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Keywords:

  • Apoptosis;
  • Bax;
  • cytochrome C;
  • ischemia-reperfusion;
  • liver transplantation;
  • mitochondrial calcium

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial-dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic-hypothermic (4 °C) or hypoxic (< 0.1% O2)-hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 μM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 °C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c-GFP expression plasmid. Immunofluorescence determined the intracellular, spatio-temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The global rise in patients on liver transplant waiting lists has forced transplant centers to increasingly use donors that in the past would have been excluded. These ‘expanded’ donors, while addressing demand, are considered marginal because factors such as donor instability, advanced age of the donor (>65 years old), and donor liver steatosis are associated with poorer outcomes. In the immediate postreperfusion setting, these grafts have an 8–20% incidence of primary nonfunction or delayed graft function, resulting in increased morbidity and mortality (1).

While it is known that these grafts are more susceptible to reperfusion injury (2), the mechanisms governing this injury response remain elusive. It is clear that reperfusion following a period of ischemia induces endothelial and Kupffer cell injury (3), which, in turn, results in the production and release of pro-inflammatory cytokines (4) and oxygen-derived free radicals (5). These factors contribute to cellular injury and the initiation of cellular apoptosis (2). It is not clear, however, whether these mechanisms are responsible for hepatocyte injury, which occurs within the first several hours following reperfusion. Little is known about the impact of hypothermic storage on postreperfusion injury and graft function. Antecedent hypothermic storage may be an underestimated factor governing early postreperfusion hepatocyte death.

Altered intracellular Ca+2 regulation during hypothermic storage may contribute to primary hepatocyte injury. Mitochondria are active participants in cellular calcium (Ca+2) homeostasis, and they have the ability to rapidly accumulate and release large quantities of Ca+2 (6). In addition, mitochondrial calcium (mCa+2) regulation plays an important function in the regulation of many apoptotic mechanisms (7). Cold ischemia is known to induce an increase in cytosolic calcium concentration (8–10), which is associated with dysfunction of Ca+2 pumps, which normally transfer Ca+2 out of the cell or into the endoplasmic reticulum (11,12). During brief periods of cold ischemia, mCa+2 uptake buffers cytosolic calcium overload and, thus, functions as a protective mechanism for the cell (13). However, when elevated cytoplasmic Ca+2 concentration are accompanied by low levels of cytoplasmic ATP during longer periods of cold ischemia, activation of the Ca+2 uniporter and subsequent mCa+2 overload occurs (14–18). Mitochondrial Ca+2 overload results in permeability transition pore (PTP) formation and loss of the mitochondrial membrane potential (19,20).

Permeability transition pore formation, regulated in part by the Bcl-2 family of proteins, plays a role in mitochondrial-dependent apoptotic pathways (9,16,18,21,22). In addition, there is evidence that Bax and Bak, when triggered by apoptotic stimuli, promote Ca+2 release from endoplasmic reticulum and subsequent mCa+2 uptake (23,24). Permeability transition pore formation leads to mitochondrial swelling, rupture of the outer membrane and release of intramitochondrial pro-apoptotic factors, such as cytochrome c (25), apoptosis-inducing factor (AIF) (26), and smac/DIABLO (27).

The mitochondrial Ca+2 uniporter is a gated ion channel (14) that can be completely inhibited by micromolar concentrations of ruthenium red (RR) (28–31), a glycoprotein stain that binds to Ca2+ binding sites. We have previously demonstrated that RR inhibits ATP-dependent Ca2+ uptake in mitochondria isolated from rat liver (32). Limiting Ca2+ transport into the mitochondria during the period of cold preservation may prevent formation of the PTP, permeabilization of mitochondrial membranes, and initiation of cellular apoptosis. In this manuscript we investigate the role of mCa+2 uptake during a period of hypothermic storage on early reperfusion cell death. We report that mCa+2 uptake during hypothermic storage triggers Bax translocation and results in caspase 3-independent T-cell death during the early reperfusion time period.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cell culture and cold ischemic conditions

The human HepG2 hepatoblastoma cell line (HepG2, ATCC, Rockville, MD, Catalog No. HB-8065) was chosen because of its stability and predictable growth, as well as retained characteristics of primary hepatocytes. These cells produce alpha-fetoprotein, albumin, alpha2-macroglobulin, alpha1-antitrypsin, transferrin, alpha1-antichymotrypsin, haptoglobin, and many other proteins produced by normal hepatocytes. Hep G2 cells have been used in the study of hepatic regeneration and oncogenesis and ischemic injury (33–36). Cells were grown at 37 °C, 5% CO2 in MEM (ATCC, Manassas, VA), with 10% fetal bovine serum (Cascade Biologicals, Winchester, MA), and 1% penicillin/streptomycin (Gibco, Grand Island, NY) to 80% confluence. Figure 1 is a flow diagram outlining the general experimental design. The media was replaced with Belzer solution (UW) and cells were incubated at 4 °C in either normoxic or hypoxic conditions for 6 h. Hypoxia was achieved by placing the cells in an airtight incubator (Forma Scientifica, Marietta, OH), which was flushed with 5% CO2 and 95% N2 until the oxygen content in the container reached < 0.1%, as verified using a dissolved O2 meter (Model 4000, VWR Scientific Products, Suwannee, GA). To render the storage solution hypoxic before experiments were carried out, UW was preincubated in the hypoxic chamber in an open sterile container for 8 h before experiments were carried out. This resulted in a final O2 concentration of < 0.1% as measured with the dissolved O2 meter.

image

Figure 1. Flow diagram demonstrating the general study design.

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Following 6 h of hypothermic storage, the UW was replaced with warm, oxygenated media, and the cells were incubated at 37 °C. Cytoplasmic extracts were harvested in lysis buffer (10 mM of KCl, 10 mM of HEPES, 1 mM of NaEDTA, 200 mM of Mannitol, 50 mM of sucrose) immediately following storage and after 60, 120, and 180 min of rewarming. HepG2 cytoplasmic extracts were harvested before exposure to storage conditions to serve as a control (untreated control). In addition HepG2 cells were stored in UW under normoxic conditions at 37 °C in order to control for any effect from UW (negative control). Ruthenium red, a mitochondrial uniporter inhibitor (Sigma, St Louis, MO; 10 μM), was added to the UW solution of selected samples before storage of cells.

Western analysis

Cytoplasmic extracts (20 μg protein for cytochrome c, 60 μg for caspase 3) were subjected to polyacrylamide gel electrophoresis and transferred overnight to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Western blotting was performed as previously reported (33) using anticytochrome c (1: 200, Santa Cruz Biotechnology, Santa Cruz, CA), anticaspase 3 (1: 500, Cell Signaling Technology Inc., Beverly, MA), and an anticaspase 3 cleavage product (19 kDa) (1: 1000, Cell Signaling Technology). Densitometry was performed to determine relative levels of cytochrome c using a Versa Doc 3000 (Bio-Rad Laboratories, Inc.). Quantitation was performed using Quality One Quantitation software v4.4.1 (Bio-Rad Laboratories, Inc.). In order to compare relative density units of samples on separate blots, untreated control cytoplasmic extracts were included on each blot and the relative densities of the untreated bands were assigned a value of 1. The sample bands were normalized to the untreated control value on their corresponding blots, allowing blot-to-blot comparison for cytochrome c densities. All experiments were performed in triplicate.

Transient transfection studies and fluorescent microscopy

HepG2 cells were transiently transfected with a cytochrome c-GFP expression plasmid (gift from Dr Nieminen) as described by Nieminen (37). The transfected cells were grown to 80% confluence on poly D-lysine-coated glass coverslips and subjected to the above-described storage conditions with or without RR. Following replacement of UW with warm, oxygenated media, cells were fixed for fluorescence microscopy immediately following storage and after 60, 120, and 180 min of rewarming. Mitotracker Red (Molecular Probes, Eugene, OR; 100 nM), which is taken up by respiring mitochondria, was added to the media for 7 min before fixation in order to study the relationship of cytochrome c-GFP to the mitochondria. Both an untreated control and a negative control (described earlier) were studied.

Fluorescent microscopy was performed using an Axio-Plan 2 microscope (Zeiss, Hallbegmoos, Germany) with an AttoArc (Zeiss) fluorescent light source. A 450–49-nm filter was used to collect GFP fluorescence images, and Mitotraker images were collected with a 546–590 nm filter. Digital images of cytochrome c-GFP fluorescence were collected and recorded using an Axiocam HRc (Zeiss) and processed using Axiovision v3.1 software (Zeiss). Processed images were imported into Adobe Photoshop v7.0 (Adobe Systems, Inc., San Jose, CA), and merged images were created to evaluate colocalization of cytochrome c-GFP and mitochondria.

Immunofluorescence for Bax

HepG2 cells were grown on poly D-lysine-coated glass coverslips and subjected to the above-described storage conditions, with or without RR. The UW was then replaced with warm oxygenated media, and cells were fixed immediately following storage and after 60 and 180 min of rewarming. Before fixation, 100 nM of Mitotracker Red was added to each sample for 7 min. Cells were then washed with PBS and fixed in 4% paraformaldehyde in PBS for 30 min at 37 °C. Fixed cells were permeablized with 0.1% Triton X-100 for 5 min at 4 °C, washed with PBS, and incubated in the dark for 2 h at room temperature in blocking solution (0.1% Triton X-100, 2% normal goat serum in PBS) containing a mouse monoclonal anti-Bax antibody (1: 50, Transduction Laboratories). After the 2-h incubation, cells were washed twice with PBS and incubated with antimouse IgG-FITC (1: 50, Jackson Immunoresearch Laboratories) for 2 h at room temperature away from light. Cells were washed twice in PBS, and the coverslips were mounted on glass slides using fluorescent mounding medium (Dako). Fluorescence microscopy was performed on each component, and overlay images were performed as described earlier. Both an untreated control and a negative control were studied.

Caspase activity assay

The CaspACE colorimetric assay system (Promega Corporation, Madison, WI) was used to determine caspase 3 activity. HepG2 cells were subjected to normoxic-hypothermic or hypoxic-hypothermic storage conditions as described earlier and harvested according to the manufacturer's recommendations either immediately following storage or after being rewarmed for 180 min. A positive control was created by UV-irradiating HepG2 cells (30 mJ/cm2 for 30 s). The negative control was HepG2 cells cultured in media containing the caspase inhibitor Z-VAD-FMK (50 μM).

TUNEL staining

TUNEL (TdT-mediated dUTP nick-end labeling) staining was performed on HepG2 cells grown on poly D-lysine-coated glass slides and subsequently subjected to the above-described storage conditions with or without RR. After 3 h of reperfusion, the cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature and washed twice with PBS. TUNEL staining was performed by the Vanderbilt Mouse Histopathology Core Facility. A positive control was created by permeabilizing untreated cells and treating with RNAse-free DNAseI (Qiagen, Valencia, CA). The negative control was untreated HepG2 cells.

Statistical analysis

Statistical significance was determined for the relative densitometry studies and for the TUNEL staining experiments using a two-tailed-homoscedastic Student's t-test. A p-value of 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Inhibition of mitochondrial calcium uptake prevents hypothermic storage-induced cytochrome c release in HepG2 cells

The etiology of graft dysfunction is related, in part, to altered cellular and mitochondrial calcium (mCa+2) homeostasis following the required cold ischemia and reperfusion of organ harvest and implantation (12,13,17). To determine whether mCa+2 uptake during the period of cold preservation was associated with cytochrome c release during reperfusion, HepG2 cells were subjected to 6 h of cold storage under normoxic or hypoxic conditions in UW with or without RR. To simulate reperfusion, UW was replaced with warm, oxygenated media after storage. Cytoplasmic extracts were prepared immediately following cold storage and during the progression of ‘reperfusion’ and then subjected to Western analysis. There was no difference in cytochrome c release between the untreated control and the negative control (data not shown), indicating that UW alone did not induce cytochrome c release. Representative Western blots are shown in Figure 2(A). Cells subjected to normoxic-hypothermic (NH) storage show a small, nonsignificant increase in cytoplasmic cytochrome c levels immediately following storage when compared with untreated cells. There was no increase in cytochrome c levels during the 180 min of reperfusion when compared with the untreated HepG2 cells (time points 30, 60, 120 min, not shown) (Figure 2B). Ruthenium red did not significantly alter the cytoplasmic levels of cytochrome c following NH storage when compared with the untreated control. In contrast, cells subjected to hypothermic-hypoxic (HH) storage demonstrate a significant increase in cytoplasmic cytochrome c levels immediately following storage (p = 0.01, compared with the untreated control), but there is no significant elevation of cytoplasmic cytochrome c levels during the period of rewarming detected by densitometry. Addition of RR to UW significantly attenuated the increase in cytoplasmic cytochrome c levels immediately following HH storage (p = 0.05) but did not alter the 180-min rewarming levels. The results of these experiments suggest that the majority of cytochrome c release occurs during the time of hypothermic storage, rather than during reperfusion.

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Figure 2. HepG2 cells were subjected to 6 h of storage under normoxic or hypoxic conditions in UW with or without ruthenium red (RR) at 4 °C before rewarming in oxygenated media. Cytoplasmic extracts were harvested immediately following storage and during the progression of ‘reperfusion’ for western analysis (A) and densitometry (B). Cells subjected to normoxic-hypothermic (NH) storage show a small but not significant increase in cytoplasmic cytochrome c levels immediately following storage. In addition, there was no significant change in levels during the 180 min of rewarming when compared with the untreated HepG2 cells. mCa+2 uptake blockade during storage did not significantly alter the cytoplasmic levels of cytochrome c observed by Western analysis immediately following NH storage, nor during the 180 min of rewarming. However, cells subjected to hypothermic-hypoxic (HH) storage demonstrate a significant increase of cytoplasmic cytochrome c levels immediately following storage compared with untreated cells (*p = 0.01), but during rewarming the cytoplasmic cytochrome c levels returned to untreated levels. Addition of RR to UW significantly attenuated the increase in cytoplasmic cytochrome c levels compared with UW alone following HH storage (†p = 0.05), but did not significantly affect the cytochrome c levels during rewarming.

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To verify the results from Western analysis and to more closely study the intracellular location of cytochrome c following the cold storage conditions, HepG2 cells transiently transfected with cytochrome c-GFP were subjected to the same storage and rewarming conditions. Mitotraker Red (MR) was added to the media before the cells were fixed. Fluorescent microscopic images were taken for GFP and MR and subsequently merged in order to demonstrate the spatial relationship of cytochrome c and mitochondria. The untreated control seen in Figure 3A demonstrates cytochrome c-GFP completely associated with mitochondria. Immediately post NH storage (Figure 3B), the majority of cytochrome c-GFP remains associated with mitochondria, but as ‘reperfusion’ progresses to 180 min (Figure 3C), cytochrome c-GFP appears in the cytoplasm separate from the mitochondria. However, when RR is added to the cold storage solution, the cytochrome c-GFP redistribution seen immediately post storage and during rewarming is prevented (Figure 3D, E, respectively).

imageimage

Figure 3. HepG2 cells were transiently transfected with cytochrome c-GFP and subjected to the described storage and rewarming conditions. Mitotraker Red (MR) was added to the media before the cells were fixed, and fluorescent microscopic images taken for cytochrome c-GFP and MR. The final merged images are shown. (A) The untreated control shows cytochrome c-GFP fluorescence concentrated at the mitochondria in the merged image. (B) Immediately post NH storage, the majority of cytochrome c-GFP remains associated with mitochondria, but as ‘reperfusion’ progresses to 180 min (C), a larger amount of cytochrome c-GFP accumulates in the cytoplasm separate from the mitochondria. Ruthenium red prevents the redistribution seen immediately post storage (D) and during rewarming (E). Cells subjected to HH storage show larger amounts of cytochrome c-GFP immediately post storage (F), and the cytoplasmic accumulation continued during our observed 180 min of rewarming (G). Again, the addition of RR to the cold-preservation solution prevents HH-induced cytochrome c release from the mitochondria both immediately post storage (H) and following 180 min of rewarming (I).

Cells subjected to HH storage show large amounts of cytochrome c-GFP in the cytoplasm immediately post storage (Figure 3F), and the increased cytoplasmic accumulation remains during the observed 180 min of rewarming (Figure 3G). The addition of RR to the cold preservation solution also prevents HH-induced cytochrome c release from the mitochondria both immediately post storage and following 180 min of rewarming (Figure 3H,I, respectively). As an additional control to these experiments, HepG2 cells were stored in UW at 37 °C normoxic conditions, and like seen with western analysis, cytochrome c release into the cytoplasm was not induced by UW storage alone (data not shown). These data corroborate the Western analysis data to illustrate that cytochrome c release is initiated during the period of hypothermic storage and is exacerbated by hypoxia (HH storage).

Ruthenium Red inhibits hypothermic storage-induced Bax translocation from cytosol to mitochondria

Multiple studies have demonstrated that mitochondrial cytochrome c release following many apoptotic stimuli is initiated by Bax translocation to the outer mitochondrial membrane (22,38–40). To determine if this mechanism plays a role in the cytochrome c release observed in HepG2 cells following hypothermic storage, we examined the spatio-temporal intracellular distribution of Bax proteins using immunofluorescence. Figure 4 shows representative examples of the fluorescence microscopy analysis of these cold-preserved cells, which were double labeled with Mitotraker Red and an antibody against Bax. Bax did not translocate to the mitochondria in the negative control (data not shown), or in the untreated control, as seen in Figure 4(A). However, immediately following storage, Bax translocation to the mitochondria was readily detectable in both the NH and HH groups (Figure 4B,C, respectively). There was no apparent difference in the magnitude of Bax translocation between the normoxic and hypoxic groups, which contrasts with the differences noted in the cytochrome c release. This redistribution of Bax was constant throughout the 180-min rewarming period following both NH and HH storage (data not shown). Addition of RR to the storage media resulted in attenuation of Bax translocation to the mitochondria following both NH and HH storage as seen in Figure 4(D,E), respectively. There were no apparent differences in magnitude of RR inhibition of Bax translocation between the NH and HH groups. In addition, Bax translocation to the mitochondria did not occur during the 180-min reperfusion period following storage in UW containing RR. These results imply that mCa2+ uptake during hypothermic storage triggers Bax translocation to the mitochondria before rewarming, but the exacerbation by hypoxia seen with cytochrome c release was not evident with Bax translocation.

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Figure 4. HepG2 cells were subjected to the described storage and rewarming conditions and then double labeled with Mitotraker Red (MR) and an antibody against Bax. Fluorescent microscopic images taken for Bax, MR, and the final merged images are shown. The untreated control demonstrates Bax located in the cytoplasm of HepG2 cells (A). However, immediately following storage, Bax translocation to the mitochondria was readily detectable in both the NH (B) and HH (C) groups. Addition of RR to the storage media resulted in an attenuation of Bax translocation to the mitochondria following both NH (D) and HH (E) storage. There was no difference in the magnitude of Bax translocation following storage between the NH and HH groups.

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Neither NH nor HH storage induced CaspACE 3 activation during the early reperfusion time period

Other investigators have noted an absence of caspase 3 activation following hypothermic storage (41,42). We studied caspase 3 activity following 3 h of rewarming in order to determine if the observed Bax translocation and cytochrome c release following hypothermic storage caused downstream activation of caspase 3. Figure 5A demonstrates the results of a caspase activity assay performed on HepG2 cells after NH or HH storage and 180 min of rewarming. There is negligible caspase activity from all samples when compared with the positive control (UV-irradiated HepG2 cells). To corroborate this finding, we obtained antibodies for both the 35 kDa uncleaved caspase 3 proform and the 19 kDa caspase 3 cleavage product. Figure 5(B,C) demonstrates that there is no cleavage of caspase 3 immediately following NH or HH storage. This is also true after 180 min of rewarming regardless of storage group (NH or HH). These data suggest that Bax translocation and cytochrome c release triggered by mCa+2 uptake during hypothermic storage either fail to activate cell death pathways, or they contribute to caspase-independent T-cell death.

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Figure 5. (A) Caspase 3 activity determined from cytoplasmic extracts of HepG2 cell subjected to NH or HH storage and subsequent rewarming for 180 min with or without ruthenium red (RR). Data is presented as nmol/h/mg protein from three separate experiments. There is no significant caspase activity following NH or HH storage nor after rewarming. (B) Western blot from cytoplasmic extracts taken following NH or HH storage immediately following storage and after 180 min of rewarming. (C) Only the UV-irradiated positive control cells show the 19 kDa caspase 3 cleavage product indicative of caspase activation.

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Ruthenium Red decreases hypothermic storage-induced cellular death during early reperfusion

We demonstrated that Bax translocation to the mitochondria and release of cytochrome c into the cytoplasm was dependent on mCa2+ uptake during hypothermic storage. However, no caspase activation occurred, and thus, we questioned whether the conditions caused cell death during the early rewarming period. Following the 6 h of NH or HH storage and 180 min of rewarming, TUNEL staining was performed. Figure 6 summarizes the results of these experiments. Normoxic hypothermia-stored HepG2 cells demonstrate approximately 27.5% cellular apoptosis after 180 min of rewarming. The percentage of apoptotic cells increases significantly to 45% (p = 0.034) when cells are rewarmed for 180 min following HH storage. Addition of RR to the UW storage solution decreases the rate of cellular apoptosis following NH storage to less than 1% (p = 0.001), while addition of RR to HH-stored cells decreased apoptosis rates to 15% (p = 0.001). These findings indicate a correlation between mCa2+ uptake blockade and prevention of early reperfusion cell death.

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Figure 6. HepG2 cells were subjected to normoxic-hypothermic (NH) or hypothermic-hypoxic (HH) storage with or without ruthenium red (RR) and subsequently rewarmed for 180 min. TUNEL staining was performed and the results are reported as percent of cells TUNEL positive (n = 4). Normoxic-hypothermic-stored cells demonstrate 27.5% cellular apoptosis after 3 h of rewarming. This increases to 45% in HH-stored cells after 3 h of rewarming. Addition of RR to the UW storage solution decreased the rate of cellular apoptosis following NH storage to less than 1% (*p = 0.001), but only to 15% (†p = 0.001) following HH storage.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We have previously shown that mCa+2 mismanagement during cold storage leads to mCa+2 overload (32). The mitochondrial uniporter inhibitor, RR, blocks mCa+2 uptake and prevents mCa+2 overload during ischemia and reperfusion in both isolated mitochondria and cell culture (32,43,44). A beneficial effect of RR has been demonstrated in postischemic reperfusion injury in rat myocardium (43). However, the mechanisms of mCa+2 overload-induced cellular injury are largely undescribed. In this study, we have used RR to show that prevention of mCa+2 overload during cold storage inhibits a pro-apoptotic cascade that includes Bax translocation to the mitochondria and cytochrome c release into the cytoplasm. The design of these experiments allowed observations to be made regarding the period of hypothermic storage and the subsequent early reperfusion period. Our results suggest that mCa+2 overload may be an early initiator of cell death following hypothermic storage.

In many models of apoptosis, Bax translocation to the mitochondria is required for cytochrome c release (9,21,22,39). Under normal conditions, Bax resides in the cytosol; but following a cell death stimulus, it localizes to the outer mitochondrial membrane where it is thought to play a role in the release of pro-apoptotic factors, including cytochrome c (21,39,45–47). Although PTP formation following a Ca+2 pulse has been shown to release small amounts of cytochrome c (48), Bax-induced cytochrome c release is independent of PTP formation (9). The manner in which these two mechanisms function together remains unclear; however, in another study using HepG2 cells it appears that a second factor (such as tBid) is required in addition to Ca+2 in order to achieve complete cytochrome c release (49,50). Bax and other Bcl-2 proteins are also implicated as regulators of intracellular Ca+2 in several studies. Bax overexpression is reported to induce endoplasmic reticulum release of Ca+2 and subsequent mCa+2 uptake in response to an apoptotic stimulus; the resulting mCa+2 uptake stimulates cytochrome c release (23,24). It remains unknown, however, whether mCa+2 uptake occurs in response to Bax redistribution to the mitochondria or secondary to increased endoplasmic reticulum release of calcium (23,24,48).

Our results indicate that Bax translocation occurs during the period of hypothermic storage. Furthermore, because Bax translocation was inhibited by RR, it appears that mCa+2 uptake precedes Bax translocation in the apoptotic cascade following hypothermic storage. This implicates mCa+2 uptake as an apoptotic trigger, signalling Bax translocation to the mitochondria. The exact mechanism of how mCa+2 mismanagement leads to Bax activation is not clear. However, these data, when combined with previous reports (23,24), suggest that during the period of hypothermia, mCa+2 uptake triggers Bax translocation, which then potentiates the mCa+2 overload either directly or indirectly, creating a cascade resulting in the release of pro-apoptotic proteins such as cytochrome c. Because we observed a strong correlation between mCa+2 uptake blockade, prevention of Bax translocation and prevention of cytochrome c release in our cellular model, Bax translocation may constitute a major initiator of early reperfusion cell death following hypothermic storage.

Interestingly, we observed no caspase activation during the period of hypothermic storage or subsequent rewarming. This suggests that a caspase-independent mechanism of cell death occurred which is dependent on mCa+2 uptake. Caspase-independent apoptosis mechanisms are well described in the literature (39,51–54). While not all caspase-independent apoptosis mechanisms are understood, emerging data indicates that these mechanisms play an important role in warm ischemia-reperfusion associated cell death (55–58). Our results implicate caspase-independent cell-death as an important component of cellular apoptosis following cold ischemia and reperfusion. Components of these mechanisms, such as apoptosis-inducing factor and endonuclease G, may remain mitochondrial dependent, and are described to be initiated by Bax and/or tBid translocation to the mitochondria (26,51–53). These mechanisms offer a plausible explanation for the observed prevention of cell death by treatment with RR during the period of hypothermic storage. This is also consistent with the observation that Bax translocation was prevented by the blockade of mCa+2 uptake.

It is debated whether cold preservation and hypoxia cause cell death via necrosis or apoptosis. A recent report asserts that hypothermic storage-induced cell death occurs via necrosis (41); however, this study only examined caspase-3 activation as a marker of apoptosis and did not search for other markers such as cytochrome c release or Bax translocation. In our study, we were able to reverse essentially all early reperfusion associated cell death following NH storage and all but 15% following HH storage by preventing mitochondrial calcium uptake during storage. The ability to reverse the occurrence of cell death suggests that the majority of the cell death occurring under our experimental conditions was owing to initiation of apoptotic pathways. This is supported by other investigators who have observed apoptosis following cold storage (42,59–61).

The addition of hypoxia to the hypothermic storage increased the magnitude of cytochrome c release from mitochondria and increased the rate of early reperfusion apoptosis. Prevention of mCa+2 uptake during storage only reduced the rate of cell death during early reperfusion to 15% following HH storage, but it prevented essentially all cell death following NH storage. This observation suggests that hypoxia initiates an alternate cell-death pathway, which would account for the 15% irreversible early reperfusion-associated cell death. The potential pathway appears to be independent of mCa2+ uptake, as RR failed to fully reverse its occurrence. We cannot exclude mitochondria-independent apoptosis or necrotic cell death secondary to hypoxia as an explanation for this observation, but other possibilities do exist. Other investigators have reported on the contribution of oxygen free radicals to the induction of apoptosis during rewarming/reperfusion following cold storage in multiple cell lines (42,59–61). While reperfusion following ischemic storage offers an ideal situation for the formation of reactive oxygen species, the role that this mechanism plays in apoptosis during the time periods studied in this report remains uninvestigated.

In summary, our results demonstrate that mCa2+ uptake during hypothermic storage, both hypoxic and normoxic, contributes to a caspase-independent mechanism of cell death during the early reperfusion period by triggering Bax translocation to the mitochondria and cytochrome c release. These findings indicate that the prevention of mCa+2 overload during the period of cold ischemia is cytoprotective.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported in part by the National Institute of Health Ruth L. Kirschstein National Research Service Award T32 DK07673 (CDA), National Institute of Health Grant 1 K08 DK59390-1 (RSC), and Vanderbilt Ingram Cancer Center Discovery Grant 5P30 CA68485-07 (RSC).

NH, normoxic hypothermia; HH, hypoxic hypothermia; Ca+2, Calcium; mCa+2, mitochondrial calcium; RR, ruthenium red; UW, Belzer storage solution; GFP, green fluorescent protein; PTP, permeability transition pore; MR, mitotracker red.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References