activity. Further, mitochondria from disused skeletal muscle possess a reduced capacity to generate ATP per gram of muscle and display an overall decrease in mitochondrial function. This has a pronounced effect on skeletal muscle performance, leading to increased fatigability and a poor endurance capacity.

Disuse-induced decrements in mitochondrial oxidative capacity also contribute to the activation of mitochondrially mediated apoptosis via the enhanced production of ROS. ROS have been shown to increase in slow-twitch and in fast-twitch muscles during 28 days of hind-limb unloading. In addition, the ROS metabolizing enzymes manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase are all reduced in their expression, and this increases the impact of the augmented ROS production. Similar results have also been demonstrated using chronic skeletal muscle denervation, demonstrating that this adaptation occurs in various models of muscle disuse. Mitochondria from denervated skeletal muscle exhibit an increased rate of mitochondrial permeability transition pore (mtPTP) opening kinetics, indicating an increase in apoptotic susceptibility. Additionally, chronic muscle disuse also alters the expression of proand antiapoptotic mitochondrial proteins. During denervation-induced muscle disuse, there is an increase in the Bax:Bcl-2 ratio, which favors the formation of the mtPTP. Thus chronic muscular inactivity leads to a number of proapoptotic adaptations, which include (1) a reduction in overall mitochondrial function, (2) an increase in ROS production, and (3) a faster rate of mtPTP formation. Interestingly, the elevated expression of Bax has further significance as this protein is able to homodimerize on the outer membrane of the mitochondria, forming the mitochondrial apoptosis channel (MAC). MAC is capable of facilitating the release of small proteins such as cytochrome c, but not larger proteins like AIF. Thus the implications of an increase in Bax protein expression are twofold: (1) Bax assists in the formation of the mtPTP, and (2) Bax can form its own pore, facilitating further proapoptotic protein release.

The disuse-induced increase in ROS production likely promotes the release of proapoptotic proteins and the fragmentation of myonuclear DNA. Using denervation as a model of disuse, a 40% increase in cytosolic cytochrome c and a 10-fold increase in AIF localized within the cytosol was observed. This increase in cytochrome c was paralleled by a 500% increase in cleaved caspase-3, indicating the activation of caspasedependent apoptosis, and a 100% increase in DNA fragmentation. Other forms of muscle disuse such as hind-limb suspension potentiate similar increases in

the caspase-independent apoptotic pathway. Within 12 hours of disuse induction, significant increases in the localization of Endo G within the nucleus and concomitant increases in DNA fragmentation were observed. Collectively, these results suggest that chronic muscle disuse induces proapoptotic changes within muscle, which lead to an increase in mitochondrial apoptotic susceptibility and myonuclear apoptosis and contribute to skeletal muscle atrophy.

4. APOPTOSIS IN MUSCLE DURING AGING AND DISEASE

In addition to muscle disuse, apoptosis in skeletal muscle is also enhanced in various pathological conditions such as aging, type 2 diabetes mellitus, chronic heart failure, and cancer. Even though skeletal muscle is not necessarily the tissue responsible for the initial clinical manifestations of these diseases, most, if not all, of these pathological conditions are associated with mitochondrial dysfunction within skeletal muscle. This elevation in apoptosis contributes to a reduction in muscle mass, and the concomitant manifestation of mitochondrial dysfunction compromises endurance capacity, thereby severely affecting the quality of life of the affected individual.

4.1. Aging

Muscle atrophy that occurs with increasing age is commonly referred to as sarcopenia. The decline in muscle mass with age has been attributed to a number of external factors such as motor unit rearrangement, hormonal status, and reduced satellite cell recruitment. Although much research has been conducted in this area, the signaling pathways that regulate this type of cell death remain elusive. Studies have documented muscle atrophy in a multitude of muscles with differing fiber type content such as the slow-twitch soleus, as well as the predominantly fast-twitch plantaris, gastrocnemius, and extensor digitorum longus muscles. Although the reduction in muscle mass is observed in each fiber type, the process is differentially regulated. Fast-twitch (type II) muscle fibers appear to be more susceptible to programmed cell death during the normal physiologic condition of aging. This likely occurs because type II fibers have less myonuclei per fiber compared with slow-twitch (type I) fibers, and/or the regular distribution of myonuclei is impaired, thus increasing transport distances of de novo gene products. Additionally, a selective loss of motor unit innervation to type II fibers occurs with advancing age. Thus an aged muscle will ultimately retain a higher proportion of type I MHC-containing

fibers compared with that same muscle before atrophy, and this is due to a preferential loss of fast-twitch type II fibers.

Recently, an increased incidence of apoptosis and enhanced expression of proapoptotic proteins has been documented in aged skeletal muscle. Skeletal muscle is more susceptible to age-induced deterioration because it is postmitotic. Although satellite cells are capable of replacing myonuclei that have been lost because of apoptosis, both the percentage of satellite cells and their proliferative capacities decrease with advancing age. Furthermore, mitochondrial dysfunction likely plays an important role in increasing the susceptibility of muscle to apoptosis, because mitochondria from senescent animals have enhanced ROS production, elevated levels of mtDNA mutations, and an augmented release of cytochrome c and Endo G as compared with their young counterparts. Transgenic mice expressing a proofreading-deficient version of mitochondrial DNA polymerase γ accumulate a high degree of mtDNA mutations and exhibit several age-related phenotypes, such as sarcopenia. Interestingly, apoptosis was observed to be one of the potential mechanisms responsible for the mtDNA mutation-induced sarcopenia in these animals. This suggests that mtDNA mutations, which lead to mitochondrial dysfunction and promote apoptosis, may play a pivotal role in age-related phenotypes such as skeletal muscle mass loss.

4.2. Type 2 diabetes mellitus

Insulin resistance and type 2 diabetes mellitus (T2DM) are common complications of obesity and a sedentary lifestyle. These conditions are characterized by elevated circulating concentrations of fatty acids (FAs) and aberrant FA metabolism, resulting in increased FA storage in nonadipose tissues such as skeletal muscle. These FAs are stored in the form of triglycerides, diacylglycerol, and ceramides. Recent evidence supports a role for FA-induced skeletal muscle apoptosis, termed lipoapoptosis, which results from FA overload. Exposure of muscle cells to the saturated FA palmitate results in apoptosis, implicating high levels of FAs in pathological conditions such as obesity and T2DM as a contributor to skeletal muscle programmed cell death. FAs can increase the production of ROS, further augmenting the susceptibility of skeletal muscle toward apoptosis. A growing body of evidence implicates oxidative stress, resulting from ROS-induced mitochondrial dysfunction, as a major contributor to skeletal muscle apoptosis. ROS-induced mtDNA damage has been

demonstrated to impair glucose utilization and insulin resistance in skeletal muscle. mtDNA mutations are elevated within skeletal muscle of T2DM patients, presumably in concert with an increase in ROS production as a result of high levels of FA. A reduction of mtDNA to levels of < 10% of normal cells using ethidium bromide has been illustrated to result in altered insulin signaling, as evidenced by a reduced expression of insulin receptor substrate-1 (IRS-1), decreased insulin-stimulated phosphorylation of IRS-1 and protein kinase B, and attenuated glucose transporter-4 translocation to the plasma membrane. This implicates a vicious cycle, whereby obesity results in FA accumulation within skeletal muscle, leading to increased ROS production. This elevation in oxidative stress can damage mtDNA and reduce the oxidative capacity of skeletal muscle by both decreasing mitochondrial function and reducing muscle mass via apoptosis. The reduction of skeletal muscle mass and oxidative capacity further exacerbates FA accumulation, thereby continuing the cycle. Additionally, because skeletal muscle is the main tissue responsible for glucose disposal from the circulation, reductions in the amount of skeletal muscle as a result of enhanced apoptosis will further propagate the clinical symptoms of T2DM.

4.3. Cancer cachexia

Severe muscle wasting, termed cachexia, occurs in the majority of cancer patients before death and accounts for nearly one-third of cancer deaths. Individuals suffering from cancer cachexia can lose up to 80% of their skeletal muscle mass. Fortunately, muscle wasting does not occur in all forms of cancer. For example, breast cancer and non-Hodgkin’s lymphoma patients rarely develop cachexia. However, gastrointestinal cancer patients are highly prone to muscle wasting. Although the loss of muscle mass observed during cancer cachexia can be jointly attributed to a reduction in protein synthesis and elevated protein degradation, the majority of muscle wasting during the advanced stages of cancer is due to increased protein degradation. The ubiquitin-proteasome pathway and apoptosis have been demonstrated to be contributing factors to skeletal muscle degeneration in cancer cachexia.

Skeletal muscle apoptosis is a secondary clinical manifestation of cancer patients. Increased DNA fragmentation, a hallmark of apoptosis, has been observed in the gastrocnemius muscle of mice and rats inoculated with tumorigenic liver and lung cells and in the rectus abdominis muscle of human gastrointestinal cancer patients. Higher levels of cytochrome c release from

mitochondria, along with enhanced caspase activation in muscle from cachectic mice, also supports a role for apoptosis during cancer cachexia. Evidence suggests that the cachectic response is mediated by inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α). TNF-α is implicated as a major contributor toward skeletal muscle apoptosis in cancer patients. This cytokine can induce apoptosis by regulating the dephosphorylation and subsequent degradation of the antiapoptotic protein Bcl-2. Additionally, TNF-α mediates caspasedependent programmed cell death via binding to its receptor on the sarcolemmal membrane of myofibrils. In conjunction with elevated levels of apoptosis, TNF-α potentiates an increase in proteolysis, as evidenced by increased expression of ubiquitin and proteosome subunits. Thus available data strongly indicate a role for apoptosis in cancer cachexia.

4.4. Chronic heart failure

Apoptosis occurs not only in the myocardium, but also in the skeletal muscle of chronic heart failure (CHF) patients. Just like individuals suffering from cancer cachexia, CHF patients also have elevated levels of inflammatory cytokines such as TNF-α. TNF-α levels have been documented to be the strongest predictors of the degree of muscle loss occurring in CHF patients. Muscle biopsies from human CHF patients exhibit elevated markers of apoptosis such as DNA fragmentation and caspase-3 cleavage. CHF patients also have elevated levels of growth hormone (GH) with concomitant decreases in insulin-like growth factor-I (IGF-1), suggesting the presence of GH resistance. IGF-1 inhibits apoptosis in cardiac cells by reducing the expression of Bax and caspase-8, as well as the activity of caspase-3. Thus the reduction of circulating IGF-1 levels in CHF patients results in an attenuation of apoptotic repression, thereby enhancing apoptotic susceptibility within skeletal muscle. Additionally, CHF patients exhibit high amounts of oxidative stress, which may further contribute to the induction of apoptosis.

A key symptom of CHF patients is exercise intolerance, which is largely due to ventricular dysfunction. Ultrastructural and functional analyses have also revealed abnormalities in skeletal muscle mitochondria from CHF patients, suggesting that mitochondrial dysfunction within skeletal muscle is a contributing determinant to the clinical symptoms, and the reduced quality of life, associated with CHF.

Given the plasticity of skeletal muscle, it is not surprising that apoptotic susceptibility and mitochon-

drial deficits observed during muscle disuse and disease can be altered by exercise. Although exercise-induced adaptations are dependent on the type, intensity, duration, and frequency of exercise training, emerging evidence indicates that exercise/chronic contractile activity can reduce apoptotic susceptibility within skeletal muscle. This exercise-mediated repression of apoptosis may prove to be an important therapeutic intervention for many diseases that are associated with apoptotic induction within this tissue.

5. EFFECT OF ENDURANCE EXERCISE/CHRONIC

CONTRACTILE ACTIVITY ON APOPTOSIS

Skeletal muscle is composed of a variety of specialized fiber types containing a continuum of mitochondrial contents, a variable that adapts readily to changing conditions of contractile activity. Indeed, the major biochemical adaptation that occurs within the muscle with exercise training is an increase in mitochondrial content, leading to functional improvements in fatigue resistance. Because mitochondria are closely associated with apoptosis, it is logical to presume that exercise may modulate rates of apoptosis. The effects of chronic contractile activity on apoptosis have been investigated using different models of endurance exercise training such as voluntary wheel running, treadmill training, and chronic low frequency electrical stimulation using both acute and chronic training models.

Although acute exercise has been demonstrated to enhance apoptosis in muscle, a plethora of data indicates that chronic exercise may have a protective effect on muscle apoptotic susceptibility. Treadmill running over a period of 8 weeks enhanced the antiapoptotic (Bcl-2, XIAP) and antioxidant (HSP70 and MnSOD) protein levels, with a simultaneous decrease in apoptotic proteins (Apaf-1 and Bax) and DNA fragmentation in the skeletal and cardiac muscle of trained compared with control animals. In agreement with these data, it was recently illustrated that chronic contractile activity, imposed via electrical stimulation, resulted in mitochondrial biogenesis and a concomitant reduction in mitochondrial apoptotic susceptibility. This was indicated by a decrease in ROS production within the IMF mitochondrial subfraction, along with reduced levels of cytochrome c and AIF release from SS and IMF mitochondria isolated from chronically stimulated muscle. As opposed to endurance exercise, resistance training does not alleviate apoptosis, despite attenuating the loss in muscle mass, during hind-limb unloading as a model of muscle atrophy.

The exercise-mediated attenuation of apoptotic susceptibility is also observed during muscle disuse conditions such as aging. Treadmill training in old rats improved exercise capacity and muscle strength and reduced the age-related increase in apoptotic markers such as an increase in the Bax:Bcl-2 ratio, caspase activation, and DNA fragmentation. However, endurance training in mitochondrial myopathy patients reduced the expression of DNA repair proteins and produced higher oxidative damage despite an increase in antiapoptotic MnSOD protein expression, indicating that more research is required to establish whether exercise training can be a beneficial therapeutic modulation for patients with mtDNA defects and other muscle myopathies.

6. CONCLUSION

Skeletal muscle is a versatile tissue that adapts phenotypically to both decreases and increases in contractile activity. During muscle disuse or disease, reductions in muscle mass, strength, mitochondrial content, and endurance performance are observed. In contrast, endurance exercise training elevates muscle mitochondrial content, and this appears to attenuate mitochondrially mediated apoptosis. Apoptosis plays a well-established role in muscle disuse conditions such as aging and in pathological conditions such as T2DM and cancer cachexia. Further research is warranted to fully elucidate the molecular regulation of apoptosis in skeletal muscle and to fortify a role for exercise training as an ideal therapeutic intervention to preserve muscle function during chronic muscle disuse and disease.

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