Bioenergetics and permeability transition pore opening in heart subsarcolemmal and interfibrillar mitochondria: Effects of aging and lifelong calorie restriction

Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca²⁺-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8-, 18-, 29- and 37-month-old male Fischer 344 × Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H₂O₂ generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca²⁺ retention capacity of SSM was not affected by age, but SSM tolerated much less Ca²⁺ than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca²⁺ retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.     

Liver mitochondrial function and redox status in an experimental model of non-alcoholic fatty liver disease induced by monosodium L-glutamate in rats

The purpose of this work was to determine if mitochondrial dysfunction is involved in the development of non-alcoholic fatty liver disease (NAFLD). Using a model of obesity induced by the neonatal treatment of rats with monosodium l-glutamate (MSG), several parameters of liver mitochondrial function and their impact on liver redox status were evaluated. Specifically, fatty acid β-oxidation, oxidative phosphorylation and Ca²⁺-induced mitochondrial permeability transition were assessed in isolated liver mitochondria, and reduced glutathione (GSH), linked thiol contents and the activities of several enzymes involved in the control of redox status were measured in the liver homogenate. Our results demonstrate that liver mitochondria from MSG-obese rats exhibit a higher β-oxidation capacity and an increased capacity for oxidising succinate, without loss in the efficiency of oxidative phosphorylation. Also, liver mitochondria from obese rats were less susceptible to the permeability transition pore (PTP) opening induced by 1.0 μM CaCl₂. Cellular levels of GSH were unaffected in the livers from the MSG-obese rats, whereas reduced linked thiol contents were increased. The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione peroxidase were increased, while catalase activity was unaffected and superoxide dismutase activity was reduced in the livers from the MSG-obese rats. In this model of obesity, liver fat accumulation is not a consequence of mitochondrial dysfunction. The enhanced glucose-6-phosphate dehydrogenase activity observed in the livers of MSG-obese rats could be associated with liver fat accumulation and likely plays a central role in the mitochondrial defence against oxidative stress.     

Effect of T3 treatment on the response to ischemia–reperfusion of heart preparations from sedentary and trained rats

We investigated whether swim training modifies the effect of T₃ treatment on rat heart response to ischemia–reperfusion. Homogenates of Langendorff preparations perfused for 25 min after 20-min ischemia were used for biochemical determinations and isolation of mitochondrial fractions. Oxidative damage and antioxidant levels of homogenates, O₂ consumption and H₂O₂ release rates, oxidative damage, and susceptibility to Ca²⁺-induced swelling of mitochondria were determined. During reperfusion, hyperthyroid hearts displayed significant tachycardia and low inotropic recovery. This pattern was improved by training, which also attenuated tissue oxidative damage and glutathione depletion. Similar training effects were shown in euthyroid preparations. Moreover, training reduced mitochondrial H₂O₂ production and oxidative damage in hyperthyroid and euthyroid hearts and susceptibility to Ca²⁺-induced swelling only in the hyperthyroid ones. Rates of mitochondrial O₂ consumption were not different in sedentary and trained hyperthyroid rats. However, determination of the oxidative capacity suggested that, in the sedentary rats, O₂ consumption was conditioned by oxidative damage mitochondria have suffered, whereas in trained rats, it was due to changes in mitochondrial characteristics. The above results suggest that moderate training is able to reduce hyperthyroid heart susceptibility to oxidative damage and dysfunction modifying mitochondrial population.     

Regulation of the mitochondrial proton gradient by cytosolic Ca2+ signals

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca²⁺ from stores and the entry of Ca²⁺ across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca²⁺ signals and the activity of Ca²⁺-regulated proteins, including Ca²⁺ entry channels that are themselves part of the Ca²⁺ circuitry. Ca²⁺ elevations in the mitochondrial matrix, in turn, activate Ca²⁺-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca²⁺ signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (??p) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (???? _m ), and a chemical component, the mitochondrial proton gradient (??pH _m ). ???? _m contributes about 190?mV to ??p and drives the entry of Ca²⁺ across a recently identified Ca²⁺-selective channel known as the mitochondrial Ca²⁺ uniporter. ??pH _m contributes ~30?mV to ??p and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ??pH _m sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ??pH _m decreases during mitochondrial Ca²⁺ elevations.     

Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes

Aim: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca²⁺ mobilizations in cold‐acclimated rats. Methods: A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca²⁺ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied. Results: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca²⁺ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) followed by mitochondrial Ca²⁺ ([Ca²⁺]_mito), the effect being transformed into an increase in [Ca²⁺]_cyto whereas a decrease in [Ca²⁺]_mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca²⁺ release from mitochondria. CCCP induced Ca²⁺ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells. Conclusions: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito, in which H⁺ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca²⁺ signalling in cold‐acclimated brown adipocytes.     

Effects of some steroid hormones on Ca2+ transport and oxidative metabolism of isolated mitochondria

Effects of prednisolone, estradiol, and testosterone on the transport of Ca²⁺ and the respiration induced by it in the heart and liver mitochondria of rats were studied. Prednisolone and testosterone were found to reduce the Ca-accumulating capacity of the mitochondria, the rates of ion entry and exit, and the rate of Ca²⁺-induced respiration. Estradiol, while inhibiting Ca²⁺ transport across mitochondrial membrane, did not influence the respiration in the phase of Ca²⁺ absorption, but accelerated it in the phase of ion exit. These data suggest that due to their lipophilic properties, the steroids become incorporated in the mitochondrial membrane, thereby changing its viscosity and permeability and limiting the mobility of transmitter proteins. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 616–618, December, 1994     

Endoplasmic reticulum protein BI-1 regulates Ca²⁺-mediated bioenergetics to promote autophagy

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²⁺ mediated by inositol triphosphate receptors (IP₃Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP₃R-dependent manner. By reducing steady-state levels of ER Ca²⁺ via IP₃Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1''s ability to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca²⁺ signaling between ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face of metabolic stress.     

Mitochondria contribute to Ca2+ removal in smooth muscle cells

Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca²⁺]_i,) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca²⁺ uptake, we have examined the contribution of mitochondria to Ca²⁺ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train of depolarizing pulses, the rate of Ca²⁺ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanideP-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cell's ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca²⁺ removal. However, experiments carried out with the mitochondrial Ca²⁺ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca²⁺ from the cytosol following stimulation. The uptake of Ca²⁺ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cell's rise in [Ca²⁺]_i;, with increased cellular ATP production.     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

Mitochondria and chromaffin cell function

Chromaffin cells are an excellent model for stimulus?Csecretion coupling. Ca²⁺ entry through plasma membrane voltage-operated Ca²⁺ channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca²⁺ signal. The endoplasmic reticulum amplifies the cytosolic Ca²⁺ ([Ca²⁺]_C) signal by Ca²⁺-induced Ca²⁺ release (CICR) and helps generation of microdomains with high [Ca²⁺]_C (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca²⁺ from HCMD and stop progression of the Ca²⁺ wave towards the cell core. On the other hand, the increase of [Ca²⁺] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca²⁺]_C decreases rapidly and mitochondria release the Ca²⁺ accumulated in the matrix through the Na⁺/Ca²⁺ exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus?Csecretion coupling while avoiding propagation of the Ca²⁺ signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders.     

Dissociation of K+-induced tension and cellular Ca2+ retention in vascular and intestinal smooth muscle in normoxia and hypoxia

In experiments on smooth muscle preparations of rabbit aorta and guinea pig taenia coli, replacement of the external Na⁺ with K⁺ produced sustained contraction. When external K⁺ concentration was increased, cellular Ca²⁺ retention as measured by a modified lanthanum technique increased. However, when K⁺ concentration was above 80 mM, the tension decreased despite an increase in Ca²⁺ retention. Maximum amount of Ca²⁺ retained was 1280 nmol/g in aorta and 980 nmol/g in taenia coli while the control values for both tissues were approximately 430 nmol/g when the external Ca²⁺ concentration was 2.5 mM. Under hypoxia (N₂ aeration), sustained contraction was induced by 80 mM K⁺ in aorta and by 45.4 mM K⁺ (and 55 mM glucose) in taenia coli. However, no increase in the cellular Ca²⁺ retention was observed under these conditions. During the K⁺-induced sustained contraction in aorta, introduction of N₂ transiently increased, while readmission of O₂ transiently decreased the muscle tension. In taenia coli, the introduction of N₂ decreased the sustained contractile tension probably because of an ATP deficiency, while the readmission of O₂ further decreased the tension trasniently. From these results, it is concluded that, in the presence of a high concentration of K⁺, external Ca²⁺ enters the cell and activates the contractile machinery. A part of the cellular Ca²⁺ is taken up by mitochondria under normoxic but not under hypoxic conditions.     

Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions

The present study investigated mitochondrial Ca²⁺ uptake and release in intact living skeletal muscle fibres subjected to bouts of repetitive activity. Confocal microscopy was used in conjunction with the Ca²⁺-sensitive dye Rhod-2 to monitor changes in mitochondrial Ca²⁺ in single Xenopus or mouse muscle fibres. A marked increase in the mitochondrial Ca²⁺ occurred in Xenopus fibres after 10 tetani applied at 4 s intervals. The mitochondrial Ca²⁺ continued to increase with increasing number of tetani. After the end of tetanic stimulation, mitochondrial Ca²⁺ declined to 50% of the maximal increase within 10 min and thereafter took up to 60 min to return to its original value. Depolarization of the mitochondria with FCCP greatly attenuated the rise in the mitochondrial Ca²⁺ evoked by repetitive tetanic stimulation. In addition, FCCP slowed the rate of decay of the tetanic Ca²⁺ transient which in turn led to an elevation of resting cytosolic Ca²⁺. Accumulation of Ca²⁺ in the mitochondria was accompanied by a modest mitochondrial depolarization. In contrast to the situation in Xenopus fibres, mitochondria in mouse toe muscle fibres did not show any change in the mitochondrial Ca²⁺ during repetitive stimulation and FCCP had no effect on the rate of decay of the tetanic Ca²⁺ transient. It is concluded that in Xenopus fibres, mitochondria play a role in the regulation of cytosolic Ca²⁺ and contribute to the relaxation of tetanic Ca²⁺ transients. In contrast to their important role in Xenopus fibres, mitochondria in mouse fast-twitch skeletal fibres play little role in Ca²⁺ homeostasis.     

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.     

Sequestration of 45Ca2+ by mitochondria from rabbit heart, liver, and kidney after doxorubicin or digoxin/doxorubicin treatment.

Doxorubicin, an antibiotic of the anthracycline group, has proven effective in treating a variety of malignant disorders. However, its use has been limited due to the cardiotoxic side effects which include myocardial necrosis that is characterized by mitochondrial calcification. The present studies were conducted to determine if treatment of rabbits with doxorubicin (an anthracycline) would affect the ability of mitochondria isolated from heart, liver, and kidney to retain ⁴⁵Ca²⁺. Increases in mitochondrial retention of ⁴⁵Ca²⁺ by all of the tissues studied were observed, although only that from the heart showed a significant increase. The changes in ⁴⁵Ca²⁺ retention and morphology (i.e., increased mitochondrial swelling and intra-mitochondrial calcium phosphate crystals) of heart mitochondria from doxorubicin-treated rabbits suggest that this anthracycline directly or indirectly affects mitochondrial flux of calcium. That liver and kidney (as compared to heart) mitochondria are relatively insensitive to the effects of doxorubicin suggests a chemical difference in the mitochondria isolated from these tissues. Digoxin/doxorubicin treatment of rabbits, however, leads to a decrease in mitochondrial retention of ⁴⁵CA²⁺, except for hear tissue, which again was significantly increased over the control.² The effects of this treatment on the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ activated ATPase of the heart, and on the accumulation of doxorubicin by the heart, were not significantly different from the control, suggesting that digoxin and doxorubicin do not compete for the same binding site.     

严重烧伤早期心肌[Ca2+]m变化及其机制研究

目的:探讨严重烧伤早期心肌线粒体Ca²⁺浓度([Ca²⁺]_m)的动态变化规律及其发生机制。方法:复制30%Ⅲ°烫伤大鼠模型,测定伤后1、3、6、12、24h大鼠心肌[Ca²⁺]_m,同时检测影响[Ca²⁺]_m的相关指标—胞浆Ca^2＋浓度(_c)及线粒体Ca^2＋转运速率。结果:烧伤后1、3、6h[Ca²⁺]_m依次升高,12、24h较6h虽有所下降,但仍高于正常对照组;_c除伤后1h无明显变化外,其余各时相点变化趋势与[Ca²⁺]_m相同,且伤后[Ca²⁺]_m与_c呈显著正相关,相关系数为0.9177(P＜0.01)。伤后1h心肌线粒体Ca^2＋摄取速率明显升高,而Ca^2＋释放速率无明显改变,但3、6、12、24h心肌线粒体Ca^2＋摄取速率与Ca^2＋释放速率均显著降低,且烧伤后3、6、12、24h[Ca²⁺]_m分别与线粒体Ca^2＋释放速率呈明显负相关。结论:烧伤后心肌线粒体存在明显的Ca^2＋超载和转运紊乱。     

Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨ_m) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨ_m. Mitochondrial density was highest in the perinuclear region, whereas ΔΨ_m tended to be higher in peripheral mitochondria. Spontaneous ΔΨ_m fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca²⁺, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca²⁺ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca²⁺ load or metabolic impairment abolished ΔΨ_m fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨ_m heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨ_m fluctuations are an indication of mitochondrial viability and are triggered by local Ca²⁺ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions.     

Frequency-dependent mitochondrial Ca2+ accumulation regulates ATP synthesis in pancreatic β cells

Pancreatic β cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K⁺ channels and electrical spiking. The latter results in oscillatory Ca²⁺ influx through voltage-gated Ca²⁺ channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca²⁺]_cyt and [Ca²⁺]_mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca²⁺-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca²⁺ pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary β cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca²⁺ and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca²⁺ uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca²⁺ oscillation frequency as stable increases in [Ca²⁺]_mit and cytosolic ATP/ADP; (3) mitochondrial Ca²⁺ uptake rates remained constant between individual spikes, arguing against activity-dependent regulation (“plasticity”) and (4) the relationship between [Ca²⁺]_cyt and [Ca²⁺]_mit is essentially unaffected by changes in endoplasmic reticulum Ca²⁺ ([Ca²⁺]_ER). Our findings thus highlight new aspects of Ca²⁺ signalling in β cells of relevance to the actions of both glucose and sulphonylureas.     

The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers

We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca²⁺ marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca²⁺ and JC-1 dye to measure mitochondria inner membrane potential (ΔΨ _m). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca²⁺ from mitochondria when ΔΨ_m is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca²⁺]_m. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mitochondrial Ca2+ homeostasis: mechanism, role, and tissue specificities

Mitochondria from every tissue are quite similar in their capability to accumulate Ca²⁺ in a process that depends on the electrical potential across the inner membrane; it is catalyzed by a gated channel (named mitochondrial Ca²⁺ uniporter), the molecular identity of which has only recently been unraveled. The release of accumulated Ca²⁺ in mitochondria from different tissues is, on the contrary, quite variable, both in terms of speed and mechanism: a Na⁺-dependent efflux in excitable cells (catalyzed by NCLX) and a H⁺/Ca²⁺ exchanger in other cells. The efficacy of mitochondrial Ca²⁺ uptake in living cells is strictly dependent on the topological arrangement of the organelles with respect to the source of Ca²⁺ flowing into the cytoplasm, i.e., plasma membrane or intracellular channels. In turn, the structural and functional relationships between mitochondria and other cellular membranes are dictated by the specific architecture of different cells. Mitochondria not only modulate the amplitude and the kinetics of local and bulk cytoplasmic Ca²⁺ changes but also depend on the Ca²⁺ signal for their own functionality, in particular for their capacity to produce ATP. In this review, we summarize the processes involved in mitochondrial Ca²⁺ handling and its integration in cell physiology, highlighting the main common characteristics as well as key differences, in different tissues.