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Am J Physiol Heart Circ Physiol 288: H2476-H2483, 2005; doi:10.1152/ajpheart.00670.2004
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Mitochondrial DNA mutations activate programmed cell survival in the mouse heart

Dekui Zhang, Justin L. Mott, Shin-Wen Chang, Melissa Stevens, Peter Mikolajczak, and H. Peter Zassenhaus

Saint Louis University Health Science Center, Department of Molecular Microbiology and Immunology, St. Louis, Missouri

Submitted 6 July 2004 ; accepted in final form 19 December 2004


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased frequencies of mitochondrial DNA (mtDNA) mutations characterize the aging heart and are also found in idiopathic dilated cardiomyopathy and end-stage heart failure. The pathogenic potential of such mutations is unclear. Transgenic mice showing accelerated accumulation of mtDNA mutations and dilated cardiomyopathy due to expression of an error-prone mtDNA polymerase specifically in the heart were characterized by Western blot analysis and immunohistochemistry for the levels of pro- and antiapoptotic proteins. By 8 wk of age, when frequencies of mtDNA mutations were ~0.01% and all transgenic mice showed four-chamber cardiac dilation, a vigorous prosurvival response was evident. Upregulated were Bcl-2, Bcl-xl, Bfl1, heat shock protein 27, and X-linked inhibitor of apoptosis protein, all of which function to inhibit apoptosis. Although translocation of Bax to mitochondria was also seen, it was not integrated into the mitochondrial membrane. Treatment of transgenic mice with doxorubicin failed to induce apoptosis, in contrast to controls, showing that the prosurvival response protected cardiomyocytes from a death stimulus. Increased apoptosis and release of cytochrome c appeared to precede the establishment of the prosurvival state suggesting that it may reflect a response to activation of programmed cell death pathways. It has been proposed that a programmed cell survival response is activated in the failing and aging heart. We show that elevated frequencies of mtDNA mutations may serve as one trigger for the activation of such a response.

aging; congestive heart failure; apoptosis; doxorubicin

HIGHER FREQUENCIES OF MITOCHONDRIAL DNA (mtDNA) mutations are found with aging and in age-related diseases (20, 23). Compared with levels found in younger individuals, frequencies may be hundreds- to thousandsfold higher, although on an absolute scale, frequencies usually do not rise above 1% (3). Such levels are considerably below the ≥20% frequencies commonly found in patients with clinical symptoms of mitochondrial oxidative phosphorylation disease (31). Age-related, low-level mtDNA mutations also differ from those found in patients, in that multiple different mutations are present that are randomly located in the genome rather than a single specific mutation. So it is more appropriate to view age-related mutations as a mutational burden. The pathogenic potential of such a burden is unclear in the human heart. With the use of a transgenic (Tg) mouse model with cardiac-specific overexpression of proofreading-deficient mtDNA polymerase (pol-{gamma}), we have previously shown that frequencies of mtDNA mutations like those commonly found in aging lead to a four-chamber dilated cardiomyopathy in the mouse heart (33).

Pathogenic mechanisms of age-related mtDNA mutations are unknown. Impaired mitochondrial respiration and increased oxidative damages may be caused by high-frequency mtDNA associated with mitochondrial respiratory disorders. Their role in the mechanisms of age-related mtDNA mutations are largely based on correlative data and speculations. On the contrary, our previous publication suggests that neither mechanism is involved in the pathology caused by low-frequency mtDNA mutations in the mouse heart (19). Associated with cardiac pathology is a low level of apoptotic loss of myocytes; furthermore, these mice show a high incidence of congestive heart failure and sudden death (34). Dilated cardiomyopathy also develops in Tg mice having low and persistent levels of apoptotic cell death in the myocardium due to chronic activation of caspase 8 (32). Loss of cardiac myocytes, possibly through apoptotic cell death, also occurs in the aging human heart (27). This loss may be responsible for the decreased cardiac reserve observed in the aging heart (12). These observations raise the possibility that the pathogenic potential of an age-related rise in mtDNA mutations may involve provocation of apoptosis in cardiomyocytes.

In this paper, evidence is presented that increased frequencies of random mtDNA mutations in the mouse heart provoke a programmed survival response. Cytochrome c release from mitochondria and increased apoptosis precede the full establishment of the survival state, suggesting that it is a response to proapoptotic signals. We discuss the roles that age-related increases in mtDNA mutations might play in cardiac pathology.


   MATERIALS AND METHODS
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Animals

The construction of Tg mice with accelerated accumulation of mtDNA mutations in the heart has been described (33). Two independent lines were used, one (line 4) on an FVB/N background and the other (line 13) as a hybrid between FVB/N and C57/Bl6. No differences in results between the lines were noted. The investigation conformed with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205].

Reagents

Sources for the primary antibodies were denatured cytochrome c and caspase 3 (Pharmingen); Bcl-2, Bcl-xl, heat shock protein (HSP)27, and HSP60 (Santa Cruz Biotechnology, Santa Cruz, CA); cytochrome c oxidase subunit 4 (COX4) (Molecular Probes, Eugene, OR); X-linked inhibitor of apoptosis protein (XIAP) (Stressgen); and Bfl-1 (R&D Systems, Minneapolis, MN). FITC-conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME) were used for immunohistochemistry, and horseradish peroxidase conjugated secondaries (Jackson Laboratories) were used for Western blot analysis. Western blots were developed by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). All other reagents were from Sigma (St. Louis, MO), except as noted.

Immunohistochemistry

Sections (5- to 7-µm) cut from formalin-fixed, paraffin-embedded tissues were deparaffinized in xylene and rehydrated with a graded series of EtOH until they were placed in PBS. For detection of cytochrome c, sections were immersed in antigen-unmasking solution (BORGdecloaker; Biocare Medical) and heated in a pressure cooker for 2 min followed by slow cooling over the next 20 min. After being washed three times in PBS, the sections were incubated in blocking solution (PBS containing 2% BSA, 0.2% nonfat dried milk, and 0.4% Triton X-100) for 1 h before incubation overnight at 4°C in blocking solution containing the primary antibody. In the morning, slides were washed five times for 5 min each in PBS/Tween (PBS with 0.1% Tween 20) and then were incubated for 1 h in blocking solution containing the FITC-conjugated secondary antibody. After five washes in TBS/Tween, the slides were dried, 10 µl antifade solution was applied, and coverslips were mounted and sealed with clear nail polish. Slides were examined with an Olympus epifluorescent microscope, and images were recorded with a SPOT digital camera. Bcl-2 and HSP27 detection used frozen sections (7–10 µm) fixed with 3% paraformaldehyde. Subsequent processing was as described above.

Western Blot Analysis

Hearts were homogenized, and mitochondria was isolated as described previously (34). The postmitochondria supernatant served as the source for the cytosolic fraction, which was further centrifuged at 100,000 g for 1 h to ensure complete clearance of mitochondrial fragments. To the resulting supernatant was added 1/10 vol 100% TCA, and the precipitated proteins were collected by centrifugation at 10,000 g for 10 min. After being washed three times in ice-cold 80% acetone, the pellet was suspended in Laemmli sample buffer (11), incubated overnight at 37°C to efficiently dissolve proteins, and then boiled for 5 min. The mitochondrial pellets, after being washed once in isolation buffer, were dissolved in sample buffer and immediately boiled. For analysis of the relative amounts of cytochrome c in the cytosolic versus mitochondrial fractions, samples were applied to polyacrylamide gels so that the mitochondrial fraction represented 1/10 of the proportionate amount compared with the cytosolic fraction. For Bax, Bcl-2, and Bcl-xl analyses, equal proportionate amounts were applied to the gels. Western blot analysis was performed as described previously (34). For analysis by Western blotting of the various proteins in the whole heart, the excised hearts were snap frozen in liquid nitrogen and pulverized while cold with a mortar and pestle, and the powder was poured into Laemmli sample buffer. After homogenization in a Dounce homogenizer with a motor-driven Teflon pestle, the sample was boiled and then centrifuged for 10 min at 10,000 g to remove insoluble material. Samples applied to gels contained 150 µg protein determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL).

Association of Bax and Bcl-2 with Mitochondrial Membranes

Bax. Isolated mitochondria (34) were suspended in 250 mM sucrose, 5 mM Tris·HCl, pH 9.0, and 1 mM EDTA at 1 mg/ml protein and subjected to five cycles of freeze/thaw in a dry ice/EtOH bath. After centrifugation at 100,000 g for 1 h, the pellet was dissolved in Laemmli sample buffer. Proteins in the supernatant were precipitated with 10% TCA before solubilization. Equal proportionate amounts of the pellet and supernatant fractions were applied to gels for Western blot analysis.

Bcl-2. Mitochondria were suspended at 1 mg/ml protein in KSCN buffer (in mM: 150 KSCN, 10 MOPS, 10 Tris·HCl, and 50 PMSF, pH 7.2, plus 0.5 mg/ml leupeptin) and subjected to five cycles of freeze/thaw in a dry ice-EtOH bath. After centrifugation at 100,000 g, the membrane fraction was resuspended in 0.2 M sodium carbonate, pH 11.5, incubated 30 min at room temperature, and then centrifuged at 100,000 g for 1 h. The pellet was solubilized in Laemmli sample buffer, whereas proteins in the supernatant were first precipitated with TCA before solubilization. Equal proportionate amounts from the supernatant and pellet fractions were applied to 12% polyacrylamide gels for Western blot analysis of Bcl-2.

Caspase 3 Activity

Excised hearts (pools from 5–10 animals) were minced and rinsed several times with ice-cold isolation buffer (in mM: 210 mannitol, 70 sucrose, 1 EGTA, 5 HEPES-KOH, pH 7.2) and then homogenized at 1 g wet wt/10 ml for 10 strokes in a tight-fitting Dounce homogenizer using a motor-driven Teflon pestle. After centrifugation of the homogenate for 10 min at 9,200 g, the supernatant was made to 1% (vol/vol) Triton X-100. Caspase 3 activity was determined at 37°C in a 1-ml reaction buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM DTT) using as substrate 2 µM acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin (Ac-DEVD-AMC; Pharmingen) and containing 1 mg homogenate protein. Reactions were incubated for 45 min to 2 h, and released AMC was measured in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength of 440 nm. Protein was measured by BCA assay.

Doxorubicin Treatment

Doxorubicin HCl (Bedford Laboratories) was reconstituted fresh with 0.9% NaCl to a final concentration of 2 mg/ml following the manufacturer's instruction. Mice were treated with a single intraperitoneal injection of doxorubicin at a dose of 25 mg/kg. Three days later, mice were killed, and the excised hearts were fixed in formalin and embedded in paraffin, and cross sections were obtained at their largest diameter. Apoptosis was assessed by terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and 4,6-diamidino-2-phenylindole (DAPI) staining as described (34). TUNEL(+) nuclei were counted in all fields from at least two sections per animal and averaged. Data are expressed as mean number of TUNEL(+) nuclei/section/animal ± SD. Statistical differences between groups were determined by Student's t-test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
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Rising Levels of mDNA Mutations Activate Apoptotic Signaling

In this Tg model, mtDNA mutations accumulated only in the heart due to tissue-specific expression of pol-{gamma} (33). The transgene was driven by myosin heavy-chain promoter. Two independent lines were generated. The total expression level of pol-{gamma} in one line (line 13) was comparable with the controls, whereas in another line (line 4), there were twofold increases. Enzymatic measurement showed that 95% of pol-{gamma} in the Tg mice was derived from the transgene in both lines (33). Expression of the error-prone pol-{gamma} began at birth, initiating a rapid accumulation of mtDNA mutations. Frequencies climbed to 1-point mutation per 10,000 bp of mtDNA by 1 mo of age; at approximately the same age, dilated cardiomyopathy wasfirst detected (34). Neither decreased mitochondrial respiration nor increased oxidative stress was detectable (19), suggesting that the rising levels of mtDNA mutations do not alter energy metabolism.

Rather, as levels rise, they appeared to initiate apoptotic signaling, as evidenced by release of mitochondrial cytochrome c, cardiomyocyte apoptosis, and upregulation of Bcl-2. The temporal relationship between apoptosis and Bcl-2, summarized in Fig. 1A, indicates that increased apoptosis in the heart precedes upregulation of Bcl-2. Rates of apoptosis declined once high levels of Bcl-2 are established, suggesting either a functional prosurvival response or a waning of the apoptotic signal. Because even at its maximum at 4 wk of age, apoptosis affects only a small fraction of myocytes calculated to be ≤1.2% based on a rate of 40 TUNEL(+) nuclei/10,000 nuclei and estimates that ~30% of the cells in the heart are cardiomyocytes (31a). We asked whether mitochondrial apoptotic signaling was widespread or confined to only a few cells.



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  		Fig. 1. Apoptotic signaling in transgenic (Tg) mice. A: relationship between rates of apoptosis and Bcl-2 levels in Tg and control hearts vs. age. CT, control. B: Western blot analysis for cytochrome c (Cyt c) levels in the mitochondrial (Mito) and cytosolic (Cyto) fractions in control and Tg animals at 4–5 wk of age. Mitochondrial fractions represented 1/10 the proportionate amount compared with cytosolic fractions. Cytochrome c oxidase subunit 4 (COX4) is a mitochondrial inner membrane integral protein; its absence in the cytosolic fraction demonstrates lack of contaminating mitochondria. Heat shock protein (HSP)60, which is found in both mitochondria and cytosol, was used as a loading control. Experiments were done on 4 separate samples, and the amount of cytosolic c was measured by densitometry. The averages for the CT samples are 1.77 ± 1.49 (arbitrary unit ± SD) and Tg 5.15 ± 0.67, P = 0.006. C: immunohistochemistry for cytochrome c in the heart from mice 3 and 5 wk of age. Note the patchy distribution of increased staining in the 3-wk-old Tg heart compared with the uniform increased staining by 5 wk (magnification, x400). D: Western blot analysis for Bax levels in the whole heart (Total) and mitochondrial fraction of control and Tg animals. Cytochrome c levels are shown as loading controls. Mild alkali treatment of Tg mitochondrial stripped Bax off the membranes so that after centrifugation, it was found in the supernatant fraction (Sup) in contrast to COX4, which remained in the membrane fraction (Mem).

 
Biochemically, the fraction of cytosolic cytochrome c in Tg mice 4 to 5 wk of age did not exceed 5% of that found in mitochondria (Fig. 1B), consistent with previous studies (19) that neither mitochondrial cytochrome c levels nor respiratory capacity were diminished in Tg mice at that age. Immunohistochemical analysis for cytochrome c, however, revealed increased staining in Tg mice (Fig. 1C). At 3 wk of age, Tg hearts showed increased staining with a patchy, salt-and-pepper distribution. By 5 wk of age nearly all cells showed heightened staining. Increased staining was not seen for either the voltage-dependent anion channel or COX4, both of which are integral membrane proteins in the mitochondrion (data not shown). The progression from a patchy to a uniform pattern of increased staining between 3- and 5 wk-old-mice is consistent with the mechanism-generating mtDNA mutations in this model. Error-prone replication would be expected to lead to a stochastic rise in mutations, so that at any given time the mutational burden between cells might be variable. The biochemical and immunohistochemical analyses for cytochrome c suggest that as the average burden rises, individual cells pass over a threshold and initiate proapoptotic signaling through release of cytochrome c. By 5 wk of age, nearly all cells appear to be engaged in such signaling.

Release of cytochrome c from mitochondria during activation of the intrinsic pathway of apoptosis is associated with the translocation and insertion of Bax into the mitochondrial outer membrane (7). Bax is a proapoptotic member of the Bcl-2 family, which may form channels in the outer membrane, allowing for release of cytochrome c (22). Tg mice at 5 wk of age showed severalfold increase of Bax associated with mitochondria, although total Bax levels in Tg mice were not changed, indicating a translocation of Bax from the cytosolic compartment into mitochondria (Fig. 1D). However, the majority of Bax appeared to be only loosely associated with the mitochondrial outer membrane, because washing of mitochondrial membranes with alkaline buffer disrupted that association (Fig. 1D). These data indicated that translocated Bax was prevented from insertion into the mitochondrial outer membrane, possibly by the prosurvival response. We cannot role out the possibility that a small amount of Bax was inserted into the mitochondrial membrane and was below detection.

Upregulation of Protective Responses Against Apoptotic Cell Death

A number of proteins have been shown to inhibit various steps in the programmed cell death pathway so as to prevent activation of caspase 3, the final executioner of apoptosis. Bcl-2 family members act at the mitochondrial membrane to inhibit cytochrome c release, IAP family members are catalytic inhibitors of caspases, and HSP27 inhibits activation of procaspases. As shown in Fig. 2, a protective response involving these proteins appears to be activated in the heart of mice with mtDNA mutations. Upregulation occurs not only in Bcl-2, but also in Bcl-xl and Bfl1, all antiapoptotic members of the Bcl-2 family (5).



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  		Fig. 2. Prosurvival responses in the Tg heart. A: Western blot analyses for whole heart (Total) and mitochondrial levels of the indicated proteins in control and Tg animals 7 wk of age. HSP60 served as a loading control. XIAP, X-linked inhibitor of apoptosis protein. B: whole heart levels of HSP27 in control and Tg animals from 2 to 7 wk of age. COX4 was used a loading control. C: protein bands from Western blot analysis were scanned and analyzed by densitometry. Intensity of the protein bands was divided by the values from the loading control, HSP60. Experiments were repeated at least 3 times, and 3–7 independent mouse hearts were used in each quantification. D: Bcl-2 (indicated by the arrow; the identity of the upper band in these blots is unknown) was associated with the membrane fraction (Mem) after disruption of Tg mitochondria by freeze/thaw cycles followed by centrifugation to give supernatant and pellet fractions. Cyclophilin D (Cyp D) is a matrix protein, a portion of which shows peripheral association with the mitochondrial inner membrane under these conditions. The adenine nucleotide transporter (ANT) is an integral inner membrane protein. Carbonate, pH 11, treatment of the membrane fraction showed that Bcl-2 remained associated with mitochondrial membranes like ANT, whereas peripherally associated Cyp D was released into the supernatant.

 
In contrast to Bax, mitochondrial Bcl-2 appears to be integrated into the mitochondrial membrane, because this association was resistant to alkaline treatment (Fig. 2B). Protein levels of Bcl-2 family members were increased in the cytoplasm (Bcl-2, Bcl-xl) as well as at mitochondria (Bcl-2). Also upregulated were HSP27 and XIAP, both of which may function to suppress activation of caspase 3 (6, 24). The rise in HSP27 levels appeared to lag that of Bcl-2. HSP27 levels were slightly elevated at 4 wk of age when the rate of apoptosis was maximal and then rose to higher levels by 7 wk of age, a time when the rate of apoptosis was declining (Fig. 2C).

It is noteworthy that even at 2 mo of age, when the protective response was at maximum, the frequency of apoptosis in the Tg heart was still significantly higher than the controls, although in a lower level. These results indicated that cell survival response may have limited the number of cells going through the apoptotic process; the initial insult was still operative inside the mitochondria.

Upregulation of Bcl-2 and HSP27 Occurs in Most Myocytes

The severalfold increase in the levels of Bcl-2 and HSP27 seen in the Tg heart suggested that a large fraction of myocytes undergo upregulation of those prosurvival proteins. Immunohistochemical analyses of 8-wk-old mice for Bcl-2 and HSP27 confirmed that interpretation. Frozen sections of Tg and control hearts were fixed with formalin and reacted with antibodies against Bcl-2 and HSP27. Figure 3 shows that most myocytes from the Tg heart have increased immunoreactivity for both proteins. As noted, control experiments staining for HSP60 showed no increase in immunoreactivity in the Tg heart, demonstrating the specificity of the increase in Bcl-2 and HSP27. These results indicate that the rising frequency of mtDNA mutations induce prosurvival responses in nearly all cardiomyocytes.



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  		Fig. 3. Immunohistochemistry for Bcl-2 and HSP27. Frozen sections from control and Tg animals 7 wk of age were reacted with antibodies against either Bcl-2 or HSP27, and bound primary antibody was detected by using FITC-conjugated secondary antibody. The increased staining of all myocytes in the Tg samples indicated that the prosurvival response was activated in a large fraction of Tg myocytes (magnification, x600).

 
Prosurvival Response is Cardioprotective

We then studied the functional effects of elevated levels of antiapoptotic proteins. To investigate this, 7-wk-old Tg and control mice were injected with doxorubicin at a dose that causes apoptosis in the mouse myocardium (17). Three days later, animals were killed, and the number of apoptotic cells were quantified by TUNEL staining. Slides were counterstained with DAPI, a DNA-binding dye, so as to confirm that TUNEL(+) cells were apoptotic by the appearance of pyknotic or fragmented nuclei showing condensed or marginalized chromatin. As expected, doxorubicin treatment caused an eightfold increase in TUNEL(+) cells in the control heart compared with vehicle treatment (Fig. 4A; 4.5 ± 1.5 vehicle vs. 33.7 ± 1.0 doxorubicin P = 0.002). In contrast, mice with mtDNA mutations in the heart showed no rise in the number of TUNEL(+) cells on doxorubicin treatment (11.1 ± 4.2 vehicle vs. 13.1 ± 3.1 doxorubicin, P value not significant). These data suggest that the prosurvival response in hearts from Tg mice is functionally able to protect against a proapoptotic stimulus. Consistent with this interpretation, no increase in caspase 3 enzymatic activity was detected in homogenates of the Tg heart (Fig. 4B). Neither increase in the amounts of proteolytically processed caspase 3 (a sign of activation) was detected by Western blot analysis (Fig. 4C), suggesting a block in the activation of downstream steps of the intrinsic pathway of apoptosis.



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  		Fig. 4. Apoptosis after doxorubicin treatment and analysis for caspase 3 by Western blot analysis and enzymatic activity. A: number of apoptotic cells/section in 7-wk-old control and Tg animals treated with 25 mg/kg doxorubicin (Dox) or vehicle (veh); mean ± SD, 3–6 animals/group. The increased apoptosis in the Dox-treated control animals compared with vehicle was highly significant by Student's t-test (P < 0.002), whereas in Tg animals, the same comparison showed a nonsignificant effect. B: Western blot analysis using an anti-caspase 3 antibody that recognized the full-length molecule (migrating here as a 37-kDa band) and the 17-kDa proteolytically cleaved fragment showed that Tg and control animals from either of the lines did not differ in the amounts of those caspase 3 forms in whole heart samples. The Bcl-2 blots showed that Tg animals from both lines demonstrated upregulation of the prosurvival state. Cytochrome c was used as a loading control. C: caspase 3 activity in heart homogenates was determined by using the fluorogenic substrate acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin. Tg animals from either of the independently derived lines did not show increased enzymatic activity (expressed as arbitrary fluorescent units) compared with littermate controls. Homogenates prepared from cell lines known to show either low or high rates of apoptosis served as negative and positive controls, respectively.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Mitochondrial DNA mutation frequency increases with age (15) with the potential to contribute to disease. We have previously shown that low-level mtDNA mutations may cause increased apoptosis in the mouse heart (33). Associated with the apoptosis, there was increased cytochrome c in the cytoplasm, indicating that the apoptosis was initiated through a mitochondrial pathway. Recently, our studies revealed that the initial process of apoptosis may involve the mitochondrial permeability transition pore, as treatment of the mice with cyclosporine A (an inhibitor of mitochondrial permeability transition pore) prevented the occurrence of apoptosis and the protective response, as well as the cardiac pathology (18). The cytoplasmic calcineurin inhibitor FK-506 had no such effect (18). Here we further show that in the mouse heart mtDNA mutations induced a prosurvival response, including upregulation of a number of proteins that have antiapoptotic functions. The increased amounts of Bcl-2 in the mitochondrial membrane indicates a suppression of the intrinsic pathway of apoptosis in which release of cytochrome c from mitochondria is an essential early step (16). Upregulation of other members of the Bcl-2 family (Bcl-xl, Bfl1) may act to counter an apoptotic signal through other mechanisms (4). These findings are significant in that low-level mtDNA mutations, as seen in aging, could induce a life-long process of apoptosis and protective responses.

Despite this concerted effort to inhibit the initiation of a mitochondrial apoptotic signal, we still see increased cytochrome c release. Thus it may be significant that we also see increased protein levels of inhibitors of the intrinsic apoptotic pathway that act downstream of cytochrome c release. Specifically, we see increased HSP27 and XIAP, which have been shown to inhibit activation of caspases 9 and 3 (6, 24). That adaptive proteins upregulated in the hearts of mice with mtDNA mutations target multiple steps along the apoptotic cascade is consistent with the idea that a program of apoptosis inhibition has been activated.

The response appears to place the heart in a state resistant to proapoptotic insults. Evidence is first apparent in the time course of cell death compared with Bcl-2 expression. Once Bcl-2 protein reaches maximal expression, apoptosis ebbs. Furthermore, Tg mice in the prosurvival state treated with an acute dose of doxorubicin were protected against cardiomyocyte apoptosis. The cardiotoxicity of doxorubicin includes activation of the intrinsic pathway of apoptosis (29), precisely the pathway activated in these mice with mtDNA mutations. Finally, evidence that the programmed cell survival response is functional is seen in the absence of caspase 3 activation in the Tg heart, despite of cytochrome c release.

Protective response seemed to be highly effective against catastrophic apoptotic events, because it had suppressed the initial wave of apoptosis, and prevented doxorubicin toxicity. However, a small number of cardiac myocytes still escaped from this protection, as low-level apoptosis persisted in the Tg heart even at the time when the protective response was at the maximum. Because caspase 3 activation is required for the typical hallmarks of apoptosis (28) and the low-level apoptosis in the Tg heart had the morphology of typical apoptosis, this may indicate that protection against caspase activation was not complete, allowing caspase 3 activation as the final executor. Because only very small numbers of cells escaped from the apoptotic process, the activated caspase 3 could be below detection. As a consequence, chronic loss of cardiac mass continued. In the meantime, low-level apoptosis may also serve as the driving force for maintaining a high level of antiapoptotic proteins. As a result, a new balance was reached between the apoptotic signaling and the antiapoptotic mechanisms.

We find several interesting parallels between our model and aging or failing hearts. With regard to aging, there appears to be an altered balance of apoptotic signaling. Several groups have found an increase in apoptosis (2, 27) as well as cytochrome c release (25). There is also modulation of the Bcl-2 family of proteins with an increase in both Bcl-2 and Bax, the ratio appearing to slightly favor survival (14). Furthermore, there is no activation of caspase 3 in the aging rat heart despite cytochrome c release (25). Thus in the aging myocardium, there may also be activation of programmed cell survival as well as death. Our study further indicated that both apoptosis and a chronic survival response in the aging heart could be caused by low-level mtDNA mutations.

In the failing heart, the similarities are even more compelling. There is again increased cytochrome c release (21) and cell death (1). Interestingly, there appears to be cytochrome c release in excess of the cell death, suggesting interruption of the death signal (8). In some studies (10, 13), apoptosis modulators are also upregulated, similar to our model, including Bcl-2, Bcl-xl, and HSP27. Others have reported conflicting data indicating Bcl-2 and XIAP downregulation, although there was still inhibition of the apoptotic process (30, 36), suggesting the existence of modifying factors, as yet unknown. The failing heart thus has activation of apoptosis, but also activation of a program of apoptosis inhibition, similar to our model. We find that this program involves Bcl-2 family members, HSP27, and XIAP.

One possible cause of the upregulation of the various prosurvival proteins is a response to signals or remodeling not directly related to the activation of the intrinsic pathway of apoptosis. However, we find this unlikely, because the initiation of the prosurvival state follows, in time, proapoptotic signaling. The prosurvival state may be a regulatory response of cardiomyocytes to activation of the intrinsic pathway of apoptosis. In this Tg model, frequencies of mtDNA mutations rise rapidly from birth onward coincident with the expression of the error-prone DNA pol-{gamma} encoded in the transgene (33). By 3 wk of age, increases in myocyte apoptosis are evident as well as immunohistochemical alteration in cytochrome c reactivity in some myocytes. By 4 to 5 wk of age, frequency of apoptosis rises to a maximum, biochemical evidence of cytochrome c release from mitochondria is seen, and nearly all myocytes show an immunohistochemical change in cytochrome c staining. The prosurvival response, as marked by levels of Bcl-2 and HSP27, appears to begin by 4 wk of age and reach a maximum by 6 to 7 wk of age, a time when the rate of apoptosis is falling. This timeline is consistent with the idea that the cell survival response is a regulatory response to intracellular apoptotic signaling. Most importantly, apoptosis and protective response from Tg mouse hearts were invoked on a background of normal cardiac function, eliminating the possibility that these responses are secondary to heart failure. This upregulation has been coined "programmed cell survival" and may reflect a common genetic response in cardiomyocytes to death-promoting stimuli (8). Signals provoking that response are poorly understood, but our results suggest that elevated frequencies of mtDNA mutations may be one.

At the same time that the activation of the programmed cell survival pathway serves to limit cell death, it may also have untoward effects on cardiac physiology. In these Tg mice, a wave of apoptosis occurs, which recedes on upregulation of the survival response. However, persistently high levels of antiapoptotic proteins are not physiological. For instance, overexpression of Bcl-2 in the mouse heart leads to inhibition of the mitochondrial permeability transition pore and the mitochondrial sodium/calcium exchanger (35). Bcl-2 may integrate into other cellular membranes besides the mitochondrial outer membrane, e.g., the sarcoplasmic reticulum, leading to potential alterations in their permeability (26). In the heart, such effects of Bcl-2 may alter calcium homeostasis and signaling. In our Tg mice, dilated cardiomyopathy persists and worsens for the life of the animal, despite the continued upregulation of the prosurvival proteins (34). It is possible that the persistence of disease may derive, in part, from physiological dysfunction related to a chronically activated survival response. The failing human heart appears also to have an activated programmed survival response, but whether it serves to support cardiac function is unclear (8).

In summary, we describe activation of a programmed cell survival pathway in mice with mtDNA mutations. This may represent a common response of the heart to distress, because similarities are seen between our model and in aging and failing hearts. The survival program follows in time the initiation of apoptotic signaling and may be the direct result of that signaling. It may be that the initiation of the cell death signal is the common denominator between hearts with increased mtDNA mutations and the aging and failing hearts. Although the survival program acts to mitigate intrinsic death signals, it may also have untoward effects on cardiac physiology. If proapoptotic signals are the immediate cause for upregulation of the programmed survival response, possible maladaptive physiological consequences may be the price to be paid for chronic suppression of apoptosis. Reversing these untoward effects may serve to improve cardiac function in the setting of disease.


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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by National Institute on Aging Grant AG-5710, National Institute of Neurological Disorders and Stroke Grant NS-41785, and American Heart Association Grant 0150375N.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. P. Zassenhaus, Saint Louis Univ. Health Science Center, Dept. of Molecular Microbiology and Immunology, 1402 S. Grand Blvd, St. Louis, MO 63104 (E-mail: Zassenp{at}slu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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