal of cellular respiration and superoxide production that was not attributed to changes in mitochondrial content not seen by the use of B12a.

Mitochondrial Mass Assessment in a Selected Cell Line under Different Metabolic Conditions.

Changes of quantity and/or morphology of cell mitochondria are often associated with metabolic modulation, pathology, and apoptosis. Exogenous fluorescent probes used to investigate changes in mitochondrial content and dynamics are strongly dependent, for their internalization, on the mitochondrial membrane potential and composition, thus limiting the reliability of measurements. To overcome this limitation, genetically encoded recombinant fluorescent proteins, targeted to different cellular districts, were used as reporters. Here, we explored the potential use of mitochondrially targeted red fluorescent probe (mtRFP) to quantify, by flow cytometry, mitochondrial mass changes in cells exposed to different experimental conditions. We first demonstrated that the mtRFP fluorescence intensity is stable during cell culture and it is related with the citrate synthase activity, an established marker of the mitochondrial mass. Incidentally, the expression of mtRFP inside mitochondria did not alter the oxygen consumption rate under both state 3 and 4 respiration conditions. In addition, using this method, we showed for the first time that different inducers of mitochondrial mass change, such as hypoxia exposure or resveratrol treatment of cells, could be consistently detected. We suggest that transfection and selection of stable clones expressing mtRFP is a reliable method to monitor mitochondrial mass changes, particularly when pathophysiological or experimental conditions change ΔΨm, as it occurs during mitochondrial uncoupling or hypoxia/anoxia conditions.

Rebuttal from Martin MacInnis, Lauren Skelly and Martin Gibala.

We commend Bishop et al. (2019) for their comprehensive summary but respectfully disagree with their conclusion that training volume is more important than exercise intensity for increasing mitochondrial content in human skeletal muscle. Their position, based on the compiled data, centres on: (1) a positive correlation between training volume and citrate synthase (CS) activity and mitochondrial volume density (MitoVD); and (2) no correlation between training intensity and CS activity or MitoVD. The analysis presented by Bishop et al. (2019) is problematic for isolating the importance of exercise intensity per se. Firstly, on average, ∼37% more training sessions were performed for the moderate-intensity continuous training (MICT) as compared to high-intensity interval training (HIIT) and sprint interval training (SIT) (Granata et al. 2018): the HIIT and SIT interventions involved ∼15 sessions (lasting ∼15 min) compared to 21 sessions (lasting ∼70 min) for MICT (Granata et al. 2018). As indicated by time-course data (e.g. Murias et al. 2011), mitochondrial content increases as a training intervention progresses, which complicates comparisons among the intervention types. Secondly, despite the interval protocols being performed at higher intensities, the training volume for MICT (n = 33) was ∼60% and ∼700% greater than for HIIT (n = 9) and SIT (n = 20), respectively (Granata et al. 2018). As Bishop et al. (2019) demonstrated that training volume was positively associated with changes in mitochondrial content, the similar increase in CS activity across training types, despite differences in training volume, is strong evidence that HIIT and SIT were more efficient than MICT for increasing mitochondrial content. Given the disparities across intervention types, the lack of correlation between exercise intensity and the change in mitochondrial content is unsurprising and is not evidence against the importance of exercise intensity. Finally, while we agree that studies comparing different training protocols are important (Daussin et al. 2008; Granata et al. 2016; MacInnis et al. 2017; Montero & Lundby, 2017; Shepherd et al. 2017), we wish to clarify that the training interventions employed by Gillen et al. (2016) elicited similar increases in CS activity despite SIT involving about one-fifth the total volume of MICT (i.e. not equal volumes), demonstrating that high intensities of exercise can ‘compensate’ for low training volumes. Thus, in addition to our initial arguments (MacInnis et al. 2019), we contend that the pooled analysis from Bishop et al. (2019) supports our position that training intensity is more important than training volume for increasing human skeletal muscle mitochondrial content. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘LastWord’. Please email your comment, including a title and a declaration of interest, to [email protected] Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The authors declare that they have no competing interests. M.J.M., L.E.S. and M.J.G. contributed equally to the writing and critical revision of this manuscript. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. The research programmes of M.J.M. (ID: RGPIN-2018-06424) and M.J.G. (ID: RGPIN-2015-04632) are funded through Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). L.E.S. was funded by an NSERC Vanier Canada Graduate Scholarship.

CrossTalk proposal: Exercise training intensity is more important than volume to promote increases in human skeletal muscle mitochondrial content.

CrossTalk proposal: Exercise training intensity is more important than volume to promote increases in human skeletal muscle mitochondrial content.

Skeletal muscle energetics are compromised only during high-intensity contractions in the Goto-Kakizaki rat model of type 2 diabetes.

Type 2 diabetes (T2D) presents with hyperglycemia and insulin resistance, affecting over 30 million people in the United States alone. Previous work has hypothesized that mitochondria are dysfunctional in T2D and results in both reduced ATP production and glucose disposal. However, a direct link between mitochondrial function and T2D has not been determined. In the current study, the Goto-Kakizaki (GK) rat model of T2D was used to quantify mitochondrial function in vitro and in vivo over a broad range of contraction-induced metabolic workloads. During high-frequency sciatic nerve stimulation, hindlimb muscle contractions at 2- and 4-Hz intensities, the GK rat failed to maintain similar bioenergetic steady states to Wistar control (WC) rats measured by phosphorus magnetic resonance spectroscopy, despite similar force production. Differences were not due to changes in mitochondrial content in red (RG) or white gastrocnemius (WG) muscles (cytochrome c oxidase, RG: 22.2 ± 1.6 vs. 23.3 ± 1.7 U/g wet wt; WG: 10.8 ± 1.1 vs. 12.1 ± 0.9 U/g wet wt; GK vs. WC, respectively). Mitochondria isolated from muscles of GK and WC rats also showed no difference in mitochondrial ATP production capacity in vitro, measured by high-resolution respirometry. At lower intensities (0.25-1 Hz) there were no detectable differences between GK and WC rats in sustained energy balance. There were similar phosphocreatine concentrations during steady-state contraction and postcontractile recovery (τ = 72 ± 6 s GK versus 71 ± 2 s WC). Taken together, these results suggest that deficiencies in skeletal muscle energetics seen at higher intensities are not due to mitochondrial dysfunction in the GK rat.

Mitochondrial Quality Control Regulation by p53 During Disuse‐Induced Atrophy

Chronic muscle disuse, as observed in conditions of aging, prolonged bedrest, or limb immobilization, contributes to progressive atrophy of the affected skeletal muscle, and a reduction in mitochondrial content. These alterations in muscle mass and aerobic potential have broader consequences on mobility, strength and overall metabolic health. At the molecular level, chronic disuse results in increased intracellular reactive oxygen species (ROS) levels and elevated protein degradation. p53 is a well‐known transcriptional regulator that responds rapidly to perturbations in the cellular environment to activate various pathways in order to reestablish homeostasis. Previous work has illustrated a role for p53 in skeletal muscle mitochondrial maintenance as well as a contribution to muscle atrophy following chronic disuse. Our investigation focused on the necessity of p53 in mediating mitochondrial quality control (MQC) and muscle atrophy following 3 and 7 days of denervation‐induced muscle disuse in both wildtype (WT) and p53 muscle‐specific knockout (mKO) mice. Tibialis anterior (TA) mass was significantly reduced by 16% following 7 days of denervation in WT animals, however this was attenuated to only a 7% reduction in the mKO mice. Genotype had no effect on changes in mitochondrial content as both WT and mKO animals exhibited similar reductions (10–13%) in COX Activity by 7 days of denervation. Despite similar changes in mitochondrial content, there were divergent responses in mitochondrial function, assessed via O‐2 consumption and ROS emission using high‐resolution respirometry. Denervation resulted in impaired respiration in both genetic backgrounds, however we observed a trend for further decrements in the mKO mice as well as concomitant blunting of ROS generation following 7 days. To examine the regulation of MQC, we measured nuclear and mitochondrial localization of the transcriptional regulator PGC‐1α, as well as markers of autophagy/mitophagy, lysosomal biogenesis, the mitochondrial unfolded protein response (UPRmt) and apoptosis. We observed no effect of 3 days of denervation in any of the MQC markers. Following 7 days, nuclear PGC‐1α protein expression was elevated nearly 2‐fold in the denervated muscle of mKO mice, whereas WT animals displayed no change. Conversely, mitochondrial PGC‐1α protein expression was reduced almost 50% in WT denervated muscle but was maintained in the mKO tissue. Moreover, chronic disuse resulted in 3–6‐fold increases in the autophagy/mitophagy proteins p62, LC3‐II, PINK1 and Parkin in isolated mitochondrial fractions from WT mice, but mKO animals showed a diminished induction of these markers. In contrast, lysosomal proteins Lamp1 and Cathepsin D were induced by 40–50% in mKO muscle compared to WT animals. Similarly, UPRmt proteins mtHSP70, LonP, and ATF5 were elevated 15–30% higher in the denervated muscle of mKO animals, while the apoptotic marker BAX and BID were attenuated relative to WT counterparts. Together, these results suggest a dysregulation of MQC in the absence of p53 within skeletal muscle subjected to denervation, and indicate a potential role of p53 in contributing to organelle maintenance during muscle atrophy.Support or Funding InformationWork supported by NSERC, Canada.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Mitochondrial Fission Improves Mitochondrial Respiratory Capacity in Primary Myotubes Derived from Obese Humans

Obesity induces mitochondrial fragmentation in skeletal muscle causing reduced mitochondrial respiratory capacity, which has been linked with the development of insulin resistance and type 2 diabetes. Recent evidence suggests that excessive mitochondrial fission is the culprit behind the mitochondrial fragmentation in skeletal muscle from obese humans. The purpose of this study was to determine if inhibiting mitochondrial fission in human skeletal muscle cells derived from severely obese individuals reduces mitochondrial fragmentation and improves mitochondrial respiratory capacity. Human skeletal muscle cells were isolated from muscle biopsies obtained from severely obese, insulin‐resistant subjects (n=6, BMI = 47.3 ± 2.8 kg/m2, HOMA‐IR = 3.4 ± 0.4), pooled and differentiated into myotubes. On day 6 of differentiation, myotubes were treated with either mitochondrial fission inhibitor Mdivi‐1 (20μM) or DMSO for 12 hrs. Mitochondrial respiratory capacity was measured using Seahorse XFp Analyzer. Live cells were stained with MitoTracker, and mitochondrial morphology was examined by confocal microscopy. Mitochondrial fission, fusion, and OXPHOS protein expressions were assessed by immunoblot. All experiments were repeated on three different occasions. Preliminary data indicate that mitochondrial fission protein dynamin related protein (Drp1) was reduced (20.9%, P = 0.093), with no changes in mitochondrial content or mitochondrial fusion proteins. Inhibiting mitochondrial fission resulted in a reduction of mitochondrial networks (9.7%) while increasing mitochondrial branch length (14.7%). Reserved mitochondrial respiratory capacity, as measured by the mitochondrial spare capacity, was significantly increased (31.4%, P &lt; 0.05) with Mdivi‐1 treatment. These results demonstrate that inhibition of mitochondrial fission improves mitochondrial morphology and respiratory capacity in skeletal muscle from obese humans, suggesting mitochondrial fission may be a novel therapeutic target for treating metabolic disorders that are typically linked to mitochondrial dysfunction.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal Muscle Mitochondrial Function in Goto‐Kakizaki Rat Model of Type 2 Diabetes

Type 2 diabetes (T2D) presents with significant comorbidities and reported exercise intolerance affecting over 30 million people in the US alone. Aerobic metabolism is the predominant pathway for fueling exercise and utilizing glucose, therefore mitochondrial oxidative phosphorylation (MOP) is important in diabetes and the future of diabetic interventions. However, studies in T2D have conflicting reports regarding the functional status of the mitochondrial pool suggesting deficits are an inherent cause of the disease but a comprehensive study has yet to be done. The Goto‐Kakizaki (GK) rat model of T2D was used to measure both mitochondrial function and density in vitro and in vivo over a broad range of metabolic challenges. Mitochondrial densities were similar in red (RG) and white gastrocnemius (WG) muscles of the GK rat (Cyt c oxidase, RG: 22.2 ± 1.6 versus 23.3 ± 1.7 μmol/min/g, WG: 10.8 ± 1.1 versus 12.1 ± 0.9 μmol/min/g; n=7–8). Mitochondria isolated from the muscles of GK rats and their Wistar controls showed no difference in mitochondrial respiration in vitro as measured by high resolution respirometry. Mitochondrial function in vivo, measured by 31Phosphorus magnetic resonance spectroscopy, was similar between GK and Wistar controls determined from phosphocreatine dynamics reaching similar exercise steady states at submaximal workloads with no difference in recovery time constant (72 ± 6 s GK vs. 71 ± 2 s Wistar). However, at exercise intensities beyond what can be sustained aerobically, the GK rat exhibited bioenergetic deficits suggesting something other than mitochondrial function is responsible at these workloads. The current work demonstrates normal mitochondrial function in a non‐obese non‐sedentary rat model of T2D in vitro and in vivo and no change in mitochondrial content. Taken together these results show when considering both density and function of mitochondria no deficit in MOP is present in the GK T2D rat model and may not be in T2D patients but these studies remain to be done.Support or Funding InformationNIH DK 095210, NIH R00HL121160, CIHR R5521A02This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Importance of TFEB and TFE3 in Mediating Lysosomal and Mitochondrial Adaptations

Autophagy is the process through which damaged cellular components are degraded by the lysosome, and it is essential for the overall maintenance of the cell. Mitochondria are recycled through a selective form of autophagy, known as mitophagy, in which dysfunctional mitochondria are poly‐ubiquitinated, engulfed in an autophagosomal membrane and transported to the lysosome for degradation. Chronic contractile activity (CCA) alters mitophagy flux in muscle, and this coincides with the activation of lysosomal biogenesis, which precedes changes in mitochondrial content. Thus, it is proposed that the clearance of dysfunctional mitochondria through the lysosomes may play an important role in mediating mitochondrial adaptations to CCA. We sought to understand the transcriptional regulation of lysosomal biogenesis brought about by CCA, by investigating the transcriptional regulators TFEB and TFE3, and their relationship to mitochondrial content. TFEB and TFE3 were silenced using siRNA in C2C12 myotubes, and subjected to 3 days of stimulation (3hr/day) to induce CA. Following a single bout, TFEB and TFE3 were elevated in both the nuclear and cytosolic fractions, which coincided with increases in Cathepsin D, preceding the increases in VDAC seen following 3 days of stimulation. The loss of TFEB or TFE3 individually had no impact on lysosomal adaptations, since LAMP1 and Cathepsin D remained responsive to CCA, demonstrating redundancy in role of TFEB and TFE3 in mediating lysosomal biogenesis. However, in the absence of both transcription factors, CCA‐induced lysosomal adaptations were reduced, illustrating their collective importance in mediating lysosomal biogenesis. Interestingly, the absence of TFEB had no effect on mitochondrial adaptations, measured by COX I and IV protein content, however this adaptation to CCA was blunted in the absence of TFE3. These results suggest that while TFEB and TFE3 may exhibit compensatory roles in mediating lysosomal biogenesis, TFE3 alone appears to be important for the mitochondrial response to CCA. Since, lysosomal adaptations proceed in the absence of TFE3, these data suggest a more direct role of TFE3 in the regulation of mitochondria in response to chronic contractile activity.Support or Funding InformationANO is funded by an Ontario Graduate Scholarship (OGS). JMM is funded by Natural Science and Engineering Research (NSERC) scholarship. DAH is the holder of a Canada Research Chair in Cell Physiology with funding from NSERC and CIHR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.