Metabolic dynamics in skeletal muscle during acute reduction in blood flow and oxygen supply to mitochondria: in-silico studies using a multi-scale, top-down integrated model

Control mechanisms of cellular metabolism and energetics in skeletal muscle that may become evident in response to physiological stresses such as reduction in blood flow and oxygen supply to mitochondria can be quantitatively understood using a multi-scale computational model. The analysis of dynamic responses from such a model can provide insights into mechanisms of metabolic regulation that may not be evident from experimental studies. For the purpose, a physiologically-based, multi-scale computational model of skeletal muscle cellular metabolism and energetics was developed to describe dynamic responses of key chemical species and reaction fluxes to muscle ischemia. The model, which incorporates key transport and metabolic processes and subcellular compartmentalization, is based on dynamic mass balances of 30 chemical species in both capillary blood and tissue cells (cytosol and mitochondria) domains. The reaction fluxes in cytosol and mitochondria are expressed in terms of a general phenomenological Michaelis-Menten equation involving the compartmentalized energy controller ratios ATP/ADP and NADH/NAD(+). The unknown transport and reaction parameters in the model are estimated simultaneously by minimizing the differences between available in vivo experimental data on muscle ischemia and corresponding model outputs in coupled with the resting linear flux balance constraints using a robust, nonlinear, constrained-based, reduced gradient optimization algorithm. With the optimal parameter values, the model is able to simulate dynamic responses to reduced blood flow and oxygen supply to mitochondria associated with muscle ischemia of several key metabolite concentrations and metabolic fluxes in the subcellular cytosolic and mitochondrial compartments, some that can be measured and others that can not be measured with the current experimental techniques. The model can be applied to test complex hypotheses involving dynamic regulation of cellular metabolism and energetics in skeletal muscle during physiological stresses such as ischemia, hypoxia, and exercise.     

Facilitated diffusion of myoglobin and creatine kinase and reaction-diffusion constraints of aerobic metabolism under steady-state conditions in skeletal muscle

The roles of creatine kinase (CK) and myoglobin (Mb) on steady-state facilitated diffusion and temporal buffering of ATP and oxygen, respectively, are assessed within the context of a reaction-diffusion model of muscle energetics. Comparison of the reaction-diffusion model with experimental data from a wide range of muscle fibers shows that the experimentally observed skeletal muscle fibers are generally not limited by diffusion, and the model further indicates that while some muscle fibers operate near the edge of diffusion limitation, no detectable effects of Mb and CK on the effectiveness factor, a measure of diffusion constraints, are observed under steady-state conditions. However, CK had a significant effect on average ATP concentration over a wide range of rates and length scales within the reaction limited regime. The facilitated diffusion functions of Mb and CK become observable in the model for larger size cells with low mitochondrial volume fraction and for low boundary O(2) concentration and high ATP demand, where the fibers may be limited by diffusion. From the transient analysis it may be concluded that CK primarily functions to temporally buffer ATP as opposed to facilitating diffusion while Mb has a small temporal buffering effect on oxygen but does not play any significant role in steady-state facilitated diffusion in skeletal muscle fibers under most physiologically relevant regions.     

Mitochondrial dysfunction in chronic ischemia and peripheral vascular disease

In peripheral vascular disease, impaired muscle energy metabolism is assumed to be due mainly to defective vascular O2 supply, the resulting cellular hypoxia inhibiting oxidative ATP synthesis. Older work suggested a compensatory increase in muscle aerobic enzymes, but more recent studies suggest a relative decrease in some mitochondrial components and an accumulation of damage in mitochondrial DNA, perhaps due to reactive oxygen species. However, to establish whether in vivo muscle mitochondria suffer from anything other than a low concentration of O2 will require more knowledge of the mitochondrial behaviour at low PO2, and the actual cell PO2 during exercise.     

Sensitivity analysis of reaction-diffusion constraints in muscle energetics

Theoretical and experimental studies of aerobic metabolism on a wide range of skeletal muscle fibers have shown that while all fibers normally function within the reaction control regime, some fibers operate near the transition region where reaction control switches to diffusion control. Thus, the transition region between reaction and diffusion control may define the limits of muscle function, and analysis of factors that affect this transition is therefore needed. In order to assess the role of all important model parameters, a sensitivity analysis (SA) was performed to define the parameter space where muscle fibers transition from reaction to diffusion control. SA, performed on a previously developed reaction–diffusion model, shows that the maximum rate for the ATPase reaction (V_max,ATPase), boundary oxygen concentration in the capillary supply (O), the mitochondrial volume fraction (ε_mito), and the diffusion coefficient of oxygen ( ) are the most sensitive parameters affecting this transition to diffusion control. It is demonstrated that fibers are not limited by diffusion for slow reactions (V_max,ATPase < 25 mM/min), high oxygen supply for the capillaries (O ≥ 35 µM), and large amounts of mitochondria (ε_mito ≥ 0.1). These conditions are applicable to muscle cells spanning a very broad range of animals. Within the diffusion‐controlled region, the overall metabolic rate and ATP concentrations have much higher sensitivity to the diffusion coefficient of oxygen than to the diffusion coefficients of the other metabolites (ATP, ADP, P_i). Biotechnol. Bioeng. 2012; 109:559–571. © 2011 Wiley Periodicals, Inc.     

Microaerobic respiration and oxidative phosphorylation by soybean nodule mitochondria: implications for nitrogen fixation

The infected cells of soybean (Glycine max) root nodules require ATP production for ammonia assimilation and purine synthesis under microaerobic conditions. It is likely that the bulk of this demand is supplied through mitochondrial oxidative phosphorylation. Mitochondria purified from root nodules respired and synthesized ATP in sub-micromolar oxygen concentrations as measured by leghaemoglobin spectroscopy and luciferase luminescence. Both oxygen uptake and the apparent ATP/O ratio declined significantly as the oxygen concentration fell below 100 μmol m^?3. Cytochrome-pathway respiration by root nodule mitochondria had a higher apparent affinity for oxygen (Km 50 μmol m^?3) than did mitochondria isolated from roots (Km 125 μmol m^?3). Electron micrographs showed that mitochondria predominated at the periphery of infected cells adjacent to gas-filled intercellular spaces, where the oxygen concentration is predicted to be highest. Calculations of oxygen concentration and nitrogen fixation rates on an infected cell basis suggest that the measured rates of ATP production by isolated mitochondria are sufficient for the quantifiable in vivo requirements of ammonia assimilation and purine synthesis. The possible roles of mitochondrial respiration in the control of infected cell metabolism are also discussed.     

Structural reaction of skeletal muscle mitochondria to deep hypothermia in the experiment

The reaction of locomotor muscle mitochondria was studied in white rats during different periods of deep hypothermia. An acute exposure to cold produced substantial changes of the muscle cellular mitochondrial apparatus, such as matrix swelling, crysts destruction with a significant decrease in mitochondrial profiles on the periphery of muscle fibers. Destructive and degenerative mitochondrial lesions were greater in prolonged hypothermia and subsequent warming. Such structural reaction of locomotor muscle mitochondria could reveal a substantial insufficiency of the muscle cellular energy apparatus during experimental hypothermia.     

Reaction–diffusion constraints in living tissue: Effectiveness factors in skeletal muscle design

A mathematical model was developed to analyze the effects of intracellular diffusion of O₂ and high‐energy phosphate metabolites on aerobic energy metabolism in skeletal muscle. We tested the hypotheses that in a range of muscle fibers from different species (1) aerobic metabolism was not diffusion limited and (2) that fibers had a combination of rate and fiber size that placed them at the brink of substantial diffusion limitation. A simplified chemical reaction rate law for mitochondrial oxidative phosphorylation was developed utilizing a published detailed model of isolated mitochondrial function. This rate law was then used as a boundary condition in a reaction–diffusion model that was further simplified using the volume averaging method and solved to determine the rates of oxidative phosphorylation as functions of the volume fraction of mitochondria, the size of the muscle cell, and the amount of oxygen delivered by the capillaries. The effectiveness factor, which is the ratio of reaction rate in the system with finite rates of diffusion to those in the absence of any diffusion limitations, defined the regions where intracellular diffusion of metabolites and O₂ may limit aerobic metabolism in both very small, highly oxidative fibers as well as in larger fibers with lower aerobic capacity. Comparison of model analysis with experimental data revealed that none of the fibers was strongly limited by diffusion, as expected. However, while some fibers were near substantial diffusion limitation, most were well within the domain of reaction control of aerobic metabolic rate. This may constitute a safety factor in muscle that provides a level of protection from diffusion constraints under conditions such as hypoxia. Biotechnol. Bioeng. 2011; 108:104–115. © 2010 Wiley Periodicals, Inc.     

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Mitophagy and the therapeutic clearance of damaged mitochondria for neuroprotection

Mitochondria are the foremost producers of the cellular energy currency ATP. They are also a significant source of reactive oxygen species and an important buffer of intracellular calcium. Mitochondrial retrograde signals regulate energy homeostasis and pro-survival elements whereas anterograde stimuli can trigger programmed cell death. Maintenance of a healthy, functional mitochondria network is therefore essential, and several mechanisms of mitochondrial quality control have been described. Mitochondrial dysfunction is linked to several neurodegenerative conditions including Parkinson, and Huntingdon diseases as well as Amyotrophic lateral sclerosis. Understanding the mechanisms governing mitochondrial quality control may reveal novel strategies for pharmacological intervention and disease therapy.     

Compartmentalized energy transfer in cardiomyocytes: use of mathematical modeling for analysis of in vivo regulation of respiration.

The mathematical model of the compartmentalized energy transfer system in cardiac myocytes presented includes mitochondrial synthesis of ATP by ATP synthase, phosphocreatine production in the coupled mitochondrial creatine kinase reaction, the myofibrillar and cytoplasmic creatine kinase reactions, ATP utilization by actomyosin ATPase during the contraction cycle, and diffusional exchange of metabolites between different compartments. The model was used to calculate the changes in metabolite profiles during the cardiac cycle, metabolite and energy fluxes in different cellular compartments at high workload (corresponding to the rate of oxygen consumption of 46 mu atoms of O.(g wet mass)-1.min-1) under varying conditions of restricted ADP diffusion across mitochondrial outer membrane and creatine kinase isoenzyme "switchoff." In the complete system, restricted diffusion of ADP across the outer mitochondrial membrane stabilizes phosphocreatine production in cardiac mitochondria and increases the role of the phosphocreatine shuttle in energy transport and respiration regulation. Selective inhibition of myoplasmic or mitochondrial creatine kinase (modeling the experiments with transgenic animals) results in "takeover" of their function by another, active creatine kinase isoenzyme. This mathematical modeling also shows that assumption of the creatine kinase equilibrium in the cell may only be a very rough approximation to the reality at increased workload. The mathematical model developed can be used as a basis for further quantitative analyses of energy fluxes in the cell and their regulation, particularly by adding modules for adenylate kinase, the glycolytic system, and other reactions of energy metabolism of the cell.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Role of nitric oxide and mitochondria in control of firefly flash

In light-producing cells (photocytes) of the firefly light organ,mitochondria are clustered in the cell periphery, positionedbetween the tracheolar air supply and the oxygen-requiring bioluminescentreactants which are sequestered in more centrally-localizedperoxisomes. This relative positioning suggests that mitochondriacould control oxygen availability for the light reaction. Wehypothesized that active cellular respiration would make theinterior regions of the photocytes relatively hypoxic, and thatthe "on" signal for production of bioluminescence might dependon inhibition of mitochondrial oxygen consumption, which wouldallow delivered oxygen to pass through the peripheral mitochondrialzone to reach peroxisomes deep in the cell interior. We publishedrecently that exogenous NO induces bioluminescence in the intactfirefly; that NO mediates octopamine-induced bioluminescencein the dissected lantern, and that nitric oxide synthase isabundant in cells of the tracheolar system of the light organ.Additional experiments showed that nitric oxide gas (NO) inhibitsrespiration in isolated lantern mitochondria. Inhibition isreversed by bright light, and this inhibition is relieved whenthe light is turned off. Altogether, the results support theidea that NO triggers light production by reversible inhibitionof mitochondrial respiration in lantern cells, and probablyin tracheolar cells as well. The data also suggest that thelight of bioluminescence itself relieves NO inhibition thuscontributing to rapid on/off switching. While other mechanismsmay be in play, NO production that is directly related to neuralinput appears to have a key role in the oxygen gating that controlsflash communication signals.     

Threshold Effects in Synaptosomal and Nonsynaptic Mitochondria from Hippocampal CA1 and Paramedian Neocortex Brain Regions

Abstract: After a brief period of global ischemia, the hippocampal CA1 region is more susceptible to irreversible damage than the paramedian neocortex. To test whether primary differences in bioenergetic parameters may be present between these regions, respiration rates and respiratory control activities were measured. In synaptosomal and nonsynaptic mitochondria isolated from the hippocampal CA1 region, state 3 respiration rates and complex IV activities were significantly lower than those present in synaptosomal and nonsynaptic mitochondria from the paramedian neocortex. These results suggest that mitochondria from the CA1 hippocampal area differ in some properties of metabolism compared with the neocortex area, which may render them more susceptible to a toxic insult such as that of ischemia. In addition, when complex I and IV activities were titrated with specific inhibitors, thresholds in ATP synthesis and oxygen respiration became apparent. Complex I and IV activities were decreased by 60% in nonsynaptic mitochondria from the hippocampal CA1 region and paramedian neocortex before oxidative phosphorylation was severely compromised; however, in synaptosomes from these regions, complex I activities had a threshold of 25%, indicating heterogenous behaviour for brain mitochondria. Reduced complex I thresholds in mitochondria, in association with other constitutive defects in energy metabolism, may induce a decreased ATP supply in the synaptic region. The implications of these findings are discussed in relation to delayed neuronal death and processes of neurodegeneration.     

Effects of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and heart

Mitochondria are chronically exposed to reactive oxygen intermediates. As a result, various tissues, including skeletal muscle and heart, are characterized by an age-associated increase in reactive oxidant-induced mitochondrial DNA (mtDNA) damage. It has been postulated that these alterations may result in a decline in the content and rate of production of ATP, which may affect tissue function, contribute to the aging process, and lead to several disease states. We show that with age, ATP content and production decreased by approximately 50% in isolated rat mitochondria from the gastrocnemius muscle; however, no decline was observed in heart mitochondria. The decline observed in skeletal muscle may be a factor in the process of sarcopenia, which increases in incidence with advancing age. Lifelong caloric restriction, which prolongs maximum life span in animals, did not attenuate the age-related decline in ATP content or rate of production in skeletal muscle and had no effect on the heart. 8-Oxo-7,8-dihydro-2'-deoxyguanosine in skeletal muscle mtDNA was unaffected by aging but decreased 30% with caloric restriction, suggesting that the mechanisms that decrease oxidative stress in these tissues with caloric restriction are independent from ATP availability. The generation of reactive oxygen species, as indicated by H2O2 production in isolated mitochondria, did not change significantly with age in skeletal muscle or in the heart. Caloric restriction tended to reduce the levels of H2O2 production in the muscle but not in the heart. These data are the first to show that an age-associated decline in ATP content and rate of ATP production is tissue specific, in that it occurs in skeletal muscle but not heart, and that mitochondrial ATP production was unaltered by caloric restriction in both tissues.     

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host‐derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (< 10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC‐1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno‐EM revealed that the ATP‐delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria‐vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP‐delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite.     

Mitochondrial coupling in vivo in mouse skeletal muscle

The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with ³¹P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 ± 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 ± 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought. P/O; oxidative phosphorylation; proton leak; optical spectroscopy     

Distinct Functional Roles of Cardiac Mitochondrial Subpopulations Revealed by a 3D Simulation Model

Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca²⁺], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25–2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca²⁺] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole.     

Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation

Mitochondria are important in the pathophysiology of several neurodegenerative diseases, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes and release of apoptogenic proteins are involved in these processes. Following brain insults, cell death often occurs in discrete regions of the brain, such as the subregions of the hippocampus. To analyse mitochondrial structure and function in such subregions, only small amounts of mitochondria are available. We developed a protocol for flow cytometric analysis of very small samples of isolated brain mitochondria, and analysed mitochondrial swelling and formation of ROS in mitochondria from the CA1 and CA3 regions of the hippocampus. Calcium-induced mitochondrial swelling was measured, and fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide) and to measure production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). We found that formation of ROS and mitochondrial permeability transition pore activation were higher in mitochondria from the CA1 than from the CA3 region, and propose that differences in mitochondrial properties partly underlie the selective vulnerability of the CA1 region to brain insults. We also conclude that flow cytometry is a useful tool to analyse the role of mitochondria in cell death processes.     

Mitochondrial filaments and clusters as intracellular power-transmitting cables

Mitochondria exist in two interconverting forms; as small isolated particles, and as extended filaments, networks or clusters connected with intermitochondrial junctions. Extended mitochondria can represent electrically united systems, which can facilitate energy delivery from the cell periphery to the cell core and organize antioxidant defence of the cell interior when O2 is consumed by mitochondrial clusters near the the outer cell membrane, and protonic potential is transmitted to the cell core mitochondria to form ATP. As to small mitochondria, they might represent a transportable form of these organelles.