Stephen E. Alway

Apoptotic adaptations from exercise training in skeletal and cardiac muscles

The effect of exercise on apoptosis in postmitotic tissues is not known. In this study, we investigated the effect of regular moderate physical activity (i.e., exercise training) on the extent of apoptosis in rat skeletal and cardiac muscles. Adult Sprague Dawley rats were trained (TR) 5 days weekly for 8 wk on treadmill. Sedentary rats served as controls (CON). An ELISA was used to detect mono‐ and oligonucleosome fragmentation as an indicator of apoptosis. Bcl‐2, Bax, Apaf‐1, AIF, cleaved PARP, cleaved caspase‐3, cleaved/active caspase‐9, heat shock protein (HSP)70, Cu/Zn‐SOD, and Mn‐SOD protein levels were determined by Western analyses. Bcl‐2 and Bax transcript contents were estimated by RT‐PCR. A spectrofluorometric assay was used to determine caspase‐3 activity. DNA fragmentation in ventricles of the TR group decreased by 15% whereas that in soleus of the TR group tended to decrease (P=0.058) when compared with CON group. Protein contents of Bcl‐2, HSP70, and Mn‐SOD increased in both soleus and ventricle muscles of TR animals when compared with CON animals. Apaf‐1 protein content in the soleus of TR animals was lower than that of CON animals. Bcl‐2 mRNA levels increased in both ventricle and soleus muscles of TR animals, and Bax mRNA levels decreased in the soleus of TR animals when compared with CON animals. Furthermore, HSP70 protein content was negatively correlated to Bax mRNA content and was positively correlated to Bcl‐2 protein and mRNA contents. Mn‐SOD protein content was negatively correlated to the apoptotic index, and caspase‐3 activity and was positively correlated to Bcl‐2 transcript content and HSP70 protein content. These data suggest that exercise training attenuates the extent of apoptosis in cardiac and skeletal muscles.

Mitochondria‐associated apoptotic signalling in denervated rat skeletal muscle

Apoptosis has been implicated in the regulation of denervation‐induced muscle atrophy. However, the activation of apoptotic signal transduction during muscle denervation has not been fully elucidated. The present study examined the apoptotic responses to denervation in rat gastrocnemius muscle. Following 14 days of denervation, the extent of apoptotic DNA fragmentation as determined by a cytosolic nucleosome ELISA was increased by 100% in the gastrocnemius muscle. RT‐PCR and immunoblot analyses indicated that Bax was dramatically upregulated while Bcl‐2 was modestly increased; however, the Bax/Bcl‐2 ratio was significantly increased in denervated muscles relative to control muscles. Analyses of ELISA and immunoblots from mitochondria‐free cytosol extracts showed a significant increase in mitochondria‐associated apoptotic factors, including cytochrome c, Smac/DIABLO and apoptosis‐inducing factor (AIF). In addition to the upregulation of caspase‐3 and ‐9 mRNA, pro‐/cleaved caspase protein and proteolytic activity levels, the X‐linked inhibitor of apoptosis (XIAP) protein level was downregulated. The cleaved product of poly(ADP‐ribose) polymerase (PARP) was detected in muscle samples following denervation. Although we did not find a difference in the inhibitor of DNA binding/differentiation‐2 (Id2) and c‐Myc protein contents between the denervated and control muscles, the protein content of tumour suppressor p53 was significantly increased in both the nuclear and the cytosolic fractions with denervation. Moreover, denervation increased the protein content of HSP70, whereas the MnSOD (a mitochondrial isoform of superoxide dismutase) protein content was diminished, which indicated that denervation might have induced cellular and/or oxidative stress. Our data show that mitochondria‐associated apoptotic signalling is upregulated during muscle denervation. We interpret these findings to indicate that apoptosis has a physiologically important role in regulating denervation‐induced muscle atrophy.

Skeletal muscle function and hypertrophy are diminished in old age

Muscle loss occurs during aging. To investigate whether the hypertrophic response is attenuated at old age, we used male Fischer 344 (26 months old; n = 5) and Fischer 344 x Brown Norway rats (6, 9, and 33 months old; n = 8, 10, and 6, respectively). Hypertrophy of the left plantaris muscle was induced by surgical denervation of its agonists. The right leg served as control. The mass and maximal tetanic force (P0) of control muscles declined by ∼30% between 9 and 26 months (P < 0.05). Fatigue resistance during intermittent isometric contractions was reduced by ∼60% at 33 months. At 33 months, the attenuated hypertrophy was accompanied by a decrease rather than an increase in P0. Yet, hypertrophy was accompanied by a 25% rise in fatigue resistance at all ages (P = 0.001). Thus, aging is associated with a decline in muscle mass and function. In addition, at advanced age, the hypertrophic response is reduced and a hypertrophic stimulus even aggravates the age‐associated muscle weakening. Muscle Nerve 27: 339–347, 2003

Vitamin E and C supplementation reduces oxidative stress, improves antioxidant enzymes and positive muscle work in chronically loaded muscles of aged rats

Aging is associated with increased oxidative stress. Muscle levels of oxidative stress are further elevated with exercise. The purpose of this study was to determine if dietary antioxidant supplementation would improve muscle function and cellular markers of oxidative stress in response to chronic repetitive loading in aging. The dorsiflexors of the left limb of aged and young adult Fischer 344 Brown×Norway rats were loaded 3 times weekly for 4.5 weeks using 80 maximal stretch-shortening contractions per session. The contra-lateral limb served as the intra-animal control. The rats were randomly assigned to a diet supplemented with Vitamin E and Vitamin C or normal non-supplemented rat chow. Biomarkers of oxidative stress were measured in the tibialis anterior muscle. Repetitive loading exercise increased maximal isometric force, negative work and positive work in the dorsiflexors of young adult rats. Only positive work increased in the aged animals that were supplemented with Vitamin E and C. Markers of oxidative stress (H(2)O(2), total GSH, GSH/GSSG ratio, malondialdehyde and 8-OHdG) increased in the tibialis anterior muscles from aged and young adult animals with repetitive loading, but Vitamin E and C supplements attenuated this increase. MnSOD activity increased with supplementation in the young adult animals. CuZnSOD and catalase activity increased with supplementation in young adult and aged animals and GPx activity increased with exercise in the non-supplemented young adult and aged animals. The increased levels of endogenous antioxidant enzymes after Vitamin E and C supplementation appear to be regulated by post-transcriptional modifications that are affected differently by age, exercise, and supplementation. These data suggest that antioxidant supplementation improves indices of oxidative stress associated with repetitive loading exercise and aging and improves the positive work output of muscles in aged rodents.

Nuclear Apoptosis Contributes to Sarcopenia

Apoptosis results in DNA fragmentation and, subsequently, destruction of cells containing a single nucleus. Our hypothesis is that multinucleated cells such as muscle fibers can experience apoptotic-induced loss of single nuclei (nuclear apoptosis) without destruction of the entire fiber. The loss of nuclei likely contributes to atrophy and sarcopenia. Furthermore, increased chronic activity attenuates apoptotic signaling, which may reduce sarcopenia.

β-Hydroxy-β-methylbutyrate reduces myonuclear apoptosis during recovery from hind limb suspension-induced muscle fiber atrophy in aged rats

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite shown to reduce protein catabolism in disease states and promote skeletal muscle hypertrophy in response to loading exercise. In this study, we evaluated the efficacy of HMB to reduce muscle wasting and promote muscle recovery following disuse in aged animals. Fisher 344×Brown Norway rats, 34 mo of age, were randomly assigned to receive either Ca-HMB (340 mg/kg body wt) or the water vehicle by gavage (n = 32/group). The animals received either 14 days of hindlimb suspension (HS, n = 8/diet group) or 14 days of unloading followed by 14 days of reloading (R; n = 8/diet group). Nonsuspended control animals were compared with suspended animals after 14 days of HS (n = 8) or after R (n = 8). HMB treatment prevented the decline in maximal in vivo isometric force output after 2 wk of recovery from hindlimb unloading. The HMB-treated animals had significantly greater plantaris and soleus fiber cross-sectional area compared with the vehicle-treated animals. HMB decreased the amount of TUNEL-positive nuclei in reloaded plantaris muscles (5.1% vs. 1.6%, P < 0.05) and soleus muscles (3.9% vs. 1.8%, P < 0.05). Although HMB did not significantly alter Bcl-2 protein abundance compared with vehicle treatment, HMB decreased Bax protein abundance following R, by 40% and 14% (P < 0.05) in plantaris and soleus muscles, respectively. Cleaved caspase-3 was reduced by 12% and 9% (P < 0.05) in HMB-treated reloaded plantaris and soleus muscles, compared with vehicle-treated animals. HMB reduced cleaved caspase-9 by 14% and 30% (P < 0.05) in reloaded plantaris and soleus muscles, respectively, compared with vehicle-treated animals. Although, HMB was unable to prevent unloading-induced atrophy, it attenuated the decrease in fiber area in fast and slow muscles after HS and R. HMBs ability to protect against muscle loss may be due in part to putative inhibition of myonuclear apoptosis via regulation of mitochondrial-associated caspase signaling.

Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of subcellular spatial location

Cardiovascular complications, such as diabetic cardiomyopathy, account for the majority of deaths associated with diabetes mellitus. Mitochondria are particularly susceptible to the damaging effects of diabetes mellitus and have been implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac mitochondria consist of two spatially distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The goal of this study was to determine whether subcellular spatial location is associated with apoptotic propensity of cardiac mitochondrial subpopulations during diabetic insult. Swiss Webster mice were subjected to intraperitoneal injection of streptozotocin or citrate saline vehicle. Ten weeks following injection, diabetic hearts displayed increased caspase-3 and caspase-9 activities, indicating enhanced apoptotic signaling (P < 0.05, for both). Mitochondrial size (forward scatter) and internal complexity (side scatter) were decreased in diabetic IFM (P < 0.05, for both) but not in diabetic SSM. Mitochondrial membrane potential (Delta(Psim)) was lower in diabetic IFM (P < 0.01) but not in diabetic SSM. Mitochondrial permeability transition pore (mPTP) opening was increased in diabetic compared with control IFM (P < 0.05), whereas no differences were observed in diabetic compared with control SSM. Examination of mPTP constituents revealed increases in cyclophilin D in diabetic IFM. Furthermore, diabetic IFM possessed lower cytochrome c and BcL-2 levels and increased Bax levels (P < 0.05, for all 3). No significant changes in these proteins were observed in diabetic SSM compared with control. These results indicate that diabetes mellitus is associated with an enhanced apoptotic propensity in IFM, suggesting a differential apoptotic susceptibility of distinct mitochondrial subpopulations based upon subcellular location.

Suppression of Oxidative Stress by Resveratrol After Isometric Contractions in Gastrocnemius Muscles of Aged Mice

This study tested the hypothesis that resveratrol supplementation would lower oxidative stress in exercised muscles of aged mice. Young (3 months) and aged (27 months) C57BL/6 mice received a control or a 0.05% trans-resveratrol-supplemented diet for 10 days. After 7 days of dietary intervention, 20 maximal electrically evoked isometric contractions were obtained from the plantar flexors of one limb in anesthetized mice. Exercise was conducted for three consecutive days. Resveratrol supplementation blunted the exercise-induced increase in xanthine oxidase activity in muscles from young (25%) and aged (53%) mice. Resveratrol lowered H(2)O(2) levels in control (13%) and exercised (38%) muscles from aged animals, reduced Nox4 protein in both control and exercised muscles of young (30%) and aged mice (40%), and increased the ratio of reduced glutathione to oxidized glutathione in exercised muscles from young (38%) and aged (135%) mice. Resveratrol prevented the increase in lipid oxidation, increased catalase activity, and increased MnSOD activity in exercised muscles from aged mice. These data show that dietary resveratrol suppresses muscle indicators of oxidative stress in response to isometric contractions in aged mice.

Resistance training increases heat shock protein levels in skeletal muscle of young and old rats

Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72-1, Hsp72-2, Hsp72-3) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (3 months) (n=10) and old (30 months) (n=9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p<0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p<0.01) by training in muscles of young rats (943.1%) and old rats (420.3%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. SOD-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was 33% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p<0.05) training by age interaction was found for SOD-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress. Furthermore, the data are consistent with the hypothesis that higher levels of oxidative stress in muscles of old animals limit HSP levels and/or function in response to high intensity contractile stress.

Age-dependent increase in oxidative stress in gastrocnemius muscle with unloading.

Oxidative stress increases during unloading in muscle from young adult rats. The present study examined the markers of oxidative stress and antioxidant enzyme gene and protein expressions in medial gastrocnemius muscles of aged and young adult (30 and 6 mo of age) Fischer 344 x Brown Norway rats after 14 days of hindlimb suspension. Medial gastrocnemius muscle weight was decreased by approximately 30% in young adult and aged rats following suspension. When muscle weight was normalized to animal body weight, it was reduced by 12% and 22% in young adult and aged rats, respectively, after suspension. Comparisons between young adult and aged control animals demonstrated a 25% and 51% decline in muscle mass when expressed as absolute muscle weight and muscle weight normalized to the animal body weight, respectively. H(2)O(2) content was elevated by 43% while Mn superoxide dismutase (MnSOD) protein content was reduced by 28% in suspended muscles compared with control muscles exclusively in the aged animals. Suspended muscles had greater content of malondialdehyde (MDA)/4-hydroxyalkenals (4-HAE) (29% and 58% increase in young adult and aged rats, respectively), nitrotyrosine (76% and 65% increase in young adult and aged rats, respectively), and catalase activity (69% and 43% increase in young adult and aged rats, respectively) relative to control muscles. Changes in oxidative stress markers MDA/4-HAE, H(2)O(2), and MnSOD protein contents in response to hindlimb unloading occurred in an age-dependent manner. These findings are consistent with the hypotheses that oxidative stress has a role in mediating disuse-induced and sarcopenia-associated muscle losses. Our data suggest that aging may predispose skeletal muscle to increased levels of oxidative stress both at rest and during unloading.