Mitochondrial DNA mutations, energy metabolism and apoptosis in aging muscle.

Locomotor functional decline and loss in muscle mass with age is virtually a universal characteristic that has been documented in several species, including worms, fruit flies, rodents, non-human primates and humans. The age-related loss of muscle mass and strength (sarcopenia) represents an important risk factor for disability and mortality in older subjects and has been linked with cellular energy deficit and increased apoptosis at old age. Many key theories on aging describing the mechanisms underlying sarcopenia are now focused on the mitochondria because of their dichotomous role in controlling life and death processes within myocytes. Mitochondria represent the main producers of cellular energy in the form of adenosine triphosphate, but are also considered a key regulatory center of apoptosis. Unknown factors leading to a decrease in aerobic energy efficiency are linked with mitochondrial mutations which may result into apoptosis. Moreover, deregulation of autophagy (degradation and recycling of long-lived protein and organelles, such as the mitochondria) in post-mitotic tissue might also be responsible for the age-associated cellular energy failure. Alterations in specific signaling pathways, such as AMP-activated protein kinases, play a role in both cell survival response and apoptotic response depending on energy depletion. Evidence supports that apoptosis occurring in aging skeletal muscle may be due, in part, to the progressive decline in mitochondrial function and the resulting energy depletion within the cell. In turn, mitochondrial dysfunction is partly due to the accumulation of oxidative damage to macromolecules, including mitochondrial DNA, RNA and proteins, essential components for optimal functioning of mitochondria. Evidence concerning these series of events leading to energy depletion and apoptosis are discussed.

Caloric restriction in humans: potential pitfalls and health concerns.

To date, the only intervention that has consistently been shown to slow the rate of aging, and to increase mean and maximum lifespan in short-lived species, is life-long calorie restriction. It is yet unclear whether long-term calorie restriction in longer lived species (i.e. primates and humans) will have a similar effect. In humans, several studies investigating short-term calorie restriction or "weight loss" programs suggest beneficial outcomes on parameters of cardiovascular disease. Studies on long-term calorie restriction are performed on a self-selected group of human subjects and show similar effects. However, few studies are currently investigating the quality of life and potential pitfalls of long-term calorie restriction in humans. It is likely that some of the physiological and psychological effects of caloric restriction that occur in animals may impact the human life very differently. For certain, calorie restriction has a plethora of health benefits in mammals, such as a reduction in age-related diseases such as cancer. However, despite the "magic" of CR, this intervention in humans may present itself with a number of health concerns, which may not be applicable to or impact the life of experimental animals, but may do so in humans. These potential pitfalls and "side effects" are not clearly addressed in the literature and will be a focus of this review.

Tumor necrosis factor alpha signaling in skeletal muscle: effects of age and caloric restriction.

Past the age of 50 years, aging individuals lose muscle mass at an approximate rate of 1-2% per year. This age-related muscle atrophy, termed sarcopenia, can have significant effects on individual health and quality of life and can also impact the socioeconomic status. Sarcopenia is due to both a decrease in the number of fibers and the atrophy of the remaining fibers. The mechanisms causing loss of fibers have not been clearly defined, but may likely involve apoptosis. Elevated levels of circulating tumor necrosis factor alpha (TNF-alpha) and adaptations in TNF-alpha signaling in aged skeletal muscle may be contributing factors for the activation of apoptosis. These adaptations may be fiber-type specific, which could explain the selective loss of type II fibers, vs. type I fibers, in the aging process. Caloric restriction, a proven antiaging intervention, is known to attenuate the loss of muscle mass and function with age. Furthermore, caloric restriction has been shown to attenuate the age-associated adaptations in TNF-alpha signaling in skeletal muscle, which may be a possible mechanism by which CR prevents apoptosis and the loss of muscle fibers with age. The potential role of TNF-alpha in the progression of sarcopenia will be discussed, as well as the effects of life-long caloric restriction on TNF-alpha signaling.

Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio.

There have been very few investigations as to whether mitochondrial-mediated apoptosis in vivo is the underlying mechanism of doxorubicin cardiotoxicity. Moreover, no investigations have been conducted to determine whether there are adaptive responses after doxorubicin treatment. We administered a single dose of doxorubicin (20 mg/kg) to male rats and isolated intact mitochondria from their hearts 4 days later. Apoptosis, as determined by the amount of cytosolic mononucleosomal and oligonucleosomal DNA fragments (180 bp or multiples), was significantly increased after doxorubicin treatment. In contrast, Troponin-T, a cardiac-specific marker for necrotic damage, was unaltered 4 days after doxorubicin treatment. Cytosolic cytochrome c increased 2-fold in the doxorubicin-treated rats and was significantly correlated (r = 0.88; P < 0.01) with the increase in caspase-3 activity observed. Moreover, the level of bleomyocin-detectable iron in serum was significantly increased and may have contributed to the increase in oxidative stress, which was indicated by an increase in cytosolic 8-iso prostaglandin F(2alpha). Cytosolic copper zinc superoxide dismutase activity also increased significantly further supporting the notion that doxorubicin increases superoxide radical production. In addition to adaptations to antioxidant defenses, other adaptive mechanisms occurred in the mitochondria such as an increase in the respiratory P/O ratio and an increase in the Bcl-2:Bax ratio. These findings demonstrate that doxorubicin induces oxidative stress and mitochondrial-mediated apoptosis, as well as adaptive responses by the mitochondria to protect cardiac myocytes in vivo.

The role of apoptosis in age-related skeletal muscle atrophy.

Skeletal myocyte atrophy and death contribute to sarcopenia, a condition associated with normal aging. By 80 years of age, it is estimated that humans generally lose 30-40% of skeletal muscle fibres. The mechanism for this loss is unknown; however, it may involve apoptosis. Mitochondrial dysfunction and sarcoplasmic reticulum (SR) stress that occurs with age may be possible stimuli inducing apoptosis. Hence, mitochondria and SR may be important organelles within skeletal myocytes responsible for apoptosis signalling. The activation of apoptosis may be partly responsible for the initiation of muscle protein degradation, loss of muscle nuclei associated with local atrophy, and cell death of the myocyte. Exercise training and caloric restriction are two interventions known to enhance skeletal muscle function. The effects of these interventions on apoptosis are discussed.

Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12.

The mechanisms of apoptosis in the loss of myocytes in skeletal muscle with age and the role of mitochondrial and sarcoplasmic reticulum-mediated pathways of apoptosis are unknown. Moreover, it is unknown whether lifelong calorie restriction prevents apoptosis in skeletal muscle and reverses age-related alterations in apoptosis signaling. We investigated key apoptotic regulatory proteins in the gastrocnemius muscle of 12 and 26 month old ad libitum fed and 26 month old calorie-restricted male Fischer-344 rats. We found that apoptosis increased with age and that calorie-restricted rats showed less apoptosis compared with their age-matched cohorts. Moreover, pro- and cleaved caspase-3 levels increased significantly with age and calorie-restricted rats had significantly lower levels than the aged ad libitum group. Neither age nor calorie restriction had any effect on muscle caspase-3 enzyme activity, but the levels of X-linked inhibitor of apoptosis, particularly an inhibitor of caspase-3, increased with age and were reduced significantly in the 26 month old calorie-restricted cohort. The apoptotic inhibitor apoptosis repressor with a caspase recruitment domain (ARC), which inhibits cytochrome c release, underwent an age-associated decline in the cytosol but increased with calorie restriction. In contrast, mitochondrial ARC levels increased with age and were lower in calorie-restricted rats than in age-matched controls, suggesting a translocation of this protein to attenuate oxidative stress. The translocation of ARC may explain the reduction in cytosolic cytochrome c levels observed with age and calorie restriction. Moreover, we found a striking approximately 350% increase in the expression of procaspase-12 (caspase located at the sarcoplasmic reticulum) with age which was significantly lower in the 26 month old calorie-restricted group. The total protein level of apoptosis-inducing factor in the plantaris muscle increased with age and was reduced calorie-restricted rats compared with age-matched controls, but there were no significant changes in this pro-apoptotic protein in the isolated nuclei. Calorie restriction is able to lower the apoptotic potential in aged skeletal muscle by altering several key apoptotic proteins toward cellular survival, thereby reducing the potential for sarcopenia.

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

Statin-induced apoptosis and skeletal myopathy.

Over 100 million prescriptions were filled for statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) in 2004. Statins were originally developed to lower plasma cholesterol in patients with hypercholesterolemia and are the most effective drugs on the market in doing so. Because of the discovered pleiotropic effects of statins, the use has expanded to the treatment of many other conditions, including ventricular arrythmias, idiopathic dilated cardiomyopathy, cancer, osteoporosis, and diabetes. The elderly population is growing. Therefore, it is estimated that the number of statin users will also increase. Fortunately, the use of statins is relatively safe with few side effects. Myopathy is the most common side effect with symptoms ranging from fatigue, weakness, and pain to symptoms associated with rhabdomyolysis which is a life-threatening condition. The development of statin-induced rhabdomyolysis is rare occurring in approximately 0.1% of patients; however, the occurrence of less severe symptoms is underreported and may be 1-5% or more. Physical exercise appears to increase the likelihood for the development of myopathy in patients taking statins. It is thought that as many as 25% of statin users who exercise may experience muscle fatigue, weakness, aches, and cramping due to statin therapy and potentially dismissed by the patient and physician. The mechanisms causing statin-induced myopathy have not been elucidated; however, research efforts suggest that apoptosis of myofibers may contribute. The mitochondrion is considered a regulatory center of apoptosis, and therefore its role in the induction of apoptosis will be discussed as well as the mechanism of statin-induced apoptosis and myopathy.