Ca<Superscript>2+</Superscript> Buffering Capacity of Mitochondria After Oxygen-Glucose Deprivation in Hippocampal Neurons

In an earlier study, we showed that mitochondria hyperpolarized after short periods of oxygen-glucose deprivation (OGD), and this response appeared to be associated with subsequent apoptosis or survival. Here, we demonstrated that hyperpolarization following short periods of OGD (30 min; 30OGD group) increased the cytosolic Ca²⁺ ([Ca²⁺]_c) buffering capacity in mitochondria. After graded OGD (0 min (control), 30 min, 120 min), rat cultured hippocampal neurons were exposed to glutamate, evoking Ca²⁺influx. The [Ca²⁺]_c level increased sharply, followed by a rapid increase in mitochondrial Ca²⁺ [Ca²⁺]_m. The increase in the [Ca²⁺]_m level accompanied a reduction in the [Ca²⁺]_c level. After reaching a peak, the [Ca²⁺]_c level decreased more rapidly in the 30OGD group than in the control group. This buffering reaction was pronounced in the 30OGD group, but not in the 120OGD group. The enhanced buffering capacity of the mitochondria may be linked to preconditioning after short-term ischemic episodes.     

Salt stress-induced programmed cell death via Ca<Superscript>2+</Superscript>-mediated mitochondrial permeability transition in tobacco protoplasts

The change in cytosolic free concentration of calcium ([Ca²⁺]_cyt) plays a key role in regulating apoptosis in animal cells. In our experiment, we tried to investigate the function of Ca²⁺ in programmed cell death (PCD) in tobacco (Nicotiana tobacum, cultivar BY-2) protoplasts induced by salt stress. An obvious increase in [Ca²⁺]_cyt was observed a few minutes after treatment and the onset of a decrease in mitochondrial membrane potential (ΔΨ_m) was also observed before the appearance of PCD, pre-treatment of protoplasts with EGTA or LaCl₃ effectively retarded the increase in [Ca²⁺]_cyt, which was concomitant with the decrease in the percentage of cell death and higher ΔΨ_m, pre-treatment with cyclosporine A (CsA) also effectively retarded the increase in [Ca²⁺]_cyt, the decrease in ΔΨ_m and the onset of PCD. All these results suggest that Ca²⁺ is a necessary element in regulating PCD and the increase in [Ca²⁺]_cyt and the opening of mitochondrial permeability transition pore (MPTP) could promote each other in regulating PCD in tobacco protoplasts induced by salt stress.Jiusheng Lin and Yuan Wang-These authors contributed equally for this work.     

Physiological role of mitochondrial Ca2+ transport

A model has been proposed in which mitochondrial Ca²⁺ ion transport serves to regulate mitochondrial matrix free Ca²⁺ ([Ca²⁺]_m), with the advantage to the animal that this allows the regulation of pyruvate dehydrogenase and the tricarboxylate cycle in response to energy demand. This article examines recent evidence for dehydrogenase activation and for increases in [Ca²⁺]_m in response to increased tissue energy demands, especially in cardiac myocytes and in heart. It critiques recent results on beat-to-beat variation in [Ca²⁺]_m in cardiac muscle and also briefly surveys the impact of mitochondrial Ca^2– transport on transient changes in cytosolic free Ca²⁺ in excitable tissues. Finally, it proposes that a failure to elevate [Ca²⁺]_m sufficiently in response to work load may underlie some cardiomyopathies of metabolic origin.     

Intracellular targeting of the photoprotein aequorin: A new approach for measuring,in living cells,Ca<Superscript>2+</Superscript> concentrations in defined cellular compartments

We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca²⁺ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a “mitochondrially-targeted” aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca²⁺, [Ca²⁺]_m, were obtained, which showed that [Ca²⁺]_m is low at rest (<0.5 μM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria “sense” very accurately the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and after cell stimulation [Ca²⁺]_m rises to values capable of activating the Ca²⁺-sensitive mitochondrial dehydrogenases.     

Mitochondrial free Ca2+ concentration in living cells

Evidence has accrued during the past two decades that mitochondrial Ca²⁺ plays an important role in the regulation of numerous cell functions such as energy metabolism. This implies that mitochondrial Ca²⁺ transport systems might be able to relay the changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_c) into mitochondrial matrix for regulating biochemical activities. To substantiate this idea, measurements of intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) become essential. In this article, we review the results from recent studies attempting to measure [Ca²⁺]_m in living cells. In addition, the significance of each study is discussed.     

Calcium and ATP handling in human NADH:Ubiquinone oxidoreductase deficiency

Proper cell functioning requires precise coordination between mitochondrial ATP production and local energy demand. Ionic calcium (Ca²⁺) plays a central role in this coupling because it activates mitochondrial oxidative phosphorylation (OXPHOS) during hormonal and electrical cell stimulation. To determine how mitochondrial dysfunction affects cytosolic and mitochondrial Ca²⁺/ATP handling, we performed life-cell quantification of these parameters in fibroblast cell lines derived from healthy subjects and patients with isolated deficiency of the first OXPHOS complex (CI). In resting patient cells, CI deficiency was associated with a normal mitochondrial ([ATP]_m) and cytosolic ([ATP]_c) ATP concentration, a normal cytosolic Ca²⁺ concentration ([Ca²⁺]_c), but a reduced Ca²⁺ content of the endoplasmic reticulum (ER). Furthermore, cellular NAD(P)H levels were increased, mitochondrial membrane potential was slightly depolarized, reactive oxygen species (ROS) levels were elevated and mitochondrial shape was altered. Upon stimulation with bradykinin (Bk), the peak increases in [Ca²⁺]_c, mitochondrial Ca²⁺ concentration ([Ca²⁺]_m), [ATP]_c and [ATP]_m were reduced in patient cells. In agreement with these results, ATP-dependent Ca²⁺ removal from the cytosol was slower. Here, we review the interconnection between cytosolic, endoplasmic reticular and mitochondrial Ca²⁺ and ATP handling, and summarize our findings in patient fibroblasts in an integrative model.     

Ageing‐associated increase in SGLT2 disrupts mitochondrial/sarcoplasmic reticulum Ca2+ homeostasis and promotes cardiac dysfunction

The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age‐associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co‐transporter gene (SGLT2) in disrupted cellular Ca²⁺‐homeostasis, and mitochondrial dysfunction in age‐associated cardiac dysfunction. In contrast to younger rats (6‐month of age), older rats (24‐month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca²⁺ ([Ca²⁺]_i) overload, indicative of disrupted cellular Ca²⁺‐homeostasis. Interestingly, increased [Ca²⁺]_i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged‐cardiomyocytes also displayed high Na⁺/Ca²⁺‐exchanger (NCX) activity and blood glucose levels compared with young‐controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age‐associated defects in [Ca²⁺]_i‐homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca²⁺‐loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca²⁺]_i‐homeostasis. Our studies support the notion that interventions that modulate SGLT2‐activity can provide benefits in maintaining [Ca²⁺]_i and cardiac function with advanced age.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

Role of mitochondrial calcium transport in the control of substrate oxidation

This paper reviews the model of the control of mitochondrial substrate oxidation by Ca²⁺ ions. The mechanism is the activation by Ca²⁺ of four mitochondrial dehydrogenases, viz: glycerol 3-phosphate dehydrogenase, the pyruvate dehydrogenase multienzyme complex (PDH), NAD-linked isocitrate dehydrogenase (NAD-IDH) and 2-oxoglutarate dehydrogenase (OGDH). This results in the increase, or near-maintenance, of mitochondrial NADH/NAD ratios in the activated state, depending upon the tissue and the degree of "downstream" activation by Ca²⁺, likely at the level of the F₁F₀ ATP-ase. Higher values of the redox span of the respiratory chain allow for greatly increased fluxes through oxidative phosphorylation with a minimal drop in protonmotive force and phosphorylation potential. As PDH, NAD-IDH and OGDH are all located within the inner mitochondrial membrane, it is changes in matrix free Ca²⁺ ( [Ca²⁺]m ) which act as a signal to these activities. In this article, we review recent work in which ([Ca²⁺]m) is measured in cells and tissues, using different techniques, with special emphasis on the question of the degree of damping of ([Ca²⁺]m) relative to changes in cytosol free Ca²⁺ in cells with rapid transients in cytosol Ca²⁺, e.g. cardiac myocytes. Further, we put forward the point of view that the failure of mitochondrial energy transduction to keep pace with cellular energy needs in some forms of heart failure may involve a failure of ([Ca²⁺]m) to be raised adequately to allow the activation of the dehydrogenases. We present new data to show that this is so in cardiac myocytes isolated from animals suffering from chronic, atreptozocin-induced diabetes. This raises the possibility of therapy based upon partial inhibition of mitochondrial Ca²⁺ efflux pathways, thereby raising ([Ca²⁺]m) at a given, time-average value of cytosol free Ca²⁺.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca²⁺ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ 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⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ 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.     

Mitochondrial Free [Ca] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca²⁺] ([Ca²⁺]_m) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca²⁺]_m is affected by H₂PO₄⁻ (P_i), Mg²⁺, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca²⁺]_m, membrane potential, redox state, matrix volume, pH_m, and O₂ consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg²⁺ and P_i. Energized mitochondria showed a dose-dependent increase in [Ca²⁺]_m after adding CaCl₂ equivalent to 20, 114, and 485 nM extramatrix free [Ca²⁺] ([Ca²⁺]_e); this uptake was attenuated at higher buffer Mg²⁺. Adding ADP transiently increased [Ca²⁺]_m up to twofold. The ADP effect on increasing [Ca²⁺]_m could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg²⁺ or P_i. Oligomycin largely reduced the increase in [Ca²⁺]_m by ADP compared to control, and [Ca²⁺]_m did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca²⁺]_m increase. Adding CaCl₂ had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca²⁺]_e. These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca²⁺]_m largely due to the interaction of matrix Ca²⁺ with ATP, ADP, P_i, and cation buffering proteins in the matrix.     

Characteristics and possible functions of mitochondrial Ca transport mechanisms

Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca²⁺ concentration ([Ca²⁺]_m) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca²⁺]_m also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca²⁺]_m can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca²⁺ is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca²⁺ pulses or transients. Mitochondria can also sequester Ca²⁺ from these transients so as to modify the shape of Ca²⁺ signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca²⁺ transport mechanisms and the PTP. The characteristics of these mechanisms of Ca²⁺ transport and a discussion of how they might function are described in this paper.     

The gatekeepers of mitochondrial calcium influx: MICU1 and MICU2

The receptor‐evoked Ca²⁺ signal is sensed and translated by mitochondria. Physiological cytoplasmic Ca²⁺ ([Ca²⁺]_c) oscillations result in mitochondrial Ca²⁺ ([Ca²⁺]_m) oscillations, while large and sustained [Ca²⁺]_c increase results in a pathologic increase in basal [Ca²⁺]_m and in Ca²⁺ accumulation. The physiological [Ca²⁺]_m signal regulates [Ca²⁺]_c and stimulates oxidative metabolism, while excess Ca²⁺ accumulation causes cell stress leading to cell death. [Ca²⁺]_m is determined by Ca²⁺ uptake mediated by the mitochondria Ca²⁺ uniporter (MCU) channel and by Na⁺‐ and H⁺‐coupled Ca²⁺ extrusion 1 .     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

The ups and downs of mitochondrial calcium signalling in the heart

Regulation of intramitochondrial free calcium ([Ca²⁺]_m) is critical in both physiological and pathological functioning of the heart. The full extent and importance of the role of [Ca²⁺]_m is becoming apparent as evidenced by the increasing interest and work in this area over the last two decades. However, controversies remain, such as the existence of beat-to-beat mitochondrial Ca²⁺ transients; the role of [Ca²⁺]_m in modulating whole-cell Ca²⁺ signalling; whether or not an increase in [Ca²⁺]_m is essential to couple ATP supply and demand; and the role of [Ca²⁺]_m in cell death by both necrosis and apoptosis, especially in formation of the mitochondrial permeability transition pore. The role of [Ca²⁺]_m in heart failure is an area that has also recently been highlighted. [Ca²⁺]_m can now be measured reasonably specifically in intact cells and hearts thanks to developments in fluorescent indicators and targeted proteins and more sensitive imaging technology. This has revealed interactions of the mitochondrial Ca²⁺ transporters with those of the sarcolemma and sarcoplasmic reticulum, and has gone a long way to bringing the mitochondrial Ca²⁺ transporters to the forefront of cardiac research. Mitochondrial Ca²⁺ uptake occurs via the ruthenium red sensitive Ca²⁺ uniporter (mCU), and efflux via an Na⁺/Ca²⁺ exchanger (mNCX). The purification and cloning of the transporters, and development of more specific inhibitors, would produce a step-change in our understanding of the role of these apparently critical but still elusive proteins. In this article we will summarise the key physiological roles of [Ca²⁺]_m in ATP production and cell Ca²⁺ signalling in both adult and neonatal hearts, as well as highlighting some of the controversies in these areas. We will also briefly discuss recent ideas on the interactions of nitric oxide with [Ca²⁺]_m.