Paul S. Brookes, Yisang Yoon, James L. Robotham, M. W. Anders, and Shey-Shing Sheu

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

ABSTRACT

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

Mitochondrial oxidative phosphorylation (ox-phos) is the major ATP synthetic pathway in eukaryotes. In this process, electrons liberated from reducing substrates are delivered to O2 via a chain of respiratory H+ pumps. These pumps (complexes I-IV) establish a H+ gradient across the inner mitochondrial membrane, and the electrochemical energy of this gradient is then used to drive ATP synthesis by complex V (ATP synthase) (136).

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.


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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

Most of the mitochondrial effects of Ca2+ require its entry across the double membrane into the matrix. Although the mitochondrial outer membrane was thought to be permeable to Ca2+, recent studies suggest that the outer membrane voltage-dependent anion channel (VDAC) is a ruthenium red (RuRed)-sensitive Ca2+ channel and thus serves to regulate Ca2+ entry to mitochondrial intermembrane space (60). Furthermore, transport across the inner membrane is highly regulated (for review see Ref. 65).

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 (Δψm), and therefore the net movement of charge due to Ca2+ uptake consumes Δψm. A recent patch-clamp study suggests the that MCU is a highly selective (Kd < 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.


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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; Δψm, membrane potential.

Das et al. (41) showed that a complex of Ca2+-polyphosphate and {beta}-hydroxybutanoate can form a Ca2+ channel indistinguishable from that in Escherichia coli, raising the possibility that the MCU (by virtue of mitochondrial/bacterial relationships) may be a nonproteinaceous entity. However, the second-order Ca2+ transport kinetics of the MCU suggest a more complex structure with separate activation and transport sites (169, 206). From a physiological perspective, a role was recently demonstrated for p38 MAP kinase in regulating RuRed-sensitive Ca2+ transport (126). Clearly, identification of the molecular nature of the MCU will aid greatly in understanding the physiological and pathological regulation of mitochondrial Ca2+ uptake.

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 Δψm 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.

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 α-ketoglutarate dehydrogenase (118), as well as stimulation of the ATP synthase (complex V) (40), α-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 {beta}-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.


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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 α-glycerophosphate (α-GP) dehydrogenase (α-GPDH), the cytosolic isoform structure is shown. Succ, succinate; α-KG, α-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; α-KGDH, α-ketoglutarate dehydrogenase; DHAP, dihydroxyacetone phosphate; CxI–V, complexes I–V; SDH, succinate dehydrogenase.

Many other mitochondrial functions are also regulated by Ca2+. For example, Ca2+ activation of N-acetylglutamine synthetase generates N-acetylglutamine (92), a potent allosteric activator of carbamoyl-phosphate synthetase, the rate-limiting enzyme in the urea cycle (119). In addition, Ca2+– and diacylglycerol-sensitive protein kinase (PKC) isoforms and calmodulin have been reported in mitochondria, although their precise targets within the organelle are less well understood (54, 160).

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

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).


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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 PT pore is triggered by high [Ca2+]m and other stimuli including oxidants and the depletion of adenine nucleotides. It is inhibited by acidic pH, antioxidants such as reduced glutathione (GSH), and cyclosporin A, which binds to Cyp-D, a matrix cis/trans-prolyl-isomerase. The ANT appears to be a key modulator, with ANT thiols proposed as a target for oxidative stress induction of the PT pore (69). Also, bongkrekic acid and attractyloside, two inhibitors of ANT, inhibit or activate PT pore opening, respectively. Recently, doubt has been cast on the role of ANT, because mitochondria isolated from double-knockout ANT1–/–, ANT2–/– mice appear capable of undergoing PT (101). However, the ANT is a mitochondrial carrier family protein, all of which share significant sequence homology, making it likely that other proteins in this family can substitute for the ANT in PT pore assembly.

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 Δψm 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.

FULL ARTICLE: http://ajpcell.physiology.org/content/287/4/C817.long

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