^*Corresponding Author:
University of Florida
Aging Biochemistry Laboratory
College of Health and Human Performance
P.O. Box 118206
Gainesville, FL 32611
352-392-0584, ext. 262
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cleeuwen@ufl.edu

Web Page: http://grove.ufl.edu/~cleeuwen/
 
 

TABLE OF CONTENTS

1. Introduction
2. Mechanisms of Apoptosis: Role of Mitochondria
3. Apoptosis and Aging
1. T cells
2. Cardiomyocytes
3. Neurons
4. Apoptosis and Exercise
5. Summary
6. Acknowledgements
7. References

In 1972, Andrew H. Wyllie, John F. R. Kerr, and Alastair R. Currie coined the term apoptosis to describe programmed cell death, a process of cell death involved in cellular development and aging distinct from necrosis. Apoptotic cells die by design whereas necrotic cells die by accident and lethal injury (1). Their findings were largely ignored in the early 1980s, but since 1987 the number of papers has been growing logarithmically. Researchers began to get interested in apoptosis after it was definitely demonstrated in the nematode Caenorhabditis elegans, followed by the identification of death gene homologs in other organisms (2).

While necrotic cells swell, apoptotic cells typically shrink and detach from surrounding parenchymal cells. Concurrently, the cell volume decreases, and the chromatin condenses at the edge of the nucleus. Next, endogenous endonucleases and ICE-like proteases are activated resulting in degradation of the nucleus and the cell itself. ICE-like proteases received their name because they structurally resemble the first member of the group discovered, i.e., interleukin-1b converting enzyme. At this point, the apoptotic cells are phagocytosed by neighboring parenchymal cells. Those not consumed typically undergo further changes: the nucleus falls apart and the cells divide into "apoptotic bodies" that may contain a piece of the nucleus.

As before, these apoptotic bodies are then phagocytosed by neighboring cells.

Aberrant regulation of apoptosis contributes to well-known pathologies, such as autoimmune diseases, cancer, and viral infections (3). In this paper, we will examine the evidence that mitochondria are the central executioners of apoptosis, and we will review the known factors that stimulate mitochondria to induce apoptosis. We will also examine the overlooked (and under-investigated) role of apoptosis in exercise-induced stress and possible mechanisms that cause apoptosis. Lastly, we will discuss the influential role apoptosis plays in development and aging, particularly of cardiomyocytes, T cells, and the brain. Our general hypothesis is that mitochondria play a central role in the regulation and control of apoptosis in most of these cell types during several physiological conditions, including aging and exercise.
 

2. Mechanisms of Apoptosis: Mitochondrial Control

Apoptosis can be divided into three non-distinct phases: an induction phase, an effector phase, and a degradation phase. The induction phase depends on death-inducing signals to stimulate pro-apoptotic signal transduction cascades. These death-inducing signals include reactive oxygen species, ceramide signaling, overactivation of Ca⁺² pathways, and Bcl-2 family proteins such as Bax and Bad. In phase two, the effector phase, the cell becomes committed to die by the action of a key regulator, which we will argue is the mitochondrion. The last phase, a degradation phase, involves both cytoplasmic and nuclear events. In the cytoplasm, a complex cascade of protein-cleaving enzymes called caspases is activated. In the nucleus, the chromatin condenses, the nuclear envelope breaks down, and the DNA fragments. Finally, the cell is fragmented into apoptotic bodies, phophatidyl serine on the membranes is recognized, and apoptotic bodies are phagocytosed by surrounding cells or macrophages (1,4).

This section of the paper will probe how mitochondria regulate the effector phase of apoptosis, particularly through the cytochrome c pathway. Also, we will investigate how different radical species can induce and/or inhibit apoptosis. Until recently, mitochondria were not assumed to be central players in the effector phase as mitochondrial morphology remains intact throughout apoptosis. But recent evidence indicates that mitochondria exhibit major functional and structural changes that serve to regulate apoptosis. First, the mitochondrial inner transmembrane potential (DY_m) collapses prior to classical morphological signs of apoptosis. Second, studies using cell-free systems suggest that mitochondrial proteins are rate-limiting for the activation of endonucleases and caspases, cysteine proteases that cleave after aspartic acid residues. Third, drugs that stabilize mitochondrial membranes have been shown to inhibit apoptosis (4). Fourth, the anti-apoptotic protein, Bcl-2, blocks the release of the intermembrane mitochondrial protein called cytochrome c, thus often blocking apoptosis as well (5,6).

Mitochondria serve as key regulators of apoptosis via this cytochrome c-mediated pathway. Many cells activate apoptosis via the cytochrome c pathway, but they also may use pathways involving other molecules that reside in the mitochondrial intermembrane space. For example, mitochondria of some cells release procaspase-3 (7). More recently, mitochondria were found to release zymogens of caspase-2 and caspase-9 that were subsequently processed to generate enzymatically active caspases (8).

Various apoptotic signals such as radicals lead to mitochondrial release of pro-apoptotic proteins like cytochrome c and apoptosis-inducing factor (AIF) (Figure 1). AIF is an approximately 50 kDa molecule and has been shown in in vitro studies to activate caspase-3 by processing procaspase-3. Intruigingly, this activity is blocked by a general caspase inhibitor (zVAD-fmk), which raises the possibility that AIF may be a caspase (7).

Moreover, cytochrome c in its holo-form (i.e., with its haem group attached), associates with Apaf-1 (a molecule associated with Bcl-2), caspase-9, and ATP to form a complex called an ‘apoptosome’. This apoptosome proteolytically activates caspase-3, which leads to the activation of the caspase cascade and the degradation phase of apoptosis (8,9). Interestingly, these proteins – Bcl-2, Apaf-1, caspase-9, cytochrome c, and caspase-3 – serve as the mammalian equivalent of the apoptosome (CED-9-CED-4-CED-3) in Caenorhabditis elegans (10).

Cytochrome c serves as the key regulator of apoptosis because once it is released from the intermembrane space, the cell is irreversibly commited to death. Either apoptosis occurs through the caspase-mediated process described above, or the cell goes through a necrosis-like death due to the collapse of electron transport. Release of cytochrome c interrupts the transfer of electrons between respiratory chain complexes III and IV, resulting in the generation of deleterious radical species and the cessation of ATP synthesis (10).

How are cytochome c and AIF released from mitochondria? As mentioned earlier, the Bcl-2 family of proteins are able to regulate apoptosis by regulating the release of cytochrome c. In mammals, the anti-apoptotic members of this family include Bcl-2, Bcl-X_L, Mcl-1, A1/Bfl-1, and Bcl-W. The pro-apoptotic members are Bax, Bcl-XS, Bak, Bad, Bik, Bim, Bid, Hrk, and Bok. Homologs have been found in birds, frogs, Caenorhabditis elegans, and human herpesviruses (11).

One model of cytochrome c release is the formation of megachannels or permeability transition pores (PT pores), which are formed when the inner transmembrane potential (DY_m) collapses (12,13). The composition and structure of such pores have yet to be elucidated, but both inner membrane proteins (e.g., adenine nucleotide translocator, ANT) and outer membrane proteins (e.g., voltage-dependent anion channel, VDAC) contribute to pore formation. These pores allow molecules less than or equal to 1.5 kDa to pass through. The effects of PT pores are many: First, the respiratory chain is uncoupled due to the equilibration of ions between the matrix and intermembrane space; this also results in the loss of the H⁺ gradient. Second, PT pores cause the matrix to expand due to its hyperosmolality. This volume expansion is then believed to cause mitochondrial outer membrane rupture with the release of cytochrome c and AIF (4). Interestingly, PT pores also can be induced by caspases. This may lead to a feedforward amplification loop that better fits circular models of apoptosis than linear models.

A few studies have suggested that cytochrome c may be released before a collapse in DY_m. This suggests a model in which PT pores may open and close repeatably at a reversible low conductance state. Such rapid opening and closing of pores may allow the outer membrane to be disrupted and cytochrome c to be released before DY_m drops (14-16). Another model involves the opening of a large outer membrane channel that would allow cytochrome c and other intermembrane space proteins to move into the cytosol. In contrast with the previous scenarios, this model would leave the outer membrance largely intact. A benefit of this model is that there is no need for the mitochondrial matrix to swell. This better fits with the evidence that mitochondrial morphology remains the same in most cell death in vivo (17).

But how does the the Bcl-2 family regulate apoptosis? Bcl-2 and Bcl-X_L prevent PT pore opening, in isolated mitochondria (18,19) and purified PT pores reconstituted in liposomes (20). Since Bcl-2 can prevent PT pore opening in a system without cytochrome c, this indicates that Bcl-2 can directly regulate PT pores. However, Bcl-2 can also maintain DY_m under conditions that do not allow permeability transitions. This suggests that Bcl-2 regulates DY_m rather than PT pores through enhancing H⁺ efflux in the presence of stimuli that collapse DY_m (21). Second, Bcl-2 was found to inhibit the release of cytochrome c (5,6). Third, Bcl-2 attenuates the PT pore promoting effects of atractyloside (an activating ligand of the adenine nucleotide translocator, ANT) and Bax (19,22,23).

Moreover, Bcl-2 homologues can form ion channels or pores in artifical membranes (24-27). In fact, they have a crystal structure similar to colicins and the B-subunit of diptheria toxin (28). The diptheria B-subunit translocates the A-subunit across membranes; likewise Bcl-2 and Bcl-X_L can potentially translocate molecules across membranes. Bcl-2 and Bcl-X_L may interact physically or functionally with the PT pore or with non-PT pore proteins that control volume regulation of the matrix (7).

It has been known for a long time that oxidative stress caused by the overproduction of radicals can induce necrosis (29). For example, the highly reactive hydroxyl radical is believed to be one of the most potent inducers of oxidative damage in necrotic cell death (30). In apoptosis, radicals still play a major role by determining the cellular redox status, and many forms of apoptosis involve reactive oxygen and nitrogen species. And in most systems, antioxidants have been shown to attenuate apoptosis.

Reactive oxygen species are generated by an impairment of the mitochondrial respiratory chain. For example, superoxide is generated when a single electron is transferred to molecular oxygen at the ubiquinone site in complex III, and approximately 1-5% of all electrons are improperly transferred in this way. Other sources, such as fatty acid metabolites derived from arachidonic acid by the lipoxygenase pathway, may also be potent mediators of apoptosis. In addition, recent studies have found that overexpressing p53 (31) or treating cells with ceramide (32) will also generate oxidants. Ceramide is a sphingolipid produced in response to a number of stimuli, e.g., signalling from Fas/Apo-1/CD95, tumor necrosis factor receptor, and non-specific stress.

Oxidants indirectly induce apoptosis by changing cellular redox potentials, depleting reduced glutathione, and decreasing reducing equivalents, such as NADH and NADPH (33-35). These changes facilitate the formation of PT pores, leading to the subsequent release of cytochrome c. These PT pores possess several redox-sensitive sites, including one in equilibrium with mitochondrial matrix glutathione, and one directly activated by oxidants (36).

Nevertheless, it appears that oxidants are not necessary to induce apoptosis because many proapoptotic stimuli still function under anoxic conditions (37). Indeed, TNF-sensitive L929 cells underwent apoptosis that was not induced by reactive oxygen species (38). Similarly, follicular B lymphoma cells underwent apoptosis under anoxic conditions, and neither ROS scavengers nor inhibitors of ROS scavengers affected cell death (39). Also, in some systems, the major increase in oxidants (e.g., in superoxide (40)) occurs relatively late, i.e., after cytochrome c is released from mitochondria activating the caspase cascade. However, since ROS can be produced by cells and mitochondrial preparations under either aerobic or virtually anaerobic conditions (41), the role of radical species as inducers of apoptosis cannot be ruled out. In addition, more research needs to be conducted to investigate if chronic conditions of oxidative stress, such as cardiovascular disease and aging, will eventually induce apoptosis.

So does Bcl-2 influence oxidant-triggered cell death? In several experimental paradigms, Bcl-2 has been shown to make the cellular redox potential more reduced (42,43). Also, free radical-induced cell death is accompanied by lipid peroxidation, which is attenuated with Bcl-2 overexpression (44). More research needs to be done to determine if intracellular redox status is a key regulator in apoptosis-signalling pathways. Alterations of Bcl-2 during the aging process may be critical in determining when a cell will undergo apoptosis.

As described earlier, oxidants like superoxide and hydrogen peroxide can act as proapoptotic stimuli by changing the cellular redox status. As an example, we will consider peroxynitrite, a potent anion oxidant generated by the reaction of nitric oxide with superoxide. In one experiment, it was shown that treating HL-60 leukemia cells with increasing concentrations of peroxynitrite induced apoptosis in a time- and concentration-dependent manner (45). This is typical of oxidant-induced apoptosis at increasing levels of oxidants.

However, at low levels researchers have found that oxidants can stimulate cells to proliferate, and mild oxidative conditions have been shown to counteract apoptotic stimuli. Low cellular levels of superoxide and hydrogen peroxide are continually being produced from the mitochondrial respiratory chain and electron transport chains in the endoplasmic reticulum and nuclear membranes. Also, low levels of H₂0₂ are produced as a by-product of the activity of gamma-glutamyl transpeptidase (GGT). This enzyme is in charge of metabolizing extracellular reduced glutathione. In recent experiments with this enzyme, the data suggest that the low levels of H₂0₂ generated by GGT serve to protect U937 cells against apoptosis and help maintain cell proliferation (46).

The free radical gas nitric oxide (NO· ) behaves similarly. Nitric oxide is known to be an important regulator of mitochondrial function, cell signaling, and gene expression (47). If one is administered high exogenous levels of NO· donating compounds, nitric oxide can induce apoptosis. For example, nitric oxide, from the NO· donor sodium nitroprusside, resulted in hepatocyte apoptosis and hepatocellular enzyme release, which indicates cell damage (48). Also, exogenous release of NO· from various NO· donors has been shown to trigger apoptosis of rat renal mesangial cells. Researchers believe the mechanism may involve an up-regulation of ceramide levels by activating sphingomyelinases while concomitantly inhibiting ceramidases (49).

In contrast, at physiological levels NO· prevents apoptosis and interferes with the activation of the caspase cascade. In one experiment, proinflammatory cytokines were used to activate inducible nitric oxide synthase (iNOS), resulting in full protection for endothelial cells undergoing UV-A radiation. The mechanism involves NO· -mediated increases in Bcl-2 expression with a concomitant decrease in the expression of Bax protein (50). Also, in vitro and in vivo experiments have shown that NO· inhibits caspase-3 by S-Nitrosation of the enzyme (51). Associated with inhibiting caspase 3, NO· has been found to suppress the self-amplification feed forward loop of apoptosis by inhibiting Bcl-2 cleavage and cytochrome c release (52). In conclusion, radicals at low physiological levels often serve as inhibitors of apoptosis. However, at high levels oxidants can induce apoptosis, and even necrosis.

3. Apoptosis and Aging

Apoptosis is highly involved throughout the aging process, from early developmental changes to senescent declines in function. One classic case of the role of apoptosis in development is the elimination of tissues, transitory organs, and phylogenetic vestiges. For example, the pronephros and mesonephros are eliminated by apoptosis in higher vertebrates. Anuran tails and gills undergo apoptosis as tadpoles change into frogs. Moreover, the roundworm Caenorhabditis elegans eliminates exactly 131 of its initial 1,090 cells as it changes into its adult form (55). Another classic example concerns apoptosis in tissue remodeling. As vertebrate limb buds develop – e.g., in chicks, ducks, and humans, webbing between digits in the hind limbs is removed by apoptosis. This indicates that the ectoderm sends signals to initiate programmed cell death (56,57). Another interesting example concerns the apoptosis of cells in spinal ganglia during the development of chick embryos. This deletion of cells is chronologically and spatially precise, and can be reduced by injections of nerve growth factor (57).

Apoptosis also influences aging through the programmed death of T cells, cardiomyocytes, neurons, and other cell types. In the following section, we will briefly survey some of the findings that connect apoptosis to the aging of these cell types and a possible link to mitochondria.

3.1 T cells

Apoptosis is a fundamental part of T-lymphocyte maturation and selection. While the T-cells are maturing in the thymus, any T-cells that bind to self-antigens or make nonfunctional receptors undergo apoptosis (55). Recent studies indicate that with advancing age, defects in T-cell apoptosis may correlate with increased autoimmune disorders and susceptibility to infections. In several studies, aging was found to increase CD8 T-cell apoptosis by hyperstimulation through the T-cell receptor, thus leading to decreased levels of CD8 T-cells (58). Further studies need to be done to determine if increased oxidative stress with age will lead to increased apoptosis in T-cells. Also, future research needs to be done to elucidate the role of reactive oxygen and nitrogen species and DY_m in the programmed death of T-cells.

In other studies, lymphocytes from elderly subjects overexpressed the apoptosis molecule CD95/Fas antigen, which induces T-cell apoptosis in the presence of Fas ligand (59). Thus, the percentage of apoptotic cells collected from the blood was increased versus young individuals. Also, mononuclear cells from elderly subjects underwent more apoptosis in response to mitogen or anti-CD3 than mononucleocytes from young controls. Moreover, experiments using interleukin-2 (IL-2) to rescue lymphocytes found that far fewer of the cells from old donors were rescued versus cells from young controls, and this effect was independent of differences in Fas expression (59).

But does this CD95 pathway have any connection to mitochondria and the Bcl-2 family of proteins? In short, some data suggest that the CD95 pathway may be independent of the Bcl-2 family. For example, the clonal deletion of autoreactive T-cells that recognized endogenous antigens was not prevented by a bcl-2 transgene (61). However, Bcl-2 has been reported to protect against CD95-induced death in some cell types (62,63). This raises the possibility that CD95 triggers apoptosis through multiple pathways. Another study identified two cell types, each using almost exclusively one of two different CD95 signaling pathways. Both cell types showed loss of mitochondrial transmembrane potential. And upon CD95 triggering, both cell types had all mitochondrial apoptogenic activities blocked by Bcl-2 or Bcl-X_L overexpression (62).

Studies so far indicate that CD95-mediated apoptosis proceeds as follows: CD95 ligation activates the adaptor protein FADD (Fas-associated protein with a Death Domain). FADD then directly activates caspase-8 and the degradation phase of apoptosis (64). This pathway is crucial because it creates an opening for Bcl-2 regulation of CD95-mediated apoptosis. Daxx, a novel signaling protein, may bind to the Fas death domain. Daxx is sensitive to Bcl-2, and its Fas-binding domain is an inhibitor of Fas-induced apoptosis (63). Thus, the Bcl-2 family of proteins can still influence CD95-mediated apoptosis.

3.2 Cardiomyocytes

Apoptosis may also have significant effects in the age-associated decline in cardiac function. During aging, the human heart loses a significant number of myocytes. In fact, the initial ventricular myocyte population may decline by 30% as the heart ages. Although necrosis of cardiomyocytes may contribute to this loss, apoptosis is also a major factor contributing to the loss of myocytes. Apoptosis of cardiomyocytes may be prevented by drugs and intervention therapies, such as exercise training and increased antioxidant intake, and thus attenuate the age-related decline in cardiac function.

In support of this hypothesis, it should be noted that superoxide and hydrogen peroxide production increased with age in isolated mitochondria and submitochondrial particles from the hearts of mongolian gerbils (65). Moreover, antioxidant defenses decline significantly with age in myocytes (66). Also, recent data suggest that tumor necrosis factor (TNF) causes a rapid rise in intracellular reactive oxygen intermediates and apoptosis in myocytes and endothelial cells. Thus, oxidative stress may mediate apoptosis and result in myocyte dysfunction and the loss of cardiomyocytes (67).

Research from other studies also supports this hypothesis. Both necrotic and apoptotic cell death occurred in the aging heart of Fischer 344 rats. Necrotic myocytes were localized and quantified by using a myosin monoclonal antibody. Apoptotic myocytes were quantified using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) assay followed by DNA laddering to confirm DNA strand breaks in myocyte nuclei. The study revealed that apoptosis of myocytes increased with age and was restricted to the left ventricular free wall (68). Our hypothesis is also supported by studies of drugs used to treat heart failure. Carvedilol, a drug used to treat hypertension, angina, and heart failure, has significant antioxidant and anti-apoptotic effects, besides its effect on beta-adrenergic receptors (69). This suggests that apoptosis causes a significant loss of cardiomyocytes. Also, long-term therapy with the drug enalapril was found to attenuate cardiomyocyte apoptosis in dogs with moderate heart failure (70). Similarly, chronic administration of enalapril in aging mice was found to decrease apoptosis in cardiomyocytes (71).

3.3 Neurons

Lastly, we consider the role of apoptosis in the death of neurons during aging. Recent research, based on new stereological techniques to estimate neuron number, has revealed that during normal aging there is no significant age-related decline in hippocampal and neocortical neurons. However, some age-related decline does occur in the hilus of the dentate gyrus and the subiculum (72). Also, it has been found that apoptosis was involved in the normal aging of

nigral dopaminergic neurons. Morphological characteristics of apoptosis, such as cell shinkage and chomatin condensation, were found in approximately 2% of the neurons studied. Also, the mitochondria were shrunken, and there were signs of oxidative stress even in neurons devoid of apoptotic features (73).

Apoptosis is highly involved in neurodegenerative diseases of aging, such as Parkinson’s disease and Alzheimer’s disease. In Parkinson’s disease, ultrastructural studies of the dopaminergic neurons revealed that they die by apoptosis. Also, an in vitro model of dopaminergic neuron death, which used differentiated PC12 cells and mesencephalic neurons, found that when the sphingomyelin-dependent signaling pathway is activated, these cells die by apoptosis, preceded by mitochondrial production of superoxide radicals (74). The role of oxidative stress in Parkinson’s disease can also be seen in the increased production of hydroxyl radicals from mitochondrial respiratory chain dysfunctions (75). Other free radicals are generated when dopamine undergoes autoxidation or is enzymatically oxidized by monoamide oxidase (76). Furthermore, a selective increase in particulate superoxide dismutase activity was found in parkinsonian substantia nigra, which indicates a protective response to elevated levels of free radicals (77).

Apoptosis has also been associated with Alzheimer’s disease. Alzheimer’s disease is clinically associated with the development of amyloid plaques. It is believed that these plaques are caused by the improper folding and processing of amyloid b -precursor protein (Ab PP). Aggregation of Ab PP may involve free radicals (78), and Ab PP can itself generate free radicals (79). Oxidative stress from radical species and other apoptosis-regulating factors have been implicated in neuronal loss in Alzheimer’s disease. Many studies have shown that both neurons and glial cells undergo apoptosis; in fact, the amyloid-b protein has been found to induce cultured hippocampal cells to degenerate in a process with biochemical and morphological characteristics of apoptosis (80). Moreover, the neuroprotective effects of estrogen in delaying the development of Alzheimer’s disease may be due to its enhancement of the expression of the anti-apoptotic protein Bcl-X_L in cultured hippocampal neurons. This estrogen-induced enhancement of Bcl-X_L was associated with a reduction in amyloid-b -induced apoptosis, inhibiting both neurotoxicity and caspase-mediated proteolysis (81).
 
 

4.0 Apoptosis and Exercise

Strenuous exercise stimulates a variety of signals, such as increases in glucocorticoid (GC) secretion, intracellular calcium levels, and reactive oxygen species, which can potentially induce apoptosis (82). In addition, glutathione depletion, thiol oxidation, DNA damage, and hypoxia have been reported during exercise and may likely contribute to programmed cell death.

We propose that apoptotic cell death induced by exercise in very metabolically active tissues, such as the heart and skeletal muscle, may be a normal process to remove partially damaged cells. However, certain forms of exercise caused by mechanical damage, followed by an inflammatory response, may lead to excessive apoptosis.

Traditionally, mitochondria have been seen as powerhouses for energy production; however, it is now clear that excessive free radical production and the loss of mitochondrial membrane potentials can lead to apoptotic cellular events, including cytochrome c release or decreased Bcl-2 expression. One of the first papers to suggest that apoptosis may play a role in post-exercised animals was by Concordet et al. (83). In this study, rats ran to exhaustion on a treadmill. The T-cells in their thymuses showed signs of programmed cell death (increased DNA fragmentation) immediately after and 24-h post exercise. Moreover, RU-486 (a potent glucocorticoid receptor antagonist) administered 2 hours and 0 hours before the run partially inhibited thymocyte DNA fragmentation. This suggests a relationship between physical stress and glucocorticoid receptor-mediated apoptosis of rat thymocytes.

Damage to myocyte mitochondria due to oxidants and a variety of other factors, such as loss of mitochondrial membrane integrity, may be important inducers of apoptosis during exercise. For example, Sandri et al. have investigated apoptosis in skeletal muscle myocytes of dystrophin-deficient (mdx) mice subjected to spontaneous exercise. Muscle analysis showed increases of apoptotic myonuclei after exercise detected by the TUNEL method using electron microscopy. It is not clear from these studies, however, whether these cells were lost entirely by an apoptotic process. Moreover, expression of ubiquitin correlated with exercise and with positive myonuclei. (Ubiquitin is a proteolytic protein expressed during apoptosis that covalently links to proteins and tags them for degradation.) Furthermore, this study demonstrated a decrease in mitochondrial Bcl-2, which may be due to exercise-induced apoptosis in the muscle of mdx mice. (84).

The same research group also found that two days after spontaneous wheel-running, both normal and dystrophin-deficient muscles of mice showed increases in fragmented DNA using the TUNEL method and gel electrophoresis. Furthermore, ubiquitin increased in muscles of both dystrophic and control exercised mice. In this study, tissues were collected two days after the exercise bout. Therefore, apoptotic events might very well reflect factors released due to inflammatory processes caused by mechanical damage to metabolically active tissues (85). Thus, factors stimulating apoptosis during or immediately following exercise may be different from those involved 24-48 h post-exercise.

Human studies on the effects of apoptosis and exhaustive exercise are scarce. One study investigating human subjects exercising on a treadmill until exhaustion found DNA strand breaks in lymphocytes immediately after exercise, but not 24 and 48-hours post-exercise (86). Moreover, flow cytometry revealed lymphocyte apoptosis in 63% of lymphocytes immediately after exercise and in 86.2% of lymphocytes 24 hours after exercise. These events could possibly explain the greater incidence of upper respiratory infections in highly trained athletes following exhaustive training periods.

Exercise training results in a variety of adaptations that may be beneficial in attenuating apoptosis. For example, we and others have shown that several critical antioxidants and antioxidant enzymes are upregulated with exercise training, thus providing additional free radical protection in myocytes (87,88,89). We therefore propose that animals that are exercise-trained should show increased resistance to apoptosis. Indeed, one study showed that resistance exercise training attenuates hindlimb unloading-induced apoptosis, which also results in muscle atrophy (90). Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance-exercise training attenuated the increase in TDT-positive nuclei and significantly decreased the number of fibers with morphologically abnormal nuclei. However, this study does not show that there is a widespread or regional degeneration of the muscle fiber. Thus, ~1,344 myonuclei per 20-m m-thick soleus cross section were lost by an apoptotic process during hindlimb suspension, but no loss was found in total fiber number. In addition, the possibility exists that some macrophages that infiltrated the skeletal muscle to assist in muscle remodeling and fiber degeneration underwent apoptosis. In general, this study shows that depletion of "survival factors" due to unloading results in apoptotic myonuclei death. GH and IGF-1 are partly responsible for the attenuation, and the contribution of exercise training was unclear.

Skeletal muscle inactivity due to chronic heart failure could potentially also influence apoptosis. Indeed, recent research showed that apoptosis occurs in skeletal muscle myocytes in about 50% of patients with chronic heart failure possibly due to inactivity (91). Importantly, patients with apoptosis-positive skeletal muscle myocytes exhibited a significantly lower VO2max, a higher iNOS expression, and lower Bcl-2 expression as compared with biopsies of healthy patients. Future research is needed to determine the molecular signals enhancing skeletal muscle apoptosis in order to develop a therapeutic rationale for muscle fiber protection and restoration of contractile force.

The possibility that mitochondrial-produced oxidants during exercise have a direct effect on apoptosis has not yet been directly investigated. It seems very plausible that disturbances in mitochondrial homeostasis, i.e., DNA damage, inner mitochondrial membrane damage, and increases in calcium, could eventually result in the release of pro-apoptotic factors, such as cytochrome c from the intermembrane space. Thus, alteration in anti-apoptotic factors, such as Bcl-2 and Bcl-X_L with aging and exercise, could increase the likelihood of an induction of mitochondrial-mediated programmed cell death (Figure 2).

Summary:

Programmed cell death has received phenomenal attention in the past few years. It is now known that mitochondria play a central regulatory role in apoptosis, particularly through the cytochrome c pathway. Also, mitochondria and radical species are intimately involved in the programmed cell death that occurs during aging and exercise. Increased oxidative stress from ROS and RNS changes the cellular redox potentials, depletes glutathione, and decreases reducing equivalents like NADP and NADPH. These intracellular changes are sufficient to induce the formation of mitochondrial permeability transition pores, leading to the subsequent release of cytochrome c and the activation of the caspase cascade. We hypothesize that during chronic conditions of oxidative stress as in aging, tissues may undergo unnecessary and increased apoptosis, leading to pathological dysfunctions from significant cell loss. Similarly, uncontrolled and unnecessary apoptosis may occur during exhaustive exercise resulting in various pathologies.
 
 

ACKNOWLEDGEMENT

We would like to thank Nichole Tripician for her technical assistance with this manuscript and Dr. P. Green, Sharon Phaneuf and Jared Wilsey for their critical reading of this manuscript. Supported by Society of Geriatric Cardiology, Merck Geriatric Cardiology Research Grant 7584. Michael Pollack is a participant in the University of Florida Scholars Program.
 
 

References

1. Kerr, J.F.R.; Wyllie, A.H.; Currie, A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer. 26:239-257; 1972.
2. Sulston, J.E.; Horvitz, H.R. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Developmental Biology. 56:110-156; 1977.
3. Carson, D.A., Ribeiro, J.M. Apoptosis and disease. Lancet. 341 (885), 1251-1254, 1993.
4. Susin, S.A.; Zamzami, N.; Kroemer, G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta. 1366:151-165; 1998.
5. Kluck, R.M.; Bossy-Wetzel, E.; Green, D.R.; Newmeyer, D.D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 275:1132-1136; 1997.
6. Yang, J.; Liu, X.; Bhalla, K.; Kim, C.N.; Ibrado, A.M.; Cai, J.; Peng, T.I.; Jones, D.P. Wang, X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 275:1129-1132;1997.
7. Green, D.R.; Reed, J.C. Mitochondria and apoptosis. Science. 281:1309-1312; 1998.
8. Susin, S.A.; Lorenzo, H.K.; Zamzami, N.; Marzo, I.; Brenner, C.; Larochette, N.; Prevost, M.C.; Alzari, P.M.; Kroemer, G. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J. Exp. Med. 189:381-394. 1999.
9. Li P.; Nijhawan D.; Budihardjo I.; Srinivasula SM.; Ahmad M.; Alnemri ES.; Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479-489; 1997.
10. Green, D.; Kroemer, G. The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biology. 8:267-271;1998.
11. Reed, J.C.; Jurgensmeier, J.M.; Matsuyama, S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta. 1366:127-137; 1997.
12. Petit, P.X.; Susin, S.A.; Zamzami, N.; Mignotte, B.; Kroemer, G. Mitochondria and programmed cell death: back to the future. FEBS Lett. 396:7-13; 1996.
13. Qian,T.; Herman, B.; Lemasters, J.; Cell Vision. 4: 166; 1997.
14. Ichas, F.; Jouaville, L.S.; Mazat, J.P. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell. 89:1145-1153. 1997.
15. Ankarcrona, M.; Dypbukt, J.M.; Orrenius, S.; Nicotera, P. Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Lett. 394: 321-324. 1996.
16. Ankarcrona, M.; Dypbukt, J.M.; Bonfoco, E.; Zhivotovsky, B.; Orrenius, S.; Lipton, S.A.; Nicotera, P. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15: 961-973. 1995.
17. Wyllie, A.H.; Kerr, J.F.; Currie, A.R. Cell death: the significance of apoptosis. Int Rev Cytol. 68: 251-306. 1980.
18. Susin, S.A.; Zamzami N.; Castedo M.; Daugas E.; Wang HG.; Geley S.; Fassy F.; Reed JC. Kroemer GThe central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 186: 25-37. 1997.
19. Susin, S.A.; Zamzami N.; Castedo M.; Hirsch T.; Marchetti P.; Macho A.; Daugas E.; Geuskens M.; Kroemer G. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184: 1331-1341. 1996.
20. Marzo, I et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J. Exp. Med. 187: 1261-1271. 1998.
21. Shimizu, S.; Eguchi, Y.; Kamiike, W.; Funahashi, Y.; Mignon, A.; Lacronique,V.; Matsuda, H.; Tsujimoto, Y. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc Natl Acad Sci U S A. 95:1455-1459. 1998.
22. Zamzami, N.; Susin, S.A.; Marchetti, P.; Hirsch, T.; Gomez-Monterrey, I.; Castedo, M.; Kroemer, G. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183: 1533-1544. 1996.
23. Xiang, J.; Chao, D.T.; Korsmeyer, S.J. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. U.S.A. 93: 14559. 1996.
24. Minn, A.J.; et al. Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature. 385: 353. 1997.
25. Schendel, S.L.; et al. Channel formation by antiapoptotic protein Bcl-2. Proc. Natl. Acad. Sci. USA. 94: 5113. 1997.
26. Antonsson, B.; et al. Inhibition of Bax channel-forming activity by Bcl-2. Science. 277: 370. 1997.
27. Schlesinger, P.; et al. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc. Natl. Acad. Sci. USA. 94: 11357. 1997.
28. Muchmore, S.W. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 381: 335-341. 1996.
29. Halliwell, B.; Gutteridge, J.M. Free radicals in biology and medicine. Oxford, Carendon Press: 1989.
30. Halliwell, B.; Gutteridge, J.M. Role of free radicals and catalytic metal ions in human disease: an overview, Methods Enzymol. 186: 1-85. 1990.
31. Polyak, K.; Xia, Y.; Zweier, J.L.; Kinzler, K.W. Vogelstein B. A model for p53-induced apoptosis. Nature. 389: 300-305. 1997.
32. Quillet-Mary, A.; Jaffrezou, J.P.; Mansat, V.; Bordier, C.; Naval, J.; Laurent, G. Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J Biol Chem. 272: 21388-21395. 1997.
33. Zoratti, M.; Szabo, I. The mitochondrial permeability transition. Biochem. Biophys. Acta. 1241:139-176. 1995.
34. Bernardi, P. The permeability transition pore. Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death. Biochem. Biophys. Acta 1275: 5-9. 1996.
35. Bernardi, P.; Petronilli, V. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J. Bioenerg Biomembr. 28: 129-136. 1996.
36. Costantini, P.; Chernyak, B.V.; Petronilli, V.; Bernardi, P. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271: 6746-6751. 1996.
37. Jacobson MD, Raff MC. Programmed cell death and Bcl-2 protection in very low oxygen. Nature 374: 814-816. 1995.
38. Schulze-Osthoff, K.; Krammer, P.H.; Droge, W. Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. EMBO J. 13: 4587-4596. 1994.
39. Shimizu, S.; Eguchi, Y.; Kosaka, H.; Kamiike, W.; Matsuda, H.; Tsujimoto Y. Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature.374: 811-813. 1995.
40. Cai, J.; Jones, D.P. Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem. 273:11401-11404. 1998.
41. Degli Esposti, M; McLennan, H. Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis FEBS Lett. 430: 338. 1998.
42. Kane, D.J.; Sarafian, T.A.; Anton, R.; Hahn, H.; Gralla, E.B.; Valentine, J.S.; Ord, T.; Bredesen, D.E. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science. 262:1274-1277. 1993.
43. Marchetti, P.; Decaudin, D.; Macho, A.; Zamzami, N.; Hirsch ,T.; Susin, S.A.; Kroemer, G .Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function. Eur J Immunol. 27: 289-296 . 1997.
44. Hockenbery DM.; Oltvai ZN.; Yin XM.; Milliman CL.; Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241-251. 1993.
45. Lin, K.T.; Xue, J.Y.; Nomen, M.; Spur, B.; Wong, P.Y. Peroxynitrite-induced apoptosis in HL-60 cells. J Biol Chem. 270:16487-90. 1995.
46. del Bello, B.; Paolicchi, A.; Comporti, M.; Pompella, A.; Maellaro, E. Hydrogen peroxide produced during gamma-glutamyl transpeptidase activity is involved in prevention of apoptosis and maintainance of proliferation in U937 cells. FASEB J. 13: 69-79.1999.
47. Beck KF, Eberhardt W, Frank S, Huwiler A, MesMer UK, MUHl H, Pfeilschifter J Inducible NO synthase: role in cellular signalling. J Exp Biol.202:645-653. 1999
48. Wang, J.H.; Redmond, H.P.; Wu, Q.D.; Bouchier-Hayes, D. Nitric oxide mediates hepatocyte injury. Am J Physio.l 275:G1117-G1126. 1998.
49. Huwiler, A.; Pfeilschifter, J.; van den Bosch, H. Nitric Oxide Donors Induce Stress Signaling via Ceramide Formation in Rat Renal Mesangial Cells. J Biol Chem. 274:7190-7195. 1999.
50. Suschek, C.V.; Krischel, V.; Bruch-Gerharz, D.; Berendji, D.; Krutmann, J.; Kroncke K.D.; Kolb-Bachofen, V. Nitric Oxide Fully Protects against UVA-induced Apoptosis in Tight Correlation with Bcl-2 Up-regulation. J Biol Chem. 274: 6130-6137. 1999.
51. Rossig, L.; Fichtlscherer, B.; Breitschopf, K.; Haendeler, J.; Zeiher, A.M. M lsch A, Dimmeler S. Nitric Oxide Inhibits Caspase-3 by S-Nitrosation in Vivo. J Biol Chem. 274: 6823-6826. 1999.
52. Kim, Y.M.; Kim, T.H.; Seol, D.W.; Talanian, R.V.; Billiar, T.R. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome crelease. J Biol Chem. 273: 31437-31441. 1998.
53. Harman, D. Free radicals in aging. Mol. Cell. Biochem. 84: 155-161. 1988.
54. Ozawa, T. Mechanism of somatic mitochondrial DNA mutations associated with age and diseases. Biochim. Biophys. Acta. 1271: 177-189. 1995.
55. Duke, R.C.; Ojcius, D.M.; Young, J.D. Cell suicide in health and disease. Scienctific American. 275: 80-87. 1996.
56. Saunders, J.W. Death in Embryonic Systems. Science. 154: 604-612. 1966.
57. Saunders, J.W., Jr. Developmental Biology. New York, Macmillan: 1982.
58. Telford, W.G.; Miller, R.A. Aging increases CD8 T cell apoptosis induced by hyperstimulation but decreases apoptosis induced by agonist withdrawal in mice. Cell Immunol. 191:131-138. 1999.
59. Potestio, M.; Caruso, C.; Gervasi, F.; Scialabba, G.; D'Anna, C.; Di Lorenzo, G.; Balistreri, C.R.; Candore, G.; Romano, G.C. Apoptosis and ageing. Mech Ageing Dev. 102: 221-237. 1998.
60. Sentman CL, Shutter JR, Hockenbery D, Kanagawa O, Korsmeyer SJ. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell: 67:879-88. 1991.
61. Strasser A, Harris AW, Huang DC, Krammer PH, Cory S. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 14: 6136-47. 1995
62. C. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17:1675-87. 1998.
63. Yang X, Khosravi-Far R, Chang HY, Baltimore D. Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell .89:1067-76. 1997.
64. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 281:1322-6. 1998
65. Sohal, R.S.; Agarwal, S.; Sohal, B.H. Oxidative stress and aging in the Mongolian gerbil (Meriones unguiculatus). Mech Ageing Dev. 81:15-25. 1995.
66. Sohal, R.S.; Weindruch, R. Oxidative stress, caloric restriction, and aging. Science. 273: 59-63. 1996.
67. Ferrari R; Agnoletti L; Comini L; Gaia G; Bachetti T; Cargnoni A; Ceconi C; Curello S; Visioli O. Oxidative stress during myocardial ischaemia and heart failure. Eur Heart J. 19 Suppl B: B2-11. 1998.
68. Kajstura, J.; Cheng, W.; Sarangarajan, R.; Lim, P.; Li, B.; Nitahara, J.A.; Chapnick, S.; Reiss, K.; Olivetti, G.; Anversa, P. Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats. Am J Physiol.271: H1215-H1228. 1996.
69. Feuerstein, G.; Yue, T.L.; Ma, X.; Ruffolo, R.R. Novel mechanisms in the treatment of heart failure: inhibition of oxygen radicals and apoptosis by carvedilol. Prog Cardiovasc Dis. 41:17-24. 1998.
70. Goussev, A.; Sharov, V.G.; Shimoyama, H.; Tanimura, M.; Lesch, M.; Goldstein, S.; Sabbah, H.N. Effects of ACE inhibition on cardiomyocyte apoptosis in dogs with heart failure. Am J Physiol. 275: H626-H631. 1998.
71. Ferder,L.; Romano, L.A.; Ercole, L.B.; Stella, I.; Inserra, F. Biomolecular changes in the aging myocardium: the effect of enalapril. Am J Hypertens.11:1297-1304. 1998.
72. Morrison, J.H.; Hof, P.R. Life and Death of Neurons in the Aging Brain. Science. 278: 412-419. 1997.
73. Anglade, P.; Vyas, S.; Hirsch, E.C.; Agid, Y. Apoptosis in dopaminergic neurons of the human substantia nigra during normal aging. Histol Histopathol.12: 603-610. 1997.
74. Ruberg, M.; France-Lanord, V.; Brugg, B.; Lambeng, N.; Michel, P.P.; Anglade, P.; Hunot, S.; Damier, P.; Faucheux, B.; Hirsch, E.; Agid, Y. Neuronal death caused by apoptosis in Parkinson disease[Article in French]. Rev Neurol. 153: 499-508. 1997.
75. Shoffner, JM; Watts, RL; Juncos, JL; Torroni, A; Wallace, DC. Mitochondrial oxidative phosphorylation defects in Parkinson’s disease. Ann Neurol30: 332-339. 1991.
76. Hirsch, EC. Why are catecholaminergic neurons more vulnerable than other cells in Parkinson’s disease? Ann Neurol 32: S88-S93. 1992.
77. Saggu, H; Cooksey, J; Dexter, D; Wells, FR; Lees, A; Jenner, P; Marsden, CD. A selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra. J Neurochem 53: 692-697. 1989.
78. Dyrks, T; Dyrks, E; Hartmann, T; Masters, C; Beyreuther, K. Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation. J Biol Chem 267: 18210-18217. 1992.
79. Hensley, K; Carney, JM; Mattson, MP; Aksenova, M; Harris, M; Wu, JF; Floyd, RA; Butterfield, DA. A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: Relevance to Alzheimer disease. Proc Natl Acad Sci USA 91: 3270-3274. 1994.
80. Ivins, K.J.; Bui, E.T.; Cotman, C.W. Beta-amyloid induces local neurite degeneration in cultured hippocampal neurons: evidence for neuritic apoptosis. Neurobiol Dis. 5:365-378. 1998.
81. Pike, CJ. Estrogen modulates neuronal Bcl-xL expression and beta-amyloid-induced apoptosis: relevance to Alzheimer's disease. J Neurochem 72: 1552-1563. 1999.
82. Carraro U.; Franceschi C. Apoptosis of skeletal and cardiac muscles and physical exercise. Aging. 1-2:19-34. 1997.
83. Concordet JP.; Ferry A. Physiological programmed cell death in thymocytes is induced by physical stress (exercise). Am J Physiol: 265:C626-629. 1993.
84. Sandri M.: Podhorska-Okolow M.; Geromel V, Rizzi.; C, Arslan P, Franceschi C.; Carraro U. Exercise induces myonuclear ubiquitination and apoptosis in dystrophin-deficient muscle of mice. J Neuropathol Exp Neurol. 56:45-57. 1997
85. Sandri M.; Carraro U.; Podhorska-Okolov M.; Rizzi C.; Arslan P.; Monti D.; Franceschi C. Apoptosis, DNA damage and ubiquitin expression in normal and mdx muscle fibers after exercise. FEBS Lett :373: 291-5. 1995
86. Mars M.; Govender S.; Weston A.; Naicker V.; Chuturgoon A. High intensity exercise: a cause of lymphocyte apoptosis? Biochem Biophys Res Commun249: 366-70. 1998.
87. Leeuwenburgh C.; J Hollander.; R Fiebig.; S Leichtweis.; M Griffith.; LL Ji. Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific. Am J Physiol 272, 363-369. 1997.
88. Powers SK.; Criswell D.; Lawler J.; Martin D.; Lieu FK.; Ji LL.; Herb RA. Rigorous exercise training increases superoxide dismutase activity in ventricular myocardium. Am J Physiol. 265:H2094-8. 1993.
89. Sen CK.; Marin E.; Kretzschmar M.; Hanninen O. Skeletal muscle and liver glutathione homeostasis in response to training, exercise, and immobilization. J Appl Physiol : 73. 1265-1272. 1992.
90. Allen DL.; Linderman JK.; Roy RR.; Bigbee AJ.; Grindeland RE.; Mukku V.; Edgerton VR. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am J Physiol 273:C579-87. 1997.
91. Adams V.; Jiang H.; Yu J.; Mobius-Winkler S.; Fiehn E, Linke A.; Weigl C.; Schuler G.; Hambrecht R. Apoptosis in skeletal myocytes of patients with chronic heart failure is associated with exercise intolerance. J Am Coll Cardiol: 33:959-65. 1999.

Figure 1. Caspases and endonucleases are activated by pro-apoptotic factors released from mitochondria; these include Apoptosis-inducing factor (AIF), cytochrome c, and Apaf-1. AIF and cytochrome c are released from the intermembrance space. And Apaf-1 is probably released from Bcl-2, a mitochondrial surface protein that plays a crucial role in regulating apoptosis

Figure 2. Aging and exercise cause changes in glutathione (GSH), ATP, NADH, calcium, and glucocorticoids, which may directly affect oxidant production and mitochondrial redox status. These changes and alterations in mitochondrial proteins, such as Bcl-2, Bcl-X_L, and Bax, may lead to a collapse of the inner transmembrane potential (DY_m) and the opening of mitochondrial transition pores (MTP) causing apoptosis.