Giant mitochondria in human liver disease

This thematic review aims to provide an overview of the current state of knowledge about the occurrence of giant mitochondria or megamitochondria in liver parenchymal cells. Their presence and accumulation are considered to be a major pathological hallmark of the health and fate of liver parenchymal cells that leads to overall tissue deterioration and eventually results in organ failure. The first description on giant mitochondria dates back to the 1960s, coinciding with the availability of the first generation of electron microscopes in clinical diagnostic laboratories. Detailed accounts on their ultrastructure have mostly been described in patients suffering from alcoholic liver disease, chronic hepatitis, hepatocellular carcinoma and non‐alcoholic fatty liver disease. Interestingly, from this extensive literature survey, it became apparent that giant mitochondria or megamitochondria present themselves with or without highly organised crystal‐like intramitochondrial inclusions. The origin, formation and potential role of giant mitochondria remain to‐date largely unanswered. Likewise, the biochemical composition of the well‐organised crystal‐like inclusions and their possible impact on mitochondrial function is unclear. Herein, concepts about the possible mechanism of their formation and three‐dimensional architecture will be approached. We will furthermore discuss their importance in diagnostics, including future research outlooks and potential therapeutic interventions to cure liver disease where giant mitochondria are implemented.


| INTRODUC TI ON
The seminal papers by Claude & Fullum in 1945 1 and Palade in 1953 2 provided the first ultrastructural depictions of mitochondria.Those historical observations are overwhelmingly impressive considering the infancy of transmission electron microscopy (TEM) and associated sample preparation. 3Around the same time, and within the same research institute, biochemical and cytological observations on rat liver cell fractions disclosed that the respiratory enzyme succinoxidase is associated with mitochondria. 4This gave way to the first important function allocated to this organelle in cellular respiration as we know today.
Almost two decades after Palade's observations on rat liver mitochondria, 2 Daems and Wisse 5 detailed the ultrastructure of mitochondria in mouse liver hepatocytes at higher TEM resolution (Figure 1A).After a long controversy among cell biologists, 3 the authors confirmed the clear structural relationship between the cristae and the inner mitochondrial membrane by narrow tubules dubbed pediculi cristae.Vastly improved fixation methods together with the meticulously reconstruction of serial sectioning TEM data allowed the authors to put forward a revised three-dimensional (3-D) model of the mitochondrion in hepatocytes that still stands today (Figure 1B; historically reviewed in Ref. 3).
To the best of our knowledge, the first description of giant mitochondria in liver parenchymal cells were reported by Ekholm and   Edlund in 1960 in patients diagnosed with cholestatic liver disease. 8 this TEM study, the authors described enlarged mitochondria that harbour inclusions of a 'crystalloid' nature.The authors put forward that the occurrence of giant mitochondria is the result of differences in the biochemical pathways between normal versus cholestatic livers.
Not long thereafter, Novikoff and Essner 9 reported enlarged mitochondria in liver parenchymal cells of rats injected with conjugated bilirubin.
Besides the loss of mitochondrial cristae, dark 'electron dense' inclusions were readily visible.At present, and notwithstanding 60 years of research, the quest to decipher the ontogeny and function of giant mitochondria remains a focus in cell biology.To date, a strict link between mitochondrial size and function has been extensively demonstrated, as well as the association between structural mitochondrial alterations and the subsequent onset of adverse cellular processes.Therefore, abnormalities such as giant mitochondria or megamitochondria represent an intriguing phenomenon in liver pathology. 10Especially since there is a clear link between alterations in mitochondrial size and the subsequent onset of adverse cellular processes within liver parenchymal cells that eventually lead to liver disease.Their presence is recognised by pathologists as an important structural hallmark to assess liver disease. 11 this thematic review, we will provide a detailed overview on the presence of abnormally large mitochondria in human liver parenchymal cells as they occur in different diseases and with a special focus on their fine structure.The relevant literature spanning over six decades of research will be discussed in-depth and elaborated in the second part of this thematic review with the latest structural observations that were attained through contemporary electron microscopy investigation.

| A SS E SS ING G IANT MITOCHONDRIA A S A S TRUC TUR AL HALLMARK OF LIVER DISE A SE (196 0 -0 0 0)
The first observations on unusually large mitochondria have primarily been reported on diseased human tissue.Upon reflection, this is a noteworthy point as it illustrates how hepatologists were captivated by this intriguing change in organelle shape and volume.
Uncoincidentally, those articles were accomplished by the availability of the first user-friendly electron microscopes in pathology departments.Pathologists immediately recognised the importance of studying tissue abnormalities with techniques that delivered superior resolution relative to light microscopy (i.e.220-nm for light microscopy versus 2-6-nm for electron microscopy). 12,13Moreover, electronic microscopy provided a far more detailed and holistic means to examine liver diseases. 14,15Table 1 provides a concise and chronological overview of the early hallmark papers that appeared between the 1960s and the millennium, documenting the occurrence of giant mitochondria in different human liver diseases, including experimental models.Notably, the earlier work on mitochondrial structural abnormalities did not go unnoticed and readily found its way into scholarly textbooks as early as the 1970s.The illustration by Krstić 6 is a classic textbook example elegantly depicting giant mitochondria and their paracrystalline inclusions within human hepatocytes (Figure 1C).Summarising these 40 years of hepatic giant mitochondria papers resulted in some interesting facts that we wish to note and discuss.
First, giant mitochondria that contain paracrystalline inclusions are observed in a range of liver diseases.While enlarged or giant mitochondria have been induced experimentally, those containing paracrystalline inclusions as seen in various human liver diseases have been challenging to produce in model organisms.This may be attributable to the fact that paracrystalline inclusions formation is a result of long-term exposure to noxious agents.Noteworthily, the study by Shapiro et al. 16 is an exception to this rule, in which giant mitochondria with inclusions occurred after mice were exposed for 19 weeks to the anti-fungal drug griseofulvin.In ALD, CHC and NALD, patients are exposed to long-lasting adverse health conditions, allowing the time for crystal-like structure to form or mature.Alternatively, paracrystalline inclusions formation may be dependent on multi-stage/factorial processes.Irrespective of the factors required for paracrystalline inclusions formation in giant mitochondria, the discrepancies between human and experimental models make it exceptionally challenging to holistically study giant mitochondria biology in laboratory settings.
Second, the biochemical composition of the intramitochondrial paracrystalline inclusions is unclear.Although attempts were made in the early days of giant mitochondria research through optical diffraction electron microscopy imaging 17 and differential histology staining on human liver sections, [18][19][20] the nature of the

Key points
This thematic paper unveils recent insights into giant mitochondria biology and pathobiology in chronic liver disease.
Their presence and accumulation are considered to be a major pathological hallmark of the health and fate of the liver.Herein the authors discuss their importance in diagnostics, including future research outlooks and potential therapeutic interventions for liver disease where giant mitochondria aka enlarged mitochondria are involved.

F I G U R E 1
Early structural and modelling studies on liver parenchymal cell mitochondria in mouse (A, B) 5 and human (C) 6 .(A) TEM image depicts mitochondria delineated by a pair of membranes which are clearly visible except where they were sectioned obliquely (*).Small arrow denotes a free crista which is open at one end and closed at the other.Note, only occasionally is a crista seen to be continuous with the inner membrane.×57 000.highly-ordered and rigid-like structure remains an enigma to date.
The paper by Sternlieb and Berger is the most comprehensive paper detailing the inclusions at the nanometre scale, including specifying their likely chemical composition. 173-D reconstructions via the aid of serial sectioning revealed that they have a typical 8-nm periodic spacing.From a biochemical point of view, the crystal-like structures are most likely not composed of crystalline phospholipid, cytochrome C or C 1 or mitochondrial structural protein complex III.
Those candidate supramolecular lipid or protein complexes were eliminated based on mathematically extrapolating the molecular weight of the different mitochondrial constituents together with the spatial EM data.From a histopathology point of view, the application of Janus Green, haematoxylin-eosin, Masson's trichrome or periodic acid-Schiff staining did not contribute any further insights about the biochemical composition of the inclusions, other than supporting the already known acidophilic nature of mitochondria. 18,21ird, the presence of giant mitochondria in human liver tissue often goes hand-in-hand with other structural hallmarks utilised by the histopathologist to score liver disease progress such as Mallory bodies and/or the presence of an increased number of discrete fat deposits within the liver parenchymal cells.Dedicated reports detailing the simultaneous occurrence of giant mitochondria, loss of mitochondrial cristae, large fat droplets and Mallory bodies within human liver parenchymal cells date back to the earliest reports of giant mitochondria. 22,23AFLP and ALD are diseases in which multiorganelle structural changes simultaneously occur.In particular, the concurrent presence of hyaline-rich Mallory bodies and giant mitochondria are more frequently reported in patients with a strong history of alcohol abuse. 18,21Whether accumulated fat or Mallory Fourth, and for completeness, paracrystalline inclusions in a small proportion of normal-sized mitochondria in healthy liver tissue regions have been reported as well. 24,25It was estimated that about 5%-10% of the total healthy human liver parenchymal cell population have one or more normal-sized mitochondria with paracrystalline inclusions.Their presence may indicate early onset liver disease, representing yet another important aspect for future investigation in healthy humans.
Lastly, and of special note, the terms megamitochondria and giant mitochondria have been used interchangeably throughout the literature.Both describe enlarged mitochondria.However, from its use, it is not clear whether they infer the presence of paracrystalline inclusions, the loss of cristae or both.This makes it all the more difficult to discern whether we are dealing with the same phenomena when exploring the literature.Two typical examples concern work by Stewart et al. 18 detailing 'giant mitochondria' in liver biopsies of both alcoholic and non-alcoholic patients, whereas Zaragozá et al. 26 use the term 'megamitochondria' in rats fed a high protein diet.Towards the end of this communication, we put forward the use of consistent terminology to describe the various forms of mitochondrial size aberrations.

| COLLEC TING G IANT MITOCHONDRIA IN S I G HTS THROUG H MODERN ELEC TRON MI CROSCOPY (2 0 01-TO DATE )
Over the past two decades, structure-function studies on the occurrence of giant mitochondria in liver parenchymal cells have been relatively sparse.The primary correlation was that giant mitochondria were present among a range of hepatic diseases, in addition to other highly metabolic tissues (e.g.neural and cardiac tissue).
Giant mitochondria are regarded as a morphological hallmark that aids in the scoring and staging of liver disease progression 32,33 and has been included in the screening for liver mitochondrial disorders in neonates and infants. 34Indeed, the dramatically enlarged and structurally altered mitochondria can be readily observed using brightfield microscopy (Figure 2). 18While giant mitochondria are regarded as an indicator of pathology, their mechanism of formation and biochemical composition is an outstanding subject of investigation to fundamental scientists.Below we discuss the literature that utilised novel tissue preparation protocols and improved imaging instrumentation-including our recent volume electron microscopy findings-that permitted the collection of new insights on the occurrence and structure of giant mitochondria.
Barbaro et al. 35  In brief, using giant mitochondria experimental models in rodents and weighing those observations against the literature data on human livers bearing enlarged mitochondria, the author put forward that mitochondria exposed to unfavourable conditions try to decrease ROS levels by all means.As such, the formation of megamitochondria is a response to decrease oxygen production.Conversely, if those mitochondria succeed to suppress ROS levels, megamitochondria return to normal, both structurally and functionally.The latter is an interesting point and a worthwhile topic of future investigation as the formation of giant mitochondria may represent an 'adaptive process' as a result of intracellular stress.
Le and co-authors 37 investigated whether there was a possible zonal distribution of giant mitochondria with crystalline inclusions in patients suffering from non-alcoholic steatohepatitis (NASH).
Using carefully chosen comparative light and electron microscopy serial sectioning techniques, permitting large-area tissue investigation across length scales, no clear evidence could be found regarding a preferred zonal tissue distribution of liver parenchymal cells bearing giant mitochondria.They concluded that the presence of those mitochondrial abnormalities occurs rather randomly, which is in accordance with the earlier observations by Stewart et al. 18 In addition, Le et al. 37 also noted within a significant proportion of liver biopsies the presence of ballooned hepatocytes and Mallory bodies.
As aforementioned, giant mitochondria are frequently co-observed with Mallory bodies and excess fat. 18,21,23Direct correlative light and electron microscopy studies may shed light on the possible structural relation between intracytoplasmic fat droplets, hyalin bodies and giant mitochondria. 38Finally, the authors put forward an interesting concept as to why crystalline inclusions are formed.They speculated that the crystal-like inclusions are composed of a ferritinlike compound together with mDNA.This is further supported by the seminal work of Wolf et al. 39 who show that E. coli under oxidative stress form identical structures to safeguard their most important cargo, bacterial DNA.Considering the evolutionary origin of mitochondria, this hypothesis should not be neglected and serves as a basis for future work.
Dedicated sample and analytical preparation methods largely contributed to our insights into giant mitochondria biology such as electron diffraction analysis, 17 directed mechanical trimming for combined large-volume microscopy 37 and more recently precisioncut human liver slices. 40The latter is an elegant example of technique innovation for ultrastructural assessment that enabled Palma et al. 40 to study giant mitochondria ex-vivo under tightly controlled physiological conditions within resected human liver tissue.Experimentally, the authors could induce giant mitochondria possessing crystalline inclusions after the tissue slices were exposed to 250 mM alcohol for 24-72 h thereby mimicking an experimental model for alcohol-induced liver disease.This finding is important as this is to the best of our knowledge the first experimental model to study clinically relevant mitochondrial abnormalities within the complex and intact multi-cellular histoarchitecture of human liver tissue.
Investigation of ultrathin-sectioned tissues through classical TEM imaging remains the first method of choice and is considered the gold standard in fundamental research and diagnostic settings.
This delivers 2-D image data at a typical resolving power of 1-3 nm.
2][43] Our team examined over 200 liver biopsies in patients presenting with different clinical manifestations (e.g.CA, HCC, NAFLD, etc.).Double-blind histopathologic scoring against patient antecedents indicated that subjects suffering from non-alcoholic fatty liver disease markedly resulted in the presence of giant mitochondria. 41,42Often those enlarged mitochondria surpassed the size of the liver parenchymal cell nuclei and changed the typical hexagonal cell boundaries into irregularly shaped hepatocytes. 43Having giant mitochondria as a structural hallmark in NAFLD patients most likely denotes the adverse organ health as a whole, and can be seen as the first structural markers and prelude for later overall liver organ impairment, and hence liver-related mortality.The exact cause of NAFLD is puzzling but underlying conditions such as obesity, metabolic syndrome, type II diabetes and high triglyceride levels manifest themselves in the clinic with insulin resistance as an important driver of disease leading to a chronic low-grade systemic inflammation. 44, while giant mitochondria with crystal-like inclusions are typically observed and well documented in patients with a history of chronic alcohol abuse (See, Table 1 [e.g.ALD, CA]), NAFLD in which excessive amounts of fat builds up in the parenchymal cells is another clinical manifestation that goes hand-in-hand with the presence of giant mitochondria.Indeed, altered mitochondria function is frequently observed in NAFLD patients.Noteworthy, there is some suggestion that these patients may not be completely abstinent of alcohol, which is an inherent challenge when analysing human samples, however, this remains controversial and requires further assessment.
When reviewing the literature, it is clear that there is no single explanation as to why giant mitochondria are observed in various liver   43 in which thousands of giant mitochondria were analysed (see, next section).In addition, during this journey, particular intermediate intramitochondrial changes were noted giving away a possible sequence of events that give rise to giant mitochondria (Figure 4).Essentially, we put forward that the following sequence of events occur: (i) initial loss of cristae arrangement; (ii) cristae on the loose; (iii) cristae meet; (iv) cristae pack; and finally (iv), cristae arrange in a crystallike arrangement.During this process, they most likely take other mitochondrial constituents along (e.g.proteins and/or ions) that eventually result in the typical mitochondrial 'crystal-like' inclusions.
The thematic review paper by Klecker and Westermann 48 indirectly supports this hypothesis for giant mitochondria formation, at least with respect to the initial loss and early rearrangement of cristae.In brief, the altered function of 'mitochondrial contact sites and cristae organising system (MICOS)' at the cristae junctions seem to be pivotal in this dynamic process of reshaping the mitochondrial inner membrane.Recently, Ma et al. 49 demonstrated lower levels of liverspecific dynamin-related protein (DRP1) in human alcoholic hepatitis, including in alcohol-fed mice.In this study, TEM investigation showed the presence of unusually enlarged mitochondria described as megamitochondria, but clear inclusions were absent.It is known that DRP1 is essential in mitochondrial fission in which the corresponding receptors are located at the outer mitochondrial membrane.This finding supports our ultrastructural modelling studies in which we put forward that the tight regulation or balance between fission and fusion are key structural processes in the formation of giant mitochondria. 43As such, decreased levels of DRP1 might result in the initial gross enlargement of mitochondria that eventually gives rise to giant mitochondria.This was further supported by Palma et al. 50where they showed that DRP1 inhibition drove megamitochondria formation, which the authors concluded was an adaptive response in alcohol-induced hepatotoxicity.Taking all this knowledge together, it is clear that more than one pathway is involved and that most likely distinct molecular processes at the inner and outer mitochondrial membrane are simultaneously implicated.
Evidently, caution should be taken when interpretating 2-D static images as they only disclose structural information close to the moment of tissue fixation.As such, it is challenging to reconstruct to a certain degree a 'dynamic (sub)cellular event' based on EM data.
Conversely, although tissues were fixed at a given timepoint, it is reasonable to assume that different structural configurations of a certain mitochondrial configuration can be captured.Conventional TEM remains a gold standard for quantifying and screening mitochondrial disorders. 51Furthermore, our findings are underpinned after studying nearly twenty-five thousand electron micrographs for over more than a decade, which were derived from over two hundred biopsies.Future work could focus on the recent work by Palma and co-authors 40 to induce giant mitochondria with crystal-like inclusions on excised human liver tissue slices by utilising well-defined experimental conditions.As a final comment, it is important to state that the quality of the human tissue samples as witnessed by our conventional 2-D TEM data exceeds the typical quality as obtained through the standard non-perfusion fixation.Fine-tuning the initial sample preparation procedures (i.e.primary fixation step) is the main contributor to the artefact-free preservation of overall liver architecture in human biopsies. 52,53This is underpinned by the fine structural preservation of subcellular matter as assessed by membrane integrity, presence of all cytoplasmic constituents and intact suborganelle detail.Either injection perfusion-fixation of liver wedge biopsies 52 or through jet-fixation for liver needle biopsies 53 present themselves as the gold standards for structural assessment at a wide range of magnifications (i.e. from the light all up to the electron microscopy level).As such, conventional immersion-fixation approaches should be avoided as they rarely result in proper tissue preservation of human biopsies for electron microscopy studies.
Recently, volume electron microscopy has proven highly beneficial in unlocking different cellular processes at the nanometre scale, 54 especially when combined with automated segmentation approaches. 55It is not a coincidence that volume electron microscopy was identified as one of the seven techniques to watch in 2023. 56ecifically, it provides holistic three-dimensional (i.e.3-D) structural information at the highest resolution, often in a semi-automated approach, when combined with serial-block face scanning electron microscopy and transmission electron tomography.In our recent EM studies, we took advantage of the latest technological advances in volume electron microscopy to map liver tissue, 57,58 including giant mitochondria, 43 at the nanoscale and within relatively large volumes (i.e.EM tomography or 3-D EM).As mentioned before, although investigations of ultrathin sections for conventional EM (i.e.2-D EM) is a powerful imaging means, it does lack the disclosure of volumetric cell information.Questions about size, shape, frequency, distribution and types of giant mitochondria per hepatocyte cannot be answered with this approach.Conversely, this can be addressed either through time-consuming serial section imaging or by employing more contemporary and semi-automated 3-D approaches such as serial blockface scanning electron microscopy, focussed ion-beam SEM or array tomography. 59It is the latter 3-D microscopy means that allowed us to build further on our earlier work as presented in Figures 3 and 4.
Applying this 3-D nanoscopic approach on wedge biopsies from NAFLD patients, permitted us to address our outstanding questions and put forward a hypothesis on the structure and possible rise of the different types of giant mitochondria as they occur in NAFLD. 43rthermore, we inferred a loss of normal mitochondrial function,  Previously, we applied transmission electron tomography (TET) to gain a better insight into the organisation of the crystal-like inclusions within giant mitochondria. 43Briefly, the individual rod-like filaments appear to be composed of globular subunits, measuring data using the latest generation of electron detection technology (Figure 6).Future studies, when combining the above-listed instrument advances together with elemental mapping, could shed light on the chemical composition of the inclusions.For example, questions could be answered whether there is an increased presence of calcium, iron or phosphate agglomerates associated with the crystal-like inclusions.Indeed, this 'multicolour EM' approach has been shown to be valuable in mapping elemental information within the nanometre window in biological soft matter such as cells and tissues. 60,61

| CONS IDER ATIONS , CON CLUS IONS AND OUTLOOK S
Although this contribution focussed on giant mitochondria present in liver parenchymal cells in different diseases, their relevance has also been extensively documented in other disease models such as skeletal muscle-related disorders, cardiomyopathies, skin ageing and neurological disease to name a few. 36,62This underpins the importance of gaining further insights regarding the pathobiology of giant mitochondria.Unlocking the mechanism of their formation, mapping their molecular composition and disclosing the mechanisms responsible for their structural changes may eventually lead to the identification of a target for future therapeutic intervention.Preventing their formation at the onset of severe disease or even reverting giant mitochondria to their normal structure and function may lead to better patient outcomes.
It is now generally accepted that the occurrence of giant mitochondria or megamitochondria is a strong histological indicator for adverse liver function, especially in alcohol-related liver diseases and drug-induced liver injury.However, also their presence in other chronic and metabolic hepatic illnesses has been widely documented.Increasingly, mitochondrial structure-function alterations have been an area of intense interest given their significance and prevalence among various hepatic diseases (for recent thematic reviews, see Ref. 63,64).Oxidative stress caused by ROS overproduction seems to be the key molecular driver.
Evidence is mounting that hepatic mitochondrial dysfunction ameliorates lipid accumulation finally resulting in liver inflammation and organ failure.Therefore, reversing the structural organisation and/or molecular micromilieu of mitochondria to their normal physiological status could be considered as an attractive alternative therapeutic approach to combat irreversible liver tissue damage.
Targeted drug strategies towards mitochondria that modulate free radicals and the biochemical pathways that initiate paracrystalline formation represent attractive nanomedicine regimens. 65Interestingly, and more recently, blocking hepatic mitochondrial fusion has been reported as a potential alternative means to prevent giant mitochondria formation and lipid accumulation within an experimental NASH mouse model. 66Noteworthy, the 'mitochondrial contact site and cristae organizing system' (MICOS) probably also forms an important part in the switch from normal-sized function to giant and dysfunctional mitochondria. 48Hence, the quest in unravelling the candidate molecular and structural targets that determines mitochondrial adversity is a worthwhile topic of future investigation and holds a promising hope to cure metabolic and chronic liver diseases.
Reviewing the literature reveals that not all enlarged mitochondria within hepatocytes contain paracrystalline inclusions.It is clear that the words 'giant mitochondria' and 'megamitochondria' are used interchangeably.Although both are utilised to describe enlarged mitochondria compared to normal conditions, the term giant mitochondria are mainly found in the clinical literature whereas cell biology papers tend to prefer megamitochondria as mainstream terminology.Furthermore, the term giant mitochondria are used, in most if not all cases, when 'paracrystalline inclusions' are present within the enlarged mitochondria.Megamitochondria on the other hand is the preferred term to denote abnormally large mitochondria and loss of cristae but without the presence of any crystal-like inclusions.In the latter, they are frequently observed under in vitro and small animal experimentation conditions as a response to toxic agents, and often go hand in hand as part of the mitochondrial death pathways (i.e.mitochondrial swelling followed by mitochondrial injury).To the best of our knowledge, no clear definition or call has been made so far when to use the scientific term giant mitochondria versus megamitochondria.This inconsistency in using both terms interchangeably not only make it difficult to search for the relevant literature but also causes some degree of confusion in the interpretation of the reported phenomena.A straightforward consensus could be to consistently use giant mitochondria or megamitochondria.As such, megamitochondria could be used to describe all phenomena in which enlarged mitochondria are observed but without the presence of crystal-like inclusions, whereas giant mitochondria would then be reserved for enlarged mitochondria with crystal-like inclusions.Of final note and for completeness, Uchida et al. 67   has not yet come to a close.On the contrary.
(B) 3-D Model of part of a mouse liver parenchymal cell mitochondrion, illustrating the shape of cristae after examining ultrathin sections obtained at different angles.The letters on the model indicate cristae mitochondriales (A), pediculus cristae (B), crista with a number of pediculi (C) and (D) indicates the thickness of an ultrathin section drawn to scale.(A and B: Journal of Ultrastructural Research by ELSEVIER.Reproduced with permission of ELSEVIER in the format Journal/Magazine via Copyright Clearance Center).(C) 2-D Schematic drawing by Radivoj Krstić of morphologically distinct structures-that is, 'crystal-like formations'-laying within the mitochondrial matrix of human liver parenchymal cells.This 1979 illustration was inspired by the work of Themann and von Bassewitz in which human liver tissue was examined via TEM. 7Number 1 portrays the parallel crystal-like organisation within the mitochondrion when sectioned longitudinally.Number 2 denotes transverse-cut crystals.Their significance at that time remains obscure.The illustration is drawn to scale: that is, ×37 000.(Reprinted and modified by permission from Springer-Verlag 6 , Copyright 1979).
reported in patients with chronic hepatitis C an increase in hepatic iron concentration.The group put forward that the excess of iron increases lipoperoxidation and activates hepatic glutathione turnover.The resulting reactive oxygen species (ROS) are most likely the cause of the structural changes observed within the mitochondria.Briefly, high-throughput TEM disclosed fusion between mitochondria and the presence of giant mitochondria.Moreover, cristae seem to rearrange into electron-dense long structures within the mitochondrial matrix.The authors concluded that patients should be treated with antioxidants and iron depletion regimes to reverse mitochondrial damage.The involvement of ROS in the formation of megamitochondria has been extensively detailed in the comparative biochemical and TEM studies by Wakabayashi.36 pathologies.Their formation appears to be a multi-step or multi-hit process in which a cascade of biochemical events is involved.It is generally acknowledged that reactive nitrogen species (RNS) and reactive oxygen species (ROS) are likely involved in NAFLD pathogenesis, eventually resulting in abnormalities of mitochondrial function and structure as this disease entity is disturbing cellular metabolism (for recent reviews, see Ref.45-47).The functional implications of giant mitochondria also continue to remain elusive.Future studies should characterise the functionality of giant mitochondria with regard to adenosine triphosphate (ATP) production, the electron transport chain and the mitochondrial permeability transition pore to better elucidate their impact on cellular physiology.

Figure 3
Figure 3 displays different giant mitochondria configurations that were frequently observed within hepatocytes through routine transmission electron microscopy (TEM) investigation of NAFLD patients.This initial observation was meticulously elaborated by the high-volume electron microscopy study by Shami et al. 43 in which

F I G U R E 2
Wide-field light optical image of a semi-thin human liver tissue section stained with toluidine blue and recorded at an intermediate magnification (×600).Arrows denote examples of the presence of giant mitochondria within the cytoplasm of liver parenchymal cells.Note the strong basophilic nature of the giant mitochondria.At the light optical level, the enlarged mitochondria present themselves in different shapes mainly ranging from a tubular, irregular and oval morphology.Scale bar, 25 μm.

F I G U R E 3
TEM images of the different types of giant mitochondria observed within human liver sections of NAFLD patients.(A) Low-Magnification overview image showing the three different morphologies of giant mitochondria.From left to right; elongated (1), irregular (2) and spheroidal (3).(B-D) Intermediate-magnification TEM data displaying elongated (B), irregular (C) and spheroidal (D) giant mitochondria (asterisks) as routinely observed in liver tissue.Note, the remarkably elongated and well-organised crystal-like inclusions within the mitochondrial matrix.Samples were prepared as detailed in the recent work by Verhaegh et al. 41 and Wisse et al. 42 Scale bars: (A) 2 μm; (B-D) 1 μm.

F I G U R E 4
TEM figure compilation depicting the possible different stages implemented in the dawn of giant mitochondria inside human liver parenchymal cells.The authors put forward the following sequence of structural changes as denoted by arrows: (A) Cristae rearrangement in discrete regions close to the inner mitochondrial membrane; (B) As a result, cristae have a random appearance within the mitochondrial matrix; (C) Cristae meet and seem to align; (D) Membranes of cristae appear to pack or stack in a well-organised manner; (E) Finally, dark electron-dense intra-mitochondrial crystal-like structures materialise typically for giant mitochondria.Note, the concurrent overall enlarged organelle size and increased electron density of the mitochondrial matrix, as the occurrence of crystal-like inclusions become more apparent.Scale bars, 0.5 μm.observing a 17.4% reduction in the surface area-to-volume ratio of the outer mitochondrial membrane between giant and normal mitochondria.This suggested a potential reduction in the efficiency of uptake and utilisation of the substrates required for ATP production.

Figure 5 F I G U R E 5
Figure5depicts the entire chondriome (thousands of mitochondria)

8. 65 ± 1 .
31 nm in diameter and are arranged in a rhomboid pattern with regular periodicities of 7.38 ± 1.61 nm.Studying the threedimensional models in depth revealed that the rod-like inclusions are organised as individual filaments rather than lamellar sheets, as they might be perceived and have previously been reported in 2-D TEM studies.Recently, we elaborated on those initial observations in more detail by taking advantage of the latest generation of electron microscopes that permits (i) imaging at higher voltage under minimal electron dose conditions, (ii) accurately controlled sample tilting over larger sample areas and (iii) capturing low-noise digital F I G U R E 6 TEM tomography data set of the paracrystalline inclusions acquired from 200 nm-thick section of human liver NAFLD tissue (See also, Video S2).(A) Reveals a transverse profile through a giant mitochondria revealing bundles of paracrystalline inclusions that project parallel relative to the longitudinal axis of the organelle.(B) Rendered model, revealing the outer mitochondrial membrane (dark blue), bundles of paracrystalline inclusions (yellow) -previously measuring 140.95 ± 16.46 nm 43 -and enlarged matrix granules (red).(C) Overlay of A and B shows the relationship between the rendered portion of the giant mitochondria and surrounding structures of interest.Scale bars, 1 μm.
classified structural changes of giant mitochondria in human ALD tissues in different types.Type I giant mitochondria show the loss of cristae and changes in their typical oval shape whereas type II and III are characterised by the presence of crystal-like inclusions.This might represent the different structural changes we recorded in our liver biopsies under Figure 4.

Figure 4A ,
Figure 4A,B might represent type I while the observations in 5D-E might reflect type II-III.For the near future, we expect that the latest developments in multimodal microscopy and associated microanalytical detection tools might contribute to further discerning the fine ultrastructural changes of mitochondria.The direct correlation of light optical observations with electron microscopy data on the same liver tissue area holds much promise.68In doing so, this approach can contribute to the outstanding enigma of the different structural observations on the same tissues across length scales.Furthermore, it may even contribute to fully mapping the fine architectural relationship of different subcellular components implemented in the rise of giant mitochondria.Likewise, the application of deep-learning image analysis algorithms69 combined with high-volume EM imaging approaches,43 will allow the detailed 3-D mapping of thousands