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Bcl-2 Regulation of Mitochondrial Energetics
Bcl-2 Regulation of Mitochondrial Energetics Elizabeth Murphy*, Ken-ichi Imahashi, and Charles Steenbergen
Recent data suggest that in addition to regulating apoptosis, Bcl-2 (an anti-apoptotic protein overexpressed in B-cell lymphoma) and Bcl-2 family members also regulate mitochondrial and cell physiology. t-Bid, a Bcl-2 family member, has been shown to modulate reorganization of mitochondrial cristae. Bcl-2 appears to regulate voltage-dependent anion channel permeability, which has important consequences for mitochondrial transport of adenine nucleotides, Ca2+, and other metabolites. BAD, a pro-apoptotic Bcl-2 family member, is required for the binding of glucokinase to a mitochondrial complex, and BAD null mice have altered glucose homeostasis. It has been suggested that Bcl-2 family members may regulate important mitochondrial/cell functions and serve as sentinels to detect abnormalities in these pathways and, when the abnormalities are severe enough, to initiate or facilitate cell death. Understanding the physiologic processes controlled by Bcl-2 will be important in understanding cell regulation, and it may also provide new insights into the regulation of apoptosis. (Trends Cardiovasc Med 2005;15:283–290) D 2005, Elsevier Inc.
Regulation of Mitochondrial Energetics
Although there has been considerable recent interest in the role of mitochondria in apoptosis, mitochondria have long been established as the major site of adenosine triphosphate (ATP) synthesis in most cells by the processes of Elizabeth Murphy and Ken-ichi Imasashi are at the Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC. Charles Steenbergen is at the Department of Pathology, Duke University, Durham, NC. * Address correspondence to: Elizabeth Murphy, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Maildrop F2-07, Research Triangle Park, NC 27709. Tel: (+1) 919-5413873; fax: (+1) 919-541-3385; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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electron transport and oxidative phosphorylation. As shown in Figure 1, NADH and/or FADH2 donate electrons to the mitochondrial electron transport chain. These electrons are transferred through a series of redox reactions with oxygen as the final electron acceptor, and the energy from the flow of electrons is coupled to extrusion of protons from the mitochondria matrix at three coupling sites. The extrusion of protons from the mitochondria results in the generation of a mitochondrial membrane potential (Dw) and a pH gradient (DpH). The Dw and DpH together comprise the protomotive force (Dp). The energy derived from the reduction of oxygen by the transfer of electrons donated by NADH is transferred to the ion gradient. The extruded protons reenter the mitochondria via the F1F0-adenosine triphosphatase (ATPase), which couples the transfer of protons down their electrochemical gradient to the generation of ATP. Electron transport
and ATP synthesis are coupled by a chemiosmotic mechanism referred to as chemiosmotic coupling. Transport of electrons back into the matrix by mechanisms other than through the F1F0-ATPase will dissipate the electrochemical gradient (Dp). Thus, unless an uncoupler is present to dissipate the proton gradient, the transfer of electrons down the electron transport chain with extrusion of protons will cease unless adenosine diphosphate (ADP) is available as a substrate for the F1F0-ATPase. Lipophilic protonophores such as DNP and FCCP were originally classified as buncouplersQ because they could uncouple electron transport and oxygen consumption from the conversion of ADP to ATP. Uncouplers stimulate oxygen consumption in the absence of ADP. However, transport of cations such as Ca2+ into the matrix via the Ca2+ uniporter will also dissipate the electrochemical gradient and stimulate oxygen consumption in the absence of ADP. Electrogenic transport of other cations such as K+ into the matrix will also dissipate Dw similar to protonophores. Obviously, the extent to which Dw is reduced depends on the number of positively charged ions that enter the matrix. Mitochondrial uncoupling proteins (UCPs), which mediate proton leak across the mitochondrial inner membrane, have also been described (Hoerter et al. 2004, Teshima et al. 2003). The precise role of these UCPs is debated, but they have been suggested to regulate thermogenesis, energy balance, and modulate production of reactive oxygen species (ROS). As shown in Figure 1, there are several inhibitors of electron transport, which inhibit at different sites. Mitochondrial electron transport is one of the primary sources of ROS in the cell, and inhibition of electron transport with different inhibitors can enhance or reduce the generation of ROS, depending on the site of inhibition (Balaban et al. 2005, Turrens 2003). As illustrated in Table 1, under conditions in which substrate, Pi, and oxygen are not limiting, addition of ADP results in an increase in oxygen consumption and a transient decrease in Dw and NADH (also see Figure 1). In contrast, addition of oligomycin, an inhibitor of the F1F0-ATPase, has the opposite effects, a decrease in oxygen consumption, and an increase in Dw. Mitochondrial uncouplers dissipate the
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Figure 1. Mitochondrial electron transport. Electrons, donated by NADH or FADH, are transferred down a series of redox reactions in which energy of the reactions are transferred to a proton gradient. This illustrates proteins involved in electron transport (complexes I, II, III, and IV), the F1F0-ATPase, which converts the proton gradient into ATP, and the ANT and the VDAC, which facilitate entry and exit of ADP and ATP from the matrix to the cytosol. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; Cyt.c, cytochrome c; CoQ, coenzyme Q.
proton gradient and maximally stimulate oxygen consumption, leading to a decrease in NADH; however, despite the high rate of electron transport, uncouplers result in loss of Dw. The ATP generated by the F 1 F 0 ATPase is generally consumed by ATPrequiring processes in the cytosol. Thus, the ATP generated in the mitochondrial matrix needs to be transported to the cytosol. In addition, the ADP generated in the cytosol needs to be transported to the F1F0-ATPase in the mitochondrial matrix. Adenosine triphosphate and ADP are transported across the inner and outer mitochondrial membranes by the adenine nucleotide translocase (ANT) and the voltage-dependent anion channel (VDAC), respectively. In heart and muscle the creatine kinase shuttle facilitates ATP/ADP transfer between the mitochondria and cytosol. In addition to their roles in adenine nucleotide transport, ANT and VDAC, along with cyclophilin D, have also been reported to form a large conductance inner membrane nonselective channel known as the mitochondrial permeability transition pore (MPTP) (Baines et al. 2005,
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Halestrap et al. 2002, Nakagawa et al. 2005). Opening of the MPTP results in loss of Dw, mitochondrial swelling, and rupture of the outer mitochondrial membrane. However, the obligatory role of ANT in the MPTP has been questioned (Kokoszka et al. 2004). ANT and VDAC form contact points between the inner and outer mitochondrial membranes (van der Klei et al. 1994), and it is suggested that intermediate filaments (IFs) can regulate the formation and/or stabilization of these mitochondrial contact sites.
Mitochondria are important regulators of apoptosis by controlling the release of cytochrome c and other recently identified apoptotic factors that reside in the intermembrane space of the mitochondria. As shown in Figure 1, cytochrome c, a protein in the intermembrane space, is a key component of electron transport. It was thus surprising when release of cytochrome c was shown to be required for the activation of caspase 9 in the cytosol. Cytochrome c release into the cytosol initiates formation of the bapoptosome,Q which consists
Table 1. Effect of substrates and inhibitors on oxygen consumption, membrane potential (#w w ), and NADH under conditions in which substrates, Pi, and oxygen are in excess Electron transport ANT/VDAC mADP Oligomycin Uncoupler inhibitors inhibitors O2 consumption Dw NADH ROS
m x x x
x m m m
m x x x
x x m a
x m m m
In an intact tissue, these condition are not always present. a Depends on the site of inhibition.
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of Apaf-1, cytchrome c, and caspase 9. The complex results in activation of caspase 9, which sets off a cascade to cleave and thereby activate effector caspases. The mechanism by which cytochrome c is released is debated and may vary depending on the cell type and the inducer of apoptosis. Opening of the MPTP, which results in mitochondrial swelling and rupture of the outer membrane, has been reported to be involved in many forms of apoptosis. As discussed later, pro-apoptotic Bcl-2 family members such as BAX may also be involved in cytochrome c release. In addition to cytochrome c, there are a number of additional intermembrane proteins, such as AIF and Smac/Diablo, which can regulate apoptosis. The mitochondrial inner membrane is highly invaginated, with a large surface area relative to matrix volume, and there is normally very little space between the inner and outer membranes. Recent models suggest that the mitochondrial cristae can be tubular or lamellar structures that merge with the inner mitochondrial membrane through tubular connections (Frey and Mannella, 2000). The matrix volume and thus the space between the inner and outer mitochondrial membranes are regulated by a K/H antiporter and a mitochondrial K channel (mito KATP channel) (Garlid 1996). Because of the high negative mitochondrial membrane potential, there is a large driving force for K entry into the mitochondrial matrix. K entry into the matrix is followed by entry of osmotically driven water, which results in mitochondrial swelling. The mitochondrial K/H antiporter, which is activated by swelling, results in K efflux followed by water, thereby restoring matrix volume. However, the efflux of K is countered by the influx of proton, thus resulting in a decrease in DpH. Furthermore, when mitochondrial volume contracts, as occurs during high ATP synthesis, there is activation of the mito KATP channel that increases matrix volume. A number of pharmacologic agents such as diazoxide have been reported to activate this mito KATP channel (Paucek et al. 1992), and its activation has been suggested to result in cardioprotection (O’Rourke 2000). Addition of diazoxide has been shown to reduce apoptosis and necrosis following ischemia and reperfusion (O’Rourke 2000). Diazoxide has also TCM Vol. 15, No. 8, 2005
been shown to reduce acidification during ischemia and to improve the recovery of phosphocreatine on reperfusion (Forbes et al. 2001). The precise mechanism by which opening of this mito KATP channel results in protection has been extensively studied, but the mechanism has not been established definitively. It has been suggested that activation of the mito K ATP channel slightly reduces the mitochondrial membrane potential which reduces mitochondrial Ca 2+ uptake during ischemia (Murata et al. 2001). Dos Santos et al. (2002) have suggested that activation of the mito KATP channel and the resultant mitochondrial matrix swelling alter the Km for ADP entry into the mitochondria, perhaps by altering the conformation of VDAC or by altering contact sites between VDAC and ANT. It has further been proposed that a change in mitochondrial matrix volume can alter metabolism (Halestrap 1994, Korge et al. 2005, Territo et al. 2001). It has also been suggested that activation of the mito KATP channel results in generation of ROS, which activates signaling pathways resulting in cardioprotection (Forbes et al. 2001, Pain et al. 2000). These mechanisms are not mutually exclusive and may all play a role. Mitochondria have been thought of as static structures; however, recent data suggest that mitochondria are dynamic organelles that undergo remodeling characterized by fission and fusion (Frank et al. 2003, Oakes and Korsmeyer 2004, Sugioka et al. 2004). The Bcl-2 family member t-Bid has been shown to cause remodeling of the cristae (Scorrano et al. 2002), resulting in fusion of individual cristae and opening of junctions between cristae and the intermembrane space. It is speculated that this remodeling may facilitate release of cytochrome c, particularly if the permeability of the outer mitochondrial membrane increases. Addition of ADP or ATP has also been shown to cause mitochondrial contraction (Klingenberg et al. 1971, Scherer and Klingenberg 1974). This contraction is not blocked by oligomycin (an inhibitor of the F1F0-ATPase) or uncoupler, so it appears to be independent of the rate of oxidative phosphorylation and of electron transport. The ANT exists in two conformations (Buchanan et al. 1976): the c-state, in which the binding pocket faces the
cytosol and binds carboxyatractylaside (CAT), and the m-state, in which the binding pocket faces the matrix and binds bongkrekic acid (BA). Addition of BA prevents dissociation of ATP/ADP from ANT and causes mitochondrial contraction similar to that observed with addition of adenine nucleotides. Addition of carboxyatractylaside binds to ANT and can reverse the ADP/ATPinduced contraction of mitochondria. These data suggest that the different conformations of ANT, which can be elicited by binding of either BA or CAT, can differentially regulate mitochondrial shape; the m-state is associated with contracted mitochondria. It is also intriguing that BA inhibits, whereas CAT stimulates MPTP opening. The number or density and localization of mitochondria are coordinated with cell metabolic needs. Mitochondrial density appears to be regulated by a number of nuclear receptors and transcription factors such as peroxisome proliferator-activated receptors (PPAR), peroxisome proliferator-activated receptor c coactivator-1a, and nuclear respiratory factor (Kelly and Scarpulla 2004). Mitochondrial localization in the cell is also highly regulated. In cardiac muscle, mitochondria are located between the myofibrils and beneath the sarcolemma, sites of high energy demand. The cytoskeleton, particularly the IFs, appears to be a key component in localization of the mitochondria. In neurons, phosphorylated neurofilaments preferentially bind to mitochondria with a high Dw (Wagner et al. 2003). Consistent with this concept are reports that axonal mitochondrial transport is dependent on mitochondrial membrane potential such that 90% of the mitochondria transported to the growth cone have a high Dw (Miller and Sheetz 2004). The IF connects to the mitochondria via outer membrane proteins such as VDAC. In hearts lacking the IF desmin, the mitochondria are swollen and disorganized spatially, and over time the mice develop a cardiomyopathy and heart failure (Capetanaki 2002, Milner et al. 1999). Interestingly, overexpression of Bcl-2 corrects the mitochondrial defects (e.g., altered localization and increased calcium sensitivity of MPTP) in the desmin null hearts (Weisleder et al. 2004). It has also been suggested that IF such as desmin plays a role in regulating ATP and ADP transport across the
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mitochondria, perhaps by regulation of VDAC. In isolated mitochondria, the Km for ADP transport into the mitochondria is in the range of 10–20 Amol/L. In muscle cells treated with saponin to permeabilize the plasma membrane, the Km for ADP transport appears to be much higher (200 – 400 Amol/L)(Appaix et al., 2003). One explanation for the higher Km for ADP transport in permeabilized cells is the increased diffusional barrier in cells. However, the observation that the mitochondrial Km for ADP in saponin-treated muscle from desmin null mice was reduced to the less than 100 Amol/L range, approaching that observed in isolated mitochondria, would suggest that diffusion is not the only explanation for the difference in the Km for ADP between isolated mitochondria and permeabilized cells (Milner et al. 2000). Voltage-dependent anion channel has also been reported to regulate uptake of Ca2+ into the mitochondria (Gincel et al. 2001, Rapizzi et al. 2002). Overexpression of VDAC was shown to increase mitochondrial Ca2+ uptake following agonist stimulation. Rapizzi et al. (2002) suggest that in the agonistmediated transfer of Ca2+ released by endoplasmic reticulum to mitochondria, the permeability of the outer mitochondrial membrane is a bottleneck, and that increasing VDAC enhances Ca2+ entry to the inner mitochondrial membrane, where it can reach concentrations high enough to approach the Km of the Ca2+ uniporter. Interestingly, the higher in situ Km for mitochondrial ADP has been reported only in muscle, and only muscle contains desmin, which appears to play a role in altering the mitochondrial Km for ADP. Muscle also contains creatine phosphate and creatine kinase (CK), which function to lower the apparent mitochondrial Km for ADP (Dzeja and Terzic 2003). Thus, the effect of desmin on the mitochondrial Km for ADP is offset by the CK system. Because cardiac myocytes have rhythmic Ca2+ transients at rapid intervals, perhaps a primary effect of desmin in these cells is to reduce Ca2+ entry through VDAC, and the concomitant effect of raising the Km for ADP is countered by the CK system.
Bcl-2
Bcl-2 is the founding member of a family of proteins that has been shown to
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regulate apoptosis. Bcl-2 was originally described in B-cell lymphomas, where it was overexpressed as a result of a chromosomal translocation, resulting in inhibition of apoptosis, which allowed the mutant lymphocytes to accumulate. In 1990, a clue to the anti-apoptotic mechanism of Bcl-2 and one of the first indications of a role for mitochondria in apoptosis was provided by a study (Hockenbery et al., 1990), which showed that Bcl-2 was a mitochondrial membrane protein, the overexpression of which blocked apoptosis. Despite an explosion of studies on Bcl-2 in the past decade, the precise mechanism by which Bcl-2 inhibits apoptosis is still somewhat unclear. Bcl-2 defines a family of proteins that contains both pro- and anti-apoptotic members. Bcl-2 family members are classified based on Bcl-2 homology (BH) domains 1– 4. Antiapoptotic Bcl-2 members such as Bcl-2w, BclXL, Mcl-1, and A1 have close homology with Bcl-2 in most BH domains. The pro-apoptotic BAX and BAK, which contain BH domains 1–3, facilitate release of cytochrome c and other intermembrane proteins and are required for apoptosis. There is also a large number of pro-apoptotic proteins that contain only the BH3 domain and are referred to as bBH3-onlyQ proteins. These BH3-only proteins, which include BAD, Bid, Bim, Bid, and others, are regulated by survival and death signals. When activated by phosphorylation or translocation for example, these BH3-only proteins can activate BAX and BAK to stimulate apoptosis. Pro-apoptotic Bcl-2 family members such as BAX or BAK, either alone or in combination with other mitochondrial proteins such as VDAC, have been suggested to form large conductance channels in the outer mitochondrial membrane. These large conductance channels are thought to mediate the release of pro-apoptotic factors such as cytochrome c. Bcl-2 also has been reported to bind to mitochondrial proteins such as VDAC and ANT (Halestrap et al. 2000, Shimizu et al. 2000b). Bcl-2 is proposed to block cytochrome c release and inhibits apoptosis either by binding and sequestering BAX or by binding to BAX binding partners such as VDAC or ANT. Interestingly, VDAC and ANT are reported to be major components of the MPTP (Baines et al. 2005, Halestrap et al. 2002, Nakagawa
et al. 2005). Bcl-2 binding to VDAC has been reported to promote the opening (Vander Heiden et al. 2000) as well as the closing of VDAC (Pastorino et al. 2002, Shimizu et al. 2000a, Shimizu et al. 1999, Tsujimoto and Shimizu 2000). Bcl-2 has also been reported to increase generation of ROS, but also enhance up-regulation of antioxidant defense mechanisms (Korsmeyer et al. 1995, Kowaltowski et al. 2004). Although a role for Bcl-2 in regulating apoptosis has been clearly established, the potential importance of Bcl-2 in physiologic regulation of mitochondrial function has received only modest attention. Bcl-2 overexpression has been shown to decrease the Ca2+ sensitivity of MPTP opening (Murphy et al. 1996). Mitochondria will accumulate added Ca2+ until a threshold level is achieved that activates the MPTP, resulting in release of the accumulated Ca2+ and loss of membrane potential. Mitochondria with overexpression of Bcl-2 can accumulate larger amounts of Ca2+ before the MPTP fully opens. However, it has been suggested that the ability of mitochondria that overexpress Bcl-2 to take up larger amounts of Ca2+ may be due in part to an increase in matrix volume (and presumably an increase in Ca2+ binding anion), as Bcl-2 overexpression has also been reported to increase mitochondrial volume (Kowaltowski et al. 2002). In mitochondria with an increase in Bcl-2, an increase in mitochondrial volume, rather than an increase in mitochondrial membrane potential, is also suggested to be responsible for the increase in the fluorescence of an indicator used to measure mitochondrial membrane potential, tetramethylrhodamine methylester (TMRE) (Kowaltowski et al. 2002). Mice with cardiac-specific overexpression of Bcl-2 have been generated and characterized (Chen et al. 2001, Imahashi et al. 2004, Zhu et al. 2001). Overexpression of Bcl-2 is reported to significantly reduce mitochondrial Na– Ca exchange, which was accompanied by increased matrix Ca2+, increased generation of NADH, and increased oxidation of pyruvate (Zhu et al. 2001). Hearts from mice with cardiac-specific Bcl-2 overexpression have been shown also to be protected during ischemia and reperfusion (Chen et al. 2001, Imahashi et al. 2004). On reperfusion following TCM Vol. 15, No. 8, 2005
ischemia, hearts with overexpression of Bcl-2 have less necrosis, less apoptosis, (Chen et al. 2001), and improved recovery of LVDP and phosphocreatine (Imahashi et al. 2004). Furthermore, Bcl-2 overexpression reduces the rate of decline in ATP and reduces the fall in intracellular pH during ischemia. Increasing mitochondrial levels of Bcl-2 have also been shown to reduce the rate of consumption of exogenously added ATP following addition of mitochondrial uncoupler (Imahashi et al. 2004). It was proposed that Bcl-2 decreased ATP entry into the mitochondria, where it would be consumed by the F1F0-ATPase attempting to regenerate the mitochondrial membrane potential. The reduced rate of decline in ATP and reduced ischemic acidification observed in hearts from Bcl-2 overexpressor mice is consistent with inhibition of consumption of glycolytically generated ATP by Bcl-2 hearts (Imahashi et al. 2004). This could be accomplished by Bcl-2-induced closure of VDAC, inhibition of the ANT, or inhibition of the F1F0-ATPase; inhibition of any of these would have similar effects on the consumption of glycolytic ATP. In the absence of aerobic metabolism, such as with ischemia, the F1F0-ATPase consumes glycolytic ATP, thus contributing to depletion of ATP and cytosolic acidification (Di Lisa et al. 1995, 1998, Leyssens et al. 1996, Rouslin et al. 1986). Consistent with this hypothesis, oligomycin and Bcl-2 have similar effects on the rate of decline in ATP and acidification during ischemia, and the effects of oligomycin and Bcl-2 are not additive as would be expected if Bcl2 and oligomycin both block consumption of ATP generated in the cytosol (Imahashi et al. 2004). The location of Bcl-2 in the outer mitochondrial membrane favors a role for Bcl-2 interaction with VDAC. In further support of this hypothesis, there is a greater association of Bcl-2 and VDAC in Bcl-2 hearts, and the association increases with ischemia (Imahashi et al. 2004), consistent with data showing that inhibition of VDAC activity by Bcl-2 family proteins occurred only under acidic conditions, not at neutral pH (Shimizu et al. 1999). Also consistent with the hypothesis, it has been reported that mitochondrial transport of ATP/ADP is inhibited during ischemia (Aw et al. 1987, Murphy et al. 1988). TCM Vol. 15, No. 8, 2005
There is controversy in the literature regarding the relationship of Bcl-2 to VDAC and ATP/ADP transport across the mitochondria. Vander Heiden et al. (2000) report that Bcl-2 reduced the rate of decline in ATP associated with growth factor withdrawal-induced apoptosis; these observations were attributed to Bcl-2 maintaining VDAC in an open conformation, thereby allowing ATP/ ADP exchange across the mitochondrial membranes. It was hypothesized that growth factor withdrawal-induced apoptosis leads to closure of VDAC, which results in decreased cytosolic ATP due to inhibition of mitochondrial ATP/ADP exchange and ultimately an inability of mitochondrial ATP to enter the cytosol (Vander Heiden et al. 2000). In growth factor withdrawal-induced apoptosis, oxygen is present and mitochondrial generation of ATP is a source of cytosolic ATP. In contrast, Shimizu et al. (1999), with the use of reconstituted liposomes and radiolabeled sucrose uptake as a measure of VDAC activity, reported that BAX enhanced VDAC opening, whereas Bcl-2 promoted VDAC closure. Additional studies by this group showed that BAX-induced apoptosis was blocked by injection of antibodies that inhibited the opening of VDAC (Shimizu et al. 2000a). Thus, there are data suggesting that Bcl-2 maintains VDAC in an open state and conflicting data, suggesting that Bcl-2 enhances closure of VDAC. It is possible that the effects of Bcl-2 on VDAC depend critically on the conditions, but all of the data suggest an interaction between Bcl-2 and VDAC. Many studies examining the relationship of Bcl-2 and mitochondrial function have used cultured cells that generate ATP primarily by glycolysis. The metabolic state and regulation in these highly glycolytic cells is likely to differ from that in a cardiomyocyte, which is dependent on oxidatively generated ATP. Bcl-2 might differentially regulate VDAC depending on factors such as pH, redox state, ROS, Dw, phosphorylation status, or binding of other proteins such as hexokinase or IFs. BAD is a pro-apoptotic Bcl-2 family member that is thought to bind Bcl-2 and thus block the anti-apoptotic function of Bcl-2, for example, the inhibition of BAX and BAK. BAD is phosphorylated by a number of growth factors and phosphorylated BAD is sequestered by
14-3-3. In liver cells, BAD has also been shown to form a mitochondrial complex with protein kinase A, protein phosphatase 1, WAVE-1 (an A kinase-anchoring protein), and glucokinase, which appears to regulate activity of mitochondrial glucokinase (Danial et al. 2003). Hepatocytes lacking BAD have reduced oxygen consumption with glucose as a substrate. Although a mutant BAD that could not be phosphorylated was still capable of forming the complex, the activity of mitochondrial glucokinase was reduced in the mitochondrial complex with nonphosphorylated BAD. Somewhat surprisingly, mice lacking BAD were shown to exhibit abnormal glucose metabolism and marked glucose intolerance (Danial et al. 2003). An association of glucokinase with liver mitochondria has been recently questioned (Bustamante et al. 2005). In addition, glucokinase is not present in myocytes; however, hexokinase II, which is expressed in myocytes, has been shown to bind to VDAC and inhibit activity of BAX (Pastorino et al. 2002).
Is There a Role for Bcl-2 in Regulation of Mitochondrial Energetics?
Data suggest that Bcl-2 family members are involved in regulation or generation of ROS (Korsmeyer et al. 1995, Kowaltowski et al. 2004), can alter mitochondrial matrix volume or structure (Kowaltowski et al. 2002, Scorrano et al. 2002), can alter mitochondrial permeability to or consumption of ATP (Imahashi et al. 2004), can alter permeability of VDAC (Tsujimoto and Shimizu 2000, Tsujimoto and Shimizu 2002, Vander Heiden et al. 2000), and can alter mitochondrial sensitivity of the MPT to Ca2+ (Murphy et al. 1996). Bcl-2 is also related to svf1, which is involved in yeast adaptation from glycolysis to oxidative metabolism (Brace et al. 2005). BAXdependent killing requires the activity of the F1F0-ATPase (Harris et al. 2000, Matsuyama et al. 1998, 2000). These data raise the possibility that Bcl-2 family members may have a role in regulating mitochondrial energetics in addition to their role in apoptosis. Given the current lack of data, one can only speculate regarding this possibility. However, it is tempting to speculate that perhaps Bcl-2 family members sense and
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}
Figure 2. Hypothesis suggesting that Bcl-2 and its family members play a role in homeostatic regulation of mitochondrial energetics. An increase in workload is typically accompanied by an increase in calcium, which could lead to activation of the mito KCa, which in turn would increase matrix volume leading to a decrease in IMS that has been shown to stimulate electron transport (ET). If matrix ADP is high, the increase in ET will activate the F1F0-ATPase and increase production of ATP. If matrix ADP is low, the increase in ET will increase Dw. This increase in Dw will result in an increase in ROS. Perhaps Bcl-2 responds to the increase in matrix volume, Dw, or ROS by an altered confirmation or association and mediates an increase the permeability of VDAC, resulting in an increase of matrix ADP and/or a decrease in matrix volume, which would decrease ET; these would both contribute to a decrease in Dw. Alternatively, Bcl-2 might mediate increased proton leak via UCPs or ANT.
regulate some aspect of mitochondrial energetics such as mitochondrial volume, ROS, or mitochondrial membrane potential. As illustrated in Figure 2, these three parameters are all related. As discussed earlier and illustrated in Figure 2, mitochondrial volume is set by K influx (via mito KATP and mito KCa) and K efflux by K/H antiporter. Alteration of matrix volume, which has been reported to occur with some Bcl-2 family members, results in a change in IMS which has been shown to alter activity of VDAC, the VDAC/ANT contact site function, CK coupling, and electron transport, all of which would alter Dw and generation of ROS. Perhaps Bcl-2 (alone or in combination with other Bcl-2 family members) is sensitive to matrix volume, Dw or ROS, and alters mitochondrial function to maintain the homeostatic level of matrix volume, Dw, and/or ROS. Appropriate regulation of mitochondrial membrane potential is essential for cellular homeostasis, as it is needed to generate ATP and also because as Dw increases, it can lead to increased generation of ROS (Aon et al. 2003, Balaban et al. 2005, Starkov and Fiskum 2003, Turrens
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2003). Reactive oxygen species can be toxic to cells, and cells have therefore developed extensive mechanisms to dissipate and limit generation of ROS. It would also be beneficial to the cell to regulate production of ROS because ROS can function as a signaling molecule. Dw has been shown to fluctuate, and ROS has been suggested to be involved in this process (Aon et al. 2003). Furthermore, ROS has been suggested to play a role in regulation of mitochondrial uncoupling, which would also modulate Dw (Echtay et al. 2003). This homeostatic regulation of Dw/ROS would complement the regulation of ATP production that occurs and has been suggested to be regulated by mitochondrial Ca2+ levels (Balaban 2002). Perhaps Bcl-2 (or a family member) responds to a change in Dw or ROS or matrix volume and mediates mitochondrial changes to restore Dw/ROS/matrix volume. For example, as illustrated in Figure 2, if Dw/ROS/matrix volume were to rise above some threshold, a decrease in Dw with the resultant decrease in ROS production could be accomplished by increasing the matrix level of ADP
(which could be mediated by VDAC/ ANT permeability), which would increase the activity of the F1F0-ATPase or by decreasing matrix volume (increasing IMS), which decreases electron transport. Interestingly, Bcl-2 has been reported to act as a K channel, providing a plausible mechanism for altering matrix volume. In addition to altering electron transport and F 1 F 0 -ATPase activity, altering matrix volume has also been suggested to alter the coupling of VDAC and ANT and thereby alter the Km for ADP entry into mitochondria (Dos Santos et al. 2002). Conversely, if Dw/ ROS/matrix volume were to fall below a threshold, a decrease in matrix ADP and/ or an increase in matrix volume with an increase in electron transport would work to increase Dw/ROS. This hypothesis would predict that high Dw/high ROS opens VDAC and low Dw/low ROS closes VDAC. This hypothesis might also explain the conflicting data regarding Bcl-2 regulation of VDAC. Imahashi et al. (2004) find that overexpression of Bcl-2 has no effect on ADP/ATP entry into the mitochondria when Dw is in the physiologic range; however, as Dw is reduced with uncoupler, the overexpression of Bcl-2 results in a more rapid and/ or a greater degree of inhibition of ATP consumption by the mitochondria, consistent with more inhibition of VDAC. In contrast, Vander Heiden et al (2000) report that Bcl-2 overexpression maintains VDAC in an open state. This would be consistent with a model in which Bcl2 senses Dw/ROS, and with high Dw, Bcl2 maintains VDAC open, but when Dw falls, Bcl-2 enhances the closure of VDAC. This would be consistent with the voltage-dependent regulation of VDAC (Lemeshko and Lemeshko 2004) and it would be consistent with the ability of Bcl-2 to replace svf1, a yeast protein involved in adaptation from glycolysis to oxidative metabolism (Brace et al. 2005). It is interesting that mild uncoupling and a slight decrease in Dw (Hausenloy et al. 2004; Minners et al. 2001), as well as overexpression of UCPs have been shown to be cardioprotective (Hoerter et al. 2004, Teshima et al. 2003); these would all enhance closure of VDAC. Clearly, additional studies will be necessary to elucidate the role of Bcl2 family members in regulating cell energetics. If this hypothesis is correct, Bcl-2 function would need to be capable TCM Vol. 15, No. 8, 2005
of sensing changes in Dw or ROS or matrix volume, Bcl-2 would also be required to initiate changes to restore Dw /ROS/matrix volume. For example, is the conformation of Bcl-2 or its binding to VDAC altered by ROS or membrane potential? There are some data in support of this hypothesis, but clearly additional studies are needed.
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PII S1050-1738(05)00166-0
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Recent data suggest that in addition to regulating apoptosis, Bcl-2 (an anti-apoptotic protein overexpressed in B-cell lymphoma) and Bcl-2 family members also regulate mitochondrial and cell physiology. t-Bid, a Bcl-2 family member, has been shown to modulate reorganization of mitochondrial cristae. Bcl-2 appears to regulate voltage-dependent anion channel permeability, which has important consequences for mitochondrial transport of adenine nucleotides, Ca2+, and other metabolites. BAD, a pro-apoptotic Bcl-2 family member, is required for the binding of glucokinase to a mitochondrial complex, and BAD null mice have altered glucose homeostasis. It has been suggested that Bcl-2 family members may regulate important mitochondrial/cell functions and serve as sentinels to detect abnormalities in these pathways and, when the abnormalities are severe enough, to initiate or facilitate cell death. Understanding the physiologic processes controlled by Bcl-2 will be important in understanding cell regulation, and it may also provide new insights into the regulation of apoptosis. (Trends Cardiovasc Med 2005;15:283–290) D 2005, Elsevier Inc.
Regulation of Mitochondrial Energetics
Although there has been considerable recent interest in the role of mitochondria in apoptosis, mitochondria have long been established as the major site of adenosine triphosphate (ATP) synthesis in most cells by the processes of Elizabeth Murphy and Ken-ichi Imasashi are at the Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC. Charles Steenbergen is at the Department of Pathology, Duke University, Durham, NC. * Address correspondence to: Elizabeth Murphy, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 111 Alexander Drive, Maildrop F2-07, Research Triangle Park, NC 27709. Tel: (+1) 919-5413873; fax: (+1) 919-541-3385; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
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electron transport and oxidative phosphorylation. As shown in Figure 1, NADH and/or FADH2 donate electrons to the mitochondrial electron transport chain. These electrons are transferred through a series of redox reactions with oxygen as the final electron acceptor, and the energy from the flow of electrons is coupled to extrusion of protons from the mitochondria matrix at three coupling sites. The extrusion of protons from the mitochondria results in the generation of a mitochondrial membrane potential (Dw) and a pH gradient (DpH). The Dw and DpH together comprise the protomotive force (Dp). The energy derived from the reduction of oxygen by the transfer of electrons donated by NADH is transferred to the ion gradient. The extruded protons reenter the mitochondria via the F1F0-adenosine triphosphatase (ATPase), which couples the transfer of protons down their electrochemical gradient to the generation of ATP. Electron transport
and ATP synthesis are coupled by a chemiosmotic mechanism referred to as chemiosmotic coupling. Transport of electrons back into the matrix by mechanisms other than through the F1F0-ATPase will dissipate the electrochemical gradient (Dp). Thus, unless an uncoupler is present to dissipate the proton gradient, the transfer of electrons down the electron transport chain with extrusion of protons will cease unless adenosine diphosphate (ADP) is available as a substrate for the F1F0-ATPase. Lipophilic protonophores such as DNP and FCCP were originally classified as buncouplersQ because they could uncouple electron transport and oxygen consumption from the conversion of ADP to ATP. Uncouplers stimulate oxygen consumption in the absence of ADP. However, transport of cations such as Ca2+ into the matrix via the Ca2+ uniporter will also dissipate the electrochemical gradient and stimulate oxygen consumption in the absence of ADP. Electrogenic transport of other cations such as K+ into the matrix will also dissipate Dw similar to protonophores. Obviously, the extent to which Dw is reduced depends on the number of positively charged ions that enter the matrix. Mitochondrial uncoupling proteins (UCPs), which mediate proton leak across the mitochondrial inner membrane, have also been described (Hoerter et al. 2004, Teshima et al. 2003). The precise role of these UCPs is debated, but they have been suggested to regulate thermogenesis, energy balance, and modulate production of reactive oxygen species (ROS). As shown in Figure 1, there are several inhibitors of electron transport, which inhibit at different sites. Mitochondrial electron transport is one of the primary sources of ROS in the cell, and inhibition of electron transport with different inhibitors can enhance or reduce the generation of ROS, depending on the site of inhibition (Balaban et al. 2005, Turrens 2003). As illustrated in Table 1, under conditions in which substrate, Pi, and oxygen are not limiting, addition of ADP results in an increase in oxygen consumption and a transient decrease in Dw and NADH (also see Figure 1). In contrast, addition of oligomycin, an inhibitor of the F1F0-ATPase, has the opposite effects, a decrease in oxygen consumption, and an increase in Dw. Mitochondrial uncouplers dissipate the
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Figure 1. Mitochondrial electron transport. Electrons, donated by NADH or FADH, are transferred down a series of redox reactions in which energy of the reactions are transferred to a proton gradient. This illustrates proteins involved in electron transport (complexes I, II, III, and IV), the F1F0-ATPase, which converts the proton gradient into ATP, and the ANT and the VDAC, which facilitate entry and exit of ADP and ATP from the matrix to the cytosol. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; Cyt.c, cytochrome c; CoQ, coenzyme Q.
proton gradient and maximally stimulate oxygen consumption, leading to a decrease in NADH; however, despite the high rate of electron transport, uncouplers result in loss of Dw. The ATP generated by the F 1 F 0 ATPase is generally consumed by ATPrequiring processes in the cytosol. Thus, the ATP generated in the mitochondrial matrix needs to be transported to the cytosol. In addition, the ADP generated in the cytosol needs to be transported to the F1F0-ATPase in the mitochondrial matrix. Adenosine triphosphate and ADP are transported across the inner and outer mitochondrial membranes by the adenine nucleotide translocase (ANT) and the voltage-dependent anion channel (VDAC), respectively. In heart and muscle the creatine kinase shuttle facilitates ATP/ADP transfer between the mitochondria and cytosol. In addition to their roles in adenine nucleotide transport, ANT and VDAC, along with cyclophilin D, have also been reported to form a large conductance inner membrane nonselective channel known as the mitochondrial permeability transition pore (MPTP) (Baines et al. 2005,
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Halestrap et al. 2002, Nakagawa et al. 2005). Opening of the MPTP results in loss of Dw, mitochondrial swelling, and rupture of the outer mitochondrial membrane. However, the obligatory role of ANT in the MPTP has been questioned (Kokoszka et al. 2004). ANT and VDAC form contact points between the inner and outer mitochondrial membranes (van der Klei et al. 1994), and it is suggested that intermediate filaments (IFs) can regulate the formation and/or stabilization of these mitochondrial contact sites.
Mitochondria are important regulators of apoptosis by controlling the release of cytochrome c and other recently identified apoptotic factors that reside in the intermembrane space of the mitochondria. As shown in Figure 1, cytochrome c, a protein in the intermembrane space, is a key component of electron transport. It was thus surprising when release of cytochrome c was shown to be required for the activation of caspase 9 in the cytosol. Cytochrome c release into the cytosol initiates formation of the bapoptosome,Q which consists
Table 1. Effect of substrates and inhibitors on oxygen consumption, membrane potential (#w w ), and NADH under conditions in which substrates, Pi, and oxygen are in excess Electron transport ANT/VDAC mADP Oligomycin Uncoupler inhibitors inhibitors O2 consumption Dw NADH ROS
m x x x
x m m m
m x x x
x x m a
x m m m
In an intact tissue, these condition are not always present. a Depends on the site of inhibition.
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of Apaf-1, cytchrome c, and caspase 9. The complex results in activation of caspase 9, which sets off a cascade to cleave and thereby activate effector caspases. The mechanism by which cytochrome c is released is debated and may vary depending on the cell type and the inducer of apoptosis. Opening of the MPTP, which results in mitochondrial swelling and rupture of the outer membrane, has been reported to be involved in many forms of apoptosis. As discussed later, pro-apoptotic Bcl-2 family members such as BAX may also be involved in cytochrome c release. In addition to cytochrome c, there are a number of additional intermembrane proteins, such as AIF and Smac/Diablo, which can regulate apoptosis. The mitochondrial inner membrane is highly invaginated, with a large surface area relative to matrix volume, and there is normally very little space between the inner and outer membranes. Recent models suggest that the mitochondrial cristae can be tubular or lamellar structures that merge with the inner mitochondrial membrane through tubular connections (Frey and Mannella, 2000). The matrix volume and thus the space between the inner and outer mitochondrial membranes are regulated by a K/H antiporter and a mitochondrial K channel (mito KATP channel) (Garlid 1996). Because of the high negative mitochondrial membrane potential, there is a large driving force for K entry into the mitochondrial matrix. K entry into the matrix is followed by entry of osmotically driven water, which results in mitochondrial swelling. The mitochondrial K/H antiporter, which is activated by swelling, results in K efflux followed by water, thereby restoring matrix volume. However, the efflux of K is countered by the influx of proton, thus resulting in a decrease in DpH. Furthermore, when mitochondrial volume contracts, as occurs during high ATP synthesis, there is activation of the mito KATP channel that increases matrix volume. A number of pharmacologic agents such as diazoxide have been reported to activate this mito KATP channel (Paucek et al. 1992), and its activation has been suggested to result in cardioprotection (O’Rourke 2000). Addition of diazoxide has been shown to reduce apoptosis and necrosis following ischemia and reperfusion (O’Rourke 2000). Diazoxide has also TCM Vol. 15, No. 8, 2005
been shown to reduce acidification during ischemia and to improve the recovery of phosphocreatine on reperfusion (Forbes et al. 2001). The precise mechanism by which opening of this mito KATP channel results in protection has been extensively studied, but the mechanism has not been established definitively. It has been suggested that activation of the mito K ATP channel slightly reduces the mitochondrial membrane potential which reduces mitochondrial Ca 2+ uptake during ischemia (Murata et al. 2001). Dos Santos et al. (2002) have suggested that activation of the mito KATP channel and the resultant mitochondrial matrix swelling alter the Km for ADP entry into the mitochondria, perhaps by altering the conformation of VDAC or by altering contact sites between VDAC and ANT. It has further been proposed that a change in mitochondrial matrix volume can alter metabolism (Halestrap 1994, Korge et al. 2005, Territo et al. 2001). It has also been suggested that activation of the mito KATP channel results in generation of ROS, which activates signaling pathways resulting in cardioprotection (Forbes et al. 2001, Pain et al. 2000). These mechanisms are not mutually exclusive and may all play a role. Mitochondria have been thought of as static structures; however, recent data suggest that mitochondria are dynamic organelles that undergo remodeling characterized by fission and fusion (Frank et al. 2003, Oakes and Korsmeyer 2004, Sugioka et al. 2004). The Bcl-2 family member t-Bid has been shown to cause remodeling of the cristae (Scorrano et al. 2002), resulting in fusion of individual cristae and opening of junctions between cristae and the intermembrane space. It is speculated that this remodeling may facilitate release of cytochrome c, particularly if the permeability of the outer mitochondrial membrane increases. Addition of ADP or ATP has also been shown to cause mitochondrial contraction (Klingenberg et al. 1971, Scherer and Klingenberg 1974). This contraction is not blocked by oligomycin (an inhibitor of the F1F0-ATPase) or uncoupler, so it appears to be independent of the rate of oxidative phosphorylation and of electron transport. The ANT exists in two conformations (Buchanan et al. 1976): the c-state, in which the binding pocket faces the
cytosol and binds carboxyatractylaside (CAT), and the m-state, in which the binding pocket faces the matrix and binds bongkrekic acid (BA). Addition of BA prevents dissociation of ATP/ADP from ANT and causes mitochondrial contraction similar to that observed with addition of adenine nucleotides. Addition of carboxyatractylaside binds to ANT and can reverse the ADP/ATPinduced contraction of mitochondria. These data suggest that the different conformations of ANT, which can be elicited by binding of either BA or CAT, can differentially regulate mitochondrial shape; the m-state is associated with contracted mitochondria. It is also intriguing that BA inhibits, whereas CAT stimulates MPTP opening. The number or density and localization of mitochondria are coordinated with cell metabolic needs. Mitochondrial density appears to be regulated by a number of nuclear receptors and transcription factors such as peroxisome proliferator-activated receptors (PPAR), peroxisome proliferator-activated receptor c coactivator-1a, and nuclear respiratory factor (Kelly and Scarpulla 2004). Mitochondrial localization in the cell is also highly regulated. In cardiac muscle, mitochondria are located between the myofibrils and beneath the sarcolemma, sites of high energy demand. The cytoskeleton, particularly the IFs, appears to be a key component in localization of the mitochondria. In neurons, phosphorylated neurofilaments preferentially bind to mitochondria with a high Dw (Wagner et al. 2003). Consistent with this concept are reports that axonal mitochondrial transport is dependent on mitochondrial membrane potential such that 90% of the mitochondria transported to the growth cone have a high Dw (Miller and Sheetz 2004). The IF connects to the mitochondria via outer membrane proteins such as VDAC. In hearts lacking the IF desmin, the mitochondria are swollen and disorganized spatially, and over time the mice develop a cardiomyopathy and heart failure (Capetanaki 2002, Milner et al. 1999). Interestingly, overexpression of Bcl-2 corrects the mitochondrial defects (e.g., altered localization and increased calcium sensitivity of MPTP) in the desmin null hearts (Weisleder et al. 2004). It has also been suggested that IF such as desmin plays a role in regulating ATP and ADP transport across the
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mitochondria, perhaps by regulation of VDAC. In isolated mitochondria, the Km for ADP transport into the mitochondria is in the range of 10–20 Amol/L. In muscle cells treated with saponin to permeabilize the plasma membrane, the Km for ADP transport appears to be much higher (200 – 400 Amol/L)(Appaix et al., 2003). One explanation for the higher Km for ADP transport in permeabilized cells is the increased diffusional barrier in cells. However, the observation that the mitochondrial Km for ADP in saponin-treated muscle from desmin null mice was reduced to the less than 100 Amol/L range, approaching that observed in isolated mitochondria, would suggest that diffusion is not the only explanation for the difference in the Km for ADP between isolated mitochondria and permeabilized cells (Milner et al. 2000). Voltage-dependent anion channel has also been reported to regulate uptake of Ca2+ into the mitochondria (Gincel et al. 2001, Rapizzi et al. 2002). Overexpression of VDAC was shown to increase mitochondrial Ca2+ uptake following agonist stimulation. Rapizzi et al. (2002) suggest that in the agonistmediated transfer of Ca2+ released by endoplasmic reticulum to mitochondria, the permeability of the outer mitochondrial membrane is a bottleneck, and that increasing VDAC enhances Ca2+ entry to the inner mitochondrial membrane, where it can reach concentrations high enough to approach the Km of the Ca2+ uniporter. Interestingly, the higher in situ Km for mitochondrial ADP has been reported only in muscle, and only muscle contains desmin, which appears to play a role in altering the mitochondrial Km for ADP. Muscle also contains creatine phosphate and creatine kinase (CK), which function to lower the apparent mitochondrial Km for ADP (Dzeja and Terzic 2003). Thus, the effect of desmin on the mitochondrial Km for ADP is offset by the CK system. Because cardiac myocytes have rhythmic Ca2+ transients at rapid intervals, perhaps a primary effect of desmin in these cells is to reduce Ca2+ entry through VDAC, and the concomitant effect of raising the Km for ADP is countered by the CK system.
Bcl-2
Bcl-2 is the founding member of a family of proteins that has been shown to
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regulate apoptosis. Bcl-2 was originally described in B-cell lymphomas, where it was overexpressed as a result of a chromosomal translocation, resulting in inhibition of apoptosis, which allowed the mutant lymphocytes to accumulate. In 1990, a clue to the anti-apoptotic mechanism of Bcl-2 and one of the first indications of a role for mitochondria in apoptosis was provided by a study (Hockenbery et al., 1990), which showed that Bcl-2 was a mitochondrial membrane protein, the overexpression of which blocked apoptosis. Despite an explosion of studies on Bcl-2 in the past decade, the precise mechanism by which Bcl-2 inhibits apoptosis is still somewhat unclear. Bcl-2 defines a family of proteins that contains both pro- and anti-apoptotic members. Bcl-2 family members are classified based on Bcl-2 homology (BH) domains 1– 4. Antiapoptotic Bcl-2 members such as Bcl-2w, BclXL, Mcl-1, and A1 have close homology with Bcl-2 in most BH domains. The pro-apoptotic BAX and BAK, which contain BH domains 1–3, facilitate release of cytochrome c and other intermembrane proteins and are required for apoptosis. There is also a large number of pro-apoptotic proteins that contain only the BH3 domain and are referred to as bBH3-onlyQ proteins. These BH3-only proteins, which include BAD, Bid, Bim, Bid, and others, are regulated by survival and death signals. When activated by phosphorylation or translocation for example, these BH3-only proteins can activate BAX and BAK to stimulate apoptosis. Pro-apoptotic Bcl-2 family members such as BAX or BAK, either alone or in combination with other mitochondrial proteins such as VDAC, have been suggested to form large conductance channels in the outer mitochondrial membrane. These large conductance channels are thought to mediate the release of pro-apoptotic factors such as cytochrome c. Bcl-2 also has been reported to bind to mitochondrial proteins such as VDAC and ANT (Halestrap et al. 2000, Shimizu et al. 2000b). Bcl-2 is proposed to block cytochrome c release and inhibits apoptosis either by binding and sequestering BAX or by binding to BAX binding partners such as VDAC or ANT. Interestingly, VDAC and ANT are reported to be major components of the MPTP (Baines et al. 2005, Halestrap et al. 2002, Nakagawa
et al. 2005). Bcl-2 binding to VDAC has been reported to promote the opening (Vander Heiden et al. 2000) as well as the closing of VDAC (Pastorino et al. 2002, Shimizu et al. 2000a, Shimizu et al. 1999, Tsujimoto and Shimizu 2000). Bcl-2 has also been reported to increase generation of ROS, but also enhance up-regulation of antioxidant defense mechanisms (Korsmeyer et al. 1995, Kowaltowski et al. 2004). Although a role for Bcl-2 in regulating apoptosis has been clearly established, the potential importance of Bcl-2 in physiologic regulation of mitochondrial function has received only modest attention. Bcl-2 overexpression has been shown to decrease the Ca2+ sensitivity of MPTP opening (Murphy et al. 1996). Mitochondria will accumulate added Ca2+ until a threshold level is achieved that activates the MPTP, resulting in release of the accumulated Ca2+ and loss of membrane potential. Mitochondria with overexpression of Bcl-2 can accumulate larger amounts of Ca2+ before the MPTP fully opens. However, it has been suggested that the ability of mitochondria that overexpress Bcl-2 to take up larger amounts of Ca2+ may be due in part to an increase in matrix volume (and presumably an increase in Ca2+ binding anion), as Bcl-2 overexpression has also been reported to increase mitochondrial volume (Kowaltowski et al. 2002). In mitochondria with an increase in Bcl-2, an increase in mitochondrial volume, rather than an increase in mitochondrial membrane potential, is also suggested to be responsible for the increase in the fluorescence of an indicator used to measure mitochondrial membrane potential, tetramethylrhodamine methylester (TMRE) (Kowaltowski et al. 2002). Mice with cardiac-specific overexpression of Bcl-2 have been generated and characterized (Chen et al. 2001, Imahashi et al. 2004, Zhu et al. 2001). Overexpression of Bcl-2 is reported to significantly reduce mitochondrial Na– Ca exchange, which was accompanied by increased matrix Ca2+, increased generation of NADH, and increased oxidation of pyruvate (Zhu et al. 2001). Hearts from mice with cardiac-specific Bcl-2 overexpression have been shown also to be protected during ischemia and reperfusion (Chen et al. 2001, Imahashi et al. 2004). On reperfusion following TCM Vol. 15, No. 8, 2005
ischemia, hearts with overexpression of Bcl-2 have less necrosis, less apoptosis, (Chen et al. 2001), and improved recovery of LVDP and phosphocreatine (Imahashi et al. 2004). Furthermore, Bcl-2 overexpression reduces the rate of decline in ATP and reduces the fall in intracellular pH during ischemia. Increasing mitochondrial levels of Bcl-2 have also been shown to reduce the rate of consumption of exogenously added ATP following addition of mitochondrial uncoupler (Imahashi et al. 2004). It was proposed that Bcl-2 decreased ATP entry into the mitochondria, where it would be consumed by the F1F0-ATPase attempting to regenerate the mitochondrial membrane potential. The reduced rate of decline in ATP and reduced ischemic acidification observed in hearts from Bcl-2 overexpressor mice is consistent with inhibition of consumption of glycolytically generated ATP by Bcl-2 hearts (Imahashi et al. 2004). This could be accomplished by Bcl-2-induced closure of VDAC, inhibition of the ANT, or inhibition of the F1F0-ATPase; inhibition of any of these would have similar effects on the consumption of glycolytic ATP. In the absence of aerobic metabolism, such as with ischemia, the F1F0-ATPase consumes glycolytic ATP, thus contributing to depletion of ATP and cytosolic acidification (Di Lisa et al. 1995, 1998, Leyssens et al. 1996, Rouslin et al. 1986). Consistent with this hypothesis, oligomycin and Bcl-2 have similar effects on the rate of decline in ATP and acidification during ischemia, and the effects of oligomycin and Bcl-2 are not additive as would be expected if Bcl2 and oligomycin both block consumption of ATP generated in the cytosol (Imahashi et al. 2004). The location of Bcl-2 in the outer mitochondrial membrane favors a role for Bcl-2 interaction with VDAC. In further support of this hypothesis, there is a greater association of Bcl-2 and VDAC in Bcl-2 hearts, and the association increases with ischemia (Imahashi et al. 2004), consistent with data showing that inhibition of VDAC activity by Bcl-2 family proteins occurred only under acidic conditions, not at neutral pH (Shimizu et al. 1999). Also consistent with the hypothesis, it has been reported that mitochondrial transport of ATP/ADP is inhibited during ischemia (Aw et al. 1987, Murphy et al. 1988). TCM Vol. 15, No. 8, 2005
There is controversy in the literature regarding the relationship of Bcl-2 to VDAC and ATP/ADP transport across the mitochondria. Vander Heiden et al. (2000) report that Bcl-2 reduced the rate of decline in ATP associated with growth factor withdrawal-induced apoptosis; these observations were attributed to Bcl-2 maintaining VDAC in an open conformation, thereby allowing ATP/ ADP exchange across the mitochondrial membranes. It was hypothesized that growth factor withdrawal-induced apoptosis leads to closure of VDAC, which results in decreased cytosolic ATP due to inhibition of mitochondrial ATP/ADP exchange and ultimately an inability of mitochondrial ATP to enter the cytosol (Vander Heiden et al. 2000). In growth factor withdrawal-induced apoptosis, oxygen is present and mitochondrial generation of ATP is a source of cytosolic ATP. In contrast, Shimizu et al. (1999), with the use of reconstituted liposomes and radiolabeled sucrose uptake as a measure of VDAC activity, reported that BAX enhanced VDAC opening, whereas Bcl-2 promoted VDAC closure. Additional studies by this group showed that BAX-induced apoptosis was blocked by injection of antibodies that inhibited the opening of VDAC (Shimizu et al. 2000a). Thus, there are data suggesting that Bcl-2 maintains VDAC in an open state and conflicting data, suggesting that Bcl-2 enhances closure of VDAC. It is possible that the effects of Bcl-2 on VDAC depend critically on the conditions, but all of the data suggest an interaction between Bcl-2 and VDAC. Many studies examining the relationship of Bcl-2 and mitochondrial function have used cultured cells that generate ATP primarily by glycolysis. The metabolic state and regulation in these highly glycolytic cells is likely to differ from that in a cardiomyocyte, which is dependent on oxidatively generated ATP. Bcl-2 might differentially regulate VDAC depending on factors such as pH, redox state, ROS, Dw, phosphorylation status, or binding of other proteins such as hexokinase or IFs. BAD is a pro-apoptotic Bcl-2 family member that is thought to bind Bcl-2 and thus block the anti-apoptotic function of Bcl-2, for example, the inhibition of BAX and BAK. BAD is phosphorylated by a number of growth factors and phosphorylated BAD is sequestered by
14-3-3. In liver cells, BAD has also been shown to form a mitochondrial complex with protein kinase A, protein phosphatase 1, WAVE-1 (an A kinase-anchoring protein), and glucokinase, which appears to regulate activity of mitochondrial glucokinase (Danial et al. 2003). Hepatocytes lacking BAD have reduced oxygen consumption with glucose as a substrate. Although a mutant BAD that could not be phosphorylated was still capable of forming the complex, the activity of mitochondrial glucokinase was reduced in the mitochondrial complex with nonphosphorylated BAD. Somewhat surprisingly, mice lacking BAD were shown to exhibit abnormal glucose metabolism and marked glucose intolerance (Danial et al. 2003). An association of glucokinase with liver mitochondria has been recently questioned (Bustamante et al. 2005). In addition, glucokinase is not present in myocytes; however, hexokinase II, which is expressed in myocytes, has been shown to bind to VDAC and inhibit activity of BAX (Pastorino et al. 2002).
Is There a Role for Bcl-2 in Regulation of Mitochondrial Energetics?
Data suggest that Bcl-2 family members are involved in regulation or generation of ROS (Korsmeyer et al. 1995, Kowaltowski et al. 2004), can alter mitochondrial matrix volume or structure (Kowaltowski et al. 2002, Scorrano et al. 2002), can alter mitochondrial permeability to or consumption of ATP (Imahashi et al. 2004), can alter permeability of VDAC (Tsujimoto and Shimizu 2000, Tsujimoto and Shimizu 2002, Vander Heiden et al. 2000), and can alter mitochondrial sensitivity of the MPT to Ca2+ (Murphy et al. 1996). Bcl-2 is also related to svf1, which is involved in yeast adaptation from glycolysis to oxidative metabolism (Brace et al. 2005). BAXdependent killing requires the activity of the F1F0-ATPase (Harris et al. 2000, Matsuyama et al. 1998, 2000). These data raise the possibility that Bcl-2 family members may have a role in regulating mitochondrial energetics in addition to their role in apoptosis. Given the current lack of data, one can only speculate regarding this possibility. However, it is tempting to speculate that perhaps Bcl-2 family members sense and
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}
Figure 2. Hypothesis suggesting that Bcl-2 and its family members play a role in homeostatic regulation of mitochondrial energetics. An increase in workload is typically accompanied by an increase in calcium, which could lead to activation of the mito KCa, which in turn would increase matrix volume leading to a decrease in IMS that has been shown to stimulate electron transport (ET). If matrix ADP is high, the increase in ET will activate the F1F0-ATPase and increase production of ATP. If matrix ADP is low, the increase in ET will increase Dw. This increase in Dw will result in an increase in ROS. Perhaps Bcl-2 responds to the increase in matrix volume, Dw, or ROS by an altered confirmation or association and mediates an increase the permeability of VDAC, resulting in an increase of matrix ADP and/or a decrease in matrix volume, which would decrease ET; these would both contribute to a decrease in Dw. Alternatively, Bcl-2 might mediate increased proton leak via UCPs or ANT.
regulate some aspect of mitochondrial energetics such as mitochondrial volume, ROS, or mitochondrial membrane potential. As illustrated in Figure 2, these three parameters are all related. As discussed earlier and illustrated in Figure 2, mitochondrial volume is set by K influx (via mito KATP and mito KCa) and K efflux by K/H antiporter. Alteration of matrix volume, which has been reported to occur with some Bcl-2 family members, results in a change in IMS which has been shown to alter activity of VDAC, the VDAC/ANT contact site function, CK coupling, and electron transport, all of which would alter Dw and generation of ROS. Perhaps Bcl-2 (alone or in combination with other Bcl-2 family members) is sensitive to matrix volume, Dw or ROS, and alters mitochondrial function to maintain the homeostatic level of matrix volume, Dw, and/or ROS. Appropriate regulation of mitochondrial membrane potential is essential for cellular homeostasis, as it is needed to generate ATP and also because as Dw increases, it can lead to increased generation of ROS (Aon et al. 2003, Balaban et al. 2005, Starkov and Fiskum 2003, Turrens
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2003). Reactive oxygen species can be toxic to cells, and cells have therefore developed extensive mechanisms to dissipate and limit generation of ROS. It would also be beneficial to the cell to regulate production of ROS because ROS can function as a signaling molecule. Dw has been shown to fluctuate, and ROS has been suggested to be involved in this process (Aon et al. 2003). Furthermore, ROS has been suggested to play a role in regulation of mitochondrial uncoupling, which would also modulate Dw (Echtay et al. 2003). This homeostatic regulation of Dw/ROS would complement the regulation of ATP production that occurs and has been suggested to be regulated by mitochondrial Ca2+ levels (Balaban 2002). Perhaps Bcl-2 (or a family member) responds to a change in Dw or ROS or matrix volume and mediates mitochondrial changes to restore Dw/ROS/matrix volume. For example, as illustrated in Figure 2, if Dw/ROS/matrix volume were to rise above some threshold, a decrease in Dw with the resultant decrease in ROS production could be accomplished by increasing the matrix level of ADP
(which could be mediated by VDAC/ ANT permeability), which would increase the activity of the F1F0-ATPase or by decreasing matrix volume (increasing IMS), which decreases electron transport. Interestingly, Bcl-2 has been reported to act as a K channel, providing a plausible mechanism for altering matrix volume. In addition to altering electron transport and F 1 F 0 -ATPase activity, altering matrix volume has also been suggested to alter the coupling of VDAC and ANT and thereby alter the Km for ADP entry into mitochondria (Dos Santos et al. 2002). Conversely, if Dw/ ROS/matrix volume were to fall below a threshold, a decrease in matrix ADP and/ or an increase in matrix volume with an increase in electron transport would work to increase Dw/ROS. This hypothesis would predict that high Dw/high ROS opens VDAC and low Dw/low ROS closes VDAC. This hypothesis might also explain the conflicting data regarding Bcl-2 regulation of VDAC. Imahashi et al. (2004) find that overexpression of Bcl-2 has no effect on ADP/ATP entry into the mitochondria when Dw is in the physiologic range; however, as Dw is reduced with uncoupler, the overexpression of Bcl-2 results in a more rapid and/ or a greater degree of inhibition of ATP consumption by the mitochondria, consistent with more inhibition of VDAC. In contrast, Vander Heiden et al (2000) report that Bcl-2 overexpression maintains VDAC in an open state. This would be consistent with a model in which Bcl2 senses Dw/ROS, and with high Dw, Bcl2 maintains VDAC open, but when Dw falls, Bcl-2 enhances the closure of VDAC. This would be consistent with the voltage-dependent regulation of VDAC (Lemeshko and Lemeshko 2004) and it would be consistent with the ability of Bcl-2 to replace svf1, a yeast protein involved in adaptation from glycolysis to oxidative metabolism (Brace et al. 2005). It is interesting that mild uncoupling and a slight decrease in Dw (Hausenloy et al. 2004; Minners et al. 2001), as well as overexpression of UCPs have been shown to be cardioprotective (Hoerter et al. 2004, Teshima et al. 2003); these would all enhance closure of VDAC. Clearly, additional studies will be necessary to elucidate the role of Bcl2 family members in regulating cell energetics. If this hypothesis is correct, Bcl-2 function would need to be capable TCM Vol. 15, No. 8, 2005
of sensing changes in Dw or ROS or matrix volume, Bcl-2 would also be required to initiate changes to restore Dw /ROS/matrix volume. For example, is the conformation of Bcl-2 or its binding to VDAC altered by ROS or membrane potential? There are some data in support of this hypothesis, but clearly additional studies are needed.
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