Skeletal muscle dysfunction is associated with derangements in mitochondrial bioenergetics (but not UCP3) in a rodent model of sepsis

Muscle dysfunction is a common feature of severe sepsis and multi-organ failure. Recent evidence implicates bioenergetic dysfunction and oxidative damage as important underlying pathophysiological mechanisms. Increased abundance of uncoupling protein-3 (UCP-3) in sepsis suggests increased mitochondrial proton leak, which may reduce mitochondrial coupling efficiency but limit ROS production. Using a murine model, we examined metabolic, cardiovascular and skeletal muscle contractile changes following induction of peritoneal sepsis in wild-type and Ucp3(-/-) mice. Mitochondrial membrane potential (Δψm) was measured using two-photon microscopy in living diaphragm, and contractile function was measured in diaphragm muscle strips. The kinetic relationship between membrane potential and oxygen consumption was determined using a modular kinetic approach in isolated mitochondria. Sepsis was associated with significant whole body metabolic suppression, hypothermia and cardiovascular dysfunction. Maximal force generation was reduced and fatigue accelerated in ex vivo diaphragm muscle strips from septic mice. Mitochondrial membrane potential was lower in the isolated diaphragm from septic mice despite normal substrate oxidation kinetics and proton leak in skeletal muscle mitochondria. Even though wild-type mice exhibited an absolute 26 ± 6% higher UCP-3 protein abundance at 24 hours, no differences were seen in whole animal or diaphragm physiology, nor in survival rates, between wild-type and Ucp3(-/-) mice. In conclusion, this murine sepsis model shows a hypometabolic phenotype with evidence of significant cardiovascular and muscle dysfunction. This was associated with lower Δψm and alterations in mitochondrial ATP turnover and phosphorylation pathway. However, UCP-3 does not play an important functional role, despite its upregulation

MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology

While the analysis of mitochondrial morphology has emerged as a key tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a challenging task and bottleneck for statistically robust conclusions. Here, we present Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet in a toolbox for Windows and Linux operating systems that combines segmentation with morphological analysis

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Summary: Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. : Maffioletti et al. generate human 3D artificial skeletal muscles from healthy donors and patient-specific pluripotent stem cells. These human artificial muscles accurately model severe genetic muscle diseases. They can be engineered to include other cell types present in skeletal muscle, such as vascular cells and motor neurons. Keywords: skeletal muscle, pluripotent stem cells, iPS cells, myogenic differentiation, tissue engineering, disease modeling, muscular dystrophy, organoid

Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca(2+) uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca(2+)-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca(2+)-induced membrane depolarisation and Ca(2+) retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature

Constitutive activation of the PI3K-Akt-mTORC1 pathway sustains the m.3243 A > G mtDNA mutation

Mutations of the mitochondrial genome (mtDNA) cause a range of profoundly debilitating clinical conditions for which treatment options are very limited. Most mtDNA diseases show heteroplasmy – tissues express both wild-type and mutant mtDNA. While the level of heteroplasmy broadly correlates with disease severity, the relationships between specific mtDNA mutations, heteroplasmy, disease phenotype and severity are poorly understood. We have carried out extensive bioenergetic, metabolomic and RNAseq studies on heteroplasmic patient-derived cells carrying the most prevalent disease related mtDNA mutation, the m.3243 A > G. These studies reveal that the mutation promotes changes in metabolites which are associated with the upregulation of the PI3K-Akt-mTORC1 axis in patient-derived cells and tissues. Remarkably, pharmacological inhibition of PI3K, Akt, or mTORC1 reduced mtDNA mutant load and partially rescued cellular bioenergetic function. The PI3K-Akt-mTORC1 axis thus represents a potential therapeutic target that may benefit people suffering from the consequences of the m.3243 A > G mutation

Mechanism of neurodegeneration of neurons with mitochondrial DNA mutations

Mutations of mitochondrial DNA are associated with a wide spectrum of disorders, primarily affecting the central nervous system and muscle function. The specific consequences of mitochondrial DNA mutations for neuronal pathophysiology are not understood. In order to explore the impact of mitochondrial mutations on neuronal biochemistry and physiology, we have used fluorescence imaging techniques to examine changes in mitochondrial function in neurons differentiated from mouse embryonic stem-cell cybrids containing mitochondrial DNA polymorphic variants or mutations. Surprisingly, in neurons carrying a severe mutation in respiratory complex I (<10% residual complex I activity) the mitochondrial membrane potential was significantly increased, but collapsed in response to oligomycin, suggesting that the mitochondrial membrane potential was maintained by the F1Fo ATPase operating in ‘reverse’ mode. In cells with a mutation in complex IV causing ∼40% residual complex IV activity, the mitochondrial membrane potential was not significantly different from controls. The rate of generation of mitochondrial reactive oxygen species, measured using hydroethidium and signals from the mitochondrially targeted hydroethidine, was increased in neurons with both the complex I and complex IV mutations. Glutathione was depleted, suggesting significant oxidative stress in neurons with a complex I deficiency, but not in those with a complex IV defect. In the neurons with complex I deficiency but not the complex IV defect, neuronal death was increased and was attenuated by reactive oxygen species scavengers. Thus, in neurons with a severe mutation of complex I, the maintenance of a high potential by F1Fo ATPase activity combined with an impaired respiratory chain causes oxidative stress which promotes cell death

In vivo imaging of flavoprotein fluorescence during hypoxia reveals the importance of direct arterial oxygen supply to cerebral cortex tissue

Live imaging of mitochondrial function is crucial to understand the important role played by these organelles in a wide range of diseases. The mitochondrial redox potential is a particularly informative measure of mitochondrial function, and can be monitored using the endogenous green fluorescence of oxidized mitochondrial flavoproteins. Here, we have observed flavoprotein fluorescence in the exposed murine cerebral cortex in vivo using confocal imaging; the mitochondrial origin of the signal was confirmed using agents known to manipulate mitochondrial redox potential. The effects of cerebral oxygenation on flavoprotein fluorescence were determined by manipulating the inspired oxygen concentration. We report that flavoprotein fluorescence is sensitive to reductions in cortical oxygenation, such that reductions in inspired oxygen resulted in loss of flavoprotein fluorescence with the exception of a preserved ‘halo’ of signal in periarterial regions. The findings are consistent with reports that arteries play an important role in supplying oxygen directly to tissue in the cerebral cortex, maintaining mitochondrial function

From the cell membrane to the nucleus: unearthing transport mechanisms for Dynein

Mutations in the motor protein cytoplasmic dynein have been found to cause Charcot-Marie-Tooth disease, spinal muscular atrophy, and severe intellectual disabilities in humans. In mouse models, neurodegeneration is observed. We sought to develop a novel model which could incorporate the effects of mutations on distance travelled and velocity. A mechanical model for the dynein mediated transport of endosomes is derived from first principles and solved numerically. The effects of variations in model parameter values are analysed to find those that have a significant impact on velocity and distance travelled. The model successfully describes the processivity of dynein and matches qualitatively the velocity profiles observed in experiments

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe