Increase In Mitochondrial Ca2 Research Articles

Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state

Rapid formation of high-Ca2+ perimitochondrial cytoplasmic microdomains has been shown to evoke mitochondrial Ca2+ signal and activate mitochondrial dehydrogenases, however, the significance of submicromolar cytoplasmic Ca2+ concentrations in the control of mitochondrial metabolism has not been sufficiently elucidated. Here we studied the mitochondrial response to application of Ca2+ at buffered concentrations in permeabilized rat adrenal glomerulosa cells, in an insulin-producing cell line (INS-1/EK-3) and in an osteosarcoma cell line (143BmA-13). Mitochondrial Ca2+ concentration was measured with the fluorescent dye rhod-2 and, using an in situ calibration method, with the mitochondrially targeted luminescent protein mt-aequorin. In both endocrine cell types, mitochondrial Ca2+ concentration increased in response to elevated cytoplasmic Ca2+ concentration (between 60 and 740 nM) and an increase in mitochondrial Ca2+ concentration could be revealed already at a cytoplasmic Ca2+ concentration step from 60–140 nM. Similar responses were observed in the osteosarcoma cell line, although a clearcut response was first observed at 280 nM extramitochondrial Ca2+ only. As examined in glomerulosa cells, graded increases in cytoplasmic Ca2+ concentration were associated with graded increases in the reduction of mitochondrial pyridine nucleotides, consistent with Ca2+-dependent activation of mitochondrial dehydrogenases. Our data indicate that in addition to therecognized role of high-Ca2+ cytoplasmic microdomains, also small Ca2+ signals may influence mitochondrialmetabolism.

Helium-Neon laser irradiation of hepatocytes can trigger increase of the mitochondrial membrane potential and can stimulate c-fos expression in a Ca2+-dependent manner.

To gain some insight into the photostimulation of isolated hepatocytes irradiated with Helium-Neon (He-Ne) laser light certain biochemical events were studied with respect to two mechanisms: i) the direct light dependent activation of certain biochemical events investigated in intact cells and isolated mitochondria, ii) the indirect stimulation of processes per se light independent. Irradiation of either isolated hepatocytes or isolated rat liver mitochondria was carried out with He-Ne laser (wavelength, 632.8 nm; fluence, 0.24 J cm-2; fluence rate, 12 mW cm-2). Changes in mitochondrial membrane potential in isolated hepatocytes were monitored using the cationic probe safranine. The c-fos expression was studied by Northern blot and immunoblot analysis. As a result of irradiation, increase of the mitochondrial membrane potential was found to occur in irradiated hepatocytes both in the presence or in the absence of CaCl2. The hyperpolarization of the mitochondrial membrane is assumed to cause an increase in mitochondrial Ca2+ uptake that was measured in isolated mitochondria. Finally, an increase in c-fos expression was found in irradiated hepatocytes when incubated in the presence of CaCl2. This paper gives additional information on the mechanism by which He-Ne laser light, either directly or in a cascade-like effect dependent on increase in cell Ca2+, can cause cell stimulation.

Characterization of Mitochondrial Function During Cell Injury

Mitochondria play an important role in apoptotic and necrotic cell death. Depending on the stimulus, a variety of mitochondrial dysfunctions can trigger onset of cell death. Assessment of mitochondrial dysfunction in living cells in situis important to understand the sequence of events producing cell injury and death. Laser scanning confocal microscopy, a technique that creates submicron thin optical slices through living cells and tissues, allows us to monitor several mitochondrial events simultaneously in intact single cells over time.During oxidative stress, we monitored mitochondrial redox status, Ca2+ increase, reactive oxygen species (ROS), the mitochondrial permeability transition, and mitochondrial depolarization in cultured hepatocytes. When an oxidant chemical, t-BuOOH, was added to cultured hepatocytes, oxidation of mitochondrial pyridine nucleotides occurred as visualized directly by ultraviolet confocal microscopy of NAD(P)H autofluorescence. This event was followed by an increase of mitochondrial Ca2+ measured by the Ca2+-indicating fluorophore, Rhod-2.

Heterogeneity of second messenger levels in living cells.

Over the last years we have utilised chimeras from aequorin and green fluorescent protein (GFP) to monitor the dynamics of second messenger levels in living cells. In this contribution we address two problems, i.e. the complexity of Ca2+ handling by mitochondria and the localization of cAMP signalling. As to the first, we here demonstrate that physiological increases in mitochondrial Ca2+, monitored with selectively localized recombinant aequorin, concern a sub-population of organelles that is stably and selectively associated with the endoplasmic reticulum. As to cAMP, we describe the use of a novel probe to monitor its changes in living cells, that takes advantage of the phenomenon of fluorescence resonance energy transfer (FRET) between suitable GFPs linked to the regulatory and catalytic subunits of protein kinase A (PKA). When cAMP is low the two fluorophores are in close proximity and generate FRET while increasing levels of cAMP determine progressive reduction of FRET as the two subunits (linked to the GFPs) diffuse apart. We also demonstrate that by using such cAMP sensor, localized increase of this second messenger can be observed upon selective stimulation of plasma membrane receptors.

Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids.

Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose- and Ca(2+)-dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca(2+) may protect the hepatocyte against bile acid-induced necrosis.

Brain Mitochondrial Responses to Postischemic Reperfusion: A Role for Calcium and Hydrogen Peroxide?

During early recirculation following global brain ischemia, mitochondria are exposed to markedly elevated Ca²⁺ concentrations and a short-lived production of reactive oxygen species, including hydrogen peroxide (H₂O₂). A brief increase in mitochondrial Ca²⁺ and a subsequent increase in mitochondrial glutathione content have been observed. In the present study, we have confirmed the increase in mitochondrial glutathione in a rat model of global forebrain ischemia. This change was not inhibited by treatment of the rats with FK506, contrasting with our previous finding that cyclosporin A partially blocked the increase. These results suggest that induction of the mitochondrial permeability transition may be necessary for the increase in glutathione content in these organelles. To further investigate possible mitochondrial responses during early postischemic reperfusion, mitochondria isolated from normal brain were exposed to Ca²⁺ and H₂O₂, under conditions similar to those in intact cells. Respiratory activity was substantially modified when the mitochondria were exposed to Ca²⁺ and H₂O₂ together. Two distinct and largely noninteracting mechanisms apparently accounted for the responses to these agents. The effects of Ca²⁺, but not H₂O₂, were inhibited by cyclosporin A, again implicating the permeability transition in some of the mitochondrial changes.

Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria.

Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)-mediated cytosolic Ca2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. To measure the amount of Ca2+ taken up by the mitochondria, we used a novel approach that involves simultaneous fluorescence imaging of mitochondrial and cytosolic [Ca2+] in permeabilized H9c2 myotubes and RBL-2H3 mast cells. Communication between sarco-endoplasmic reticulum (SR/ER) and mitochondria is maintained in these permeabilized cells, as evidenced by the large RyR/IP3R-driven mitochondrial matrix [Ca2+] and NAD(P)H signals and also by preservation of the morphology of the SR/ER-mitochondrial junctions. Ca2+ was released from the SR/ER by addition of saturating caffeine or IP3 and subsequently thapsigargin (Tg), an inhibitor of SR/ER Ca2+ pumps. The amount of Ca2+ transmitted to the mitochondria was determined by measuring increases of global [Ca2+] in the incubation medium (cytosolic [Ca2+] ([Ca2+]c)). Mitochondrial Ca2+ uptake was calculated from the difference between [Ca2+]c responses recorded in the absence and presence of uncoupler or from [Ca2+]c elevations evoked by uncoupler or ionophore applied after complete Ca2+ mobilization from the SR/ER. [Ca2+]c increases were calibrated by adding Ca2+ pulses to the permeabilized cells. In H9c2 cells, caffeine induced partial mobilization of SR Ca2+ and mitochondria accumulated 26% of the released Ca2+. Sequential application of caffeine and Tg elicited complete discharge of SR Ca2+ without further increase in mitochondrial Ca2+ uptake. In RBL-2H3 mast cells, IP3 by itself elicited complete discharge of the ER Ca2+ store and the increase of the ionophore-releasable mitochondrial Ca2+ content reached 50% of the Ca2+ amount mobilized by IP3 + Tg. Thus, RyR/IP3R direct a substantial fraction of released Ca2+ to the mitochondria.

Elevation of Mitochondrial Calcium by Ryanodine-sensitive Calcium-induced Calcium Release

Calcium is an important regulator of mitochondrial function. Since there can be tight coupling between inositol 1,4, 5-trisphosphate-sensitive Ca(2+) release and elevation of mitochondrial calcium concentration, we have investigated whether a similar relationship exists between the release of Ca(2+) from the ryanodine receptor and the elevation of mitochondrial Ca(2+). Perfusion of permeabilized A10 cells with inositol 1,4, 5-trisphosphate resulted in a large transient elevation of mitochondrial Ca(2+) to about 8 microm. The response was inhibited by heparin but not ryanodine. Perfusion of the cells with Ca(2+) buffers in excess of 1 microm leads to large increases in mitochondrial Ca(2+) that are much greater than the perfused Ca(2+). These increases, which average around 10 microm, are enhanced by caffeine and inhibited by ryanodine and depletion of the intracellular stores with either orthovanadate or thapsigargin. We conclude that Ca(2+)-induced Ca(2+) release at the ryanodine receptor generates microdomains of elevated Ca(2+) that are sensed by adjacent mitochondria. In addition to ryanodine-sensitive stores acting as a source of Ca(2+), Ca(2+)-induced Ca(2+) release is required to generate efficient elevation of mitochondrial Ca(2+).

Mitochondrial Ca2+ uptake, efflux, and sarcolemmal damage in Ca2+-overloaded cultured rat cardiomyocytes.

The purpose of this study was to determine mitochondrial Ca2+ accumulation and its possible role in initiation of mitochondrial permeability transition (MPT) and sarcolemmal damage in Ca2+-overloaded cardiomyocytes. Cellular Ca2+ overload, generated secondary to ouabain or p-chloromercuribenzoate-stimulated cell Na+ concentration increase, induced Ca2+ accumulation in mitochondria (approximately 3/4 of total net uptake) as identified by kinetic analysis and verified by use of mitochondrial inhibition. Mitochondrial Ca2+ uptake was followed by a rapid Ca2+ efflux ( approximately 1 mmol . kg dry wt-1 . min-1) that can be best explained by efflux via Ca2+-dependent nonspecific pores. Cell ATP concentration was stable during mitochondrial Ca2+ uptake and decreased in parallel with Ca2+ efflux. In addition, sarcolemmal damage was not related to the increase in mitochondrial Ca2+ concentration per se, but rather connected with the extent of Ca2+ efflux from the mitochondria. A decrease in the rate of this Ca2+ efflux, indicating also a decrease in a subpopulation of mitochondria with open pores, was followed by decreased sarcolemmal damage. Both dithiothreitol and cyclosporin A decreased rapid Ca2+ efflux and inhibited sarcolemmal damage, implicating MPT as an important component in the mechanism of sarcolemmal damage.

Model of beta-cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables.

In the preceding article [Am. J. Physiol. 274 (Cell Physiol. 43): C1158-C1173, 1998], we describe the development of a kinetic model for the interaction of mitochondrial Ca2+ handling and electrical activity in the pancreatic beta-cell. Here we describe further results of those simulations, focusing on mitochondrial variables, the rate of respiration, and fluxes of metabolic intermediates as a function of D-glucose concentration. Our simulations predict relatively smooth increases of O2 consumption, adenine nucleotide transport, oxidative phosphorylation, and ATP production by the tricarboxylic acid cycle as D-glucose concentrations are increased from basal to 20 mM. On the other hand, we find that the active fraction of pyruvate dehydrogenase saturates, due to increases in matrix Ca2+, near the onset of bursting electrical activity and that the NADH/NAD+ ratio in the mitochondria increases by roughly an order of magnitude as glucose concentrations are increased. The mitochondrial ATP/ADP ratio increases by factor of < 2 between the D-glucose threshold for bursting and continuous spiking. According to our simulations, relatively small changes in mitochondrial membrane potential (approximately 1 mV) caused by uptake of Ca2+ are sufficient to alter the cytoplasmic ATP/ADP ratio and influence ATP-sensitive K+ channels in the plasma membrane. In the simulations, these cyclic changes in the mitochondrial membrane potential are due to synchronization of futile cycle of Ca2+ from the cytoplasm through mitochondria via Ca2+ uniporters and Na+/Ca2+ exchange. Our simulations predict steady mitochondrial Ca2+ concentrations on the order of 0.1 microM at low glucose concentrations that become oscillatory with an amplitude on the order of 0.5 microM during bursting. Abrupt increases in mitochondrial Ca2+ concentration > 5 microM may occur during continuous electrical activity.

Effects of trimetazidine on the calcium transport and oxidative phosphorylation of isolated rat heart mitochondria.

Trimetazidine (TMZ) added in vitro to isolated cardiac mitochondria at concentrations 10-100 microM in the presence of 25-100 nM extramitochondrial Ca2+ increased Ca2+ uptake and matrix Ca2+ concentration. This effect was less evident in the presence of physiologically Na+ and Mg2+ extramitochondrial concentrations since only 100 microM TMZ was able to increase mitochondrial Ca2+ entry in the presence of 100 nM Ca2+. The drug stimulated a Ca(2+)-cooperative effect on mitochondrial Ca2+ transport, but did not modify the rate of Ca2+ egress stimulated by 10 mM NaCl. An increase in mitochondrial Ca2+ level produced by TMZ enhanced oxoglutarate dehydrogenase activity and then ATP synthesis, particularly when 50 nM extramitochondrial Ca2+ was used. These data suggest that a possible cardiac mechanism of action of TMZ at mitochondrial level could support ATP synthesis by elevating the mitochondrial Ca2+ level.

The mitochondrial permeability transition in toxic, hypoxic and reperfusion injury.

Opening of a non-specific, high conductance permeability transition pore or megachannel in the inner mitochondrial membrane causes onset of the mitochondrial permeability transition, which is characterized by mitochondrial swelling, depolarization and uncoupling. Inducers of the permeability transition include Ca2+, oxidant stress and a permissive pH greater than 7.0. Blockers include cyclosporin A, trifluoperazine and pH < 7. Using laser scanning confocal microscopy, we developed techniques to visualize onset of the mitochondrial permeability transition in situ in living cells. In untreated cells, the permeability transition pore is continuously closed and does not 'flicker' open. By contrast, the pore opens in liver and heart cells after exposure to oxidant chemicals, calcium ionophore, hypoxia and ischemia/reperfusion, causing mitochondrial uncoupling and aggravation of ATP depletion. In injury to hepatocytes from tert-butylhydroperoxide, an analog of lipid hydroperoxides generated during oxidative stress, onset of the mitochondrial permeability transition is preceded by oxidation of mitochondrial pyridine nucleotides, mitochondrial generation of oxygen radicals and an increase of mitochondrial Ca2+, all inducers of the mitochondrial permeability transition. In ischemia, the acidosis of anaerobic metabolism protects strongly against cell death. During reperfusion, recovery of pH to normal levels is a stress that actually precipitates cell killing. Onset of the mitochondrial permeability transition may be responsible, in part, for this pH-dependent injury, or pH paradox. The mitochondrial permeability transition may also be responsible for a variety of pathological phenomena. In particular, the mitochondrial permeability transition may underlie Reye's syndrome and Reye's-like drug toxicities. In conclusion, multiple mechanisms contribute to cell injury after hypoxia, ischemia/reperfusion and toxic chemicals, but a common final pathway leading to acute cellular necrosis may be ATP depletion after mitochondrial failure. One important mechanism causing mitochondrial failure is the mitochondrial permeability transition, which both uncouples oxidative phosphorylation and accelerates ATP hydrolysis. Interventions that block this pH-dependent phenomenon protect against onset of cell death.

A Role for Calcium Influx in the Regulation of Mitochondrial Calcium in Endothelial Cells

By using an endothelial cell line (ECV304), derived from human umbilical vein and transfected with recombinant aequorin targeted to the mitochondrial matrix, we find that stimulation with ATP evokes long lasting increases in mitochondrial Ca2+ ([Ca2+]m) that largely depend on Ca2+ influx. In these cells, the release of stored Ca2+ is inefficient at elevating [Ca2+]m. Consequently it appears that in ECV304 cells, bulk cytosolic Ca2+ ([Ca2+]c) is the main determinant of [Ca2+]m changes. In ECV304 cells < 4% of mitochondria are within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa cells, whereas 14% are within 700 nm of the inner surface of the plasma membrane, compared with < 6% in HeLa cells. Following Ca2+ depletion, readdition of extracellular Ca2+ evokes an increase in [Ca2+]m but not in [Ca2+]c. Under these conditions, microdomains of high [Ca2+]c may occur beneath the plasma membrane of ECV304 cells resulting in the preferential elevation of Ca2+ in mitochondria located in this region. A model is discussed in which the localization of mitochondria with respect to Ca2+ sources is the main determinant of their in situ Ca2+ uptake kinetics. Thus, in any given cell type mitochondria may be localized to suit the energy and metabolic demands of their physiological actions.

The Role of Mitochondria in Ischemic Heart Disease

Summary Mitochondria in the heart play two roles essential for cell survival: ATP synthesis and maintenance of Ca2+ homeostasis. These two processes are driven by the same energy source, the H+ electrochemical gradient (ΔμH). Under aerobic physiologic conditions, mitochondria do not contribute to the beat-to-beat regulation of cytosolic Ca2+, although a Ca2+ transient in mitochondrial matrix has been described. Micromolar increases in mitochondrial Ca2+ concentration stimulate the Krebs cycle and the NADH redox potential and, therefore, ATP synthesis. Trimetazidine has been shown to improve the calcium transient and, in so doing, the overall myocardial energy production. Under pathologic conditions, mitochondrial Ca2+ overload causes a series of vicious cycles that lead to irreversible cell damage. During ischemia, an alteration in intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca2+-transporting capacity. Accordingly, once isolated, even after prolonged ischemia the majority of the mitochondria are able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, and produce an excess of oxygen free radicals. Their membrane pores are stimulated and the capacity for oxidative phosphorylation is irreversibly disrupted. The role of mitochondrial DNA damage in progressive human diseases such as coronary atherosclerosis is receiving growing interest. The sequence of ischemia and reperfusion, through increased production of oxygen free radicals, causes mitochondrial deletions in several areas of the mitochondrial genome. This cumulative mitochondrial DNA damage is associated with induction of nuclear oxidative phosphorylation gene mRNA. These observations support the hypothesis that mitochondria and mitochondrial DNA damage play important roles in ischemic heart disease.

The role of mitochondria in ischemic heart disease.

Mitochondria in the heart play two roles essential for cell survival: ATP synthesis and maintenance of Ca2+ homeostasis. These two processes are driven by the same energy source, the H+ electrochemical gradient (delta microH). Under aerobic physiologic conditions, mitochondria do not contribute to the beat-to-beat regulation of cytosolic Ca2+, although a Ca2+ transient in mitochondrial matrix has been described. Micromolar increases in mitochondrial Ca2+ concentration stimulate the Krebs cycle and the NADH redox potential and, therefore, ATP synthesis. Trimetazidine has been shown to improve the calcium transient and, in so doing, the overall myocardial energy production. Under pathologic conditions, mitochondrial Ca2+ overload causes a series of vicious cycles that lead to irreversible cell damage. During ischemia, an alteration in intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca(2+)-transporting capacity. Accordingly, once isolated, even after prolonged ischemia the majority of the mitochondria are able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, and produce an excess of oxygen free radicals. Their membrane pores are stimulated and the capacity for oxidative phosphorylation is irreversibly disrupted. The role of mitochondrial DNA damage in progressive human diseases such as coronary atherosclerosis is receiving growing interest. The sequence of ischemia and reperfusion, through increased production of oxygen free radicals, causes mitochondrial deletions in several areas of the mitochondrial genome. This cumulative mitochondrial DNA damage is associated with induction of nuclear oxidative phosphorylation gene mRNA. These observations support the hypothesis that mitochondria and mitochondrial DNA damage play important roles in ischemic heart disease.

Calcium ion-dependent signalling and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal stimulation in intact liver cells and its implication for the mitochondrial permeability transition

Hormones that elevate cytosolic Ca 2+ concentrations ([Ca 2+] cyt) often use Ca 2+ as a messenger to activate intramitochondrial metabolic processes. However, the mitochondrial Ca 2+ level also regulates the activation of the mitochondrial permeability transition (MPT), a process that involves the assembly of a high conductance proteinaceous pore across the inner and outer membrane. Studies on intact liver cells indicate that the MPT is a critical step in the cell killing induced by anoxia or respiratory inhibitors. In this study, we used freshly isolated hepatocytes to investigate to what extent the elevation of [Ca 2+] cyt by vasopressin or other agonists causes Ca 2+ accumulation in the mitochondria and how this treatment affects the mitochondrial susceptibility to undergo the MPT. Hepatocytes were incubated with vasopressin, glucagon, or with thapsigargin (an inhibitor of the endoplasmic reticulum Ca 2+ pump) prior to permeabilization with digitonin. Mitochondrial Ca 2+ accumulation was determined by following the ionomycin-induced Ca 2+ release in permeabilized cells and mitochondrial swelling was studied by following cyclosporin A-sensitive light scattering changes induced by phenylarsenoxide and rotenone. The results indicate that agents that elevate [Ca 2+] cyt cause a significant Ca 2+ accumulation in the mitochondria. Excessive Ca 2+ accumulation (> 10-fold increase over basal levels) was obtained with the combination of vasopressin and glucagon or with incubations containing thapsigargin. These conditions were also associated with a marked increase in rotenone-induced mitochondrial swelling. However, the more modest increase in mitochondrial Ca 2+ content after treating cells with vasopressin alone did not enhance the swelling response; instead, vasopressin suppressed mitochondrial swelling compared to control incubations. Vasopressin also partly suppressed the swelling associated with thapsigargin treatment, although it did not significantly affect the Ca 2+ accumulation under these conditions. This effect of vasopressin was mimicked by phorbol ester, suggesting a role for protein kinase C. The data indicate that mitochondrial Ca 2+ accumulation following elevation of [Ca 2+] cyt enhances the susceptibility for activation of the MPT, a response that may increase cell injury during anoxia or in response to other challenges. However, hormones also activate protective responses in the cell that suppress the MPT.

Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes.

1. Shortening and intracellular Ca2+ (Ca2+i) transients were measured in isolated rabbit ventricular myocytes during paired contractures induced by rapid application of 10 mM caffeine. 2. Caffeine-induced contractures relax despite maintained presence of caffeine. In control solution, a second phasic caffeine contracture failed to appear, unless the sarcoplasmic reticulum (SR) was refilled by a series of electrically stimulated twitches during the interval between caffeine exposures. 3. The relaxation of caffeine-induced contractures in 0 Na(+)-0 Ca2+ solution has previously been shown to rely on mitochondrial Ca2+ uptake and sarcolemmal Ca2(+)-ATPase. Thus, a second caffeine contracture (T2) while still in 0 Na(+)-0 Ca2+ was greatly reduced compared to the first one (T1). However, the amplitude of T2 increased exponentially with the time interval, attaining a maximum of approximately 50% of T1 for an interval of 180-300 s, with a time constant (tau) of 41.2 s. Similar results were found for Ca2+i transients (tau = 45 s). 4. Inhibition of the mitochondrial Ca2+ uptake by the oxidative phosphorylation uncoupler, FCCP during T1 dramatically depressed T2. On the other hand, inhibition of the sarcolemmal Ca2(+)-ATPase (by increasing extracellular Ca2+ concentration, [Ca2+]o) resulted in increase of T2. Spermine inclusion during T1 also increased T2, possibly by an increase of mitochondrial Ca2+ uptake. 5. We conclude that Ca2+ taken up by mitochondria during the decline of T1 moves back to the SR after caffeine is removed, with a tau approximately 40 s. 6. Partial intracellular Na+ depletion by prolonged (3 min) perfusion with 0 Na(+)-0 Ca2+ solution before T1 (a) accelerated relaxation and [Ca2+]i decline during T1, and (b) slowed, but did not abolish, the recovery of T2 as the interval was increased. This effect was particularly pronounced when choline was used instead of Li+ as the Na+ substitute. 7. We further conclude that the mitochondrial Na(+)-Ca2+ antiporter influences the rate of net Ca2+ uptake by mitochondria and is also important in Ca2+ efflux from mitochondria during rest.

Mitochondrial energy production and cation control in myocardial ischaemia and reperfusion.

In the heart mitochondria exert two roles essential for cell survival: ATP synthesis and maintainance of Ca2+ homeostasis. These two processes are driven by the same energy source: the H+ electrochemical gradient (delta microH) which is generated by electron transport along the inner mitochondrial membrane. Under aerobic physiological condition mitochondria do not contribute to the beat to beat regulation of cytosolic Ca2+, although Ca2+ transient in mitochondrial matrix has been described. Increases in mitochondrial Ca2+ of mumolars concentration stimulate the Krebs cycle and NADH redox potential and, therefore, ATP synthesis. Under pathological conditions, however, mitochondrial Ca2+ transport and overload might cause a series of vicious cycles leading to irreversible cell damage. Mitochondrial Ca2+ accumulation causes profound alterations in permeability of the inner membrane to solutes, leading to severe mitochondrial swelling. In addition Ca2+ transport takes precedence over ATP synthesis and inhibits utilization of delta microH for energy production. These processes are important to understand the sequence of the molecular events occurring during myocardial reperfusion after prolonged ischaemia which lead to irreversible cell damage. During ischaemia an alteration of intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca2+ transporting capacity. Accordingly, once isolated, even after prolonged ischaemia, the majority of the mitochondria is able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, produce excess of oxygen free radicals, their membrane pores are stimulated and the oxidative phosphorylation capacity is irreversibly disrupted. Most likely, reperfusion provides oxygen to reactivate mitochondrial respiration but also causes large influx of Ca2+ in the cytosol as result of sarcolemmal damage. Mitochondrial Ca2+ transport is therefore stimulated at maximal rates and, as consequence, the equilibrium between ATP synthesis and Ca2+ influx is shifted towards Ca2+ influx with loss of the ability of ATP synthesis.

A digitized-fluorescence-imaging study of mitochondrial Ca2+ increase by doxorubicin in cardiac myocytes.

The objective of the present study was to investigate the role of mitochondrial Ca2+ in doxorubicin-induced cell injury. The effect of doxorubicin on cultured cells was investigated by digitized fluorescence imaging. The Ca2+ sensitive fluorescent dye fura-2 was used to estimate cytosolic, mitochondrial and total cellular Ca2+. Rhodamine 123 was used to estimate the mitochondrial membrane potential, and cellular ATP was determined by h.p.l.c. The data showed that doxorubicin induced greater-than-2-fold increases in mitochondrial Ca2+ before changes in cytosolic Ca2+ could be detected. An increase in mitochondrial Ca2+ paralleled the observed dissipation in mitochondrial membrane potential. Cellular ATP levels appeared to decrease as a result of mitochondrial dysfunction, which in turn produced greater-than-2-fold increases in cytosolic Ca2+. The data suggest that doxorubicin-induced alterations in mitochondrial Ca2+ homoeostasis are associated with a dissipation in energy conservation, which may result in cell injury.

Activation of pyruvate dehydrogenase by electrical stimulation, and low-Na + perfusion of guinea-pig heart

(1) Electrical stimulation (2 Hz) of guinea-pig hearts, perfused with medium containing 11 mM D-glucose plus 0.1 mM octanoate as substrate, resulted in an increase in the percentage of pyruvate dehydrogenase in the active form (PDH a) from 16 to 68%. (2) Rapid isolation of mitochondria by a technique designed to minimize net loss or gain of Ca 2+ revealed an increase in mitochondrial Ca 2+ content of the stimulated hearts, as measured with 45Ca (2.74 ± 0.27 versus 1.37 ± 0.11 nmol/mg protein; stimulated versus rested). (3) Perfusion of rested hearts with a medium containing a reduced Na + concentration (20 mM, with the remainder replaced with Li +) also gave increased values of PDH a content (30.9% versus 16% for the normal, physiological medium). This procedure is known to raise cytosol Ca 2+ concentrations and would be expected to give mitochondrial Ca 2+ loading. (4) These results are consistent with a role of mitochondrial Ca 2+ in activating pyruvate dehydrogenase in the intact heart.