Adenylate kinase AK2 isoform integral in embryo and adult heart homeostasis.

Adenylate kinase2 (AK2) catalyzes trans-compartmental nucleotide exchange, but the functional implications of this mitochondrial intermembrane isoform is only partially understood. Here, transgenic AK2-/- null homozygosity was lethal early in embryo, indicating a mandatory role for intact AK2 in utero development. In the adult, conditional organ-specific ablation of AK2 precipitated abrupt heart failure with Krebs cycle and glycolytic metabolite buildup, suggesting a vital contribution to energy demanding cardiac performance. Depressed pump function recovered to pre-deletion levels overtime, suggestive of an adaptive response. Compensatory upregulation of phosphotransferase AK1, AK3, AK4 isozymes, creatine kinase isoforms, and hexokinase, along with remodeling of cell cycle/growth genes and mitochondrial ultrastructure supported organ rescue. Taken together, the requirement of AK2 in early embryonic stages, and the immediate collapse of heart performance in the AK2-deficient postnatal state underscore a primordial function of the AK2 isoform. Unsalvageable in embryo, loss of AK2 in the adult heart was recoverable, underscoring an AK2-integrated bioenergetics system with innate plasticity to maintain homeostasis on demand.

Correction: A line-broadening free real-time 31P pure shift NMR method for phosphometabolomic analysis.

Correction for 'A line-broadening free real-time 31P pure shift NMR method for phosphometabolomic analysis' by Karl Kristjan Kaup et al., Analyst, 2021, 146, 5502-5507, DOI: 10.1039/D1AN01198G.

A line-broadening free real-time 31P pure shift NMR method for phosphometabolomic analysis.

Phosphometabolomics by 31P NMR can be challenging, since overlapping multiplets of homonuclear coupled phosphorus nuclei complicate spectral analysis. Pure shift NMR allows to simplify such spectra by collapsing multiplets into singlets, but most pure shift methods require substantially elongated measurement times or cause disturbing spectral line broadening. Herein, we combine established pure shift NMR and artefact suppression techniques to record 31P pure shift NMR spectra without penalties in measurement time or line width. Examples are demonstrated in resolution of a mixture of nucleotide triphosphates and a biological sample of 18O labelled ATP isotopomers.

Cardiac metabolism as a driver and therapeutic target of myocardial infarction.

Reducing infarct size during a cardiac ischaemic-reperfusion episode is still of paramount importance, because the extension of myocardial necrosis is an important risk factor for developing heart failure. Cardiac ischaemia-reperfusion injury (IRI) is in principle a metabolic pathology as it is caused by abruptly halted metabolism during the ischaemic episode and exacerbated by sudden restart of specific metabolic pathways at reperfusion. It should therefore not come as a surprise that therapy directed at metabolic pathways can modulate IRI. Here, we summarize the current knowledge of important metabolic pathways as therapeutic targets to combat cardiac IRI. Activating metabolic pathways such as glycolysis (eg AMPK activators), glucose oxidation (activating pyruvate dehydrogenase complex), ketone oxidation (increasing ketone plasma levels), hexosamine biosynthesis pathway (O-GlcNAcylation; administration of glucosamine/glutamine) and deacetylation (activating sirtuins 1 or 3; administration of NAD+ -boosting compounds) all seem to hold promise to reduce acute IRI. In contrast, some metabolic pathways may offer protection through diminished activity. These pathways comprise the malate-aspartate shuttle (in need of novel specific reversible inhibitors), mitochondrial oxygen consumption, fatty acid oxidation (CD36 inhibitors, malonyl-CoA decarboxylase inhibitors) and mitochondrial succinate metabolism (malonate). Additionally, protecting the cristae structure of the mitochondria during IR, by maintaining the association of hexokinase II or creatine kinase with mitochondria, or inhibiting destabilization of FO F1 -ATPase dimers, prevents mitochondrial damage and thereby reduces cardiac IRI. Currently, the most promising and druggable metabolic therapy against cardiac IRI seems to be the singular or combined targeting of glycolysis, O-GlcNAcylation and metabolism of ketones, fatty acids and succinate.

On the role of tubulin, plectin, desmin, and vimentin in the regulation of mitochondrial energy fluxes in muscle cells.

Mitochondria perform a central role in life and death of the eukaryotic cell. They are major players in the generation of macroergic compounds and function as integrated signaling pathways, including the regulation of Ca2+ signals and apoptosis. A growing amount of evidence is demonstrating that mitochondria of muscle cells use cytoskeletal proteins (both microtubules and intermediate filaments) not only for their movement and proper cellular positioning, but also to maintain their biogenesis, morphology, function, and regulation of energy fluxes through the outer mitochondrial membrane (MOM). Here we consider the known literature data concerning the role of tubulin, plectin, desmin and vimentin in bioenergetic function of mitochondria in striated muscle cells, as well as in controlling the permeability of MOM for adenine nucleotides (ADNs). This is of great interest since dysfunctionality of these cytoskeletal proteins has been shown to result in severe myopathy associated with pronounced mitochondrial dysfunction. Further efforts are needed to uncover the pathways by which the cytoskeleton supports the functional capacity of mitochondria and transport of ADN(s) across the MOM (through voltage-dependent anion channel).

Comparative analysis of the bioenergetics of human adenocarcinoma Caco-2 cell line and postoperative tissue samples from colorectal cancer patients.

The aim of this work was to explore the key bioenergetic properties for mitochondrial respiration in the widely-used Caco-2 cell line and in human colorectal cancer (HCC) postoperational tissue samples. Oxygraphy and metabolic control analysis (MCA) were applied to estimate the function of oxidative phosphorylation in cultured Caco-2 cells and HCC tissue samples. The mitochondria of Caco-2 cells and HCC tissues displayed larger functional activity of respiratory complex (C)II compared with CI, whereas in normal colon tissue an inverse pattern in the ratio of CI to CII activity was observed. MCA showed that the respiration in Caco-2 and HCC tissue cells is regulated by different parts of electron transport chain. In HCC tissues, this control is performed essentially at the level of respiratory chain complexes I-IV, whereas in Caco-2 cells at the level of CIV (cytochrome c oxidase) and the ATP synthasome. The differences we found in the regulation of respiratory chain activity and glycose index could represent an adaptive response to distinct growth conditions; this highlights the importance of proper validation of results obtained from in-vitro models before their extrapolation to the more complex in-vivo systems.

The complexity of mitochondrial outer membrane permeability and VDAC regulation by associated proteins.

Previous studies have shown that class II ß-tubulin plays a key role in the regulation of oxidative phosphorylation (OXPHOS) in some highly differentiated cells, but its role in malignant cells has remained unclear. To clarify these aspects, we compared the bioenergetic properties of HL-1 murine sarcoma cells, murine neuroblastoma cells (uN2a) and retinoic acid - differentiated N2a cells (dN2a). We examined the expression and possible co-localization of mitochondrial voltage dependent anion channel (VDAC) with hexokinase-2 (HK-2) and ßII-tubulin, the role of depolymerized ßII-tubuline and the effect of both proteins in the regulation of mitochondrial outer membrane (MOM) permeability. Our data demonstrate that neuroblastoma and sarcoma cells are prone to aerobic glycolysis, which is partially mediated by the presence of VDAC bound HK-2. Microtubule destabilizing (colchicine) and stabilizing (taxol) agents do not affect the MOM permeability for ADP in N2a and HL-1 cells. The obtained results show that ßII-tubulin does not regulate the MOM permeability for adenine nucleotides in these cells. HL-1 and NB cells display comparable rates of ADP-activated respiration. It was also found that differentiation enhances the involvement of OXPHOS in N2a cells due to the rise in their mitochondrial reserve capacity. Our data support the view that the alteration of mitochondrial affinity for ADNs is one of the characteristic features of cancer cells. It can be concluded that the binding sites for tubulin and hexokinase within the large intermembrane protein supercomplex Mitochondrial Interactosome, could be different between muscle and cancer cells.

Intracellular Energy-Transfer Networks and High-Resolution Respirometry: A Convenient Approach for Studying Their Function.

Compartmentalization of high-energy phosphate carriers between intracellular micro-compartments is a phenomenon that ensures efficient energy use. To connect these sites, creatine kinase (CK) and adenylate kinase (AK) energy-transfer networks, which are functionally coupled to oxidative phosphorylation (OXPHOS), could serve as important regulators of cellular energy fluxes. Here, we introduce how selective permeabilization of cellular outer membrane and high-resolution respirometry can be used to study functional coupling between CK or AK pathways and OXPHOS in different cells and tissues. Using the protocols presented here the ability of creatine or adenosine monophosphate to stimulate OXPHOS through CK and AK reactions, respectively, is easily observable and quantifiable. Additionally, functional coupling between hexokinase and mitochondria can be investigated by monitoring the effect of glucose on respiration. Taken together, high-resolution respirometry in combination with permeabilization is a convenient approach for investigating energy-transfer networks in small quantities of cells and tissues in health and in pathology.

The impact of cardiac ischemia/reperfusion on the mitochondria-cytoskeleton interactions.

Cardiac ischemia-reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation (OxPhos) and compartmentalized intracellular energy transfer via the phosphocreatine/creatine kinase (CK) network. The restriction of ATP/ADP diffusion at the level of the mitochondrial outer membrane (MOM) is an essential element of compartmentalized energy transfer. In adult cardiomyocytes, the MOM permeability to ADP is regulated by the interaction of voltage-dependent anion channel with cytoskeletal proteins, particularly with ß tubulin II. The IR-injury alters the expression and the intracellular arrangement of cytoskeletal proteins. The objective of the present study was to investigate the impact of IR on the intracellular arrangement of ß tubulin II and its effect on the regulation of mitochondrial respiration. Perfused rat hearts were subjected to total ischemia (for 20min (I20) and 45min (I45)) or to ischemia followed by 30min of reperfusion (I20R and I45R groups). High resolution respirometry and fluorescent confocal microscopy were used to study respiration, ß tubulin II and mitochondrial arrangements in cardiac fibers. The results of these experiments evidence a heterogeneous response of mitochondria to IR-induced damage. Moreover, the intracellular rearrangement of ß tubulin II, which in the control group colocalized with mitochondria, was associated with increased apparent affinity of OxPhos for ADP, decreased regulation of respiration by creatine without altering mitochondrial CK activity and the ratio between octameric to dimeric isoenzymes. The results of this study allow us to highlight changes of mitochondrial interactions with cytoskeleton as one of the possible mechanisms underlying cardiac IR injury.

Changes in the mitochondrial function and in the efficiency of energy transfer pathways during cardiomyocyte aging.

The role of mitochondria in alterations that take place in the muscle cell during healthy aging is a matter of debate during recent years. Most of the studies in bioenergetics have a focus on the model of isolated mitochondria, while changes in the crosstalk between working myofibrils and mitochondria in senescent cardiomyocytes have been less studied. The aim of our research was to investigate the modifications in the highly regulated ATP production and energy transfer systems in heart cells in old rat cardiomyocytes. The results of our work demonstrated alterations in the diffusion restrictions of energy metabolites, manifested by changes in the apparent Michaelis-Menten constant of mitochondria to exogenous ADP. The creatine kinase (CK) phosphotransfer pathway efficiency declines significantly in senescence. The ability of creatine to stimulate OXPHOS as well as to increase the affinity of mitochondria for ADP is falling and the most critical decline is already in the 1-year group (middle-age model in rats). Also, a moderate decrease in the adenylate kinase phosphotransfer system was detected. The importance of glycolysis increases in senescence, while the hexokinase activity does not change during healthy aging. The main result of our study is that the decline in the heart muscle performance is not caused by the changes in the respiratory chain complexes activity but mainly by the decrease in the energy transfer efficiency, especially by the CK pathway.

2102Ep embryonal carcinoma cells have compromised respiration and shifted bioenergetic profile distinct from H9 human embryonic stem cells.

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells, it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system, aerobic glycolysis, and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells, while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass, increased proton leak, and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference, and highlight the importance of further research concerning energy metabolism of stem cells.

Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells.

The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between non-transformed cells and tumor cells.

Formation of highly organized intracellular structure and energy metabolism in cardiac muscle cells during postnatal development of rat heart.

Adult cardiomyocytes have highly organized intracellular structure and energy metabolism whose formation during postnatal development is still largely unclear. Our previous results together with the data from the literature suggest that cytoskeletal proteins, particularly ßII-tubulin, are involved in the formation of complexes between mitochondria and energy consumption sites. The aim of this study was to examine the arrangement of intracellular architecture parallel to the alterations in regulation of mitochondrial respiration in rat cardiomyocytes during postnatal development, from 1 day to 6 months. Respirometric measurements were performed to study the developmental alterations of mitochondrial function. Changes in the mitochondrial arrangement and cytoarchitecture of ßII- and αIV-tubulin were examined by confocal microscopy. Our results show that functional maturation of oxidative phosphorylation in mitochondria is completed much earlier than efficient feedback regulation is established between mitochondria and ATPases via creatine kinase system. These changes are accompanied by significant remodeling of regular intermyofibrillar mitochondrial arrays aligned along the bundles of ßII-tubulin. Additionally, we demonstrate that formation of regular arrangement of mitochondria is not sufficient per se to provide adult-like efficiency in metabolic feed-back regulation, but organized tubulin networks and reduction in mitochondrial outer membrane permeability for ADP are necessary as well. In conclusion, cardiomyocytes in rat heart become mature on the level of intracellular architecture and energy metabolism at the age of 3 months.

Role of mitochondria-cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles.

The aim of this work was to study the regulation of respiration and energy fluxes in permeabilized oxidative and glycolytic skeletal muscle fibers, focusing also on the role of cytoskeletal protein tubulin ßII isotype in mitochondrial metabolism and organization. By analyzing accessibility of mitochondrial ADP, using respirometry and pyruvate kinase-phosphoenolpyruvate trapping system for ADP, we show that the apparent affinity of respiration for ADP can be directly linked to the permeability of the mitochondrial outer membrane (MOM). Previous studies have shown that MOM permeability in cardiomyocytes can be regulated by VDAC interaction with cytoskeletal protein, ßII tubulin. We found that in oxidative soleus skeletal muscle the high apparent Km for ADP is associated with low MOM permeability and high expression of non-polymerized ßII tubulin. Very low expression of non-polymerized form of ßII tubulin in glycolytic muscles is associated with high MOM permeability for adenine nucleotides (low apparent Km for ADP).

Comparative analysis of some aspects of mitochondrial metabolism in differentiated and undifferentiated neuroblastoma cells.

The aim of the present study is to clarify some aspects of the mechanisms of regulation of mitochondrial metabolism in neuroblastoma (NB) cells. Experiments were performed on murine Neuro-2a (N2a) cell line, and the same cells differentiated by all-trans-retinoic acid (dN2a) served as in vitro model of normal neurons. Oxygraphy and Metabolic Control Analysis (MCA) were applied to characterize the function of mitochondrial oxidative phosphorylation (OXPHOS) in NB cells. Flux control coefficients (FCCs) for components of the OXPHOS system were determined using titration studies with specific non-competitive inhibitors in the presence of exogenously added ADP. Respiration rates of undifferentiated Neuro-2a cells (uN2a) and the FCC of Complex-II in these cells were found to be considerably lower than those in dN2a cells. Our results show that NB is not an exclusively glycolytic tumor and could produce a considerable part of ATP via OXPHOS. Two important enzymes - hexokinase-2 and adenylate kinase-2 can play a role in the generation of ATP in NB cells. MCA has shown that in uN2a cells the key sites in the regulation of OXPHOS are complexes I, II and IV, whereas in dN2a cells complexes II and IV. Results obtained for the phosphate and adenine nucleotide carriers showed that in dN2a cells these carriers exerted lower control over the OXPHOS than in undifferentiated cells. The sum of FCCs for both types of NB cells was found to exceed significantly that for normal cells suggesting that in these cells the respiratory chain was somehow reorganized or assembled into large supercomplexes.

The role of tubulin in the mitochondrial metabolism and arrangement in muscle cells.

Tubulin, a well-known component of the microtubule in the cytoskeleton, has an important role in the transport and positioning of mitochondria in a cell type dependent manner. This review describes different functional interactions of tubulin with cellular protein complexes and its functional interaction with the mitochondrial outer membrane. Tubulin is present in oxidative as well as glycolytic type muscle cells, but the kinetics of the in vivo regulation of mitochondrial respiration in these muscle types is drastically different. The interaction between VDAC and tubulin is probably influenced by such factors as isoformic patterns of VDAC and tubulin, post-translational modifications of tubulin and phosphorylation of VDAC. Important factor of the selective permeability of VDAC is the mitochondrial creatine kinase pathway which is present in oxidative cells, but is inactive or missing in glycolytic muscle and cancer cells. As the tubulin-VDAC interaction reduces the permeability of the channel by adenine nucleotides, energy transfer can then take place effectively only through the mitochondrial creatine kinase/phosphocreatine pathway. Therefore, closure of VDAC by tubulin may be one of the reasons of apoptosis in cells without the creatine kinase pathway. An important question in tubulin regulated interactions is whether other proteins are interacting with tubulin. The functional interaction may be direct, through other proteins like plectins, or influenced by simultaneous interaction of other complexes with VDAC.

Smoking cessation only partially reverses cardiac metabolic and structural remodeling in mice.

AIMS: Active cigarette smoking is a major risk factor for chronic obstructive pulmonary disease that remains elevated after cessation. Skeletal muscle dysfunction has been well documented after smoking, but little is known about cardiac adaptations to cigarette smoking. The underlying cellular and molecular cardiac adaptations, independent of confounding lifestyle factors, and time course of reversibility by smoking cessation remain unclear. We hypothesized that smoking negatively affects cardiac metabolism and induces local inflammation in mice, which do not readily reverse upon 2-week smoking cessation. METHODS: Mice were exposed to air or cigarette smoke for 14 weeks with or without 1- or 2-week smoke cessation. We measured cardiac mitochondrial respiration by high-resolution respirometry, cardiac mitochondrial density, abundance of mitochondrial supercomplexes by electrophoresis, and capillarization, fibrosis, and macrophage infiltration by immunohistology, and performed cardiac metabolome and lipidome analysis by mass spectrometry. RESULTS: Mitochondrial protein, supercomplex content, and respiration (all p < 0.03) were lower after smoking, which were largely reversed within 2-week smoking cessation. Metabolome and lipidome analyses revealed alterations in mitochondrial metabolism, a shift from fatty acid to glucose metabolism, which did not revert to control upon smoking cessation. Capillary density was not different after smoking but increased after smoking cessation (p = 0.02). Macrophage infiltration and fibrosis (p < 0.04) were higher after smoking but did not revert to control upon smoking cessation. CONCLUSIONS: While cigarette-impaired smoking-induced cardiac mitochondrial function was reversed by smoking cessation, the remaining fibrosis and macrophage infiltration may contribute to the increased risk of cardiovascular events after smoking cessation.

Regulation of respiration in muscle cells in vivo by VDAC through interaction with the cytoskeleton and MtCK within Mitochondrial Interactosome.

This review describes the recent experimental data on the importance of the VDAC-cytoskeleton interactions in determining the mechanisms of energy and metabolite transfer between mitochondria and cytoplasm in cardiac cells. In the intermembrane space mitochondrial creatine kinase connects VDAC with adenine nucleotide translocase and ATP synthase complex, on the cytoplasmic side VDAC is linked to cytoskeletal proteins. Applying immunofluorescent imaging and Western blot analysis we have shown that ß2-tubulin coexpressed with mitochondria is highly important for cardiac muscle cells mitochondrial metabolism. Since it has been shown by Rostovtseva et al. that αß-heterodimer of tubulin binds to VDAC and decreases its permeability, we suppose that the ß-tubulin subunit is bound on the cytoplasmic side and α-tubulin C-terminal tail is inserted into VDAC. Other cytoskeletal proteins, such as plectin and desmin may be involved in this process. The result of VDAC-cytoskeletal interactions is selective restriction of the channel permeability for adenine nucleotides but not for creatine or phosphocreatine that favors energy transfer via the phosphocreatine pathway. In some types of cancer cells these interactions are altered favoring the hexokinase binding and thus explaining the Warburg effect of increased glycolytic lactate production in these cells. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Matters of the heart in bioenergetics: mitochondrial fusion into continuous reticulum is not needed for maximal respiratory activity.

Mitochondria are dynamic structures for which fusion and fission are well characterized for rapidly dividing cells in culture. Based on these data, it has recently been proposed that high respiratory activity is the result of fusion and formation of mitochondrial reticulum, while fission results in fragmented mitochondria with low respiratory activity. In this work we test the validity of this new hypothesis by analyzing our own experimental data obtained in studies of isolated heart mitochondria, permeabilized cells of cardiac phenotype with different mitochondrial arrangement and dynamics. Additionally, we reviewed published data including electron tomographic investigation of mitochondrial membrane-associated structures in heart cells. Oxygraphic studies show that maximal ADP-dependent respiration rates are equally high both in isolated heart mitochondria and in permeabilized cardiomyocytes. On the contrary, these rates are three times lower in NB HL-1 cells with fused mitochondrial reticulum. Confocal and electron tomographic studies show that there is no mitochondrial reticulum in cardiac cells, known to contain 5,000-10,000 individual, single mitochondria, which are regularly arranged at the level of sarcomeres and are at Z-lines separated from each other by membrane structures, including the T-tubular system in close connection to the sarcoplasmic reticulum. The new structural data in the literature show a principal role for the elaborated T-tubular system in organization of cell metabolism by supplying calcium, oxygen and substrates from the extracellular medium into local domains of the cardiac cells for calcium cycling within Calcium Release Units, associated with respiration and its regulation in Intracellular Energetic Units.

Mysterious Ca(2+)-independent muscular contraction: déjà vu.

The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca(2+)-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30-37°C). We show that very similar strong spontaneous Ca(2+)-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca(2+)-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca(2+)-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.