Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro

The role of Ca(2+) as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca(2+) sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa(3) ratio. The half-maximal concentration (K(1/2)) of SR ATPase was 335 nM Ca(2+). The ATP synthesis dependence was similar with a K(1/2) of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca(2+) increased thapsigargin-sensitive ATP production (maximum approximately 5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca(2+) concentrations (0 to approximately 500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca(2+) that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca(2+) is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.     

The mitochondrial Na(+)/Ca(2+) exchanger

Powered by the steep mitochondrial membrane potential Ca(2+) permeates into the mitochondria via the Ca(2+) uniporter and is then extruded by a mitochondrial Na(+)/Ca(2+) exchanger. This mitochondrial Ca(2+) shuttling regulates the rate of ATP production and participates in cellular Ca(2+) signaling. Despite the fact that the exchanger was functionally identified 40 years ago its molecular identity remained a mystery. Early studies on isolated mitochondria and intact cells characterized the functional properties of a mitochondrial Na(+)/Ca(2+) exchanger, and showed that it possess unique functional fingerprints such as Li(+)/Ca(2+) exchange and that it is displaying selective sensitivity to inhibitors. Purification of mitochondria proteins combined with functional reconstitution led to the isolation of a polypeptide candidate of the exchanger but failed to molecularly identify it. A turning point in the search for the exchanger molecule came with the recent cloning of the last member of the Na(+)/Ca(2+) exchanger superfamily termed NCLX (Na(+)/Ca(2+)/Li(+) exchanger). NCLX is localized in the inner mitochondria membrane and its expression is linked to mitochondria Na(+)/Ca(2+) exchange matching the functional fingerprints of the putative mitochondrial Na(+)/Ca(2+) exchanger. Thus NCLX emerges as the long sought mitochondria Na(+)/Ca(2+) exchanger and provide a critical molecular handle to study mitochondrial Ca(2+) signaling and transport. Here we summarize some of the main topics related to the molecular properties of the Na(+)/Ca(2+) exchanger, beginning with the early days of its functional identification, its kinetic properties and regulation, and culminating in its molecular identification.     

An integrated model of cardiac mitochondrial energy metabolism and calcium dynamics

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an important role in matching energy supply with demand in cardiac myocytes.     

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.     

Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.     

Mitochondrial Ca2+ homeostasis in human NADH:ubiquinone oxidoreductase deficiency

NADH:ubiquinone oxidoreductase or complex I is a large multisubunit assembly of the mitochondrial inner membrane that channels high-energy electrons from metabolic NADH into the electron transport chain (ETC). Its dysfunction is associated with a range of progressive neurological disorders, often characterized by a very early onset and short devastating course. To better understand the cytopathological mechanisms of these disorders, we use live cell luminometry and imaging microscopy of patient skin fibroblasts with mutations in nuclear-encoded subunits of the complex. Here, we present an overview of our recent work, showing that mitochondrial membrane potential, Ca(2+) handling and ATP production are to a variable extent impaired among a large cohort of patient fibroblast lines. From the results obtained, the picture emerges that a reduction in cellular complex I activity leads to a depolarization of the mitochondrial membrane potential, resulting in a decreased supply of mitochondrial ATP to the Ca(2+)-ATPases of the intracellular stores and thus to a reduced Ca(2+) content of these stores. As a consequence, the increase in cytosolic Ca(2+) concentration evoked by a Ca(2+) mobilizing stimulus is decreased, leading to a reduction in mitochondrial Ca(2+) accumulation and ensuing ATP production and thus to a hampered energization of stimulus-induced cytosolic processes.     

Recombinant expression of the Ca(2+)-sensitive aspartate/glutamate carrier increases mitochondrial ATP production in agonist-stimulated Chinese hamster ovary cells

The Ca(2+)-sensitive dehydrogenases of the mitochondrial matrix are, so far, the only known effectors to allow Ca2+ signals to couple the activation of plasma membrane receptors to the stimulation of aerobic metabolism. In this study, we demonstrate a novel mechanism, based on Ca(2+)-sensitive metabolite carriers of the inner membrane. We expressed in Chinese hamster ovary cells aralar1 and citrin, aspartate/glutamate exchangers that have Ca(2+)-binding sites in their sequence, and measured mitochondrial Ca2+ and ATP levels as well as cytosolic Ca2+ concentration with targeted recombinant probes. The increase in mitochondrial ATP levels caused by cell stimulation with Ca(2+)-mobilizing agonists was markedly larger in cells expressing aralar and citrin (but not truncated mutants lacking the Ca(2+)-binding site) than in control cells. Conversely, the cytosolic and the mitochondrial Ca2+ signals were the same in control cells and cells expressing the different aralar1 and citrin variants, thus ruling out an indirect effect through the Ca(2+)-sensitive dehydrogenases. Together, these data show that the decoding of Ca2+ signals in mitochondria depends on the coordinate activity of mitochondrial enzymes and carriers, which may thus represent useful pharmacological targets in this process of major pathophysiological interest.     

Uncoupling protein 3 (UCP3) modulates the activity of Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) by decreasing mitochondrial ATP production

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.     

Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.     

Molecular identity and functional properties of the mitochondrial na+/ca2+ exchanger

The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca(2+) shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca(2+) signaling, and determines cell fate. Mitochondrial Ca(2+) transport is mediated primarily by two major transporters: a Ca(2+) uniporter that mediates Ca(2+) uptake and a Na(+)/Ca(2+) exchanger that subsequently extrudes mitochondrial Ca(2+). In this minireview, we focus on the specific role of the mitochondrial Na(+)/Ca(2+) exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.     

Metabolic network control of oxidative phosphorylation: multiple roles of inorganic phosphate

Phosphate (Pi) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that Pi controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F1F0-ATPase in heart and skeletal muscle mitochondria. In these studies the effect of Pi was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. Pi enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. Pi oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of Pi was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [Pi]. These results demonstrate that Pi signaling results in the balanced modulation of oxidative phosphorylation, by influencing both deltaGH+ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems.     

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.     

Essential role of aralar in the transduction of small Ca2+ signals to neuronal mitochondria

Aralar, the neuronal Ca(2+)-binding mitochondrial aspartate-glutamate carrier, has Ca(2+) binding domains facing the extramitochondrial space and functions in the malate-aspartate NADH shuttle (MAS). Here we showed that MAS activity in brain mitochondria is stimulated by extramitochondrial Ca(2+) with an S(0.5) of 324 nM. By employing primary neuronal cultures from control and aralar-deficient mice and NAD(P)H imaging with two-photon excitation microscopy, we showed that lactate utilization involves a substantial transfer of NAD(P)H to mitochondria in control but not aralar-deficient neurons, in agreement with the lack of MAS activity associated with aralar deficiency. The increase in mitochondrial NAD(P)H was greatly potentiated by large [Ca(2+)](i) signals both in control and aralar-deficient neurons, showing that these large signals activate the Ca(2+) uniporter and mitochondrial dehydrogenases but not MAS activity. On the other hand, small [Ca(2+)](i) signals potentiate the increase in mitochondrial NAD(P)H only in control but not in aralar-deficient neurons. We concluded that neuronal MAS activity is selectively activated by small Ca(2+) signals that fall below the activation range of the Ca(2+) uniporter and plays an essential role in mitochondrial Ca(2+) signaling.     

Vascular smooth muscle mitochondria at the cross roads of Ca(2+) regulation

Mitochondria play an essential role in the regulation of vascular smooth muscle Ca(2+) signaling being simultaneously integrated in the regulation of ion channels and Ca(2+) transporters, oxygen radical production, metabolite recycling and intracellular redox potential. Mitochondria buffer Ca(2+) from cytoplasmic microdomains to alter the spatio-temporal pattern of Ca(2+) gradients following Ca(2+)-influx and Ca(2+)-release, and thus control site-specific, Ca(2+)-dependent ion channel activation and inactivation. The sub-cellular localization of mitochondria in conjunction with tissue-specific channel expression is fundamental to vascular heterogeneity. The mitochondrial electron transport chain recycles metabolic intermediates that modulate cellular redox potential and produces oxygen radicals in proportion to oxygen tension. Perturbation of specific complexes within the transport chain can affects NADH:NAD and ATP:ADP ratios and radical production, which can in turn influence second messenger metabolism, ion channel gating and Ca(2+)-transporter activity. Mitochondria thus provide the common ground for cross-talk between these regulatory systems that are mutually sensitive to one another. This cross-talk between signaling systems provides a means to render the physiological regulation of vascular tone responsive to complex stimulation by paracrine and endocrine factors, blood pressure and flow, tissue oxygenation and metabolic state.     

Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.     

Simulation of cardiac work transitions, in vitro: effects of simultaneous Ca2+ and ATPase additions on isolated porcine heart mitochondria

During increases in cardiac work there are net increases in cytosolic [Ca(2+)] and ATP hydrolysis by myofiliments and ion transport ATPases. However, it is still unclear what role Ca(2+)or the ATP hydrolysis products, ADP and Pi, have on the regulation of mitochondrial ATP production. In this study, work jumps were simulated by simultaneous additions of Ca(2+) and ATPase to porcine heart mitochondria. The net effects on the mitochondrial ATP production were monitored by simultaneously monitoring respiration (mVo2), [NADH], [ADP] and membrane potential (deltapsi) at 37 degrees C. Addition of exogenous ATPase (300 mlU.ml(-1))]ATP (3.4 mM) was used to generate a 'resting' background production of ADP. This resting metabolic rate was 200% higher than the quiescent rate while [NADH] and deltapsi were reduced. Subsequent ATPase additions (1.3IU.ml(-)) were made with varying amounts of Ca(2+)(0 to 535 nM) to simulate step increases in cardiac work. Ca(2+) additions increased mVo2 and depolarized deltapsi, and were consistent with an activation of Fo/F1)ATPase. In contrast, Ca(2+) reduced the [NADH] response to the ATPase addition, consistent with Ca(2+)-sensitive dehydrogenase activity (CaDH). The calculated free ADP response to ATPase decreased \2-fold in the presence of Ca(2+). The addition of 172nM free Ca(2+)] ATPase increased mVo2 by 300% (P<0.05, n=8) while deltapsi decreased by 14.9+/-0.1 mV without changes in [NADH] (P > or =0.05, n=8), consistent with working heart preparations. The addition of Ca(2+) and ATPase combined increased the mitochondrial ATP production rate with changes in deltapsi, NADH and [ADP], consistent with an activation of CaDH and F o /F(1)ATPase activity. These balancing effects of ATPase activity and [Ca(2+)] may explain several aspects of metabolic regulation in the heart during work transitions in vivo.     

The role of Ca signaling in the coordination of mitochondrial ATP production with cardiac work

The heart is capable of balancing the rate of mitochondrial ATP production with utilization continuously over a wide range of activity. This results in a constant phosphorylation potential despite a large change in metabolite turnover. The molecular mechanisms responsible for generating this energy homeostasis are poorly understood. The best candidate for a cytosolic signaling molecule reflecting ATP hydrolysis is Ca²⁺. Since Ca²⁺ initiates and powers muscle contraction as well as serves as the primary substrate for SERCA, Ca²⁺ is an ideal feed-forward signal for priming ATP production. With the sarcoplasmic reticulum to cytosolic Ca²⁺ gradient near equilibrium with the free energy of ATP, cytosolic Ca²⁺ release is exquisitely sensitive to the cellular energy state providing a feedback signal. Thus, Ca²⁺ can serve as a feed-forward and feedback regulator of ATP production. Consistent with this notion is the correlation of cytosolic and mitochondrial Ca²⁺ with work in numerous preparations as well as the localization of mitochondria near Ca²⁺ release sites. How cytosolic Ca²⁺ signaling might regulate oxidative phosphorylation is a focus of this review. The relevant Ca²⁺ sensitive sites include several dehydrogenases and substrate transporters together with a post-translational modification of F1-FO-ATPase and cytochrome oxidase. Thus, Ca²⁺ apparently activates both the generation of the mitochondrial membrane potential as well as utilization to produce ATP. This balanced activation extends the energy homeostasis observed in the cytosol into the mitochondria matrix in the never resting heart.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.