Mitochondria in PET studies
Mitochondrion is a cell organelle in eukaryotes, originally endosymbiotic prokaryotic cell. Mitochondria are present in all cells, except mature red blood cells, but their number, size, and shape is highly variable. The highest mitochondrial content is found in the heart and kidneys, which also are the organs with the highest resting metabolic rates. Mitochondria are dynamic organelles, constantly undergoing fusion and fission, in association with the cytoskeleton and endoplasmic reticulum. Mitophagy is the process of selective removal of dysfunctional mitochondria from cells by autophagy. Mitochondrial homeostasis requires that these processes are in dynamic balance, as required by changing metabolic conditions.
Mitochondrion has two membranes, the outer membrane, and and the inner membrane, which is highly compartmentalized with infoldings (cristae), with surface area several folds larger than the outer membrane. The space within the inner membrane (matrix) contains mitochondrial ribosomes, several copies of mitochondrial DNA, and a highly concentrated mixture of hundreds of enzymes.
The outer mitochondrial membrane resembles cellular plasma membranes, except that the large number of porins allow free diffusion of small molecules and even small proteins across it. Therefore the solute concentrations in the cytoplasm and in mitochondrial intermembrane space (perimitochondrial space) are the same, but protein contents are different; for example cytochrome c is located in the intermembrane space only. Endoplasmic reticulum (ER) associates with large fraction of the mitochondrial outer membrane (mitochondria-associated ER-membrane, MAM); it has a critical role in cellular homeostasis, for example via involvement in Ca2+ signalling; it is also crucial in lipid and lipid intermediate transfer between mitochondria and ER.
The inner mitochondrial membrane has unique phospholipid content, including cardiolipin, that is not found elsewhere. Proteins make up about 75% of the mass of the inner membrane, regulating strictly the transport of solutes between perimitochondrial space and matrix.
The mitochondrial genomes in animal cells are small, less than 1/10 of the size in plants. Human mitochondrial DNA (mtDNA, mDNA) contains only 37 genes, which encode for rRNAs, tRNAs, and 13 proteins. Most of the components of mitochondria are encoded by nuclear DNA.
All mitochondria, and thus also mtDNA, are contributed by the oocyte, not by the sperm (maternal inheritance); mutations in mtDNA are therefore transmitted to the next generation by the mother. It is possible that only part of the mitochondria, or part of the mtDNA inside one mitochondrion, are defective. During cell division, the mitochondria segregate randomly between the two new cells. During embryonic development the cells carrying defective mitochondria may end up in different organs, causing variable symptoms, like in MELAS. Some mutations are seldom inherited but occur spontaneously, such as mtDNA deletions in Kearns-Sayre syndrome.
Progressive accumulation of mutations in mtDNA may contribute to the ageing process. Mitochondrial genome lacks histones and introns. Mitochondria have less effective DNA error checking capability than nuclear DNA. However, mtDNA is well protected against DNA damage by proteins and the multiple copies of mtDNA.
Mitochondrial disorders can also be caused by mutations of the nuclear DNA, in genes that code for mitochondrial components.
Fatty acids are broken down in beta oxidation in the inner mitochondrial membrane to produce acetyl-CoA for the citric acid cycle. Glycolysis in the cytoplasm produces pyruvate, which is oxidized and converted to acetyl-CoA, also in the inner mitochondrial membrane and matrix.
Citric acid cycle (CAC, tricarboxylic acid cycle, Kreb cycle) occurs in the mitochondrial matrix, oxidizing the acetyl in acetyl-CoA to CO2. CAC provides cells with essential precursors for synthesis of amino acids and other molecules, and NADH, FADH2, and succinate for the oxidative phosphorylation pathway.
Oxidative phosphorylation pathway produces ATP. Inner mitochondrial membrane contains the components of electron transport chain, where electrons are transferred in redox reactions from NADH to oxygen, and the energy is used to create an electrochemical gradient across the membrane by pumping H+ (protons) out of the matrix, creating negative charge in the inner side of the membrane. ATP synthase uses the H+ gradient to produce ATP. The solubility of O2 is highest in the centre of lipid bilayer, and the diffusion of oxygen to the binding site of cytochrome c oxidase (COX) at the centre of the inner membrane bilayer is the last step of oxygen transport in the respiratory cycle.
Cells can regulate the number of proton leaking ion channels on the inner membrane. These uncoupling proteins (UCPs) reduce the proton gradient and production of ATP, thus uncoupling the ATP synthesis from substrate oxidation. UCP1 is present in brown adipose tissue, required for its thermogenic function. UCP2 is present in tissues such as kidneys that have high ATP production. Oxidative stress and hyperglycaemia induce expression of UCP2 and UCP3. In cardiomyocytes the expression patterns of UCP2 and UCP3 reflect the metabolic type (Hilse et al., 2018).
Reduction of oxygen in the oxidative phosphorylation pathway involves potentially harmful intermediates, reactive oxygen species (ROS). Under normal conditions about 0.1-0.2% of oxygen consumption in mitochondria results into production of ROS. ROS have also signalling functions, affecting for example vascular tone. Mitochondria have an important role in regulation of inflammation (Meyer et al., 2018) and apoptosis.
Cardiolipin in the inner mitochondrial membrane contains four fatty acids, and is otherwise found only in bacterial cell membranes. Cardiolipin is involved in regulation of electron transport chain assembly and function, ATP synthesis, and apoptosis. Peroxidation of cardiolipin destabilizes electron transport chain, increasing ROS production further. Elamipretide (Bendavia) is a tetrapeptide drug that selectively binds to and stabilizes cardiolipin, reduces ROS production, and prevents apoptosis.
NADPH oxidases are the main producers of ROS in mitochondria. Mitochondrial SOD-1 and SOD-2 scavenges superoxide into oxygen and hydrogen peroxide, but high ROS production can disrupt mitochondrial membranes, leading to decreased activity of SODs.
Mitochondrial outer membrane contains monoamine oxidase (MAO), which oxidizes monoamines, such as dopamine and noradrenaline, and produces hydrogen peroxide in the process.
The inner membrane of mitochondria contain angiotensin II type 2 receptors (components of RAAS), which, when activated, increases mitochondrial membrane potential and ROS production.
64Cu- or 62Cu-labelled Cu-ATSM is used to measure tissue hypoxia, but since its accumulation mechanism is based on the electron rich environment induced by mitochondrial impairment, it can represent the oxidative stress state (Okazawa et al., 2014). It has been used for example in a PET study of ALS patients (Ikawa et al., 2015).
Mitochondrial complex I (MC-I, NADH dehydrogenase, NADH-coenzyme Q oxidoreductase) is the first and largest component in the electron transport chain. Oxidative phosphorylation, and thus MC-I, is usually very active in metabolically active cells, but activated inflammatory cells and cancer cells typically have reduced oxidative phosphorylation, and produce ATP by converting glucose into lactate instead, despite of availability of oxygen (aerobic glycolysis, Warburg effect).
Pyridaben is an insecticide which competes for MC-1 binding with ubiquinone. [18F]Flurpiridaz (BMS-747158-02, [18F]BMS-747158-01, [18F]BMS) is a structural analogue of pyridaben. In the heart oxidative phosphorylation is very active and the density of mitochondria is high; the uptake of [18F]Flurpiridaz is nearly irreversible, and limited by blood flow, in the myocardium. Therefore, this tracer has been used to measure myocardial perfusion. However, in other organs, the uptake of [18F]Flurpiridaz may represent the activity (or concentration) of MC-I. Mitochondrial dysfunction in the mouse model of NASH can be seen as reduced [18F]Flurpiridaz uptake in the liver (Rokugawa et al., 2017). Neuronal damage after ischemia can be observed with this tracer, although relatively high nonspecific uptake in the brain has led the researchers to develop other analogues (Tsukada et al., 2014).
[18F]-BCPP-EF has shown promise in the brain imaging of stroke, ageing, and dementia (Tsukada et al., 2014a, 2014b, 2014c, 2016; Nishiyama et al., 2015; Fukuta et al., 2016; Mansur et al., 2020), and in early detection of radiotherapy effect (Murayama et al., 2017). [18F]-BCPP-BF may be useful in detection of impaired MC-I activity in liver and kidneys (Ohba et al., 2016; Sakai et al., 2018).
Rotenone is another lipophilic competitive inhibitor of MC-I, used as an organic pesticide. 18F-labeled rotenone derivative, [18F]FDHR, has been developed to be used as myocardial perfusion tracer (Marshall et al., 2004).
Mitochondrial membrane potential (voltage sensors)
The electrical gradient across plasma membrane (membrane potential, ΔΨm) is ∼30-60 mV, negative inside, which drives accumulation of lipophilic cations inside cells. Mitochondrial membrane potential (MMP) is much higher, ∼130-150 mV, and according to Nernst equation, every 60-mV difference results in a 10-fold increase in accumulation of a lipophilic cation. Therefore 90-95% of intracellular lipophilic cations are localized in mitochondria (Murphy & Smith, 2000). Labelled lipophilic cations can be used to assess mitochondrial density and membrane potential.
Tetraphenylphosphonium and triphenylphosphonium (TPP) cations accumulate in mitochondria driven by the negative potential in the matrix (Zielonka et al., 2017). Several 18F-, 11C-, and 64Cu-labelled TPP derivatives have been synthesized, mainly to measure myocardial perfusion (Kim and Min, 2016; Zeng et al., 2016; Tominaga et al., 2016; Kim et al., 2018). Selective dissipation of ΔΨm can lead to 80% decrease in cellular uptake of [18F]FBnTP (Madar et al., 2007). [18F]FBnTP can be used to detect brown adipose tissue due to its high mitochondrial density, and to assess cold-induced reduction in MPP (Madar et al., 2011; Madar et al., 2015).
Rhodamines are another group of lipophilic cations, also accumulating in the mitochondria in proportion to MMP. Several rhodamine derivatives for PET imaging have been synthesized, but the main aim has been to study myocardial perfusion, and not the membrane potential (Bartholomä et al., 2013 and 2015; Gottumukkala et al., 2010; Al Jammaz et al., 2015 and 2019).
MMP is high in healthy cardiomyocytes, but is decreased in ischaemia, which cause bias when MMP radioligands are used as perfusion imaging agents. However, radioligands such as [18F]TPP+ can be used to assess myocardial MMP (Gurm et al., 2012; Kim & Min, 2016; Alpert et al., 2018 and 2020; Pelletier-Galarneau et al., 2021a and 2021b).
MMP radioligands have been used to label mitochondria in order to follow the integration of transplanted mitochondria in living tissue (Cowan et al., 2016).
Cancer cell lines that have high plasma and mitochondrial membrane potential can be detected with these radioligands (Zhou et al., 2011; Momcilovic et al., 2019). However, lipophilic cations generally are substrates for P-glycoprotein, and may therefore not be suitable for imaging the brain, or tumours that have high P-glycoprotein expression.
The translocator protein 18kDa (TSPO), earlier called peripheral benzodiazepine receptor (PBR) and mitochondrial benzodiazepine receptor, is mainly situated in the outer mitochondrial membrane, but also in other cell organelles. Secretory and glandular tissues, such as adrenal glands, pineal gland, salivary glands, and olfactory epithelium contain high levels of TSPO; intermediate levels in renal and myocardial tissues, and low levels in the brain and liver. In the brain parenchyma TSPO is located in glial cells, and has thus been used as a biomarker of activated glial cells. It can also be found in other inflammatory cells. Generally, TSPO imaging might have potential in studying the concentration of viable mitochondria in tissues. For instance, regional uptake of TSPO radioligand [11C]PBR28 was lower in subjects with autism spectrum disorder in brain regions associated with sociocognitive processes (Zürcher et al., 2020). Abnormal brain uptake of [11C]-(R)-PK11195 has been seen in patients with mitochondrial disease (van den Ameele et al., 2021). In rat myocardial ischaemia model, the uptake of TSPO radioligand is markedly reduced, consistent with mitochondrial dysfunction (Luo et al., 2021). Later the macrophage-driven inflammation in infarct region leads to increased uptake of TSPO radioligand (MacAskill et al., 2021).
P2X7 ionotropic purinoceptor is expressed on cell membranes and also on the outer membrane of mitochondria, with its ATP-binding site facing the cytosol, where it can participate in regulation of oxidative phosphorylation.
- Cell death
- Reactive oxygen species (ROS)
- Skeletal muscle
- Brown adipose tissue
- Translocator protein (TSPO)
- Fatty acids
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Updated at: 2021-12-22
Created at: 2017-09-20
Written by: Vesa Oikonen