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INVITED REVIEW

Calcium, ATP, and ROS: a mitochondrial love-hate triangle

Departments of Anesthesiology, Pharmacology, and Physiology and Mitochondrial Research Interest Group, University of Rochester Medical Center, Rochester New York 14642

	ABSTRACT

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

 
The mitochondrion is at the core of cellular energy metabolism, being the site of most ATP generation. Calcium is a key regulator of mitochondrial function and acts at several levels within the organelle to stimulate ATP synthesis. However, the dysregulation of mitochondrial Ca²⁺ homeostasis is now recognized to play a key role in several pathologies. For example, mitochondrial matrix Ca²⁺ overload can lead to enhanced generation of reactive oxygen species, triggering of the permeability transition pore, and cytochrome c release, leading to apoptosis. Despite progress regarding the independent roles of both Ca²⁺ and mitochondrial dysfunction in disease, the molecular mechanisms by which Ca²⁺ can elicit mitochondrial dysfunction remain elusive. This review highlights the delicate balance between the positive and negative effects of Ca²⁺ and the signaling events that perturb this balance. Overall, a "two-hit" hypothesis is developed, in which Ca²⁺ plus another pathological stimulus can bring about mitochondrial dysfunction.

mitochondria; reactive oxygen species; free radicals; apoptosis; neurodegeneration; ischemia; permeability transition

	CA2+ AND MITOCHONDRIAL PHYSIOLOGY

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

Chemically, the stepwise reduction of O₂ (O₂ O₂^–· H₂O₂ OH· H₂O) proceeds via several reactive oxygen species (ROS). These ROS can damage cellular components such as proteins, lipids, and DNA (70), but recent evidence also highlights a specific role in redox cell signaling for mitochondrial ROS (55, 203). In the fine balancing act of aerobic metabolism, mitochondrial ox-phos accomplishes the reduction of O₂ to H₂O while maximizing ATP synthesis and maintaining ROS production to only the amounts required for microdomain cell signaling (19, 87).

In addition to ATP synthesis, mitochondria are the site of other important metabolic reactions, including steroid hormone and porphyrin synthesis, the urea cycle, lipid metabolism, and interconversion of amino acids (39, 141). Mitochondria also play central roles in xenobiotic metabolism, glucose sensing/insulin regulation (113), and cellular Ca²⁺ homeostasis (65, 66), which affects numerous other cell signaling pathways.

Despite these critical metabolic roles of mitochondria, classic "mitochondriology" was considered a mature field as recently as 1990. However, several important observations have fueled a renaissance in mitochondrial research, including 1) mitochondrial ROS are not just damaging by-products of respiration, but important for cell signaling (19, 23); 2) mitochondrial release of factors such as cytochrome c is an important step in programmed cell death (100, 110, 112); 3) nitric oxide (NO·) is an potent regulator of mitochondrial function (19, 23, 34); 4) mitochondrial morphology is far from static, with the organelles being subject to fission, fusion, and intracellular movement on a rapid timescale (95, 218); and 5) mitochondria actively orchestrate the spatiotemporal profiles of intracellular Ca²⁺, under both physiological and pathological conditions (65, 66). Together these observations suggest an extensive regulatory role for mitochondria in both normal and pathological cell function.

The interplay between the conventional and novel roles of mitochondria has received little consideration, and an examination of recent mitochondrial science reveals several incompatibilities with classic bioenergetic viewpoints. An example is the requirement of ATP for apoptosis (137). How does the cell maintain ATP synthesis in the face of mitochondrial disassembly that occurs during apoptosis? Another example, which is the focus of this review, is the role of Ca²⁺ in regulating organelle function and dysfunction. How can Ca²⁺, a physiological stimulus for ATP synthesis (5, 72, 118), become a pathological stimulus for ROS generation, cytochrome c release, and apoptosis? As will be discussed extensively, this apparent mitochondrial Ca²⁺ paradox revolves around a "two-hit" hypothesis (Fig. 1) in which a concurrent pathological stimulus can turn Ca²⁺ from a physiological to a pathological effector.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Two-hit hypothesis for mitochondrial Ca2+ in physiology and pathology. Under physiological conditions, Ca2+ is beneficial for mitochondrial function. However, in the presence of an overriding pathological stimulus, Ca2+ is detrimental. Similarly, Ca2+ can potentiate a subthreshold pathological stimulus, resulting in pathogenic consequences. See text for full explanation. [Ca2+]m, mitochondrial matrix Ca2+ concentration; ROS, reactive oxygen species.

	MITOCHONDRIAL CA2+ UPTAKE AND RELEASE PATHWAYS

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

Figure 2 outlines the major mechanisms for mitochondrial Ca²⁺ transport, with Ca²⁺ uptake achieved primarily via the mitochondrial Ca²⁺ uniporter (MCU). Uptake is driven by the membrane potential (_m), and therefore the net movement of charge due to Ca²⁺ uptake consumes _m. A recent patch-clamp study suggests the that MCU is a highly selective (K_d < 2 nM) Ca²⁺ channel (99), but attempts to define its molecular nature have been largely unsuccessful. The channel is known mostly for its pharmacological sensitivity to RuRed (127), and a colorless component of RuRed (Ru360) is the active MCU-binding agent (156, 216). Saris et al. (165) identified a 40-kDa glycoprotein of the intermembrane space as an MCU regulatory component, although the transmembrane component of the MCU has been more difficult to isolate, with limited reports of such an entity (124). Interestingly, reverse MCU transport (Ca²⁺ export) was shown to be regulated by Ca²⁺ binding to the outer surface of the inner membrane (86) and was also linked to a soluble intermembrane space component.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Pathways of mitochondrial Ca2+ uptake and export. The respiratory chain is shown with (left to right) complexes I, III, IV, and V. The outer mitochondrial membrane and complex II are omitted for clarity. Where possible, known 3-dimensional (3D) structures obtained from the Protein Data Bank (http://www.rcsb.org/pdb) are shown. UP, Ca2+ uniporter; RaM, rapid-mode Ca2+ uptake; RyR, ryanodine receptor; PTP, permeability transition pore; m, membrane potential.

Two additional mechanisms of Ca²⁺ entry into mitochondria have also been identified. The first, called "rapid-mode" uptake (RaM), occurs on a millisecond timescale and allows fast changes in mitochondrial matrix Ca²⁺ concentration ([Ca²⁺]_m) to mirror changes in the cytosol ([Ca²⁺]_c) (186). Second, we have found (11) that ryanodine receptor isoform (RyR)1 is localized to the inner membrane of mitochondria in excitable cells and have termed this channel "mRyR." Kinetic analysis of the MCU predicts a tetrameric structure like RyR, which exists as a tetramer of 500-kDa subunits (17). Together, mRyR and RaM are thought to underlie the phenomenon of excitation-metabolism coupling, in which [Ca²⁺]_c-induced contraction is matched by [Ca²⁺]_m stimulation of ox-phos (see below).

A fast response of [Ca²⁺]_m to [Ca²⁺]_c requires rapid Ca²⁺ efflux from the mitochondrial matrix, and several mechanisms exist for this purpose (65). Primarily, Ca²⁺ efflux is achieved by exchange for Na⁺, which is in turn pumped out of the matrix in exchange for protons (Fig. 2). Thus both Ca²⁺ uptake and efflux from mitochondria consume _m and are therefore reliant on H⁺ pumping by the respiratory chain to maintain this driving force. In addition to these pathways of Ca²⁺ efflux, an additional mechanism exists in the form of the permeability transition (PT) pore (10). The PT pore is assembled from a group of preexisting proteins in the mitochondrial inner and outer membranes (38), with Ca²⁺ binding sites on the matrix side of the inner membrane believed to regulate pore activity. Normally, "flickering" of the PT pore between open and closed states serves to release Ca²⁺ from the matrix (84, 141, 205). However, prolonged PT pore opening due to [Ca²⁺]_m overload can result in pathological consequences (38).

	CA2+ AS POSITIVE EFFECTOR OF MITOCHONDRIAL FUNCTION.

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

 
The primary role of mitochondrial Ca²⁺ is the stimulation of ox-phos (5, 40, 72, 118, 123). As shown in Fig. 3, this occurs at many levels, including allosteric activation of pyruvate dehydrogenase, isocitrate dehydrogenase, and -ketoglutarate dehydrogenase (118), as well as stimulation of the ATP synthase (complex V) (40), -glycerophosphate dehydrogenase (211), and the adenine nucleotide translocase (ANT) (123). Overall the effect of elevated [Ca²⁺]_m is the coordinated upregulation of the entire ox-phos machinery, resulting in faster respiratory chain activity and higher ATP output. Thus mitochondrial ATP output can be changed to meet the cellular ATP demand. An example of this is -adrenergic stimulation in cardiomyocytes signaling the demand for increased contractility. The concomitant upregulation of ox-phos via [Ca²⁺]_m elevation provides the ATP needed for increased contractile force.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Ca2+ activation of the TCA cycle and oxidative phosphorylation. Thin arrows represent metabolic pathways/reactions; thick arrows represent actions of Ca2+. The outer membrane is omitted for clarity. Where possible, known 3D structures obtained from the Protein Data Bank are shown. For -glycerophosphate (-GP) dehydrogenase (-GPDH), the cytosolic isoform structure is shown. Succ, succinate; -KG, -ketoglutarate; Isocit, isocitrate; Cit, citrate; OAA, oxaloacetate; Mal, malate; Fum, fumarate; Ac-CoA, acetyl coenzyme A; Pyr, pyruvate; PDH, pyruvate dehydrogenase; Acon, aconitase; CS, citrate synthase; MDH, malate dehydrogenase; ICDH, isocitrate dehydrogenase; -KGDH, -ketoglutarate dehydrogenase; DHAP, dihydroxyacetone phosphate; CxI–V, complexes I–V; SDH, succinate dehydrogenase.

Overall, it appears that Ca²⁺ is a global positive effector of mitochondrial function, and thus any perturbation in mitochondrial or cytosolic Ca²⁺ homeostasis will have profound implications for cell function, for example, at the level of ATP synthesis. Also, it cannot be ignored that Ca²⁺, particularly at the high concentrations experienced in pathology, appears to have several negative effects on mitochondrial function, as discussed in the following sections.

	CA2+ OVERLOAD AND PERMEABILITY TRANSITION PORE

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

 
In contrast to the beneficial effects of Ca²⁺, the PT pore embodies the pathological effects of Ca²⁺ on mitochondria. The PT pore, as described nearly 25 years ago by Haworth and Hunter (81–83), is an assembly of preexisting proteins of the inner and outer mitochondrial membranes into a large conductance channel permeable to solutes of <1,500 Da. Debate still surrounds the composition of the PT pore, and although a full discussion is beyond the scope of this review, Fig. 4 shows the key components. Along with VDAC, ANT, and cyclophilin D (Cyp-D), several proteins are believed to regulate the pore (for review see Ref. 38).

View larger version (25K): [in this window] [in a new window]   Fig. 4. The mitochondrial permeability transition pore. The putative components of the pore are shown, although the exact arrangement and stoichiometry are not known. Where possible, known 3D structures from the Protein Data Base are shown. In the case of cyclophilin D (Cyp-D), the structure of Cyp-A is shown. ANT, adenine nucleotide translocase; PBR, peripheral benzodiazepine receptor; VDAC, voltage-dependent anion channel; RSH, reduced thiol; RSSR, thiol disulfide.

The role of the PT pore in pathological cell injury and death has been cemented by the discovery that opening of this pore is mechanistically linked to cytochrome c release, a key event in apoptosis (112). Despite the recent popularity of this research topic, it is worth noting that Knyazeva et al. (100) discovered mitochondrial cytochrome c release in ischemic liver nearly 30 years ago! Several studies suggest a role for the PT pore in this process, including findings that 1) PT pore inhibitors (e.g., cyclosporin A) inhibit cytochrome c release and apoptosis (176, 222), 2) the Bcl family proteins have been shown to functionally interact with PT pore components such as VDAC (132, 177, 200), and 3) the loss of _m is a hallmark of apoptotic cell death and is thought to signal the recruitment of Bcl family proteins to the mitochondrion (44).

Despite strong evidence linking the PT pore, cytochrome c release, and apoptosis, the precise mechanism of cytochrome c release is still unknown and is likely to be dependent on cell type, apoptotic stimulus, and precise cellular conditions. Several non-PT pore-mediated mechanisms of cytochrome c release may exist, and it is important to emphasize that cytochrome c does not exit through the PT pore itself. Also, although in vitro PT pore opening results in mitochondrial swelling and outer membrane rupture (38, 143), this is unlikely to occur in vivo, because mitochondrial swelling is not typically observed in apoptosis (although it is in necrosis) (111). This is in agreement with our data from time-course experiments in isolated mitochondria showing that cytochrome c release is temporally unrelated to swelling (24). Overall, the PT pore can be considered an important signaling pathway leading to cytochrome c release, but its involvement in the physical mechanism of cytochrome c release is still debated.

Cytochrome c is highly positively charged and binds to negatively charged cardiolipin on the outside of the inner membrane. There are also binding sites on respiratory complexes III and IV, and it has been shown that cytochrome c release is a two-step process (143), involving release of the protein from its inner membrane binding sites followed by outer membrane translocation. In addition, PT pore opening appears to be accompanied by a burst of ROS (61, 62), and this phenomenon is proposed to be involved in the autoamplification phase of the pore (62, 102). Because ROS can cause oxidation of cardiolipin, changing its physical properties (204), this may also enhance cytochrome c release (62, 143). Therefore, it is possible that high [Ca²⁺] in the intermembrane space may enhance cytochrome c release by competing it off binding sites (89), through a mechanism involving ROS oxidation of cardiolipin. Within the overall context of Ca²⁺ as a mitochondrial pathological stimulus, we have shown (21) that PT pore triggering by ROS is potentiated by Ca²⁺. This is an example of the two-hit hypothesis (Fig. 1), in which the combination of Ca²⁺ plus a pathological stimulus such as ROS can elicit mitochondrial dysfunction.

In addition, recent evidence has suggested that cytochrome c can bind to the endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP₃R), rendering the channel insensitive to autoinhibition by high [Ca²⁺]_c and resulting in enhanced ER Ca²⁺ release (14, 15). Thus Ca²⁺-induced mitochondrial cytochrome c release may propagate apoptotic signaling by promoting further Ca²⁺ overload. The close proximity between the ER and mitochondria (115) facilitates this cross-talk and is discussed in the next section.

	MITOCHONDRIAL MORPHOLOGY, DYNAMICS, AND ER COORDINATION

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

 
Morphological alterations of mitochondria have been observed in multiple pathologies, implying a close relationship between mitochondrial structure and function. However, even in normal cells (95) mitochondria display dynamic tubular networks undergoing constant fission, fusion, and cytoskeletal trafficking (8, 9, 138). These networks appear to intertwine closely with tubules of the ER. Although the reason for dynamic mitochondrial behavior is not fully understood, it is believed to ensure appropriate mitochondrial distribution to provide ATP to localized cytosolic regions.

Mitochondrial fission requires the dynamin-like protein DLP1, a large GTPase that is transiently recruited to mitochondria by interactions with the outer membrane protein hFis1 (13, 142, 151, 179, 180, 217–219). Fusion is also mediated by GTPases; mitofusin (Mfn) is an outer membrane anchored GTPase and a homolog of the Drosophila "fuzzy onion" protein (31, 67, 85, 104, 158, 163, 164). Two mammalian Mfn isoforms (Mfn1 and Mfn2) have both redundant and distinct functions, forming homo- and heteromeric complexes (31). The coordinated fusion of inner and outer membranes is thought to occur when Fzo1p (a yeast homolog of fuzzy onion) forms a complex with Mgm1p, which is an inner membrane-associated dynamin family GTPase (174, 213).

Mitochondrial fission and fusion must occur at balanced levels to maintain normal morphology (173), and an imbalance results in excessive fragmentation or tubulation, with pathological consequences. Increased fission appears to be a prerequisite for cytochrome c release, because mitochondrial morphology in many apoptotic cells changes from tubular networks to a fragmented phenotype (95). For example, hFis1 overexpression causes mitochondrial fragmentation, cytochrome c release, and apoptosis (90, 217), whereas a dominant-negative DLP1 prevents cytochrome c release in staurosporine-induced apoptosis (57). Similarly, Mfn^–/– mice die at midgestation (31), and the human homolog of Mgm1p has been identified as OPA1, which causes hereditary blindness when mutated (4, 46). Notably, DLP1 and Mfn2 have been shown to closely appose to proapoptotic Bax (94), and the development of mitochondrial fragmentation has been demonstrated in several apoptotic cell systems, indicating that the mitochondrial fission/fusion machinery directly or indirectly interacts with apoptotic components to regulate cell death (18).

Recent studies have suggested coordination between Ca²⁺ signaling of the ER and mitochondria, facilitated by strategic location of mitochondria at sites of ER Ca²⁺ release (115). The ER is the major intracellular Ca²⁺ store, and regulated Ca²⁺ release from this organelle is essential for cellular signaling. However, in pathological situations this may be detrimental to mitochondrial function, and Ca²⁺ released during the ER stress response may promote mitochondrial fragmentation and apoptosis. As noted above, it has also been reported that cytochrome c can bind to the IP₃R, causing further ER Ca²⁺ release (14, 15). Because mitochondrial cytochrome c release during apoptosis is an "all-or-nothing" event occurring within a rapid time frame (116), it has been suggested that Ca²⁺ is the coordinating signal for cytochrome c release (15).

Several questions arise from these studies, such as the following: How can the cell avoid cytochrome c release during normal fission/fusion events? Is cytochrome c release in apoptosis merely an accidental by-product of mitochondrial fission (leakage?), or is fission a deliberate part of the mechanism of cytochrome c release? It is possible that when the distance between two fission sites is extremely close, the short lipid bilayer may not be able to provide sufficient curvature for membrane sealing, causing membrane rupture and leakage. In support of this notion, cytochrome c release on hFis1 overexpression is not inhibited by classic inhibitors of PT pore opening (90), suggesting that the PT pore is not involved in the mechanism of cytochrome c release under these conditions.

An additional link between ER Ca²⁺, mitochondria, and apoptosis is provided by studies showing that overexpression of antiapoptotic Bcl-2 lowers the steady-state ER [Ca²⁺] ([Ca²⁺]_ER), leading to lower mitochondrial Ca²⁺ uptake on IP₃R activation (56, 150). In addition, proapoptotic Bax^–/–-Bak^–/– fibroblasts also display lower [Ca²⁺]_ER (172). These data suggest that increased ER Ca²⁺ release is a key event in apoptosis regulated by both pro- and antiapoptotic Bcl proteins. Furthermore, a direct role for ER Ca²⁺ in regulating mitochondrial fission has been proposed; ectopic expression of the p20 fragment of BAP31 (an ER protein that binds to Bcl-2 and Bcl-X_L) causes mitochondrial translocation of DLP1, resulting in fragmentation and cytochrome c release (18). The signal for mitochondrial recruitment of DLP1 is likely to be elevated [Ca²⁺]_m, because inhibition of mitochondrial Ca²⁺ uptake attenuates the fragmentation (18).

In summary, coordination between ER and mitochondria occurs both spatiotemporally and biochemically and has the potential to regulate mitochondrial morphology. Perturbation of the ER can lead to [Ca²⁺]_m overload, fragmentation, and apoptosis. This is a further example of the two-hit hypothesis (Fig. 1), in which a normal mitochondrial process (fission/fusion) can have pathological consequences in the presence of high [Ca²⁺].

	MITOCHONDRIAL ROS GENERATION

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

 
Mitochondria are a significant source of ROS, although the assumption that 1–2% of all O₂ consumed ends up as ROS is likely an overestimate. Although an extensive review of mitochondrial ROS generation is beyond the scope of this review (see Refs. 88 and 202), an outline of the major sources within the organelle serves to highlight that this is both a physiological and a pathological phenomenon and will aid in the understanding of its regulation by Ca²⁺.

The primary ROS made by mitochondria is superoxide (O₂^–·), which is converted to H₂O₂ either by spontaneous dismutation or by the enzyme superoxide dismutase (SOD). H₂O₂ can be further transformed to OH· in the presence of metal ions by Fenton chemistry, although metal chaperone proteins in the mitochondrial matrix (37, 106) likely prevent this from occurring in the organelle.

The main source of O₂^–· in mitochondria is the ubisemiquinone radical intermediate (QH·), formed during the Q cycle at the Q_O site of complex III (128, 192, 201). Generation of ROS is accelerated by complex III inhibitors distal to this sitg (e.g., antimycin A), although evidence that inhibition further down the respiratory chain (e.g., at complex IV) can also elevate ROS is sparse (19). The majority of O₂^–· from Q_O is made facing the intermembrane space (192), leading to suggestions that O₂^–· may be released through VDAC into the cytosol (71). However, the intermembrane space contains both Cu/Zn-SOD (139) and 20 mM cytochrome c, which can be reduced by O₂^–· and pass the electrons on to complex IV (114). Indeed, exogenous O₂^–· is an excellent substrate for generation of _m and ATP synthesis (114), and mitochondria have been championed as a significant cellular ROS sink (63, 147, 224). Some HO₂· is probably also made from Q_O at the intermembrane space, because the acidic dissociation constant (pK_a) of O₂^–· is 4.8 (45). Uncharged HO₂· can pass through membranes but is highly reactive and therefore likely initiates membrane lipid oxidation (45). Furthermore, the spontaneous dismutation of HO₂· (to H₂O₂) is five to eight orders of magnitude faster than for O₂^–·. Under certain conditions, it has been shown that some O₂^–·/HO₂· from Q_O may reach the matrix space (128, 192), but Mn-SOD in this compartment would rapidly convert it to H₂O₂.

Complex I is also a source of ROS, although the mechanism of generation is less clear than for complex III. Rotenone and other distal complex I inhibitors can cause O₂^–· generation facing the matrix side of the inner membrane (201, 192, 108), where Mn-SOD would convert it to H₂O₂. Recent reports suggest that glutathionylation of complex I (197) or phosphorylation by PKA (145, 153) can elevate ROS generation, but the physiological or pathological significance of this is unclear. In vitro, electrons entering at complex II (succinate dehydrogenase) can flow backward through complex I to make ROS (201, 202). In vivo this would be prevented by forward electron flow through complex I from NADH, except under pathological conditions in which NADH is depleted (see below).

Another important regulator of mitochondrial ROS is _m. The generation of ROS is exponentially dependent on _m (187), and both chemical uncouplers (e.g., 2,4-dinitrophenol) (140) and the novel uncoupling proteins (UCPs) (133) appear to decrease mitochondrial ROS generation in whole cells and organs, although in vitro experiments with isolated mitochondria have revealed opposing effects (27). Interestingly, as we originally hypothesized in 1998 (20), it has now been shown that ROS can stimulate mitochondrial uncoupling (51, 130) and that the processes of uncoupling and ROS generation exist in a feedback loop (20, 130, 125).

Our understanding of the roles of mitochondrially derived ROS has been transformed in recent years. Previously ROS were thought to be damaging by-products of respiration, responsible for oxidative damage and contributing to aging (for review see Ref. 75). However, recent evidence has shown that mitochondrially derived ROS are important for a multitude of cell signaling processes (23). Illustratively, cytosolic Cu/Zn-SOD^–/– mice are viable, whereas mitochondrial Mn-SOD^–/– mice are not (79). The fact that Cu/Zn-SOD cannot compensate for the loss of Mn-SOD (121) suggests a much more subtle, compartmentalized role for these enzymes than mere damage limitation and ROS detoxification. Several recent studies have shown that perturbations in mitochondrial ROS generation can affect diverse redox signaling pathways such as the cell cycle (167), cell proliferation (98), apoptosis (108), metalloproteinase function (155), oxygen sensing (30), protein kinases (154, 155), phosphatases (152), and transcription factors (78).

	CA2+ AND MITOCHONDRIAL ROS

TOP ABSTRACT Ca2+ AND MITOCHONDRIAL... MITOCHONDRIAL Ca2+ UPTAKE AND... Ca2+ AS POSITIVE EFFECTOR... Ca2+ OVERLOAD AND PERMEABILITY... MITOCHONDRIAL MORPHOLOGY,... MITOCHONDRIAL ROS GENERATION Ca2+ AND MITOCHONDRIAL ROS Ca2+ AND PT PORE... CLOSING THE... EXAMPLES OF PATHOLOGICAL [Ca2+]M... EXAMPLES OF PATHOLOGICAL [Ca2+]M... THERAPEUTIC STRATEGIES CONCLUSIONS GRANTS REFERENCES

 
At the heart of understanding how Ca²⁺ can be both a physiological and a pathological effector of mitochondrial function is the issue of how Ca²⁺ modulates mitochondrial ROS generation. In this section, the theoretical and experimental considerations underlying this "mitochondrial Ca²⁺ paradox" are discussed. Because the QH· intermediate in the Q cycle is a significant source of ROS (vide supra), two related parameters that can regulate ROS generation are 1) the effective concentration of QH·, which is increased when the distal respiratory chain is inhibited, and 2) the frequency of QH· occurrence, which is increased when the respiratory chain turns over more quickly. Thus both stimulation and inhibition of mitochondria can result in enhanced ROS generation.

Figure 5 shows the theoretical mechanisms by which Ca²⁺ can enhance ROS generation. Stimulation of the TCA cycle and ox-phos by Ca²⁺ would enhance ROS output by making the whole mitochondrion work faster and consume more O₂. Indeed, mitochondrial ROS generation correlates well with metabolic rate (148, 184), suggesting that a faster metabolism simply results in more respiratory chain electron leakage. In addition, Ca²⁺ stimulation of nitric oxide synthase (NOS) (3) generates NO·, which inhibits complex IV (34), and this would enhance ROS generation at Q_O. Indeed, we hypothesized (19) that a major physiological role for NO· is the regulation of mitochondrial ROS output. Thus mitochondria can act as a "redox signaling box," converting an NO· signal into an ROS signal (19). This signaling axis operates within a physiological window of NO· concentrations, and disruption of this axis by pathological levels of NO· is detrimental to mitochondrial ATP synthesis and cell function (178). Furthermore, NO·, in conjunction with high [Ca²⁺]_m, can inhibit mitochondrial complex I (91), and this is another example of the two-hit hypothesis (Fig. 1) because NO· and Ca²⁺ are not detrimental to complex I function. Although other inhibitors of complex I are known to enhance its ROS generation (108, 197), it is not known whether this is the case for NO· + Ca²⁺. Furthermore, Ca²⁺ can enhance cytochrome c dislocation from the mitochondrial inner membrane, either by competing for cardiolipin binding sites or by inducing the PT pore, and this results in an effective block of the respiratory chain at complex III, which would enhance ROS generation (62, 143). Another possibility is that Ca²⁺ can perturb mitochondrial antioxidant status. We have observed (unpublished data) that mitochondrial GSH is released very early in Ca²⁺-induced PT pore opening, suggesting that Ca²⁺-exposed mitochondria may generate more ROS because of diminished GSH levels.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Mechanisms for Ca2+ stimulation of mitochondrial ROS generation. Ca2+ stimulation of the TCA cycle (1) will enhance electron flow into the respiratory chain, and Ca2+ stimulation of nitric oxide synthase (NOS) and subsequent nitric oxide (NO·) generation (2) would inhibit respiration at complex IV (3). These events would enhance ROS generation from the Q cycle (4). In addition, NO· and Ca2+ can inhibit complex I, possibly enhancing ROS generation from this complex (5). Ca2+ also dissociates cytochrome c (cyt-c) from the inner membrane cardiolipin (6) and at high concentrations triggers PTP opening and cytochrome c release across the outer membrane (7). The subsequent inhibition at complex III (8) would enhance ROS generation at the Q cycle (4) Complex II is omitted from this diagram for clarity.

Addition of Ca²⁺ to rat heart mitochondria with a complex III inhibitor (antimycin A) caused a sharp increase in ROS generation, similar to that observed with FCCP (27). Because Ca²⁺ uptake causes mild uncoupling of mitochondria (_m dissipation), some of its effects on ROS generation may be due to this uncoupling effect. The mechanism by which uncouplers enhance ROS generation is unclear (201) but may involve perturbation of the pH gradient (pH) across the mitochondrial membrane, which would affect the topology of HO₂·/O₂^–· generation (45).

In contrast, addition of Ca²⁺ to brain mitochondria in the presence of antimycin A did not stimulate ROS generation from complex III, but Ca²⁺ did elicit a complex I ROS generation when rotenone was present (185). Notably, this effect was reversible by addition of EGTA, suggesting that the PT pore (irreversible cytochrome c loss) is not involved and hinting at a reversible Ca²⁺ binding site that may modulate ROS generation. This is supported by data showing that in liver mitochondria with antimycin A the ability of Ca²⁺ to stimulate ROS generation is inhibited by the local anesthetic dibucaine and by Mg²⁺ (102). The authors attribute this effect to the ability of these species to displace Ca²⁺ from its membrane protein binding sites (102).

A further study on Ca²⁺ and brain mitochondria showed that in the presence of rotenone plus complex I substrates, Ca²⁺ decreased ROS generation (188). This was not due to uncoupling, because Ca²⁺-induced loss of _m lasted only a few seconds whereas ROS generation was depressed for several minutes. With oligomycin present, Ca²⁺ caused large-scale uncoupling that lasted for several minutes. Notably, this was reversed by EGTA, again suggesting a reversible Ca²⁺ binding site that regulates ROS (188).

Overall these experiments suggest that in a tissue-specific manner, Ca²⁺ diminishes ROS from both complexes I and III under normal conditions and enhances ROS when these complexes are inhibited. The exact mechanism of Ca²⁺-induced ROS generation is unclear, although it may involve changes in the three-dimensional conformation of the respiratory complexes. Indeed, Ca²⁺ is reported to alter the spectrum of cytochromes a/a₃ in isolated complex IV (212), and we have shown (21) that Ca²⁺ exposes novel mitochondrial targets for nitration by ONOO^–, consistent with protein conformational changes. Ultimately, differences in the subunit composition of the respiratory complexes between tissues may underlie the tissue specificity of Ca²⁺ effects on ROS generation.

Together, Ca²⁺-mediated events may have additive effects on ROS generation. For example, Ca²⁺ stimulation of the TCA cycle would increase electron flux into the proximal respiratory chain while Ca²⁺-induced cytochrome c release would simultaneously inhibit the distal respiratory chain. Although thus far our two-hit model (Fig. 1) has concentrated on the convergence of Ca²⁺ with other pathological stimuli, it should be recognized that Ca²⁺ itself is a pathological stimulus and in some cases Ca²⁺ is both the first and second "hit," i.e., Ca²⁺ can modulate its own pathological effects.

	CA2+ AND PT PORE OPENING—A CAUSE OR EFFECT OF ROS GENERATION?

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Apoptosis is reportedly accompanied by a burst of ROS (29, 61, 62, 102), although the recent discovery that the ROS fluorescent probe dichlorofluorescin (DCF) can be oxidized directly by cytochrome c raises the possibility of experimental artifacts due to apoptotic cytochrome c release (25). Studies on isolated mitochondria with other ROS probes have provided more information on this scenario, showing that a complex interplay exists between PT pore opening and ROS generation in response to Ca²⁺ (29, 61, 102, 204). What is still unclear is whether ROS generation is merely a consequence of PT pore opening and cytochrome c release or is an integral part of the signaling machinery of PT pore opening. Although evidence abounds that exogenously added ROS can trigger the PT pore (21, 29, 144), it is unclear whether endogenously generated ROS can perform this function. In support of a role for endogenous ROS in the PT pore, antioxidants and thiol reagents such as DTT are able to inhibit Ca²⁺-induced PT pore opening (38). Furthermore, Castilho et al. (29) report that Ca²⁺-induced PT pore opening is sensitive to the concentration of O₂ (the substrate for O₂^–· generation) and is inhibited by catalase, suggesting a role for H₂O₂ generation.

Theoretically, PT pore-mediated cytochrome c release would enhance ROS generation by inhibiting complex III, but experimental evidence for this is limited. A key issue is how much cytochrome c is required to sustain respiration. Studies on isolated complex IV have shown that very low levels of cytochrome c (1 µM) can sustain activity (7), suggesting that with millimolar levels of cytochrome c in the intermembrane space (39, 171), significant loss would not result in respiratory inhibition. Indeed, it appears that mitochondrial electron transport and ATP synthesis are maintained during apoptosis (208), presumably to allow execution of the apoptotic program, which requires ATP (137). However, it is worth noting that cytochrome c is not the only factor lost from mitochondria on PT pore opening. Nearly 100 proteins are lost from the intermembrane space (146), as well as GSH and other matrix solutes. Any of these components (especially GSH) could be responsible for enhanced ROS generation during apoptosis. Another idea developed within this context is that cytochrome c is an antioxidant, because it is an effective scavenger of O₂^–· and is readily diffusible and recycled in the cell (147). This raises the possibility that cytochrome c release during Ca²⁺-/ROS-mediated mitochondrial dysfunction may have evolved as a protective strategy to scavenge extramitochondrial ROS before they reach the organelle.

Overall, the interplay among Ca²⁺, ROS, and ATP in both the life and the death of the cell is extremely complex. This interplay is illustrated in Fig. 6, showing that the roles of each player in this "love-hate triangle" are different depending on the physiological/pathological status of the cell.

View larger version (31K): [in this window] [in a new window]   Fig. 6. The mitochondrial Ca2+/ATP/ROS triangle. Solid arrows denote physiological effects; dotted arrows denote pathological effects. Words alongside the arrows describe specific events mediating the positive or negative effect. At the center of the triangle are factors that can orchestrate these effects. For full details, see text. SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; ox-phos, oxidative phosphorylation; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; mRyR, mitochondrial RyR; IP3R, inositol 1,4,5-trisphosphate.

	CLOSING THE TRIANGLE—ATP/ROS/CA2+

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A further level of cross-talk is suggested by Zoccarato et al. (224), who report that mitochondrial scavenging of external H₂O₂ is inhibited by Ca²⁺. They attribute this to a Ca²⁺-induced inactivation of glutathione reductase (GR) and glutathione peroxidase (GPX). The authors speculate that succinate-driven reverse electron flow through complex I, generating NADH (the substrate for GR and GPX), may underlie this phenomenon (224).

Finally, the importance of ATP in Ca²⁺ homeostasis cannot be understated, because the maintenance of ion gradients in all cell types depends on ATP (26). Therefore, any disturbance in ATP levels will have a large impact on SR, ER, and plasma membrane (PM) Ca²⁺ pumps. Overall, as highlighted in Fig. 6, it is clear that ATP, ROS, and Ca²⁺ exist in a triangular network, with each having the ability to control the others. The following sections discuss two pathological examples of this triangle in action.

	EXAMPLES OF PATHOLOGICAL [CA2+]M OVERLOAD—CARDIAC ISCHEMIA-REPERFUSION INJURY

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Overload of [Ca²⁺]_m is a key event in cardiac ischemia-reperfusion (I/R) injury (189, 190), with the end point being opening of the PT pore, cytochrome c release, and apoptosis/necrosis (38, 68). Figure 7 outlines the events preceding [Ca²⁺]_m overload. Under normal conditions, the PM Na⁺/H⁺ exchanger (NHE) equilibrates internal and external Na⁺ and H⁺ and the PM Na⁺/Ca²⁺ exchanger keeps [Ca²⁺]_c low. During ischemia, lactic acidosis forces the NHE to import external Na⁺, resulting in cytosolic Na⁺ overload (96). Subsequently, the PM Na⁺/Ca²⁺ exchanger is forced into reverse mode to dispose of excess Na⁺, resulting in [Ca²⁺]_c overload. This Ca²⁺ is then taken up by mitochondria, resulting in [Ca²⁺]_m overload and all its consequences as described above (for reviews see Refs. 38 and 68).

View larger version (36K): [in this window] [in a new window]   Fig. 7. Mitochondria, Ca2+, and ROS in myocardial ischemia-reperfusion (I/R) injury. A: the events leading up to [Ca2+]m overload during I/R. Briefly, a drop in pH triggers Na+/H+ exchanger (NHE)-1-mediated Na+ influx, resulting in Na+ overload. Reverse-mode Na+/Ca2+ exchange across the plasma membrane (PM) then results in Ca2+ overload and subsequent [Ca2+]m overload. Ca2+ cycling across the mitochondrial membrane can lead to propagation and amplification of [Ca2+]m overload. In addition, ATP deficiency prevents adequate Ca2+ export by Ca2+-ATPases. B: cross-talk between the pathways of mitochondrial dysfunction, ROS, and Ca2+ overload in I/R injury. The precipitating events (boxes) are acidosis, elevated AMP, and ROS generation. A feed-forward loop is hypothesized, encompassing mitochondrial ROS generation, MAPKs, and [Ca2+]m overload. For further explanation, see text. RNS, reactive nitrogen species; AMPK, AMP-dependent protein kinase.

In support of a role for MAP kinases in the cross-talk between ROS and Ca²⁺, JNK has been shown to be elevated within mitochondria during I/R (77), raising the possibility of direct phosphorylation of mitochondrial respiratory complexes, with subsequent effects on ROS generation. In addition, p38 can inhibit the mitochondrial RuRed-sensitive Ca²⁺ uniporter (126), suggesting a protective role for this MAP kinase. However, in neurons, p38 can mediate the translocation of proapoptotic BH₃-containing proteins to mitochondria (59), suggesting opposing roles for p38 in different cell types.

Mitochondrial Ca²⁺ overload does not have to result in PT pore opening and cytochrome c release to be detrimental to heart function. For example, the inhibition of complex I that occurs in I/R injury is dependent on [Ca²⁺]_m, because it is prevented by RuRed (73, 74). Several other mitochondrial enzymes are inhibited in response to I/R or ROS exposure, including aconitase, -ketoglutarate dehydrogenase, and complexes III and IV (105, 161). Because the heart is almost entirely dependent on mitochondrially generated ATP for its contractile energy, a defect at any level of the mitochondrial ox-phos machinery can have profound implications for contractile function on reperfusion.

Another important mitochondrial consequence of I/R injury is an increase in the H⁺ leak (uncoupling) of the inner membrane (Fig. 7B; Ref. ). This may involve the generation of RNS such as ONOO^–, which we demonstrated (22) can increase H⁺ leak. In addition, O₂^–· and lipid oxidation products can activate mitochondrial UCPs (51, 125, 130), although this remains controversial (36). Alternatively, it has been shown that allosteric activation of the ANT by AMP turns it into a H⁺ channel (28). Notably, AMP is elevated in I/R, and inhibitors of adenylate kinase (which catalyzes the reaction 2ADP ATP + AMP) are of therapeutic benefit in cardiac I/R (81, 168). In addition, AMP-dependent protein kinase is known to activate endothelial NOS (eNOS) (3, 32), thereby leading to generation of NO· and possibly additional effects on mitochondria, MAP kinases, and other signaling pathways. Although at first glance mitochondrial uncoupling might be seen as detrimental to cardiac function because it would inhibit ATP generation, there may be benefits, too. Uncoupling would decrease -dependent Ca²⁺ accumulation and would also decrease ROS generation. Indeed, several groups have demonstrated the efficacy of mitochondrial uncoupling (either chemical or by transfection of UCPs) as a protective strategy in I/R injury (12, 58, 117, 199). In summary, Fig. 7B outlines the roles of [Ca²⁺]_m overload, ROS generation, and diminished ATP in the progression of cardiac mitochondrial dysfunction in I/R.

	EXAMPLES OF PATHOLOGICAL [CA2+]M OVERLOAD—NEURONAL EXCITOTOXICITY

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Excitotoxicity is a phenomenon in which neuronal cells undergo necrosis or apoptosis in response to overexposure to excitatory amino acids such as glutamate (166). In particular, prolonged activation of the N-methyl-D-aspartate (NMDA) receptor leads to massive Ca²⁺ influx, resulting in [Ca²⁺]_c overload and cell death. Presently there are two schools of thought regarding the mechanisms of excitotoxicity: the mitochondrial Ca²⁺ hypothesis and the nuclear poly-ADP-ribose polymerase (PARP) hypothesis, with both theories emphasizing a key role for ROS.

The mitochondrial Ca²⁺ hypothesis of excitotoxicity centers on [Ca²⁺]_m overload as a central event in cell death (50, 135). In support of this, inhibition of [Ca²⁺]_m accumulation with the protonophore FCCP prevents excitotoxic cell death (191). Moreover, during the progression to excitotoxic cell death, a late secondary increase in [Ca²⁺]_c occurs, termed "delayed Ca²⁺ deregulation" (DCD) (134, 135). Although the mechanism of DCD is still under debate, it is agreed that the speed of DCD is strongly dependent on the magnitude of [Ca²⁺]_m accumulation, suggesting that mitochondrial Ca²⁺ release causes DCD (134).

Parallel to the mitochondrial Ca²⁺ hypothesis, a role for PARP-1 in excitotoxicity has been established by showing that PARP-1 inhibitors or genetic knockouts prevent excitotoxicity (149, 194). The mechanism of PARP-1-mediated excitotoxicity has been proposed to involve NMDA-receptor-mediated Ca²⁺ influx, leading to activation of neuronal NOS (nNOS) and excessive production of NO· (43). Overload of [Ca²⁺]_c also activates the protease calpain (33), which converts xanthine dehydrogenase