Mitochondria: isolation, structure and function Picard, Martin; Taivassalo, Tanja; Gouspillou, Gilles ...
The Journal of physiology,
September 2011, Letnik:
589, Številka:
18
Journal Article
Recenzirano
Odprti dostop
Mitochondria are complex organelles constantly undergoing processes of fusion and fission, processes that not only modulate their morphology, but also their function. Yet the assessment of ...mitochondrial function in skeletal muscle often involves mechanical isolation of the mitochondria, a process which disrupts their normally heterogeneous branching structure and yields relatively homogeneous spherical organelles. Alternatively, methods have been used where the sarcolemma is permeabilized and mitochondrial morphology is preserved, but both methods face the downside that they remove potential influences of the intracellular milieu on mitochondrial function. Importantly, recent evidence shows that the fragmented mitochondrial morphology resulting from routine mitochondrial isolation procedures used with skeletal muscle alters key indices of function in a manner qualitatively similar to mitochondria undergoing fission in vivo. Although these results warrant caution when interpreting data obtained with mitochondria isolated from skeletal muscle, they also suggest that isolated mitochondrial preparations might present a useful way of interrogating the stress resistance of mitochondria. More importantly, these new findings underscore the empirical value of studying mitochondrial function in minimally disruptive experimental preparations. In this review, we briefly discuss several considerations and hypotheses emerging from this work.
Limb muscle dysfunction is prevalent in chronic obstructive pulmonary disease (COPD) and it has important clinical implications, such as reduced exercise tolerance, quality of life, and even ...survival. Since the previous American Thoracic Society/European Respiratory Society (ATS/ERS) statement on limb muscle dysfunction, important progress has been made on the characterization of this problem and on our understanding of its pathophysiology and clinical implications.
The purpose of this document is to update the 1999 ATS/ERS statement on limb muscle dysfunction in COPD.
An interdisciplinary committee of experts from the ATS and ERS Pulmonary Rehabilitation and Clinical Problems assemblies determined that the scope of this document should be limited to limb muscles. Committee members conducted focused reviews of the literature on several topics. A librarian also performed a literature search. An ATS methodologist provided advice to the committee, ensuring that the methodological approach was consistent with ATS standards.
We identified important advances in our understanding of the extent and nature of the structural alterations in limb muscles in patients with COPD. Since the last update, landmark studies were published on the mechanisms of development of limb muscle dysfunction in COPD and on the treatment of this condition. We now have a better understanding of the clinical implications of limb muscle dysfunction. Although exercise training is the most potent intervention to address this condition, other therapies, such as neuromuscular electrical stimulation, are emerging. Assessment of limb muscle function can identify patients who are at increased risk of poor clinical outcomes, such as exercise intolerance and premature mortality.
Limb muscle dysfunction is a key systemic consequence of COPD. However, there are still important gaps in our knowledge about the mechanisms of development of this problem. Strategies for early detection and specific treatments for this condition are also needed.
The normal decline in skeletal muscle mass that occurs with aging is exacerbated in patients with chronic obstructive pulmonary disease (COPD) and contributes to poor health outcomes, including a ...greater risk of death. There has been controversy about the causes of this exacerbated muscle atrophy, with considerable debate about the degree to which it reflects the very sedentary nature of COPD patients vs. being precipitated by various aspects of the COPD pathophysiology and its most frequent proximate cause, long-term smoking. Consistent with the latter view, recent evidence suggests that exacerbated aging muscle loss with COPD is likely initiated by decades of smoking-induced stress on the neuromuscular junction that predisposes patients to premature failure of muscle reinnervation capacity, accompanied by various alterations in mitochondrial function. Superimposed upon this are various aspects of COPD pathophysiology, such as hypercapnia, hypoxia, and inflammation, that can also contribute to muscle atrophy. This review will summarize the available knowledge concerning the mechanisms contributing to exacerbated aging muscle affect in COPD, consider the potential role of comorbidities using the specific example of chronic kidney disease, and identify emerging molecular mechanisms of muscle impairment, including mitochondrial permeability transition as a mechanism of muscle atrophy, and chronic activation of the aryl hydrocarbon receptor in driving COPD muscle pathophysiology.
Master athletes (MAs) prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear.
We performed ...muscle biopsies in 15 octogenarian world-class track and field MAs and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mitochondrial DNA (mtDNA) copy number, and proteomics by liquid-chromatography mass spectrometry.
MA exhibited markedly better performance on clinical function tests and greater cross-sectional area of the vastus lateralis muscle. Proteomics analysis revealed marked differences, where most of the ~800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage-dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and that are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA.
We conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with underrepresentation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging.
Funding for this study was provided by operating grants from the Canadian Institutes of Health Research (MOP 84408 to TT and MOP 125986 to RTH). This work was supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Baltimore, MD, USA.
PGC-1α regulates critical processes in muscle physiology, including mitochondrial biogenesis, lipid metabolism and angiogenesis. Furthermore, PGC-1α was suggested as an important regulator of fiber ...type determination. However, whether a muscle fiber type-specific PGC-1α content exists, whether PGC-1α content relates to basal levels of mitochondrial content, and whether such relationships are preserved between humans and classically used rodent models are all questions that have been either poorly addressed or never investigated. To address these issues, we investigated the fiber type-specific content of PGC-1α and its relationship to basal mitochondrial content in mouse, rat and human muscles using in situ immunolabeling and histochemical methods on muscle serial cross-sections. Whereas type IIa fibers exhibited the highest PGC-1α in all three species, other fiber types displayed a hierarchy of type IIx>I>IIb in mouse, type I = IIx> IIb in rat, and type IIx>I in human. In terms of mitochondrial content, we observed a hierarchy of IIa>IIx>I>IIb in mouse, IIa >I>IIx> IIb in rat, and I>IIa> IIx in human skeletal muscle. We also found in rat skeletal muscle that type I fibers displayed the highest capillarization followed by type IIa >IIx>IIb. Finally, we found in human skeletal muscle that type I fibers display the highest lipid content, followed by type IIa>IIx. Altogether, our results reveal that (i) the fiber type-specific PGC-1α and mitochondrial contents were only matched in mouse, (ii) the patterns of PGC-1α and mitochondrial contents observed in mice and rats do not correspond to that seen in humans in several respects, and (iii) the classical phenotypes thought to be regulated by PGC-1α do not vary exclusively as a function of PGC-1α content in rat and human muscles.
Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca(2+) handling and apoptotic signaling. Two classical methods exist to study ...mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca(2+) challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H(2)O(2) production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.
Mitochondrial dysfunction is implicated in skeletal muscle atrophy and dysfunction with aging, with strong support for an increased mitochondrial‐mediated apoptosis in sedentary rodent models. ...Whether this applies to aged human muscle is unknown, nor is it clear whether these changes are caused by sedentary behavior. Thus, we examined mitochondrial function respiration, reactive oxygen species (ROS) emission, and calcium retention capacity (CRC) in permeabilized myofibers obtained from vastus lateralis muscle biopsies of healthy physically active young (23.7±2.7 yr; mean±sd) and older (71.2±4.9 yr) men. Although mitochondrial ROS and maximal respiratory capacity were unaffected, the acceptor control ratio was reduced by 18% with aging, suggesting mild uncoupling of oxidative phosphorylation. CRC was reduced by 50% with aging, indicating sensitization of the mitochondrial permeability transition pore (mPTP) to apoptosis. Consistent with the mPTP sensitization, older muscles showed a 3‐fold greater fraction of endonuclease G (a mitochondrial proapoptotic factor)‐positive myonuclei. Aged muscles also had lower mitophagic potential, based on a 43% reduction in Parkin to the voltage‐dependent anion channel (VDAC) protein ratio. Collectively, these results show that mitochondrial‐mediated apoptotic signaling is increased in older human muscle and suggest that accumulation of dysfunctional mitochondria with exaggerated apoptotic sensitivity is due to impaired mitophagy.—Gouspillou, G., Sgarioto, N., Kapchinsky, S., Purves‐Smith, F., Norris, B., Pion, C. H., Barbat‐Artigas, S., Lemieux, R, Taivassalo, T., Morais, J. A., Aubertin‐Leheudre, M., Hepple, R. T. Increased sensitivity to mitochondrial permeability transition and myonuclear translocation of endonuclease G in atrophied muscle of physically active older humans. FASEB J. 28, 28–1621 (1633). www.fasebj.org
COPD is a significant public health challenge, notably set to become the third leading cause of death and fifth leading cause of chronic disability worldwide by the next decade. Skeletal muscle ...impairment is now recognized as a disabling, extrapulmonary consequence of COPD that is associated with reduced quality of life and premature mortality. Because COPD typically manifests in older individuals, these clinical features may overlie normal age-associated declines in muscle function and performance. Although physical inactivity, oxidative stress, inflammation, hypoxia, malnutrition, and medications all likely contribute to this comorbidity, a better understanding of the underlying mechanism is needed to develop effective therapies. Mitochondrial alterations have been described; these alterations include reductions in density and oxidative enzyme activity, increased mitochondrial reactive oxygen species production, and induction of muscle proteolysis including autophagy. This review focuses on the perspective that mitochondrial alterations contribute to impaired locomotor muscle performance in patients with COPD by reducing oxidative capacity and thus endurance, as well as by triggering proteolysis and thus contributing to atrophy and weakness. We discuss how the potential underlying mechanisms converge on mitochondria by targeting the peroxisome proliferator-activated receptor γ-coactivator-1α signaling pathway (thereby reducing mitochondrial biogenesis and muscle oxidative capacity and potentially increasing fiber atrophy) and how taking advantage of normal muscle plasticity and mitochondrial biogenesis may reverse this pathophysiology. We propose recent therapeutic strategies aimed at increasing peroxisome proliferator-activated receptor γ-coactivator-1α levels, such as endurance training and exercise mimetic drugs, with the strong rationale for increasing mitochondrial biogenesis and function and thus improving the muscle phenotype in COPD.
Mechanical ventilation (MV) is associated with adverse effects on the diaphragm, but the cellular basis for this phenomenon, referred to as ventilator-induced diaphragmatic dysfunction (VIDD), is ...poorly understood.
To determine whether mitochondrial function and cellular energy status are disrupted in human diaphragms after MV, and the role of mitochondria-derived oxidative stress in the development of VIDD.
Diaphragm and biceps specimens obtained from brain-dead organ donors who underwent MV (15-176 h) and age-matched control subjects were compared regarding mitochondrial enzymatic function, mitochondrial DNA integrity, lipid content, and metabolic gene and protein expression. In addition, diaphragmatic force and oxidative stress after exposure to MV for 6 hours were evaluated in mice under different conditions.
In human MV diaphragms, mitochondrial biogenesis and content were down-regulated, with a more specific defect of respiratory chain cytochrome-c oxidase. Laser capture microdissection of cytochrome-c oxidase-deficient fibers revealed mitochondrial DNA deletions, consistent with damage from oxidative stress. Diaphragmatic lipid accumulation and responses of master cellular metabolic sensors (AMP-activated protein kinase and sirtuins) were consistent with energy substrate excess as a possible stimulus for these changes. In mice, induction of hyperlipidemia worsened diaphragmatic oxidative stress during MV, whereas transgenic overexpression of a mitochondria-localized antioxidant (peroxiredoxin-3) was protective against VIDD.
Our data suggest that mitochondrial dysfunction lies at the nexus between oxidative stress and the impaired diaphragmatic contractility that develops during MV. Energy substrate oversupply relative to demand, resulting from diaphragmatic inactivity during MV, could play an important role in this process.
Key points
Mitochondria are frequently implicated in the ageing of skeletal muscle, although the role of denervation in modulating mitochondrial function in ageing muscle is unknown.
We show that ...increased sensitivity to apoptosis initiation occurs prior to evidence of persistent denervation and is thus a primary mitochondrial defect in ageing muscle worthy of therapeutic targeting.
However, at more advanced age, mitochondrial function changes are markedly impacted by persistent sporadic myofibre denervation, suggesting the mitochondrion may be a less viable therapeutic target.
Experimental denervation modulates mitochondrial function, where changes in both reactive oxygen species (ROS) and sensitivity to permeability transition are implicated in the resultant muscle atrophy. Notably, although denervation occurs sporadically in ageing muscle, its impact on ageing muscle mitochondria is unknown. Because this information has important therapeutic implications concerning targeting the mitochondrion in ageing muscle, we examined mitochondrial function in skeletal muscle from four groups of humans, comprising two active (mean ± SD age: 23.7 ± 2.7 years and 71.2 ± 4.9 years) and two inactive groups (64.8 ± 3.1 years and 82.5 ± 4.8 years), and compared this with a murine model of sporadic denervation. We tested the hypothesis that, although some alterations of mitochondrial function in aged muscle are attributable to a primary organelle defect, mitochondrial dysfunction would be impacted by persistent denervation in advanced age. Both ageing in humans and sporadic denervation in mice increased mitochondrial sensitivity to permeability transition (humans, P = 0.004; mice, P = 0.01). To determine the contribution of sporadic denervation to mitochondrial function, we pharmacologically inhibited the denervation‐induced ROS response. This reduced ROS emission by 60% (P = 0.02) in sporadically denervated mouse muscle, which is similar to that seen in humans older than 75 years (–66%, P = 0.02) but not those younger than 75 years. We conclude that an increased sensitivity to permeability transition is a primary mitochondrial defect in ageing muscle. However, at more advanced age, when muscle atrophy becomes more clinically severe, mitochondrial function changes are markedly impacted by persistent sporadic denervation, making the mitochondrion a less viable therapeutic target.
Key points
Mitochondria are frequently implicated in the ageing of skeletal muscle, although the role of denervation in modulating mitochondrial function in ageing muscle is unknown.
We show that increased sensitivity to apoptosis initiation occurs prior to evidence of persistent denervation and is thus a primary mitochondrial defect in ageing muscle worthy of therapeutic targeting.
However, at more advanced age, mitochondrial function changes are markedly impacted by persistent sporadic myofibre denervation, suggesting the mitochondrion may be a less viable therapeutic target.
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