Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection

Background - Transient (low-conductance) opening of the mitochondrial permeability transition pore (mPTP) may limit mitochondrial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. We hypothesize that transient mPTP opening and ROS mediate the protection associated with myocardial preconditioning and mitochondrial uncoupling.Methods and Results - Isolated perfused rat hearts were subjected to 35 minutes of ischemia/ 120 minutes of reperfusion, and the infarct-risk-volume ratio was determined by tetrazolium staining. Inhibiting mPTP opening during the preconditioning phase with cyclosporine-A (CsA, 0.2 mumol/L) or sanglifehrin-A (SfA, 1.0 mumol/L) abolished the protection associated with ischemic preconditioning (IPC) ( 20.2 +/- 3.6% versus 45.9 +/- 2.5% with CsA, 49.0 +/- 7.1% with SfA; P < 0.001); and pharmacological preconditioning with diazoxide (Dzx, 30 mu mol/L) (22.1 +/- 2.7% versus 46.3 +/- 3.0% with CsA, 48.4 +/- 5.5% with SfA; P < 0.001), CCPA ( the adenosine A1-receptor agonist, 200 nmol/L) (24.9 +/- 4.5% versus 54.4 +/- 6.6% with CsA, 42.6 +/- 9.0% with SfA; P < 0.001), or 2,4-dinitrophenol (DNP, the mitochondrial uncoupler, 50 mu mol/L) (15.7 +/- 2.7% versus 40.8 +/- 5.5% with CsA, 34.3 +/- 3.1% with SfA; P < 0.001), suggesting that mPTP opening during the preconditioning phase is required to mediate protection in these settings. Inhibiting ROS during the preconditioning protocols with N-mercaptopropionylglycine (MPG, 1 mmol/L) also abolished the protection associated with IPC (20.2 +/- 3.6% versus 47.1 +/- 3.8% with MPG; P < 0.001), diazoxide (22.1 +/- 2.7% versus 56.3 +/- 3.8% with MPG; P < 0.001), and DNP (15.7 +/- 2.7% versus 50.7 +/- 6.6% with MPG; P < 0.001) but not CCPA (24.9 +/- 4.5% versus 26.5 +/- 8.4% with MPG; P = NS). Further experiments in adult rat myocytes demonstrated that diazoxide induced CsA-sensitive, low-conductance transient mPTP opening (represented by a 28 +/- 3% reduction in mitochondrial calcein fluorescence compared with control; P < 0.01).Conclusions - We report that the protection associated with IPC, diazoxide, and mitochondrial uncoupling requires transient mPTP opening and ROS

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis

Heterogeneity and restricted state selection in FRET with fluorescent proteins

Most fluorescent proteins exhibit multi-exponential fluorescence decays, indicating the presence of a heterogeneous excited state population. In the analysis of FRET to and between fluorescent proteins, it is often convenient to assume that a single interaction pathway is involved. However, in recent work we have shown that this assumption does not hold. Moreover, certain pathways can be highly constrained, leading to the potential misinterpretation of experimental data concerning protein-protein interactions. FRET and single-photon absorption both obey the same global electric dipole selection rules but differ greatly in the mechanism of the acceptor photoselection. In an isotropic medium, single-photon excitation accesses all acceptor transition dipole moment orientations with an equal probability. However, the FRET rate depends on the relative orientation of the donor and acceptor through the κ2 orientation parameter. We show how time- and spectrally- resolved fluorescence intensity and anisotropy decay measurements following direct acceptor excitation, combined with those of the interacting FRET pair, can be used to identify restricted FRET state selection and thus provide accurate measurements of protein-protein interaction dynamics

'Modelling mitochondrial dysfunction in Alzheimer's disease using human induced pluripotent stem cells

Alzheimer’s disease (AD) is the most common form of dementia. To date, only five pharmacological agents have been approved by the Food and Drug Administration for clinical use in AD, all of which target the symptoms of the disease rather than the cause. Increasing our understanding of the underlying pathophysiology of AD will facilitate the development of new therapeutic strategies. Over the years, the major hypotheses of AD etiology have focused on deposition of amyloid beta and mitochondrial dysfunction. In this review we highlight the potential of experimental model systems based on human induced pluripotent stem cells (iPSCs) to provide novel insights into the cellular pathophysiology underlying neurodegeneration in AD. Whilst Down syndrome and familial AD iPSC models faithfully reproduce features of AD such as accumulation of Aβ and tau, oxidative stress and mitochondrial dysfunction, sporadic AD is much more difficult to model in this way due to its complex etiology. Nevertheless, iPSC-based modelling of AD has provided invaluable insights into the underlying pathophysiology of the disease, and has a huge potential for use as a platform for drug discovery

Inhibition of NAADP signalling on reperfusion protects the heart by preventing lethal calcium oscillations via two-pore channel 1 and opening of the mitochondrial permeability transition pore

Aims In the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca2+ oscillations that can cause lethal cell injury. These signals represent attractive cardioprotective targets, but the underlying mechanisms of genesis are ill-defined. Here, we investigated the role of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which is known in several cell types to induce Ca2+ oscillations that initiate from acidic stores such as lysosomes, likely via two-pore channels (TPCs, TPC1 and 2). Methods and results An NAADP antagonist called Ned-K was developed by rational design based on a previously existing scaffold. Ned-K suppressed Ca2+ oscillations and dramatically protected cardiomyocytes from cell death in vitro after ischaemia and reoxygenation, preventing opening of the mitochondrial permeability transition pore. Ned-K profoundly decreased infarct size in mice in vivo. Transgenic mice lacking the endo-lysosomal TPC1 were also protected from injury. Conclusion NAADP signalling plays a major role in reperfusion-induced cell death and represents a potent pathway for protection against reperfusion injury

Protection of cerebral microcirculation, mitochondrial function and electrocortical activity by small-volume resuscitation with terlipressin in a model of haemorrhagic shock

Background During early treatment of haemorrhagic shock, cerebral perfusion pressure can be restored by small-volume resuscitation with vasopressors. Whether this therapy is improved with additional fluid remains unknown. We assessed the value of terlipressin and lactated Ringer’s solution (LR) on the early recovery of the microcirculation, tissue oxygenation, and mitochondrial and electrophysiological function in the rat cerebral cortex. Methods Animals treated with LR replacing three times (3x) the volume bled (n=26), terlipressin (n=27), terlipressin plus LR of 1x (n=26), 2x (n=16), or 3x (n=15) were compared with untreated (n=36) and sham-operated rats (n=17). In vivo confocal microscopy was used to assess cortical capillary perfusion, changes in tissue oxygen concentration, and mitochondrial membrane potential and redox state. Electrophysiological function was assessed by cortical somatosensory evoked potentials (SEPs), spinal cord dorsum potential, and peripheral electromyography. Results Compared with sham, the mean (SD) area of perfused vessels was lower in rats subjected to haemorrhagic shock: 82 (10)% vs. 38 (12)%; P<0.001) and impaired oxygen concentration, mitochondrial redox state (99±4 vs. 59±15 % of baseline; P<0.001), and SEPs (97±13% vs. 27±19% of baseline). Adminstration of terlipressin plus 1X or 2X LR was able to recover these measures, but terlipressin+3LR or 3LR alone were not as effective. Spinal cord dorsum potential was preserved in all groups, but no therapy protected electromyographic function. Conclusion Resuscitation from haemorrhagic shock using terlipressin with small-volume LR was superior to high-volume LR, with regard to cerebral microcirculation, and mitochondrial and electrophysiological function

Inference of evolutionary jumps in large phylogenies using Lévy processes

Although it is now widely accepted that the rate of phenotypic evolution may not necessarily be constant across large phylogenies, the frequency and phylogenetic position of periods of rapid evolution remain unclear. In his highly influential view of evolution, G. G. Simpson supposed that such evolutionary jumps occur when organisms transition into so-called new adaptive zones, for instance after dispersal into a new geographic area, after rapid climatic changes, or following the appearance of an evolutionary novelty. Only recently, large, accurate and well calibrated phylogenies have become available that allow testing this hypothesis directly, yet inferring evolutionary jumps remains computationally very challenging. Here, we develop a computationally highly efficient algorithm to accurately infer the rate and strength of evolutionary jumps as well as their phylogenetic location. Following previous work we model evolutionary jumps as a compound process, but introduce a novel approach to sample jump configurations that does not require matrix inversions and thus naturally scales to large trees. We then make use of this development to infer evolutionary jumps in Anolis lizards and Loriinii parrots where we find strong signal for such jumps at the basis of clades that transitioned into new adaptive zones, just as postulated by Simpson’s hypothesis

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 48h 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