Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca ²⁺) dysregulation followed by rapid ...loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the F O of the F ₁F O ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca ²⁺ enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F ₁, providing a mechanism for mPTP opening. In contrast, recombinant F ₁ beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca ²⁺-induced IMM depolarization and inhibits Ca ²⁺ and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.
Cyclophilin D (CyPD) is an important mitochondrial chaperone protein whose mechanism of action remains a mystery. It is well known for regulating mitochondrial function and coupling of the electron ...transport chain and ATP synthesis by controlling the mitochondrial permeability transition pore (PTP), but more recent evidence suggests that it may regulate electron transport chain activity. Given its identification as a peptidyl-prolyl,
-
isomerase (PPIase), CyPD, is thought to be involved in mitochondrial protein folding, but very few reports demonstrate the presence of this activity. By contrast, CyPD may also perform a scaffolding function, as it binds to a number of important proteins in the mitochondrial matrix and inner mitochondrial membrane. From a clinical perspective, inhibiting CyPD to inhibit PTP opening protects against ischemia⁻reperfusion injury, making modulation of CyPD activity a potentially important therapeutic goal, but the lack of knowledge about the mechanisms of CyPD's actions remains problematic for such therapies. Thus, the important yet enigmatic nature of CyPD somehow makes it a master regulator, yet a troublemaker, for mitochondrial function.
C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by ...under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.
Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca
2+
exchange/extrusion. The energy needs of these events are met in ...large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F
1
F
O
ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.
Coronary artery disease (CAD) is a leading cause of death worldwide and frequently associated with mitochondrial dysfunction. Detailed understanding of abnormalities in mitochondrial function that ...occur in patients with CAD is lacking. We evaluated mitochondrial damage, energy production, and mitochondrial complex activity in human non-CAD and CAD hearts. Fresh and frozen human heart tissue was used. Cell lysate or mitochondria were isolated using standard techniques. Mitochondrial DNA (
DNA), NAD + and ATP levels, and mitochondrial oxidative phosphorylation capacity were evaluated. Proteins critical to the regulation of mitochondrial metabolism and function were also evaluated in tissue lysates. PCR analysis revealed an increase in
DNA lesions and the frequency of mitochondrial common deletion, both established markers for impaired mitochondrial integrity in CAD compared to non-CAD patient samples. NAD
and ATP levels were significantly decreased in CAD subjects compared to Non-CAD (NAD
fold change: non-CAD 1.00 ± 0.17 vs. CAD 0.32 ± 0.12* and ATP fold change: non-CAD 1.00 ± 0.294 vs. CAD 0.01 ± 0.001*; N = 15, P < 0.005). We observed decreased respiration control index in CAD tissue and decreased activity of complexes I, II, and III. Expression of ETC complex subunits and respirasome formation were increased; however, elevations in the de-active form of complex I were observed in CAD. We observed a corresponding increase in glycolytic flux, indicated by a rise in pyruvate kinase and lactate dehydrogenase activity, indicating a compensatory increase in glycolysis for cellular energetics. Together, these results indicate a shift in mitochondrial metabolism from oxidative phosphorylation to glycolysis in human hearts subjects with CAD.
Hypoxic ischemic encephalopathy (HIE) is associated with acute kidney injury (AKI) in neonates with birth asphyxia. This study aimed to utilize urinary biomarkers to characterize AKI in an ...established neonatal rat model of HIE. Day 7 Sprague–Dawley rat pups underwent HIE using the Rice–Vannucci model (unilateral carotid ligation followed by 120 mins of 8% oxygen). Controls included no surgery and sham surgery. Weights and urine for biomarkers (NGAL, osteopontin, KIM‐1, albumin) were collected the day prior, daily for 3 days post‐intervention, and at sacrifice day 14. Kidneys and brains were processed for histology. HIE pups displayed histological evidence of kidney injury including damage to the proximal tubules, consistent with resolving acute tubular necrosis, and had significantly elevated urinary levels of NGAL and albumin compared to sham or controls 1‐day post‐insult that elevated for 3 days. KIM‐1 significantly increased for 2 days post‐HIE. HIE did not significantly alter osteopontin levels. Seven days post‐start of experiment, controls were 81.2% above starting weight compared to 52.1% in HIE pups. NGAL and albumin levels inversely correlated with body weight following HIE injury. The AKI produced by the Rice–Vannucci HIE model is detectable by urinary biomarkers, which can be used for future studies of treatments to reduce kidney injury.
Although animal models of brain injury resulting from HIE are well established, the kidney injury that occurs is not well studied. This study aims to characterize AKI in an established rat model of HIE and track injury progression using sensitive urinary biomarkers. This provides an experimental approach utilizable for testing effectiveness of therapies that treat AKI.
Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, ...we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•Dantrolene and ryanodine block mitochondrial Ca2+ uptake in striated neurons.•Ryanodine receptor (RyR) is expressed in the inner mitochondrial membrane in neurons.•Brain mitochondria bind ...3Hryanodine both in Ca2+- and caffeine-sensitive manner.•Mitochondrial RyR takes part in the mitochondrial Ca2+ influx mechanism in neurons.
Mitochondrial Ca2+ controls numerous cell functions, such as energy metabolism, reactive oxygen species generation, spatiotemporal dynamics of Ca2+ signaling, cell growth and death in various cell types including neurons. Mitochondrial Ca2+ accumulation is mainly mediated by the mitochondrial Ca2+ uniporter (MCU), but recent reports also indicate that mitochondrial Ca2+-influx mechanisms are regulated not only by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor (RyR), which is a one of the main Ca2+-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) in excitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca2+ uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as a Ca2+-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyR and, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we show that neuronal mitochondria express RyR at IMM and accumulate Ca2+ through this channel in response to cytosolic Ca2+ elevation, which is similar to what we observed in another excitable cell-type, cardiomyocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca2+ uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrial Ca2+ uptake mechanism in response to the elevation of Ca2+c in neurons, suggesting that this channel may play a critical role in mitochondrial Ca2+-mediated functions such as energy metabolism.
Ca2+ channels that underlie mitochondrial Ca2+ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that ...mitochondrial Ca2+ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca2+ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca2+ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca2+ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca2+ channels.
Heart mitochondria utilize multiple Ca2+ transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca2+ uptake pathway across the inner membrane to control “excitation ...and metabolism coupling.” In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ∼100, ∼225, ∼700, and ∼1,000 picosiemens (pS) in symmetrical 150 mm CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels.
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