Short Communication - (2023) Volume 13, Issue 6
In nucleated eukaryotic cells, mitochondria are active organelles that are constantly present and serve a variety of metabolic tasks, including cellular ATP production. Oxidative phosphorylation, also known as OXPHOS. Five transmembrane respiratory chain enzyme complexes (RC) make up the OXPHOS mechanism. Mitochondrial disorders (mtD) are caused by defective OXPHOS. At least in part, the RC dual genetic control (nuclear DNA [nDNA] and mitochondrial DNA [mtDNA]) and the intricate interplay between the two genomes are responsible for the extraordinary phenotypic and genetic variety of mtD. The current clinical practise for investigating mtD essentially involves a multifaceted approach including clinical assessment, metabolic screening, imaging, pathological, biochemical, and functional testing to guide molecular genetic analysis, despite the growing use of Next-Generation Sequencing (NGS) and various -omics platforms in unravelling novel mtD genes and pathomechanisms. This review discusses the entire spectrum of muscle pathology, including genotype-phenotype relationships in adult and paediatric mtD, the contribution of immunodiagnostics to the understanding of some of the patho-mechanisms underlying the canonical aspects of mtD, and current developments in the field of diagnostics.
•Mitochondrial• Muscle Biopsy• Ragged Red• COXnegative• Subsarcolemmal• Immunohistochemistry
Due to the remarkable phenotypic and genetic variety linked to these disorders, mtD diagnosis is difficult. This is caused in part by the RC's dual genetic control (nDNA and mtDNA), the complexity of intergenomic signaling, and the functional ramifications of that signaling. X-linked, mitochondrial (i.e. maternal), autosomal dominant, and autosomal recessive inheritance patterns are all possible for mtD. 13 RC subunits, 22 mitochondrial tRNAs, and 2 ribosomal RNAs are all encoded by the circular mtDNA. In addition, the production, assembly, and support of the five multimeric OXPHOS RC (I-V), as well as auxiliary mitochondrial activities, depend on over 1300 nucleus encoded proteins.mtD frequently affects tissues and organs that have high energy needs. Clinical symptoms can appear at any age, can affect one organ or many organ systems, and can impact any age4. Usually, the more severe phenotypes appear earlier in life, and the milder traits appear later5. Leigh syndrome (subacute necrotizing encephalomyopathy), MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), and Alpers disease (epilepsy and liver failure) are examples of classic clinical syndromes having stereotypical symptoms. However, a lot of children exhibit generalised signs of developmental delay or regression, making a precise diagnosis more difficult.
Laboratory investigations and the rationale for muscle biopsy
Due to the complexity of mtD phenotypes and genetics, accurate diagnosis frequently requires a battery of invasive and noninvasive tests, such as imaging, neurophysiology, metabolic and biochemical studies, muscle pathology, and functional testing, followed by molecular genetic confirmation. A useful, if non-specific, screening method for mtD is the rise in blood lactate during rest and exercise. However, in some conditions, such as Complex I deficiency, Leber Hereditary Optic Neuropathy (LHON), Leigh disease, and mitochondrial Polymerase Gamma (POLG1) associated diseases, this rise in blood lactate may be normal or only mildly elevated.
Inborn flaws in the mitochondrial respiratory chain may cause an increase in the blood lactate/pyruvate ratio9. Poor collection or handling techniques, subsequent mitochondrial dysfunction in a variety of systemic and metabolic illnesses, and dietary thiamine shortage can all lead to spurious elevations of plasma lactate and/or pyruvate. Defects in pyruvate metabolism may result in an increase in blood and/or CSF pyruvate levels. Similarly, in mtD with significant CNS manifestations [1], CSF lactate and/or pyruvate levels may rise without blood elevation. Elevated plasma/CSF amino acids, urine organic acids, and plasma acylcarnitines all point to underlying mitochondrial dysfunction. Unless rhabdomyolysis is present, CK levels are normal or slightly elevated.When a myopathy or neuropathy is present, neurophysiology may exhibit ambiguous symptoms of these conditions, or it may be normal.There is no one 'gold standard' laboratory test for the detection of mtD. The screening tests mentioned above serve to raise or lower the clinical suspicion of mtD by widely confirming the existence of malfunction in a number of organ systems. Direct morphological, biochemical and molecular genetic evidence of mitochondrial malfunction must be established through more invasive testing [2] The relevant tissue for research is one that, in theory, displays a condition clinically. The preferred tissue is still skeletal muscle, which is routinely sampled in part due to the relative safety and simplicity of taking tissue samples. Even without clinically overt myopathic involvement, it can often provide useful diagnostic information14–16. Skeletal muscle is a terminally differentiated postmitotic tissue with a very low capacity for regeneration via satellite cell transformation. The mutant to wildtype mtDNA ratio is fairly stable throughout life as a result of terminal differentiation (heteroplasmy), in contrast to nucleated blood cells where this ratio might drop due to selection pressure, masking signs of mitochondrial dysfunction16.Determining individual or paired respiratory chain enzyme complexes is a common step in biochemical testing of respiratory chain enzyme failure. Activity in tissue homogenates or mitochondrial fractions made from fresh or frozen muscle tissue. Since there is no systematic programme in place to interchange samples and standardise procedures among diagnostic laboratories, biochemical assays have low inter-laboratory repeatability. A low-level heteroplasmy-induced concealment of an RC deficiency in tissue homogenates and a physiologicallydriven compensatory mitochondrial proliferative response6 are further complicating variables. Parallel histological analysis of skeletal muscle can give the only histochemical proof of RC deficiency in this situation cellular level. In the clinical differential diagnosis, a number of diseases that can mimic a mitochondrial myopathy or cause secondary mitochondrial dysfunction can be evaluated concurrently during the biopsy. This covers muscular dystrophies, endocrine, congenital, and inflammatory myopathies, as well as abnormalities in fatty acid oxidation and glycogen storage. Numerous classification schemes for diagnosing mtD in adults and children17–20 now include morphological and histochemical abnormalities in skeletal muscle as main and minor criteria due to the validity of this method of detection. The primary purpose of performing a skin biopsy concurrently with a muscle biopsy is to establish fibroblast cultures. Patients with OXPHOS abnormalities in skeletal muscle frequently have normal RC activity in fibroblasts, despite the fact that this is less invasive. This is partly because fibroblasts regenerate tissues more quickly than skeletal muscle and have altered heteroplasmy. Recognising the limitations of muscle biopsy analysis while looking into mtD is equally crucial. The kind of mutation and the peculiarities of mitochondrial genetics are additional factors influencing RC deficits, which are typically tissue specific, especially if sporadic and somatic. As a result, it's possible for muscle samples with known mtD mutations or phenotypes to lack pathological or biochemical signs of mitochondrial dysfunction [2].
Technical considerations
To enable the widest possible spectrum of tissue investigations into mitochondrial dysfunction, muscle and skin biopsies must be carried out and processed in a way that optimally retains mitochondrial shape, enzymatic/functional activity, protein, and DNA/ RNA content. This calls for effective coordination and communication between pathologists, surgeons, and clinicians [3]. An array of artefacts brought on by poor sampling and processing reduce the potential of uncertainty in the interpretation of data when a standard biopsy protocol is rigorously followed. Below are some of these topics discussed. Depending on the institutional choice, limb muscles such the vastus lateralis, gastrocnemius, deltoid, or biceps brachii are used for sampling. Patients with external ophthalmoplegia may occasionally have samples taken from their extraocular muscles.However, compared to limb muscles from the third decade of life, these muscles have a higher predominance of Ragged Red Fibres (RRF) and COX-negative fibres, which are characteristics thought to be "myopathic" for limb muscles.
A needle biopsy or an open biopsy can be used to remove skeletal muscle.The latter has the benefit of leaving a smaller scar and being carried out under local anaesthesia and/or severe sedation. By establishing techniques that use the modified Bergström needle for sampling, concerns concerning tissue fragmentation and reduced tissue yield that would not be sufficient for biochemical testing have been addressed. The material should ideally be checked for suitability and alignment in the operation room using a dissecting microscope. For biochemical and genetic testing, a portion of fresh, unfixed muscle is immediately placed in RNase-free tubes and quickly frozen in liquid nitrogen.For histology, histochemistry, and immunohistochemistry, the best-oriented section can be frozen in isopentane and cooled in liquid nitrogen, or it can be placed in a closed petridish on a piece of gauze that has been lightly moistened in saline [4]. For electron microscopy, a brief longitudinal portion of 0.5 mm in length is put in cold 2% glutaraldehyde.Fresh, unfixed tissue is needed for functional assays, such as polarographic studies.Morphology and histochemistry may be difficult to interpret due to excessive mechanical stress to the collected tissue, local anaesthetic infiltration into the fascicles, drying out, and excessive contact with saline.
When measuring complexes I, II, and III in biochemical tests, isopentane might cause artificially low activity readings [5]. A piece of muscle is frequently fixed in 10% formalin in laboratories before being paraffin embedded. With the exception of high-risk infectious samples, this practise has various drawbacks other from expanding the sampling area and should be discouraged from being used regularly. Inadequate muscle morphology, enhanced autofluorescence, and the need for time-consuming antigen retrieval procedures for protein immunohistochemistry are all consequences of formalin fixation. When performing big open biopsies, an additional block can be made in isopentane for freezing and any extra tissue can be quickly frozen in liquid nitrogen.
Histochemical assays for detecting RC defects
The inner mitochondrial membrane is home to the mitochondrial RC, which consists of five complexes and is responsible for cellular ATP production. NADHcoenzyme CI Q reductase, CII, succinate-CoQ reductase, which also contains iron-sulfur proteins and Succinate Dehydrogenase (SDH), CIII, reduced CoQ-cytochrome C reductase, CIV, cytochrome C oxidase, and CV, ATP synthase31. Protons are pumped through CI, CIII, and CIV, and CV allows protons to flow back into the mitochondrial matrix, creating a transmembrane proton gradient. The free energy created by redox reactions involving electron transfer across the complexes to molecular oxygen is used to synthesise ATP [6]. There are histochemical stains that can show the CI (NADH-TR), CII (SDH), and CIV (COX) activities. The substrate for the NADH-TR stain is reduced nicotinamide adenine dinucleotide, which is oxidised by NADH-dependent enzymes. When a tetrazolium salt (NBT) is added, a reduced, insoluble blue formazan product is deposited at the reaction site. Tetrazolium reductase is designated as TR. However, the sarcoplasmic reticulum (SR) NADH-oxidizing enzymes also contribute to the RC's CI's ability to oxidise NADH. This stain is not specific for CI and always masks any CI malfunction in the absence of particular inhibitors of the 'non-mitochondrial' NADH-oxidase activity32.The advantage is that this stain is great at showing structural flaws like cores or minicores because it can be used as a general marker of mitochondria and SR.
CII activity can be shown by SDH stain. As a substrate, Na-succinate is utilised. When NBT is present, it is oxidised by CII to fumarate, which is then reduced at the reaction site32 to insoluble blue formazan. All of the CII subunits are encoded by nuclear genes, hence conditions with primary mtDNA abnormalities seldom affect CII. Cytochrome C oxidase activity is demonstrated by the COX stain.To decrease cytochrome C in this process, Diaminobenzidine (DAB) serves as an electron donor. For the creation of water, the haeme units of CIV catalyse the passage of electrons from reduced cytochrome C to molecular oxygen [7].
Canonical pathological features
Depending on the underlying genotype, skeletal muscle biopsies from people with mtD can exhibit a variety of pathological alterations. Rugged red fibres (RRF) and COX-negative fibres are often recognised as the canonical hallmarks of mtD pathology, although not being completely specific SDH deficiency is another helpful diagnostic marker, albeit being uncommon.
Ragged red fibres
RRF has long been recognised as a morphological sign of mtD dating back before the molecular era. When thorough histochemical and ultrastructural studies revealed an excessive proliferation of normal or abnormal-looking mitochondria in the skeletal muscle of patients with weakness or exercise intolerance in the 1960s mitochondrial myopathies were first identified. The abnormal fibres in these conditions showed up as bright red accumulation of staining and "cracking" of the fibre edges, corresponding to the massive irregular proliferation of mitochondria, and were dubbed "ragged red" after the development of the Modified Gomori Trichrome (MGT) stain that allowed visualising connective tissue (light green), nuclei (red/purple), mitochondria, sarcoplasmic reticulum, sarcolemma, and myofibrils.
COX-negative fibres
The majority of people who have mitochondrial myopathies, which result in single or combined CIV insufficiency due to mtDNA mutations, have heteroplasmy, which means that each myofibre has a mixture of wild-type and mutant mtDNA molecules, with the amount of mutant mtDNA varying between different myofibres. When the amount of mutant mtDNA reaches a certain point, a myofibre segment will experience biochemical OXPHOS insufficiency [8]. This is a hallmark of mitochondrial disease and can be seen histochemically in transverse sections of frozen skeletal muscle as a mosaic pattern of COX-positive and COX-negative fibres, which equally affect slow-twitch (oxidative) and fast-twitch (glycolytic) muscle fibers.
SDH deficiency
Due to autosomal recessive mutations in the nuclear-encoded structural sub-units and assembly factor genes of CII (SDHA, SDHB, SDHD, SDHAF1)88-91, isolated CII deficiency is a rare Mendelian mitochondrial illness. With the exception of an autosomal dominant mutation in SDHA that manifests as late-onset optic atrophy, ataxia, and myopathy [9], the majority of documented cases are of early onset and appear with Leigh syndrome, cardiomyopathy, leukodystrophy, or encephalomyopathy.
Associated pathological features
Muscle biopsy results that are early in the course of the disease or in patients with CI deficiency brought on by recessive mutations in nuclear-encoded subunits may appear histologically normal. There may not be much abnormalities in situations with heteroplasmic mtDNA mutations other than the presence of classical traits. When present, myopathic modifications including increased fiber-size variation and internal nucleation are often of mild-to-moderate intensity. Except in mitochondrial myopathies presenting with rhabdomyolysis, inflammation is absent, and necrosis and regeneration are not visible.
Myopathology in novel mitochondrial diseases
Choline kinase, an enzyme that catalyses the first step in the biosynthesis of phosphatidylcholine, the most prevalent mitochondrial membrane phospholipid, is encoded by the CHKB gene [10]. Recessive loss-of-function mutations in CHKB lead to congenital muscular dystrophy with elevated serum CK, severe intellectual disability with skeletal and cardiac muscle involvement, and characteristic symptoms.
Vascular pathology
Microangiopathy, or small blood vessel dysfunction, can be a symptom of mitochondrial vasculopathy and can affect capillaries, small arteries, arterioles, venules, and venules. Premature atherosclerosis, arterial ectasia, vascular malformation, spontaneous rupture, and decreased flow-mediated vasodilation are a few of the clinical symptoms of macroangiopathy121.A fatal spontaneous aortic rupture in a 15-yearold female with the m.3243 A G mutation was linked to disorganisation, decreased COX staining in the Vascular Smooth Muscle Cells (VSMCs) of the aortic vasa vasora, and an 85% mutation load in the arteries compared to a 40% mutation load in blood lymphocytes122.Leukoencephalopathy, migraine-like headaches, stroke-like episodes, or peripheral retinopathy are some of the clinical manifestations of microangiopathy. A subclinical microangiopathy may be indicated by VSMC, pericytes, or endothelial cell morphological abnormalities found in skeletal muscle or other tissues.
Muscle biopsy is still an important tool for providing tissue for diagnostic and functional studies that guide molecular genetic testing, despite the rapid advancements in genetic technologies and the growing use of high-throughput Next-Generation Sequencing (NGS) platforms in the diagnostic pipeline for patients with suspected mtD.
Evidence for a heteroplasmic mtDNA illness is crucially provided by the demonstration of histochemical mosaic COX deficiency. When diagnosing mitochondrial pathology in muscle samples, the pathologist must take developmental, ageing-related, and secondary mitochondrial alterations into account. The diagnostic yield of biopsies is maximised by the proper handling and processing of tissue. As NGS platforms are used more often in diagnostic laboratories, the difficulty of functional testing to establish pathogenicity for variants of unknown relevance is growing.
Citation: Nicoldi. F Adult and paediatric mitochondrial myopathies: Pathology. Prim Health Care, 2023, 13(6), 510
Received: 08-Jun-2023, Manuscript No. jphc-23-102007; Editor assigned: 12-Jun-2023, Pre QC No. jphc-23-102007 (PQ); Reviewed: 14-Jun-2023, QC No. jphc-23-102007 (Q); Revised: 15-Jun-2023, Manuscript No. jphc-23-102007 (R); Published: 25-Jun-2023, DOI: 10.35248/2332 2594.23.13(6).51 0
Copyright: ©2023 Nicoldi.F, This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
