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Regulation of Sodium–Calcium Exchange and Mitochondrial Energetics by Bcl-2 in the Heart of Transgenic Mice
J Mol Cell Cardiol 33, 2135–2144 (2001) doi:10.1006/jmcc.2001.1476, available online at http://www.idealibrary.com on
Regulation of Sodium–Calcium Exchange and Mitochondrial Energetics by Bcl-2 in the Heart of Transgenic Mice Liping Zhu1, Yingjie Yu1, Balvin H. L. Chua2, Ye-Shih Ho3 and Tuan H. Kuo1 1
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan; James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee; 3 Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 2
(Received 9 August 2001, accepted for publication 14 September 2001, published electronically 30 October 2001) L. Z, Y. Y, B. H. L. C, Y.-S. H T. H. K. Regulation of Sodium–Calcium Exchange and Mitochondrial Energetics by Bcl-2 in the Heart of Transgenic Mice. Journal of Molecular and Cellular Cardiology (2001) 33, 2135–2144. Our previous work in cultured cells has shown that the maintenance of mitochondrial Ca2+ homeostasis is essential for cell survival, and that the anti-apoptotic protein Bcl-2 is able to maintain a threshold level of mitochondrial Ca2+ by the inhibition of permeability transition. To test whether Bcl-2 also affects the mitochondrial Na+–Ca2+ exchange (NCE), a major efflux pathway for mitochondrial Ca2+, studies using transgenic mice that overexpress Bcl-2 in the heart have been performed. NCE activity was determined as the Na+-dependent Ca2+ efflux in the isolated mitochondria. Overexpression of Bcl-2 led to a significant reduction of NCE activity as well as increased resistance to permeability transition in the mitochondria of transgenic heart. This was accompanied by increased matrix Ca2+ level, enhanced formation of NADH and enhanced oxidation of pyruvate, an NAD+-linked substrate. Furthermore, there was induction of cellular Ca2+ transport proteins including the Na+–Ca2+ exchanger of the sarcolemma (NCX). Bcl-2 not only stimulates NCX expression in the sarcolemma but also attenuates the Na+–Ca2+ exchange in the mitochondria. These results are consistent with the protection 2001 Academic Press by Bcl-2 against apoptosis in heart following ischemia/reperfusion. K W: Sodium–calcium exchange; Mitochondria; Bcl-2; Transgenic mice; Apoptosis; Necrosis; Calcium homeostasis.
Introduction Altered calcium homeostasis is commonly observed in vitro during apoptotic or necrotic cell death.1–6 Excessive Ca2+ influx is known to be a key component of ischemic injury in neuronal cells7 and cardiomyocytes.8 However, apoptosis can be attenuated by increased calcium entry,9 raising the question of how cytosolic Ca2+ overload kills cells. Recently, we have found that mitochondrial Ca2+ (Cam) plays an important role in the regulation of cell death.10,11 We showed that high concentrations
of cytosolic Ca2+ lead to mitochondrial Ca2+ overload, which in turn activates the permeability transition (MPT) pore, resulting in the loss of matrix Ca2+ through the pore.11 The reduction of Cam (FCCP-releasable) then leads to ATP depletion and cell death by necrosis.11 Interestingly, reduced Ca2+ entry through the capacitative channel has been correlated with apoptosis.6 Reduced Ca2+ entry (by decreasing extracellular Ca2+) also correlates with lower level of mitochondrial Ca2+.10 Combined with our kinetic analysis of cell death, this evidence suggests that Cam depletion is a cause rather than
Please address all correspondence to: T. H. Kuo, Department of Pathology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA. E-mail: [email protected]
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a consequence of cell death.10,11 While it is long recognized that lowering Cam levels reduces the rate of oxidative phosphorylation and thus affects the performance of work in the heart,12–14 the finding that reducing mitochondrial Ca2+ below a threshold level is detrimental to cell survival has only recently been made.10 Thus, it is important to understand how the basal Cam level is controlled in the cell and how it may affect cell survival. The steady state level of Cam is the result of a kinetic balance between uptake and efflux.14 While Cam uptake is achieved primarily via the ruthenium red-sensitive uniporter, there are at least two mechanisms that mediate Cam efflux in the mitochondria.14,15 One is the mitochondrial Na+–Ca2+ exchange (NCE) which represent a specific efflux pathway,16,17 and the other is the permeability transition (MPT) pore which represent a non-specific efflux pathway for mitochondrial Ca2+.18–20 Because Cam uptake is a fast reaction and Cam efflux is a slower process, it has been suggested that the levels of matrix Ca2+ are determined by efflux mechanisms.14 This hypothesis is supported by our initial study showing the control of Cam load by inhibition of both the MPT pore and NCE.11 Since ectopic expression of Bcl-2 enables cells to retain more Ca2+ in the mitochondria in the resting state,10,21 it has been assumed that the anti-apoptotic effect of Bcl-2 is due to the inhibition of the MPT pore.22,23 Inhibition of the pore by Bcl-2 or cyclosporin A leads to accumulation of Cam.10,18 However, whether Bcl-2 expression also affects the NCE system is unknown. In this regard, the study of mitochondrial Ca2+ homeostasis has been hampered by the lack of cloning of the uniporter or NCE genes. In the present study, we have investigated the effect of Bcl-2 on NCE activity and the associated mitochondrial functions in the heart of transgenic mice. Comparison of bioenergetics from hearts of transgenic and non-transgenic mice indicates that Bcl-2 expression not only inhibits permeability transition, but also enhances the rate of oxidative phosphorylation by attenuating NCE activity. Thus both MPT pore and NCE, two efflux mechanisms for mitochondrial Ca2+, are modulated by Bcl-2. Most interestingly, Bcl-2 stabilizes cellular Ca2+ homeostasis by upregulating the expression of the sarcolemma Na+–Ca2+ exchange, a major efflux mechanism in cardiomyocytes.
construct, containing the human Bcl-2 gene under the control of the cardiac--myosin heavy chain promoter, was used for the injection into pronuclei of fertilized C57BL/6xC3HF1 mouse oocytes. These mice were well characterized.24 Bcl-2 RNA and protein overexpression in the heart was confirmed by Northern and Western blot, respectively.24 Overexpression of Bcl-2 was shown to attenuate apoptosis and protect against myocardial ischemia/ reperfusion injury in the transgenic mice.24 Eighteen heterozygous transgenic mice of 4–8 months old were used. Eighteen age-matched and sex-matched non-transgenic littermates were used as experimental controls. All animals were treated in accordance with NIH guidelines.
Materials and Methods
Mitochondrial Ca2+ retention capacity, oxygen consumption, and swelling
Isolation of cardiac mitochondria Mitochondria were isolated using the procedure of Sordahl et al.25 as described previously.26 We have shown that this procedure yielded pure mitochondria as characterized by electron microscopy.26 Briefly, hearts from four mice were pooled, rinsed to remove blood, and weighed. The hearts were cut into halves in 12 volumes of KEA medium (0.18 KCl, 10 m EGTA, 0.5% bovine serum albumin, pH 7.2–7.4) and homogenized with a Polytron (Brinkman). The homogenate was fractionated by differential centrifugation. The resulting mitochondria pellet was washed twice and resuspended in 180 m KCl, 50 EGTA to yield approximately 20 mg protein per ml. Protein concentration was determined by the Biuret method.26
Western blot analysis For detecting Bcl-2 protein expression, the mitochondrial fraction as well as whole heart extract (50 g) were solubilized and samples separated on 12% polyacrylamide gel for immuno-blotting as described previously.27 The detection of Ca2+ transport proteins was carried out in whole heart lysate using 7.5% polyacrylamide gel. Bcl-2 antibody was obtained from Dako; PMCA, SERCA2, and NCX-1 were from Affinity Bioreagents.
Animals The generation of Bcl-2 transgenic mice has been described by Chen and co-workers.24 The transgene
Extramitochondrial Ca2+ concentration was measured with a spectrofluorometer (Photon Technology International) in the presence of 1 Calcium
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Green-5N as described by Murphy et al.21 using excitation-emission wavelengths: 503–535 nm. Calibration of the signal was achieved by the addition of known amounts of Ca2+. Mitochondrial oxygen consumption was measured polarographically at 30 °C using a Clarke-type oxygen electrode.26 Mitochondrial swelling was monitored as the decrease in light absorbance at 540 nm with a Shimadzu spectrophotometer. Incubations were carried out at 25 °C with 0.25 mg of mitochondrial protein per ml in the buffer containing 0.25 sucrose, 10 m Tris-MOPS, pH 7.4, 5 m glutamate, 2.5 m malate, and 1 m Pi–Tris. MPT pore opening was induced by the successive addition of Ca2+ (25 for the retention assay, and 50 for the swelling assay).
2 m K+-phosphate and 0.5 m -ketoglutarate).17 Ruthenium red (1 ) was added approximately 1 min after addition of mitochondria, followed by addition of 0.5 m ADP to initiate state 3 respiration. Immediately after the addition of ADP, KCN (1 m) was added to block the electron transport. Absorbance at 340 nm was measured with a Shimadzu spectrophotometer. The rate of NADH formation upon blockade of electron transport was calculated by converting the rate of increase in absorbance (OD/mg/min) to nanomoles of NADH formed per mg/min, assuming a millimolar absorptivity for NADH of 6.22.
Assay of mitochondrial Na+–Ca2+ exchange (NCE)
Protein bands were scanned and the optical density of the bands quantified using the NIH image 1.61 software. Data (mean±..) were analyzed by Student’s t-test and significance defined as P<0.05.
The assay of NCE activity was performed according to Cox and Matlib.17 Isolated heart mitochondria were loaded with fura-2 AM (10 ) for 5 min at 30 °C. After dye loading and washing, the mitochondria (0.25 mg/ml) was added to assay buffer (120 m KCl, 10 m MOPS, pH 7.2, 2 m K+phosphate and 0.5 m -ketoglutarate). Time-resolved measurements were performed to follow the changes in fluorescence. Fura-2 fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength at 500 nm.10 The ratio of emitted light from the two wavelengths (340/380) provided a measure of Ca2+. A standard curve was constructed relating the ratio to Ca2+ concentration. The assay began with the addition of ruthenium red (RR, 1 ) that blocks the Ca2+ uptake pathway, thus leaving the NCE as the only major route for Ca2+ transport in these mitochondria. In order to quench the fluorescence of any extramitochondrial fura-2 that may have been present, MnCl2 (1 ) was routinely added after addition of RR. The addition of 10 m NaCl then resulted in a Na+-dependent decrease in the 340/ 380 nm fluorescence ratio, indicating that activation of the NCE by Na+ led to Cam efflux, and a decrease in matrix-free Ca2+. Calculation of the initial rate of this Na+-dependent Ca2+ efflux provided a measure of NEC activity.
Measurement of the rate of NADH production in intact mitochondria Mitochondria (1 mg) were assayed at 25 °C in 2 ml of assay buffer (120 m KCl, 10 m MOPS, pH 7.2,
Densitometry and data analysis
Results Cardiac mitochondria from Bcl-2 transgenic mice are resistant to Ca2+-induced swelling We have examined the effect of Bcl-2 overexpression on the mitochondrial permeability transition (MPT) induced by Ca2+ as assessed by the swelling assay. For this purpose, heart mitochondria from four transgenic mice were compared side by side with that from four non-transgenic littermate controls. Our previous electron microscopy study has verified that the procedure for isolating pure and intact mitochondria was satisfactory, thus allowing meaningful comparison.26 Figure 1A shows a representative experiment of Western blot analysis of Bcl-2 protein in the mitochondrial fraction prepared from hearts of transgenic mice v controls. The immuno-blot was overexposed to show that Bcl-2 was undetectable in the isolated mitochondria of the control group, while the level of Bcl-2 was significantly enhanced in the transgenic group (two independent experiments). In addition to mitochondria, increased expression of Bcl-2 was also found in the ER fraction (not shown) such that the resulting Bcl-2 level in the whole heart lysate was 4.6-fold higher in the transgenic group as compared to control group (Fig. 6A, and reference 24). Induction of the permeability pore opening was evaluated as the Ca2+-induced mitochondrial swelling due to uptake of sucrose (Fig. 1B). Exposure of mitochondria to successive additions of Ca2+ (50
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Figure 1 Bcl-2 delays the MPT pore opening induced by Ca2+ as assessed by mitochondrial swelling. A. Western blot showing increased level of Bcl-2 protein in the heart mitochondria of Bcl-2 transgenic mice as compared with non-transgenic mice. B. MPT pore opening was monitored as the absorbance decrease at 540 nm. The assay medium (2 ml) contained 250 m sucrose, 1 m Pi–Tris, 10 m Tris–MOPS, 5 m glutamate–Tris, 2.5 m malate–Tris, pH 7.4, 25 °C. Experiments were started by the addition of 1 mg of mitochondria followed by successive additions of 50 Ca2+ pulses (arrows) for samples from Bcl-2 transgenic mice and non-transgenic controls. Results are typical of two independent experiments.
each time) led to opening of the pore as indicated by a decrease in absorbance at 540 nm (see Materials and Methods). Figure 1B shows that the Bcl-2 group was more resistant to Ca2+-induced MPT. While the requirement of Ca2+ for the pore opening was 150 in the control group, this Ca2+ threshold was increased to 200 or higher in the Bcl-2 group. The extent of swelling, as indicated by the maximal decrease in A540, was also reduced in the Bcl-2 group such that it was only 66% of the control. Increased capacity of the mitochondrial calcium store by Bcl-2 overexpression Our previous studies in cultured neuroblastoma cells and cardiomyocytes10,11 have indicated an en-
Figure 2 Bcl-2 enhances the Ca2+ retention capacity of mouse heart mitochondria. Assay medium was the same as in Figure 1 except 1 Calcium Green-5N was included. Mitochondria (1 mg) was added in the assay medium and the experiments began with successive addition of 25 Ca2+ pulses (arrows) for non-transgenic mice (trace A) and Bcl-2 transgenic mice (trace B). After mitochondria were loaded with 75 Ca2+, 250 atractyloside were added (at the upward arrow) to the medium, followed by additional 25 Ca2+ pulses for non-transgenic mice (trace C) and Bcl-2 transgenic mice (trace D). Results are typical of three independent experiments.
hanced capacity of mitochondrial Ca2+ (Cam) in these cells by Bcl-2 overexpression. Cells with ectopic expression of Bcl-2 were able to retain more FCCP-releasable Ca2+ in the mitochondria than control cells with endogenous Bcl-2.10,11 Here, we have examined this phenomenon directly using mitochondria isolated from hearts of transgenic mice and non-transgenic controls. Heart mitochondria energized with glutamate plus malate in the presence of 1 m Pi were loaded with a series of 25 Ca2+ pulses at 1-min intervals (Fig. 2). Under these conditions, the control group took up and retained Ca2+ until the load reached a threshold of 150 (the accumulation of 6 pulses of Ca2+), at which point the mitochondria underwent a fast process of Ca2+ release (Fig. 2A). The precipitous Ca2+ release was due to the opening of the MPT pore, because the critical Ca2+ load for Ca2+ release was increased by the pore inhibitor cyclosporin A (data not shown). A parallel study for the Bcl-2 group (Fig. 2B) indicated an increase of Ca2+ retention capacity to 11 pulses. Complete opening of
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the pore occurred at 275 of Ca2+. Results from three independent experiments indicated that the Ca2+ retention capacity for the control group was 150±12 , while that for the Bcl-2 group was 243±32 (n=3, P<0.02). Thus, Bcl-2 (like cyclosporin A) enabled the heart mitochondria to retain more Ca2+. Further studies were carried out with the addition of the pore activator atractyloside. Figure 2C shows that addition of atractyloside (Atr, 250 ) to the control sample after 3 pulses of Ca2+ (at the upward arrow) triggered a premature opening of the pore, which in turn prevented subsequent pulses of Ca2+ from being retained. In contrast, addition of Atr to the Bcl-2 group (Fig. 2D) after 3 pulses did not open the pore fully. Instead, Ca2+ retention continued for three more pulses, and a total of 175 of Ca2+ was needed to trigger the MPT. These results are in complete agreement with our previous studies showing an increased capacity for the FCCP-releasable Cam by Bcl-2 in cultured cardiomyocytes and neuroblastoma cells.10,11 Mitochondrial Na+-Ca2+ exchange as a major efflux pathway for matrix Ca2+ Although the presence of a Na+–Ca2+ exchange system (NCE) in heart mitochondria is well known, its role in the regulation of matrix Ca2+ level and cell survival remains unclear. For this reason, we have investigated the NCE activity in normal hearts as well as in hearts that overexpress Bcl-2. Figure 3A shows a routine assay for NCE, using isolated mitochondria from a normal rat heart. This assay was based on the use of fura-2 loaded mitochondria to follow the release of matrix Ca2+ in response to the addition of NaCl (see Materials and Methods). To ensure that the measurement was specific to NCE, ruthenium red (RR) was used to block the Ca2+ uptake by the uniporter, and MnCl2 (Mn) was used to quench any excess fura-2 that may be present on the outside of mitochondria.17 The assay (Fig. 3A) indicated a stable baseline upon addition of RR. Addition of MnCl2 did not cause a decline of fluorescence, indicating that all of the fura-2 was located in the matrix. After the equilibration of baseline, NaCl (10 m) was added to release the matrix Ca2+ as indicated by an immediate decrease in fura-2 fluorescence ratio. The initial rate of Ca2+ efflux was calculated from the slope of this decline. Figure 3B shows that the rate of Ca2+ efflux was dependent on Na+ concentration. Results from two independent experiments indicated a linear response between 0–10 m followed by a plateau.
Figure 3 Na+–Ca2+ exchange activity of rat heart mitochondria. Fura-2 loaded mitochondria (0.5 mg) were added to assay medium (2 ml) supplemented with 120 m KCl, 10 m MOPS, pH 7.2, 2 m potassium phosphate and 0.5 m -ketoglutarate (KG) at 37 °C. Approximately 1 min after addition of mitochondria, 1 RR was added to block Ca2+ uptake, followed by the addition of 1 MnCl2 to quench fluorescence of extramitochondral fura-2. Then NaCl (10 m) was added to activate the Na+–Ca2+ exchange (NCE). The rate of decrease in fluorescence ratio (340/380 nm) represents NCE activity. A. Trace displays the time course of the NCE assay. B. The rate of decrease in matrix Ca2+ (Ca2+ efflux) was dependent on Na+ concentration. The fluorescence ratio was converted to Ca2+ concentration by a calibration curve. C. Inhibition of mitochondrial Na+–Ca2+ exchange activity by indicated concentration of clonazepam (Clo). Mitochondria were pre-incubated with Clo for 3 min before the assay with 10 m NaCl.
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Figure 5 Bcl-2 increases the rate of KG-supported NADH formation in mouse heart mitochondria. Experimental conditions were the same as in Figure 3 except that 1 mg mitochondria was added. Approximately 1 min after adding mitochondria, 1 RR was added followed by addition of 0.5 m ADP and immediately addition of 1 m KCN. The rate of NADH formation following KCN addition was calculated as described in Materials and Methods. Values denote mean±.. of three samples (∗ P<0.001).
Figure 4 Na+–Ca2+ exchange activity of mouse heart mitochondria is inhibited by Bcl-2. Experimental conditions were the same as in Figure 3; 10 m NaCl was added to activate the Na+–Ca2+ exchange. A. The rate of decrease in matrix free [Ca2+] in Bcl-2 transgenic v non-transgenic mice mitochondria (∗ P<0.02). B. The resting level of Cam in Bcl-2 transgenic v non-transgenic mice mitochondria. Results are typical of four or more independent experiments (∗ P<0.002).
Therefore, 10 m NaCl was chosen as the standard assay condition. Figure 3C shows the dose–response inhibition of NCE by a specific inhibitor clonazepam (Clo).28 At concentrations between 75 and 100 , Clo completely inhibited the NCE activity. It should be mentioned that, at this concentration, Clo also blocked the Ca2+-induced necrotic cell death in cultured cardiomyocytes.11 Thus, inhibition of NCE promotes cell survival.
Downregulation of NCE activity by Bcl-2 overexpression To test the hypothesis that inhibition of NCE is beneficial for cell survival further, we determined the NCE activity of cardiac mitochondria from the transgenic mice that overexpressed Bcl-2. Figure 4A shows that, indeed, the NCE activity of the Bcl2 group was depressed significantly by 30% as
compared to controls. Results from four experiments indicated the value of 415±41 n/mg/min for the control group as compared to 265±11 n/mg/min for the Bcl-2 group (P<0.02). This depressed NCE activity is also consistent with the study showing the protective effect of Bcl-2 against ischemia reperfusion injury in the transgenic mice.24 We have also estimated the matrix Ca2+ concentration from the baseline of the NCE assay before the addition of Na+. Figure 4B indicates higher matrix Ca2+ level in the Bcl-2 group as compared to control group (125±0.7 n v 111±2.0, n=4, P<0.002). The results support our earlier finding of increased basal level of Cam in cultured cells that overexpress Bcl-2.10,11
Bcl-2 increases the rate of NADH formation in mouse heart mitochondria It has been shown by Cox and Matlib17 that alterations in mitochondrial matrix Ca2+ concentration induced by changes in NCE activity were translated into changes in the rate of NADH production and the overall rate of oxidative phosphorylation. NCE inhibitors such as diltiazem and clonazepam were shown to enhance NADH formation and oxidative phosphorylation rates.17 These observations prompted us to determine the effect of Bcl-2 on NADH formation in mitochondria from the transgenic mouse heart. Figure 5 shows that there was a significant increase in the relative
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rate of NADH formation (supported by -ketoglutarate) in the Bcl-2 group as compared to controls (1.0±0.04 v 1.63±0.02, n=3, P<0.001). Study of oxidative phosphorylation also indicated a 16% increase in the respiratory control ratio for the Bcl-2 group v control group (not shown). The results suggest more efficient ATP synthesis in the Bcl-2 group.
Upregulation of genes involved in cellular Ca2+ homeostasis Previous studies have indicated that overexpression of Bcl-2 in the heart of transgenic mice did not induce any changes in the expression of pro-apoptotic protein Bax, or heat shock proteins (HSP70 and HSP25).24 However, Bcl-2 is known to alter Ca2+ homeostasis of the ER compartment,29 and local changes of Ca2+ in the microdomain have profound effect on the expression of several Ca2+ transport proteins.27 It is therefore important to test the possibility that the lower NCE activity may be related to altered expression of Ca2+ transport proteins. Figure 6A shows the Western analyses of the heart extracts from the transgenic and the nontransgenic mice. Comparison of the two groups indicated altered expression of several major Ca2+ transport proteins in the Bcl-2 mice. Results from two independent experiments indicated that the expression of Bcl-2 in the transgenic heart was approximately 4.6-fold higher than controls. This was accompanied by increased expression in the sarcolemma Na+–Ca2+ exchanger isoform 1 (NCX1, 7.8-fold), plasma membrane calcium pump (PMCA, 2-fold), and ER calcium pump (SERCA, 1.4-fold) in the transgenic heart (Fig. 6B). The Coomassie blue staining of the gel samples indicated equal loading of the protein samples (not shown). Interestingly, the expression of the -type Ca2+ channel (DHP receptor, -2 subunit) involved in Ca2+ entry was unchanged. Comparison of the mitochondrial fraction with the whole heart extract (on equal basis of protein) also indicated that the majority of Bcl2 was found in the cytosol (in the ER fraction), while only a minor portion of Bcl-2 was associated with the mitochondria (Fig. 1A). The results suggest that Bcl-2 may modulate cellular Ca2+ homeostasis directly or by upregulating the expression of NCX1, PMCA and SERCA. Since NCX1 is the principal Ca2+ efflux mechanism in cardiac myocytes and plays a critical role in regulating the force of cardiac muscle contraction, the large increase in NCX1 protein level in the sarcolemma could influence the mitochondrial Ca2+ transport and NCE activity.
Figure 6 Bcl-2 upregulates the expression of sarcolemma Na+–Ca2+ exchanger. A. Western blot analysis of Bcl-2, NCX1, DHP2, PMCA and SERCA2 in heart homogenates from transgenic (Bcl-2) and non-transgenic (control) mice. Note that the molecular weights of PMCA, SERCA, and NCX1 are smaller than their rat counterpart (mouse cDNAs are also smaller than rat). B. Densitometry analysis of the protein bands indicates a striking upregulation in NCX1 expression than other transporters. Results are obtained from four pooled hearts in each group.
Discussion Using transgenic mice overexpressing Bcl-2, Chen et al.24 showed the cardioprotective effect of Bcl-2 in apoptosis induced by ischemia-reperfusion injury. Similar in-vivo protection by Bcl-2 has been reported in the ischemic injury of brain30 and I/R injury of intestine.31 However, the precise mechanism for the inhibition of myocardial I/R injury in the transgenic mice is not known; and many possibilities were considered.24 Our previous study using cultured cells have suggested that the pro-survival effect of Bcl-2 may be linked to its ability to modulate mitochondrial as well as cellular Ca2+ homeo-
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stasis.10,29 Here, we have further tested this hypothesis in the in-vivo setting of transgenic mice. We show that cardiac mitochondria from Bcl-2 transgenic mice are more resistant to Ca2+-induced swelling (Fig. 1B). The increased resistance is related to increased retention of Ca2+ in the mitochondria (Fig. 2). The increased level of matrix Ca2+ is then translated to enhanced NADH production as well as more efficient respiration and ATP generation in the mitochondria of the transgenic heart (Fig. 5). The more efficient respiration is also known to reduce the risk in the generation of superoxide anion, thereby diminishing the danger of oxidative damage to the mitochondria.32 Thus the present study supports the notion that the major physiological function of mitochondrial Ca2+ is the regulation of mitochondrial metabolism.12–14 The increased loading of Cam by Bcl-2 could be a result of either increased uptake by the uniporter or decreased efflux by the exchanger. We show for the first time that the higher basal level of matrix Ca2+ is at least partly due to the downregulation of NCE activity by Bcl-2 (Fig. 4). In contrast, reduced basal level of matrix Ca2+ is a common characteristic of the pro-apoptotic cells such as Bax-overexpressing cells.10 Current literature has emphasized the importance of Bcl-2 as a blocker of MPT and cytochrome c release.22,33,34 However, almost no information is available about the role of Bcl-2 in the control of mitochondrial Ca2+ efflux and the subsequent effect on bioenergetics. Our results suggest that the maintenance of mitochondrial bioenergetics via the regulation of Cam is an important consequence of Bcl-2 overexpression in the transgenic mouse heart. A link between excessive Ca2+ influx into the cell (Ca2+ overload) and cell death has long been proposed, but how Ca2+ kill cells is not clear. Our previous study of the kinetics of cell death indicates that high Ca2+ influx leads to mitochondrial Ca2+ overload which then trigger the MPT, resulting in the loss of Cam through the pore before cell death ensues.11 This kinetic study indicates that Cam depletion is a cause rather than a consequence of cell death; furthermore, it shows that Bcl-2 protects cells from unwanted death by maintenance of a threshold level of Cam.10,11 This finding in cultured cells is reinforced here by the study in transgenic mice. It is suggested that MPT and Cam depletion may account (at least in part) for the apoptotic cell death observed during ischemia-reperfusion injury in the transgenic mouse heart.24 In addition to the release of Cam through the MPT pore observed here (Fig. 2 and reference 20), other laboratories have shown the loss of cytochrome c35 and NAD+36
through the MPT pore. Therefore, cytochrome c is not the only factor that is released during permeability transition; loss of other factors such as Cam and NAD+ through the pore can also contribute to cell death. This is especially important in tissues enriched with mitochondria, such as heart and brain tissue. Normal mitochondrial energetics is vital for cardiomyocyte function during excitationcontraction coupling. Therefore, MPT pore opening is a causative event in reperfusion damage of the heart.36 It is possible that the severity in the loss in Cam and NAD+ may explain the difference between apoptotic and necrotic death. The present results indicate the importance of NCE in the control of basal Cam level. In addition to MPT, NCE activity is also important in the regulation of cell death. This hypothesis is supported by a previous study showing that inhibition of NCE by clonazepam protects cultured cardiomyocytes from necrotic death induced by high Ca2+.11 It has been shown that at 100 , clonazepam targets primarily to NCE without affecting Ca2+ channels or SR transport.37 We also have evidence of increased NCE activity in the development of myocyte death that occurs spontaneously in the heart of Syrian hamsters with a mutation in dystrophin-associated protein (Kuo et al., to be submitted). Treatment of these hamsters with diltiazem, an NCE antagonist, ameliorates cell death and prevents the development of cardiomyopathy.38 Thus, enhanced NCE activity may be a final common step leading to cell death in cardiac diseases of diverse etiology. The role of the MPT in the release of cytochrome c is controversial (reviewed in reference 39). It has been suggested that during Bid or Bax-induced apoptosis, only the outer membrane is permeabilized with no involvement of the inner membrane.39,40 In support of this possibility, Bax itself has been suggested to form the pore by oligomerization process, leading to the release of cytochrome c.41 Therefore, Bcl-2 may protect cells by simply preventing Bax oligomerization42 without the participation of the MPT pore.39 However, the present results suggest a more complex action of Bcl-2, not limited to the blocking of Bid or Bax on the outer membrane. Indeed, Bcl-2 has profound effects on cellular Ca2+ homeostasis that include mitochondria and ER compartments.10,29 The downregulation of NCE activity suggests that Bcl-2 exerts impact on this inner membrane protein. In addition, Bcl-2 increases the expression of major Ca2+ transport proteins (Fig. 6) without altering the level of Bax protein or mitochondrial heat shock proteins.24 Taken together, the results suggest that the protective effect of Bcl-2 in the transgenic mice against
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I/R injury is related to its ability to maintain the mitochondrial Ca2+ homeostasis. It appears that the regulation of both MPT pore and NCE activity are involved. This is supported by our previous results, which showed complete inhibition of myocyte death in the presence of inhibitors of both MPT and NCE, while only partial protection was obtained with one inhibitor.11 The mechanism of Bcl-2 induced downregulation of NCE activity is not clear. One possibility is a direct effect of Bcl-2 on mitochondrial physiology. However, our preliminary study has indicated that incubation of mitochondria with recombinant Bcl2 did not lead to inhibition of the NCE activity (data not shown). Therefore, it is likely that Bcl-2 affects NCE activity (at least in part) through indirect means such as gene expression. Previous studies in epithelial cell culture have indicated upregulation of SERCA expression by Bcl-2.29 We show here that, in addition to SERCA, Bcl-2 also upregulates the expression of PMCA and Na+–Ca2+ exchanger of the sarcolemma (NCX), both of which function to reduce cytoplasmic Ca2+ level by moving excess Ca2+ out of the myocytes. Interestingly, the expression of the -type channel that mediates Ca2+ entry in cardiomyocytes is not affected by Bcl-2, suggesting that Bcl-2 modulate Ca2+ efflux rather than influx. The stimulation of PMCA level is consistent with our observation of a protective effect of PMCA against thapsigargin-induced apoptosis in endothelial cells (Kuo et al., unpublished results). The striking increase of the sarcolemmal NCX1 (7.8-fold) in the transgenic mice (Fig. 6) is consistent with a dominant role of NCX in mediating Ca2+ efflux in cardiac myocytes. The simultaneous upregulation of several Ca2+ transport proteins is in agreement with our previous observation that the genes of the major Ca2+ network are regulatorally linked,27 and that Ca2+ itself may provide this regulation at the level of transcription and/or translation.27 Similarly, Ca2+ may also regulate the NCE gene expression. However, this possibility could not be determined presently, due to the lack of purification or cloning of this protein. Since Bcl-2 protein possesses ion-channel activity,41 and cytoprotection by Bcl-2 requires the pore-forming portion of the molecule,43 it is reasonable to speculate that Bcl-2 regulates Ca2+ levels in the vicinity (microdomain) of the ER and mitochondria which in turn can regulate NCE expression and/or activity. This hypothesis is supported by the fact that Bcl-2 modulates mitochondrial Ca2+ as well as ER Ca2+.10,29 Expression of Bcl-2 on both ER- and mitochondrial-membrane may facilitate the cross talk between these two compartments.10 Al-
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ternatively, Bcl-2 may affect NCE activity secondarily through the regulation of NCX activity. A link between mitochondria and the regulation of sarcolemmal NCX activity has been reported.44 Further studies at the cellular level are required to distinguish these possibilities.
Acknowledgements This work was supported by National Institutes of Health (HL-39481, to T.H.K), the American Heart Association Midwest Affiliate (Grant in Aid to T.H.K) and the Department of Veterans Affairs Medical Research Fund and the National American Heart Association (to B.H.L. Chua).
References 1. J S, C SC, N P, O S. Intracellular Ca2+ signals activate apoptosis in thymocytes: Studies using the Ca2+-ATPase inhibitor Thapsigargin. Exp Cell Res 1994; 212: 84–92. 2. MG KM, W KK, G ME. Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells. J Neurochem 1999; 72: 1853– 1863. 3. Z WH, L TT. Roles of calcium in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. Life Sci 1995; 57: 2091–2099. 4. B X, H FM, H Y, C JA, P JW. Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells. Am J Physiol 1997; 272: C1241–C1249. 5. P GA, B JC, B JA, M E. Effects of alterations in calcium homeostasis on apoptosis during neoplastic progression. Cancer Res 1997; 57: 537–542. 6. J S, P JG, C SK, B JA, B JC, M E. Reduce capacitative calcium entry correlates with vesicle accumulation and apoptosis. J Biol Chem 1999; 274: 8261–8268. 7. L JM, Z GJ, C DW. The changing landscape of ischaemic brain injury mechanisms. Nature 1999; 399 (Suppl. A7–A14: 6738. 8. H A, I S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res 1998; 82: 1111–1129. 9. J EM J, D TL. Molecular mechanisms of developmental neuronal death. Annu Rev Neurosci 1993; 16: 31–46. 10. Z L, L S, Y XD, V LK, S T, C G, K TH. Modulation of mitochondrial Ca2+ homeostasis by Bcl-2. J Biol Chem 1999; 274: 33267–33273. 11. Z LP, Y XD, L S, B RA, K TH. Mitochondrial Ca2+ homeostasis in the regulation of apoptotic and necrotic cell deaths. Cell Calcium 2000; 28: 107–117. 12. H RG. Physiological role of mitochondrial
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transport. J Bioenerg Biomembr 1994; 26: 495–508. 13. N BJ, D RM. Towards the molecular basis for the regulation of mitochondrial dehydrogenases by calcium ions. Mol Cell Biochem 1995; 149/150: 203–212. 14. G TE, G KK, S S-S, G CE. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol 1994; 267: C313–C339. 15. B P. Mitochondrial transport of cations: Channels, exchangers and permeability transition. Physiol Rev 1999; 79: 1127–1155. 16. H LH, C M. Evidence for the existence of regulatory sites for Ca2+ on the Na+/Ca2+ carrier of cardiac mitochondria. Biochem J 1982; 202: 509–518. 17. C DA, M MA. A role for the mitochondrial Na+-Ca2+ exchanger in the regulation of oxidative phosphorylation in isolated heart mitochondria. J Biol Chem 1993; 268: 938–947. 18. A RA, H CM, C LC, G AA, S RC, B GP. Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes. Am J Physiol 1992; 262: H1699–H1704. 19. I F, J LS, M J-P. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 1997; 89: 1145–1153. 20. B P, P V. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J Bioenerg Biomembr 1996; 28: 131–138. 21. M AN, B DE, C G, W E, F G. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc Natl Acad Sci USA 1996; 93: 9893–9898. 22. G DR, R JC. Mitochondria and apoptosis. Science 1998; 281: 1309–1312. 23. K G, R JC. Mitochondrial control of cell death. Nature Med 2000; 6: 513–519. 24. C Z, C CC, H YS, H RC, C BHL. Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial ischemia/reperfusion injury in transgenic heart. Am J Physiol 2001; 280: H2313–H2320. 25. S LA, J C, B ZR, S A. In: Methods in Pharmacology (Schwartz A, ed.). Vol. 1. New York: Appleton-Century-Crofts, 247–286. 26. K TH, G F, W J. Oxidative metabolism of polytron versus nagarse mitochondria in hearts of genetically diabetic mice. Biochim Biophys Acta 1985; 806: 9–15. 27. L BF, X X, F R, M S, K TH. Consequences of functional expression of the plasma membrane calcium pump isoform 1a. J Biol Chem 1996; 271: 5536–5544. 28. C DA, M MA. Modulation of intramitochondrial free Ca2+ concentration by antagonists of Na+-Ca2+ exchange. Trends Pharmacol Sci 1993; 14: 408–413. 29. K TH, K H-RC, Z L, Y Y, L HM, T W.
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Modulation of endoplasmic reticulum calcium pump by Bcl-2. Oncogene 1998; 17: 1903–1910. M J-C, D-D M, S JK, R I, F H, M M, A P, T D, C S, P C, H J. Overexpression of Bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 1994; 13: 1017– 1031. C CM, O’D D, G JL. Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice. Am J Physiol 1999; 276: G677–G686. W DC. Mitochondrial disease in man and mouse. Science 1999; 283: 1482–1488. G A, MD JM, K SJ. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13: 1899–1911. C P, J E, D D, K G. Mitochondrion as a novel target of anticancer chemotherapy. J Natl Cancer Inst 2000; 92: 1042– 1053. H KM, B MB, W H-W, M J, N AL. J Biol Chem 1999; 274: 5654–5658. D L F, M R, C M, B M, B P. Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD and is a causative event in the death of myocytes in postischemic reperfusion of the heart. J Biol Chem 2001; 276: 2571–2575. M MA, D JD, S N, R-N F. Clonazepam and diltiazem both inhibit sodiumcalcium exchange of mitochondia, but only diltiazem inhibits the slow action potentials of cardiac muscles. Biochem Biophys Res Commun 1985; 128: 290–296. J PL, B SK. Regulation of membrane-mediated chronic muscle degeneration in dystrophic hamsters by calcium-channel blockers: diltiazem, nifedipine and verapamil. J Neurol Sci 1993; 115: 76–90. C M. Bax, Bid and the permeabilization of the mitochondrial outer membrane in apoptosis. Curr Opin Cell Biol 2000; 12: 414–419. K RM, E MD, P G, R C, K T, B-W E, G M, B MJ, G DR, N DD. The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J Cell Biol 1999; 147: 809–822. S SL, M M, R JC. Bcl-2 family proteins as ion-channels. Cell Death Differ 1998; 5: 372–380. M V, M M, P DJ, D Z, V MA, S P. Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. J Biol Chem 2001; 276: 18361–18374. M S, S SL, X Z, R JC. Cytoprotection by Bcl-2 requires the pore-forming alpha5 and alpha6 helices. J Biol Chem 1998; 273: 30995– 31001. O K, R JP. Feedback inhibition of sodium/ calcium exchange by mitochondrial calcium regulation. J Biol Chem 2000; 275: 21549–21554.
Regulation of Sodium–Calcium Exchange and Mitochondrial Energetics by Bcl-2 in the Heart of Transgenic Mice Liping Zhu1, Yingjie Yu1, Balvin H. L. Chua2, Ye-Shih Ho3 and Tuan H. Kuo1 1
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan; James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee; 3 Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 2
(Received 9 August 2001, accepted for publication 14 September 2001, published electronically 30 October 2001) L. Z, Y. Y, B. H. L. C, Y.-S. H T. H. K. Regulation of Sodium–Calcium Exchange and Mitochondrial Energetics by Bcl-2 in the Heart of Transgenic Mice. Journal of Molecular and Cellular Cardiology (2001) 33, 2135–2144. Our previous work in cultured cells has shown that the maintenance of mitochondrial Ca2+ homeostasis is essential for cell survival, and that the anti-apoptotic protein Bcl-2 is able to maintain a threshold level of mitochondrial Ca2+ by the inhibition of permeability transition. To test whether Bcl-2 also affects the mitochondrial Na+–Ca2+ exchange (NCE), a major efflux pathway for mitochondrial Ca2+, studies using transgenic mice that overexpress Bcl-2 in the heart have been performed. NCE activity was determined as the Na+-dependent Ca2+ efflux in the isolated mitochondria. Overexpression of Bcl-2 led to a significant reduction of NCE activity as well as increased resistance to permeability transition in the mitochondria of transgenic heart. This was accompanied by increased matrix Ca2+ level, enhanced formation of NADH and enhanced oxidation of pyruvate, an NAD+-linked substrate. Furthermore, there was induction of cellular Ca2+ transport proteins including the Na+–Ca2+ exchanger of the sarcolemma (NCX). Bcl-2 not only stimulates NCX expression in the sarcolemma but also attenuates the Na+–Ca2+ exchange in the mitochondria. These results are consistent with the protection 2001 Academic Press by Bcl-2 against apoptosis in heart following ischemia/reperfusion. K W: Sodium–calcium exchange; Mitochondria; Bcl-2; Transgenic mice; Apoptosis; Necrosis; Calcium homeostasis.
Introduction Altered calcium homeostasis is commonly observed in vitro during apoptotic or necrotic cell death.1–6 Excessive Ca2+ influx is known to be a key component of ischemic injury in neuronal cells7 and cardiomyocytes.8 However, apoptosis can be attenuated by increased calcium entry,9 raising the question of how cytosolic Ca2+ overload kills cells. Recently, we have found that mitochondrial Ca2+ (Cam) plays an important role in the regulation of cell death.10,11 We showed that high concentrations
of cytosolic Ca2+ lead to mitochondrial Ca2+ overload, which in turn activates the permeability transition (MPT) pore, resulting in the loss of matrix Ca2+ through the pore.11 The reduction of Cam (FCCP-releasable) then leads to ATP depletion and cell death by necrosis.11 Interestingly, reduced Ca2+ entry through the capacitative channel has been correlated with apoptosis.6 Reduced Ca2+ entry (by decreasing extracellular Ca2+) also correlates with lower level of mitochondrial Ca2+.10 Combined with our kinetic analysis of cell death, this evidence suggests that Cam depletion is a cause rather than
Please address all correspondence to: T. H. Kuo, Department of Pathology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA. E-mail: [email protected]
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a consequence of cell death.10,11 While it is long recognized that lowering Cam levels reduces the rate of oxidative phosphorylation and thus affects the performance of work in the heart,12–14 the finding that reducing mitochondrial Ca2+ below a threshold level is detrimental to cell survival has only recently been made.10 Thus, it is important to understand how the basal Cam level is controlled in the cell and how it may affect cell survival. The steady state level of Cam is the result of a kinetic balance between uptake and efflux.14 While Cam uptake is achieved primarily via the ruthenium red-sensitive uniporter, there are at least two mechanisms that mediate Cam efflux in the mitochondria.14,15 One is the mitochondrial Na+–Ca2+ exchange (NCE) which represent a specific efflux pathway,16,17 and the other is the permeability transition (MPT) pore which represent a non-specific efflux pathway for mitochondrial Ca2+.18–20 Because Cam uptake is a fast reaction and Cam efflux is a slower process, it has been suggested that the levels of matrix Ca2+ are determined by efflux mechanisms.14 This hypothesis is supported by our initial study showing the control of Cam load by inhibition of both the MPT pore and NCE.11 Since ectopic expression of Bcl-2 enables cells to retain more Ca2+ in the mitochondria in the resting state,10,21 it has been assumed that the anti-apoptotic effect of Bcl-2 is due to the inhibition of the MPT pore.22,23 Inhibition of the pore by Bcl-2 or cyclosporin A leads to accumulation of Cam.10,18 However, whether Bcl-2 expression also affects the NCE system is unknown. In this regard, the study of mitochondrial Ca2+ homeostasis has been hampered by the lack of cloning of the uniporter or NCE genes. In the present study, we have investigated the effect of Bcl-2 on NCE activity and the associated mitochondrial functions in the heart of transgenic mice. Comparison of bioenergetics from hearts of transgenic and non-transgenic mice indicates that Bcl-2 expression not only inhibits permeability transition, but also enhances the rate of oxidative phosphorylation by attenuating NCE activity. Thus both MPT pore and NCE, two efflux mechanisms for mitochondrial Ca2+, are modulated by Bcl-2. Most interestingly, Bcl-2 stabilizes cellular Ca2+ homeostasis by upregulating the expression of the sarcolemma Na+–Ca2+ exchange, a major efflux mechanism in cardiomyocytes.
construct, containing the human Bcl-2 gene under the control of the cardiac--myosin heavy chain promoter, was used for the injection into pronuclei of fertilized C57BL/6xC3HF1 mouse oocytes. These mice were well characterized.24 Bcl-2 RNA and protein overexpression in the heart was confirmed by Northern and Western blot, respectively.24 Overexpression of Bcl-2 was shown to attenuate apoptosis and protect against myocardial ischemia/ reperfusion injury in the transgenic mice.24 Eighteen heterozygous transgenic mice of 4–8 months old were used. Eighteen age-matched and sex-matched non-transgenic littermates were used as experimental controls. All animals were treated in accordance with NIH guidelines.
Materials and Methods
Mitochondrial Ca2+ retention capacity, oxygen consumption, and swelling
Isolation of cardiac mitochondria Mitochondria were isolated using the procedure of Sordahl et al.25 as described previously.26 We have shown that this procedure yielded pure mitochondria as characterized by electron microscopy.26 Briefly, hearts from four mice were pooled, rinsed to remove blood, and weighed. The hearts were cut into halves in 12 volumes of KEA medium (0.18 KCl, 10 m EGTA, 0.5% bovine serum albumin, pH 7.2–7.4) and homogenized with a Polytron (Brinkman). The homogenate was fractionated by differential centrifugation. The resulting mitochondria pellet was washed twice and resuspended in 180 m KCl, 50 EGTA to yield approximately 20 mg protein per ml. Protein concentration was determined by the Biuret method.26
Western blot analysis For detecting Bcl-2 protein expression, the mitochondrial fraction as well as whole heart extract (50 g) were solubilized and samples separated on 12% polyacrylamide gel for immuno-blotting as described previously.27 The detection of Ca2+ transport proteins was carried out in whole heart lysate using 7.5% polyacrylamide gel. Bcl-2 antibody was obtained from Dako; PMCA, SERCA2, and NCX-1 were from Affinity Bioreagents.
Animals The generation of Bcl-2 transgenic mice has been described by Chen and co-workers.24 The transgene
Extramitochondrial Ca2+ concentration was measured with a spectrofluorometer (Photon Technology International) in the presence of 1 Calcium
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Green-5N as described by Murphy et al.21 using excitation-emission wavelengths: 503–535 nm. Calibration of the signal was achieved by the addition of known amounts of Ca2+. Mitochondrial oxygen consumption was measured polarographically at 30 °C using a Clarke-type oxygen electrode.26 Mitochondrial swelling was monitored as the decrease in light absorbance at 540 nm with a Shimadzu spectrophotometer. Incubations were carried out at 25 °C with 0.25 mg of mitochondrial protein per ml in the buffer containing 0.25 sucrose, 10 m Tris-MOPS, pH 7.4, 5 m glutamate, 2.5 m malate, and 1 m Pi–Tris. MPT pore opening was induced by the successive addition of Ca2+ (25 for the retention assay, and 50 for the swelling assay).
2 m K+-phosphate and 0.5 m -ketoglutarate).17 Ruthenium red (1 ) was added approximately 1 min after addition of mitochondria, followed by addition of 0.5 m ADP to initiate state 3 respiration. Immediately after the addition of ADP, KCN (1 m) was added to block the electron transport. Absorbance at 340 nm was measured with a Shimadzu spectrophotometer. The rate of NADH formation upon blockade of electron transport was calculated by converting the rate of increase in absorbance (OD/mg/min) to nanomoles of NADH formed per mg/min, assuming a millimolar absorptivity for NADH of 6.22.
Assay of mitochondrial Na+–Ca2+ exchange (NCE)
Protein bands were scanned and the optical density of the bands quantified using the NIH image 1.61 software. Data (mean±..) were analyzed by Student’s t-test and significance defined as P<0.05.
The assay of NCE activity was performed according to Cox and Matlib.17 Isolated heart mitochondria were loaded with fura-2 AM (10 ) for 5 min at 30 °C. After dye loading and washing, the mitochondria (0.25 mg/ml) was added to assay buffer (120 m KCl, 10 m MOPS, pH 7.2, 2 m K+phosphate and 0.5 m -ketoglutarate). Time-resolved measurements were performed to follow the changes in fluorescence. Fura-2 fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength at 500 nm.10 The ratio of emitted light from the two wavelengths (340/380) provided a measure of Ca2+. A standard curve was constructed relating the ratio to Ca2+ concentration. The assay began with the addition of ruthenium red (RR, 1 ) that blocks the Ca2+ uptake pathway, thus leaving the NCE as the only major route for Ca2+ transport in these mitochondria. In order to quench the fluorescence of any extramitochondrial fura-2 that may have been present, MnCl2 (1 ) was routinely added after addition of RR. The addition of 10 m NaCl then resulted in a Na+-dependent decrease in the 340/ 380 nm fluorescence ratio, indicating that activation of the NCE by Na+ led to Cam efflux, and a decrease in matrix-free Ca2+. Calculation of the initial rate of this Na+-dependent Ca2+ efflux provided a measure of NEC activity.
Measurement of the rate of NADH production in intact mitochondria Mitochondria (1 mg) were assayed at 25 °C in 2 ml of assay buffer (120 m KCl, 10 m MOPS, pH 7.2,
Densitometry and data analysis
Results Cardiac mitochondria from Bcl-2 transgenic mice are resistant to Ca2+-induced swelling We have examined the effect of Bcl-2 overexpression on the mitochondrial permeability transition (MPT) induced by Ca2+ as assessed by the swelling assay. For this purpose, heart mitochondria from four transgenic mice were compared side by side with that from four non-transgenic littermate controls. Our previous electron microscopy study has verified that the procedure for isolating pure and intact mitochondria was satisfactory, thus allowing meaningful comparison.26 Figure 1A shows a representative experiment of Western blot analysis of Bcl-2 protein in the mitochondrial fraction prepared from hearts of transgenic mice v controls. The immuno-blot was overexposed to show that Bcl-2 was undetectable in the isolated mitochondria of the control group, while the level of Bcl-2 was significantly enhanced in the transgenic group (two independent experiments). In addition to mitochondria, increased expression of Bcl-2 was also found in the ER fraction (not shown) such that the resulting Bcl-2 level in the whole heart lysate was 4.6-fold higher in the transgenic group as compared to control group (Fig. 6A, and reference 24). Induction of the permeability pore opening was evaluated as the Ca2+-induced mitochondrial swelling due to uptake of sucrose (Fig. 1B). Exposure of mitochondria to successive additions of Ca2+ (50
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Figure 1 Bcl-2 delays the MPT pore opening induced by Ca2+ as assessed by mitochondrial swelling. A. Western blot showing increased level of Bcl-2 protein in the heart mitochondria of Bcl-2 transgenic mice as compared with non-transgenic mice. B. MPT pore opening was monitored as the absorbance decrease at 540 nm. The assay medium (2 ml) contained 250 m sucrose, 1 m Pi–Tris, 10 m Tris–MOPS, 5 m glutamate–Tris, 2.5 m malate–Tris, pH 7.4, 25 °C. Experiments were started by the addition of 1 mg of mitochondria followed by successive additions of 50 Ca2+ pulses (arrows) for samples from Bcl-2 transgenic mice and non-transgenic controls. Results are typical of two independent experiments.
each time) led to opening of the pore as indicated by a decrease in absorbance at 540 nm (see Materials and Methods). Figure 1B shows that the Bcl-2 group was more resistant to Ca2+-induced MPT. While the requirement of Ca2+ for the pore opening was 150 in the control group, this Ca2+ threshold was increased to 200 or higher in the Bcl-2 group. The extent of swelling, as indicated by the maximal decrease in A540, was also reduced in the Bcl-2 group such that it was only 66% of the control. Increased capacity of the mitochondrial calcium store by Bcl-2 overexpression Our previous studies in cultured neuroblastoma cells and cardiomyocytes10,11 have indicated an en-
Figure 2 Bcl-2 enhances the Ca2+ retention capacity of mouse heart mitochondria. Assay medium was the same as in Figure 1 except 1 Calcium Green-5N was included. Mitochondria (1 mg) was added in the assay medium and the experiments began with successive addition of 25 Ca2+ pulses (arrows) for non-transgenic mice (trace A) and Bcl-2 transgenic mice (trace B). After mitochondria were loaded with 75 Ca2+, 250 atractyloside were added (at the upward arrow) to the medium, followed by additional 25 Ca2+ pulses for non-transgenic mice (trace C) and Bcl-2 transgenic mice (trace D). Results are typical of three independent experiments.
hanced capacity of mitochondrial Ca2+ (Cam) in these cells by Bcl-2 overexpression. Cells with ectopic expression of Bcl-2 were able to retain more FCCP-releasable Ca2+ in the mitochondria than control cells with endogenous Bcl-2.10,11 Here, we have examined this phenomenon directly using mitochondria isolated from hearts of transgenic mice and non-transgenic controls. Heart mitochondria energized with glutamate plus malate in the presence of 1 m Pi were loaded with a series of 25 Ca2+ pulses at 1-min intervals (Fig. 2). Under these conditions, the control group took up and retained Ca2+ until the load reached a threshold of 150 (the accumulation of 6 pulses of Ca2+), at which point the mitochondria underwent a fast process of Ca2+ release (Fig. 2A). The precipitous Ca2+ release was due to the opening of the MPT pore, because the critical Ca2+ load for Ca2+ release was increased by the pore inhibitor cyclosporin A (data not shown). A parallel study for the Bcl-2 group (Fig. 2B) indicated an increase of Ca2+ retention capacity to 11 pulses. Complete opening of
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the pore occurred at 275 of Ca2+. Results from three independent experiments indicated that the Ca2+ retention capacity for the control group was 150±12 , while that for the Bcl-2 group was 243±32 (n=3, P<0.02). Thus, Bcl-2 (like cyclosporin A) enabled the heart mitochondria to retain more Ca2+. Further studies were carried out with the addition of the pore activator atractyloside. Figure 2C shows that addition of atractyloside (Atr, 250 ) to the control sample after 3 pulses of Ca2+ (at the upward arrow) triggered a premature opening of the pore, which in turn prevented subsequent pulses of Ca2+ from being retained. In contrast, addition of Atr to the Bcl-2 group (Fig. 2D) after 3 pulses did not open the pore fully. Instead, Ca2+ retention continued for three more pulses, and a total of 175 of Ca2+ was needed to trigger the MPT. These results are in complete agreement with our previous studies showing an increased capacity for the FCCP-releasable Cam by Bcl-2 in cultured cardiomyocytes and neuroblastoma cells.10,11 Mitochondrial Na+-Ca2+ exchange as a major efflux pathway for matrix Ca2+ Although the presence of a Na+–Ca2+ exchange system (NCE) in heart mitochondria is well known, its role in the regulation of matrix Ca2+ level and cell survival remains unclear. For this reason, we have investigated the NCE activity in normal hearts as well as in hearts that overexpress Bcl-2. Figure 3A shows a routine assay for NCE, using isolated mitochondria from a normal rat heart. This assay was based on the use of fura-2 loaded mitochondria to follow the release of matrix Ca2+ in response to the addition of NaCl (see Materials and Methods). To ensure that the measurement was specific to NCE, ruthenium red (RR) was used to block the Ca2+ uptake by the uniporter, and MnCl2 (Mn) was used to quench any excess fura-2 that may be present on the outside of mitochondria.17 The assay (Fig. 3A) indicated a stable baseline upon addition of RR. Addition of MnCl2 did not cause a decline of fluorescence, indicating that all of the fura-2 was located in the matrix. After the equilibration of baseline, NaCl (10 m) was added to release the matrix Ca2+ as indicated by an immediate decrease in fura-2 fluorescence ratio. The initial rate of Ca2+ efflux was calculated from the slope of this decline. Figure 3B shows that the rate of Ca2+ efflux was dependent on Na+ concentration. Results from two independent experiments indicated a linear response between 0–10 m followed by a plateau.
Figure 3 Na+–Ca2+ exchange activity of rat heart mitochondria. Fura-2 loaded mitochondria (0.5 mg) were added to assay medium (2 ml) supplemented with 120 m KCl, 10 m MOPS, pH 7.2, 2 m potassium phosphate and 0.5 m -ketoglutarate (KG) at 37 °C. Approximately 1 min after addition of mitochondria, 1 RR was added to block Ca2+ uptake, followed by the addition of 1 MnCl2 to quench fluorescence of extramitochondral fura-2. Then NaCl (10 m) was added to activate the Na+–Ca2+ exchange (NCE). The rate of decrease in fluorescence ratio (340/380 nm) represents NCE activity. A. Trace displays the time course of the NCE assay. B. The rate of decrease in matrix Ca2+ (Ca2+ efflux) was dependent on Na+ concentration. The fluorescence ratio was converted to Ca2+ concentration by a calibration curve. C. Inhibition of mitochondrial Na+–Ca2+ exchange activity by indicated concentration of clonazepam (Clo). Mitochondria were pre-incubated with Clo for 3 min before the assay with 10 m NaCl.
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Figure 5 Bcl-2 increases the rate of KG-supported NADH formation in mouse heart mitochondria. Experimental conditions were the same as in Figure 3 except that 1 mg mitochondria was added. Approximately 1 min after adding mitochondria, 1 RR was added followed by addition of 0.5 m ADP and immediately addition of 1 m KCN. The rate of NADH formation following KCN addition was calculated as described in Materials and Methods. Values denote mean±.. of three samples (∗ P<0.001).
Figure 4 Na+–Ca2+ exchange activity of mouse heart mitochondria is inhibited by Bcl-2. Experimental conditions were the same as in Figure 3; 10 m NaCl was added to activate the Na+–Ca2+ exchange. A. The rate of decrease in matrix free [Ca2+] in Bcl-2 transgenic v non-transgenic mice mitochondria (∗ P<0.02). B. The resting level of Cam in Bcl-2 transgenic v non-transgenic mice mitochondria. Results are typical of four or more independent experiments (∗ P<0.002).
Therefore, 10 m NaCl was chosen as the standard assay condition. Figure 3C shows the dose–response inhibition of NCE by a specific inhibitor clonazepam (Clo).28 At concentrations between 75 and 100 , Clo completely inhibited the NCE activity. It should be mentioned that, at this concentration, Clo also blocked the Ca2+-induced necrotic cell death in cultured cardiomyocytes.11 Thus, inhibition of NCE promotes cell survival.
Downregulation of NCE activity by Bcl-2 overexpression To test the hypothesis that inhibition of NCE is beneficial for cell survival further, we determined the NCE activity of cardiac mitochondria from the transgenic mice that overexpressed Bcl-2. Figure 4A shows that, indeed, the NCE activity of the Bcl2 group was depressed significantly by 30% as
compared to controls. Results from four experiments indicated the value of 415±41 n/mg/min for the control group as compared to 265±11 n/mg/min for the Bcl-2 group (P<0.02). This depressed NCE activity is also consistent with the study showing the protective effect of Bcl-2 against ischemia reperfusion injury in the transgenic mice.24 We have also estimated the matrix Ca2+ concentration from the baseline of the NCE assay before the addition of Na+. Figure 4B indicates higher matrix Ca2+ level in the Bcl-2 group as compared to control group (125±0.7 n v 111±2.0, n=4, P<0.002). The results support our earlier finding of increased basal level of Cam in cultured cells that overexpress Bcl-2.10,11
Bcl-2 increases the rate of NADH formation in mouse heart mitochondria It has been shown by Cox and Matlib17 that alterations in mitochondrial matrix Ca2+ concentration induced by changes in NCE activity were translated into changes in the rate of NADH production and the overall rate of oxidative phosphorylation. NCE inhibitors such as diltiazem and clonazepam were shown to enhance NADH formation and oxidative phosphorylation rates.17 These observations prompted us to determine the effect of Bcl-2 on NADH formation in mitochondria from the transgenic mouse heart. Figure 5 shows that there was a significant increase in the relative
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rate of NADH formation (supported by -ketoglutarate) in the Bcl-2 group as compared to controls (1.0±0.04 v 1.63±0.02, n=3, P<0.001). Study of oxidative phosphorylation also indicated a 16% increase in the respiratory control ratio for the Bcl-2 group v control group (not shown). The results suggest more efficient ATP synthesis in the Bcl-2 group.
Upregulation of genes involved in cellular Ca2+ homeostasis Previous studies have indicated that overexpression of Bcl-2 in the heart of transgenic mice did not induce any changes in the expression of pro-apoptotic protein Bax, or heat shock proteins (HSP70 and HSP25).24 However, Bcl-2 is known to alter Ca2+ homeostasis of the ER compartment,29 and local changes of Ca2+ in the microdomain have profound effect on the expression of several Ca2+ transport proteins.27 It is therefore important to test the possibility that the lower NCE activity may be related to altered expression of Ca2+ transport proteins. Figure 6A shows the Western analyses of the heart extracts from the transgenic and the nontransgenic mice. Comparison of the two groups indicated altered expression of several major Ca2+ transport proteins in the Bcl-2 mice. Results from two independent experiments indicated that the expression of Bcl-2 in the transgenic heart was approximately 4.6-fold higher than controls. This was accompanied by increased expression in the sarcolemma Na+–Ca2+ exchanger isoform 1 (NCX1, 7.8-fold), plasma membrane calcium pump (PMCA, 2-fold), and ER calcium pump (SERCA, 1.4-fold) in the transgenic heart (Fig. 6B). The Coomassie blue staining of the gel samples indicated equal loading of the protein samples (not shown). Interestingly, the expression of the -type Ca2+ channel (DHP receptor, -2 subunit) involved in Ca2+ entry was unchanged. Comparison of the mitochondrial fraction with the whole heart extract (on equal basis of protein) also indicated that the majority of Bcl2 was found in the cytosol (in the ER fraction), while only a minor portion of Bcl-2 was associated with the mitochondria (Fig. 1A). The results suggest that Bcl-2 may modulate cellular Ca2+ homeostasis directly or by upregulating the expression of NCX1, PMCA and SERCA. Since NCX1 is the principal Ca2+ efflux mechanism in cardiac myocytes and plays a critical role in regulating the force of cardiac muscle contraction, the large increase in NCX1 protein level in the sarcolemma could influence the mitochondrial Ca2+ transport and NCE activity.
Figure 6 Bcl-2 upregulates the expression of sarcolemma Na+–Ca2+ exchanger. A. Western blot analysis of Bcl-2, NCX1, DHP2, PMCA and SERCA2 in heart homogenates from transgenic (Bcl-2) and non-transgenic (control) mice. Note that the molecular weights of PMCA, SERCA, and NCX1 are smaller than their rat counterpart (mouse cDNAs are also smaller than rat). B. Densitometry analysis of the protein bands indicates a striking upregulation in NCX1 expression than other transporters. Results are obtained from four pooled hearts in each group.
Discussion Using transgenic mice overexpressing Bcl-2, Chen et al.24 showed the cardioprotective effect of Bcl-2 in apoptosis induced by ischemia-reperfusion injury. Similar in-vivo protection by Bcl-2 has been reported in the ischemic injury of brain30 and I/R injury of intestine.31 However, the precise mechanism for the inhibition of myocardial I/R injury in the transgenic mice is not known; and many possibilities were considered.24 Our previous study using cultured cells have suggested that the pro-survival effect of Bcl-2 may be linked to its ability to modulate mitochondrial as well as cellular Ca2+ homeo-
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stasis.10,29 Here, we have further tested this hypothesis in the in-vivo setting of transgenic mice. We show that cardiac mitochondria from Bcl-2 transgenic mice are more resistant to Ca2+-induced swelling (Fig. 1B). The increased resistance is related to increased retention of Ca2+ in the mitochondria (Fig. 2). The increased level of matrix Ca2+ is then translated to enhanced NADH production as well as more efficient respiration and ATP generation in the mitochondria of the transgenic heart (Fig. 5). The more efficient respiration is also known to reduce the risk in the generation of superoxide anion, thereby diminishing the danger of oxidative damage to the mitochondria.32 Thus the present study supports the notion that the major physiological function of mitochondrial Ca2+ is the regulation of mitochondrial metabolism.12–14 The increased loading of Cam by Bcl-2 could be a result of either increased uptake by the uniporter or decreased efflux by the exchanger. We show for the first time that the higher basal level of matrix Ca2+ is at least partly due to the downregulation of NCE activity by Bcl-2 (Fig. 4). In contrast, reduced basal level of matrix Ca2+ is a common characteristic of the pro-apoptotic cells such as Bax-overexpressing cells.10 Current literature has emphasized the importance of Bcl-2 as a blocker of MPT and cytochrome c release.22,33,34 However, almost no information is available about the role of Bcl-2 in the control of mitochondrial Ca2+ efflux and the subsequent effect on bioenergetics. Our results suggest that the maintenance of mitochondrial bioenergetics via the regulation of Cam is an important consequence of Bcl-2 overexpression in the transgenic mouse heart. A link between excessive Ca2+ influx into the cell (Ca2+ overload) and cell death has long been proposed, but how Ca2+ kill cells is not clear. Our previous study of the kinetics of cell death indicates that high Ca2+ influx leads to mitochondrial Ca2+ overload which then trigger the MPT, resulting in the loss of Cam through the pore before cell death ensues.11 This kinetic study indicates that Cam depletion is a cause rather than a consequence of cell death; furthermore, it shows that Bcl-2 protects cells from unwanted death by maintenance of a threshold level of Cam.10,11 This finding in cultured cells is reinforced here by the study in transgenic mice. It is suggested that MPT and Cam depletion may account (at least in part) for the apoptotic cell death observed during ischemia-reperfusion injury in the transgenic mouse heart.24 In addition to the release of Cam through the MPT pore observed here (Fig. 2 and reference 20), other laboratories have shown the loss of cytochrome c35 and NAD+36
through the MPT pore. Therefore, cytochrome c is not the only factor that is released during permeability transition; loss of other factors such as Cam and NAD+ through the pore can also contribute to cell death. This is especially important in tissues enriched with mitochondria, such as heart and brain tissue. Normal mitochondrial energetics is vital for cardiomyocyte function during excitationcontraction coupling. Therefore, MPT pore opening is a causative event in reperfusion damage of the heart.36 It is possible that the severity in the loss in Cam and NAD+ may explain the difference between apoptotic and necrotic death. The present results indicate the importance of NCE in the control of basal Cam level. In addition to MPT, NCE activity is also important in the regulation of cell death. This hypothesis is supported by a previous study showing that inhibition of NCE by clonazepam protects cultured cardiomyocytes from necrotic death induced by high Ca2+.11 It has been shown that at 100 , clonazepam targets primarily to NCE without affecting Ca2+ channels or SR transport.37 We also have evidence of increased NCE activity in the development of myocyte death that occurs spontaneously in the heart of Syrian hamsters with a mutation in dystrophin-associated protein (Kuo et al., to be submitted). Treatment of these hamsters with diltiazem, an NCE antagonist, ameliorates cell death and prevents the development of cardiomyopathy.38 Thus, enhanced NCE activity may be a final common step leading to cell death in cardiac diseases of diverse etiology. The role of the MPT in the release of cytochrome c is controversial (reviewed in reference 39). It has been suggested that during Bid or Bax-induced apoptosis, only the outer membrane is permeabilized with no involvement of the inner membrane.39,40 In support of this possibility, Bax itself has been suggested to form the pore by oligomerization process, leading to the release of cytochrome c.41 Therefore, Bcl-2 may protect cells by simply preventing Bax oligomerization42 without the participation of the MPT pore.39 However, the present results suggest a more complex action of Bcl-2, not limited to the blocking of Bid or Bax on the outer membrane. Indeed, Bcl-2 has profound effects on cellular Ca2+ homeostasis that include mitochondria and ER compartments.10,29 The downregulation of NCE activity suggests that Bcl-2 exerts impact on this inner membrane protein. In addition, Bcl-2 increases the expression of major Ca2+ transport proteins (Fig. 6) without altering the level of Bax protein or mitochondrial heat shock proteins.24 Taken together, the results suggest that the protective effect of Bcl-2 in the transgenic mice against
Mitochondrial Na+–Ca2+ Exchange and Bcl-2
I/R injury is related to its ability to maintain the mitochondrial Ca2+ homeostasis. It appears that the regulation of both MPT pore and NCE activity are involved. This is supported by our previous results, which showed complete inhibition of myocyte death in the presence of inhibitors of both MPT and NCE, while only partial protection was obtained with one inhibitor.11 The mechanism of Bcl-2 induced downregulation of NCE activity is not clear. One possibility is a direct effect of Bcl-2 on mitochondrial physiology. However, our preliminary study has indicated that incubation of mitochondria with recombinant Bcl2 did not lead to inhibition of the NCE activity (data not shown). Therefore, it is likely that Bcl-2 affects NCE activity (at least in part) through indirect means such as gene expression. Previous studies in epithelial cell culture have indicated upregulation of SERCA expression by Bcl-2.29 We show here that, in addition to SERCA, Bcl-2 also upregulates the expression of PMCA and Na+–Ca2+ exchanger of the sarcolemma (NCX), both of which function to reduce cytoplasmic Ca2+ level by moving excess Ca2+ out of the myocytes. Interestingly, the expression of the -type channel that mediates Ca2+ entry in cardiomyocytes is not affected by Bcl-2, suggesting that Bcl-2 modulate Ca2+ efflux rather than influx. The stimulation of PMCA level is consistent with our observation of a protective effect of PMCA against thapsigargin-induced apoptosis in endothelial cells (Kuo et al., unpublished results). The striking increase of the sarcolemmal NCX1 (7.8-fold) in the transgenic mice (Fig. 6) is consistent with a dominant role of NCX in mediating Ca2+ efflux in cardiac myocytes. The simultaneous upregulation of several Ca2+ transport proteins is in agreement with our previous observation that the genes of the major Ca2+ network are regulatorally linked,27 and that Ca2+ itself may provide this regulation at the level of transcription and/or translation.27 Similarly, Ca2+ may also regulate the NCE gene expression. However, this possibility could not be determined presently, due to the lack of purification or cloning of this protein. Since Bcl-2 protein possesses ion-channel activity,41 and cytoprotection by Bcl-2 requires the pore-forming portion of the molecule,43 it is reasonable to speculate that Bcl-2 regulates Ca2+ levels in the vicinity (microdomain) of the ER and mitochondria which in turn can regulate NCE expression and/or activity. This hypothesis is supported by the fact that Bcl-2 modulates mitochondrial Ca2+ as well as ER Ca2+.10,29 Expression of Bcl-2 on both ER- and mitochondrial-membrane may facilitate the cross talk between these two compartments.10 Al-
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ternatively, Bcl-2 may affect NCE activity secondarily through the regulation of NCX activity. A link between mitochondria and the regulation of sarcolemmal NCX activity has been reported.44 Further studies at the cellular level are required to distinguish these possibilities.
Acknowledgements This work was supported by National Institutes of Health (HL-39481, to T.H.K), the American Heart Association Midwest Affiliate (Grant in Aid to T.H.K) and the Department of Veterans Affairs Medical Research Fund and the National American Heart Association (to B.H.L. Chua).
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