Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions.

The mitochondrial permeability transition (mPT) describes a Ca-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP).

Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.

Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11.

Interleukin-33 regulates metabolic reprogramming of the retinal pigment epithelium in response to immune stressors.

It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE.

Hexokinase II dissociation alone cannot account for changes in heart mitochondrial function, morphology and sensitivity to permeability transition pore opening following ischemia.

We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria.

The role of succinate and ROS in reperfusion injury - A critical appraisal.

We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation.

Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning.

Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function.

Mechanism of antineoplastic activity of lonidamine.

Lonidamine (LND) was initially introduced as an antispermatogenic agent. It was later found to have anticancer activity sensitizing tumors to chemo-, radio-, and photodynamic-therapy and hyperthermia. Although the mechanism of action remained unclear, LND treatment has been known to target metabolic pathways in cancer cells.

Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity.

Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca(2+)], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP.

The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters.

Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). In the present study, we test these possibilities directly.

Differences in the profile of protection afforded by TRO40303 and mild hypothermia in models of cardiac ischemia/reperfusion injury.

The mode of protection against cardiac reperfusion injury by mild hypothermia and TRO40303 was investigated in various experimental models and compared to MitoQ in vitro. In isolated cardiomyocytes subjected to hypoxia/reoxygenation, TRO40303, MitoQ and mild hypothermia delayed mPTP opening, inhibited generation of mitochondrial superoxide anions at reoxygenation and improved cell survival. Mild hypothermia, but not MitoQ and TRO40303, provided protection in a metabolic starvation model in H9c2 cells and preserved respiratory function in isolated rat heart mitochondria submitted to anoxia/reoxygenation.

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity.

The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4.

The 'mitoflash' probe cpYFP does not respond to superoxide.

Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial. There have been a number of studies suggesting that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of 3-day old correlated inversely with lifespan.

Switching back to normal diet following high-fat diet feeding reduces cardiac vulnerability to ischaemia and reperfusion injury.

Background: We have recently shown that hearts of mice fed high-fat diet exhibit increased vulnerability to ischaemia and reperfusion (I/R) in parallel to changes in catalase protein expression, mitochondrial morphology and intracellular diastolic Ca(2+).

Aims: To determine whether switching from high-fat back to normal diet alters vulnerability to I/R and to investigate cardiac cellular remodelling in relation to the mechanism(s) underlying I/R injury.

Methods And Results: Male C57BL/6J mice were fed a high-fat diet for 19-22 weeks; after which a subset of mice was switched back to normal diet for 4-6 weeks.

The role of hexokinase in cardioprotection - mechanism and potential for translation.

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion.

The mitochondrial permeability transition: a current perspective on its identity and role in ischaemia/reperfusion injury.

The mitochondrial permeability transition pore (MPTP) is a non-specific pore that opens in the inner mitochondrial membrane (IMM) when matrix [Ca(2+)] is high, especially when accompanied by oxidative stress, high [Pi] and adenine nucleotide depletion. Such conditions occur during ischaemia and subsequent reperfusion, when MPTP opening is known to occur and cause irreversible damage to the heart. Matrix cyclophilin D facilitates MPTP opening and is the target of its inhibition by cyclosporin A that is cardioprotective.

Hearts from mice fed a non-obesogenic high-fat diet exhibit changes in their oxidative state, calcium and mitochondria in parallel with increased susceptibility to reperfusion injury.

Rationale: High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown.

Objectives: To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury.

Clinically-relevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion.

Background: Consecutive treatment of normal heart with a high dose of isoproterenol and adenosine (Iso/Ade treatment), confers strong protection against ischaemia/reperfusion injury. In preparation for translation of this cardioprotective strategy into clinical practice during heart surgery, we further optimised conditions for this intervention using a clinically-relevant dose of Iso and determined its cardioprotective efficacy in hearts isolated from a model of surgically-induced heart failure.

Methods: Isolated Langendorff-perfused rat hearts were treated sequentially with 5 nM Iso and 30 μM Ade followed by different durations of washout prior to 30 min global ischaemia and 2 hrs reperfusion.

Monocarboxylic acid transport.

Monocarboxylates such as lactate, pyruvate, and the ketone bodies play major roles in metabolism and must be transported across both the plasma membrane and mitochondrial inner membrane. A family of five proton-linked MonoCarboxylate Transporters (MCTs) is involved in the former and the mitochondrial pyruvate carrier (MPC) mediates the latter. In the intestine and kidney, two Sodium-coupled MonoCarboxylate Transporters (SMCTs) provide active transport of monocarboxylates across the apical membrane of the epithelial cells with MCTs on the basolateral membrane transporting the accumulated monocarboxylate into the blood.

The neuroplastin adhesion molecules are accessory proteins that chaperone the monocarboxylate transporter MCT2 to the neuronal cell surface.

Background: The neuroplastins np65 and np55 are two synapse-enriched immunoglobulin (Ig) superfamily adhesion molecules that contain 3 and 2 Ig domains respectively. Np65 is implicated in long term, activity dependent synaptic plasticity, including LTP. Np65 regulates the surface expression of GluR1 receptor subunits and the localisation of GABA(A) receptor subtypes in hippocampal neurones.

The SLC16 gene family - structure, role and regulation in health and disease.

The SLC16 gene family has fourteen members. Four (SLC16A1, SLC16A3, SLC16A7, and SLC16A8) encode monocarboxylate transporters (MCT1, MCT4, MCT2, and MCT3, respectively) catalysing the proton-linked transport of monocarboxylates such as l-lactate, pyruvate and ketone bodies across the plasma membrane. SLC16A2 encodes a high affinity thyroid hormone transporter (MCT8) and SLC16A10 an aromatic amino acid transporter (TAT1).

Hexokinase II and reperfusion injury: TAT-HK2 peptide impairs vascular function in Langendorff-perfused rat hearts.

Rationale: Mitochondrial-bound hexokinase II (HK2) was recently proposed to play a crucial role in the normal functioning of the beating heart and to be necessary to maintain mitochondrial membrane potential. However, our own studies confirmed that mitochondria from ischemic rat hearts were HK2-depleted, yet showed no indication of depolarization and responded normally to ADP.

Objective: To establish whether the human TAT-HK2 peptide used to dissociate mitochondrial-bound HKII in the Langendorff-perfused heart may exert its effects indirectly by impairing coronary function.

Mitochondrial 'flashes': a radical concept repHined.

Mitochondrial free radicals and redox poise are central to metabolism and cell fate. Their measurement in living cells remains a major challenge and their in vivo dynamics are poorly understood. Reports of 'superoxide flashes' in single mitochondria have therefore been perceived as a major breakthrough: single mitochondria expressing the genetically encoded sensor circularly permuted yellow fluorescent protein (cpYFP) display spontaneous flashes of fluorescence that are responsive to metabolic changes and stressors.