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Apoptotic adaptations from exercise training in skeletal and cardiac muscles

Laboratory of Muscle Biology and Sarcopenia, Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia, USA

¹Correspondence: Division of Exercise Physiology, School of Medicine, Robert C. Byrd Health Science Center, West Virginia University, Morgantown WV 26506-9227, USA. E-mail: salway{at}hsc.wvu.edu

SPECIFIC AIMS

Since exercise training has been consistently shown to increase antioxidant defense capacity and elevate the expression of heat shock protein-70 in skeletal and cardiac muscle, the aim of this study was to test the hypothesis that exercise training would decrease the level of apoptosis in cardiac and skeletal muscles.

PRINCIPAL FINDINGS

1. Apoptotic index decreases after exercise training
Rats were randomly assigned to control (CON, n=8) or training (TR, n=8) groups. TR animals were trained by running on a motorized rodent treadmill 5 days weekly for 8 wk. The speed of the treadmill and duration of the training sessions were gradually increased from 10 m·min^–1 for 10 min to a running speed of 28 m·min^–1 for 55 min by the end of the 4th wk. For the next 4 wk, a 5 min warm-up session at a speed of 20 m·min^–1 was followed by a 55 min training session at a speed of 28 m·min^–1. Soleus and ventricular muscle samples were obtained 48 h after the last exercise bout. DNA fragmentation as estimated by a Cell Death ELISA was 15% lower in the ventricles and 33% lower in soleus of TR animals than in CON animals. DNA fragmentation qualitatively analyzed by DNA gel electrophoresis was inconclusive.

2. Bcl-2 and Bax contents
Transcriptional expression of Bcl-2 and Bax genes was analyzed by semiquantitative RT-PCR. Bcl-2 transcript content was 48% and 35% higher in soleus and ventricle samples, respectively, of TR animals than in muscles of CON animals (Fig. 1 A, P<0.01). The transcript content of Bax in soleus muscles of TR animals decreased by 35% vs. CON animals (P<0.01). No difference was found in Bax transcript content in ventricle muscles between CON and TR groups (P>0.05, Fig. 1B ). Western blot analysis showed that Bcl-2 protein content was 64% and 68% higher in soleus and ventricle samples, respectively, of TR compared with CON animals (P<0.05, Fig. 1C ). However, no difference was found in Bax protein contents in soleus or ventricle muscles when CON and TR samples were compared (P>0.05, Fig. 1D ).

View larger version (40K): [in this window] [in a new window]   Figure 1. A) Bcl-2 mRNA content was estimated by RT-PCR. Insert: a representative result of PCR in soleus and ventricle muscles isolated from CON and TR animals. PCR products were visualized with ethidium bromide staining. Quantification of PCR signals was obtained by densitometric analysis of the signal product optical density (OD) x resulting band area. Gene expression is normalized to the ribosomal 18S signal from the same RT product. Normalized data are presented as means ± SE. Data were run in duplicate on different gels for each gene. *P < 0.01, data significantly different from CON animals. B) Bax mRNA content was estimated by RT-PCR. Insert: PCR results from soleus and ventricle muscles isolated from CON/TR animals. PCR products were visualized with ethidium bromide staining. Quantification of PCR signals was by densitometric analysis of the signal product optical density (OD) x resulting band area. Gene expression is normalized to the ribosomal 18S signal from the same RT product. Normalized data are means ± SE. Data were run in duplicate on different gels. *P < 0.01; data significantly different from CON animals. C) Bcl-2 protein content was determined by Western blot analysis. Data are expressed as optical density (OD) x resulting band area expressed in arbitrary units x 107. Insert: representative blot for Bcl-2 in soleus and ventricle muscles isolated from CON and TR animals. Data are means ± SE. *P < 0.05, data significantly different from CON animals. D) Bax protein content was determined by Western blot analysis. Data are expressed as optical density (OD) x resulting band area in arbitrary units x 108. Insert shows a representative blot for Bax in soleus and ventricle muscles isolated from CON and TR animals. Data are presented as means ± SE. CON, control; TR, trained, rats.

3. Apaf-1 and AIF protein content
An immunoreactive band of 67 kDa corresponding to the predicted molecular mass of AIF protein was detected in the cytoplasmic protein fraction of soleus and ventricle muscles. In the soleus of TR animals, Apaf-1 protein content (estimated by Western blot) was 49% lower in the soleus of CON animals than in TR animals (P<0.05). No difference was found in the Apaf-1 protein content in ventricle muscles between TR and CON groups (P>0.05). AIF protein content levels were similar in soleus and ventricle muscles of TR animals vs. that of CON animals (P>0.05).

4. Caspase activity
Caspase-3 activity was measured using a fluorometric assay. Caspase-3 activity in soleus and ventricle muscles was not different in TR and CON groups.

5. HSP70, Mn-SOD, and Cu/Zn-SOD protein contents
Western blot analyses demonstrated that HSP70 protein content was 170% greater in the soleus and 2100% greater in ventricles (P<0.01) of TR vs. CON animals. Relative to total muscle protein content, Mn-SOD protein content increased by 64% (P<0.05) and 39% (P<0.05) in soleus and ventricle muscles, respectively, of the TR group compared with the CON group. However, protein content of Cu/Zn-SOD in soleus and ventricle muscles of TR animals was similar to that of CON animals (P>0.05).

6. Relationship of HSP70 and apoptotic factors
HSP70 protein content was positively correlated with Bcl-2 protein content (r=0.483, P=0.005, n=32). HSP70 protein content was correlated with Bcl-2 protein content in ventricle muscle samples when CON and TR animals were pooled as a group (r=0.589, P=0.016, n=16). A positive relationship was also found between HSP70 protein content and Bcl-2 protein content when soleus and ventricle muscles of CON animals were pooled (r=0.621, P=0.010, n=16). We found that HSP70 protein content was positively correlated with Bcl-2 protein content in ventricle muscle of CON animals (r=0.742, P=0.035, n=8). HSP70 protein content was positively correlated with Bcl-2 transcript content in the soleus muscles of CON animals (r=0.739, P=0.036, n=8). When CON and TR animals were pooled as a single group, HSP70 protein content was negatively correlated with the Bax transcript content in soleus muscles (r=–0.525, P=0.037, n=16).

7. Relationship of Mn-SOD and apoptotic factors
Mn-SOD protein content was negatively correlated with the apoptotic index from ventricle muscle samples of CON animals (r=–0.961, P=0.0001, n=8). When ventricle muscle samples of CON and TR groups were collapsed and treated as a single group, Mn-SOD protein content was negatively correlated with the apoptotic index (r=–0.539, P=0.031, n=16). A negative relationship was found between Mn-SOD protein content and caspase-3 activity when soleus and ventricle muscles of CON animals were pooled (r=–0.535, P=0.033, n=16). Mn-SOD protein content was positively correlated with Bcl-2 transcript content when soleus and ventricle muscles of CON and TR animals were pooled (r=0.381, P=0.032, n=32). When pooled as a group, Mn-SOD protein content was positively correlated with HSP70 protein content (r=0.729, P=0.001, n=16).

DISCUSSION AND SIGNIFICANCE

In this study we show that regular moderate physical activity (i.e., exercise training) influences apoptosis in skeletal and cardiac muscles of young adult rats. Positive relationships exist between the protein level of HSP70 and Bcl-2 protein/mRNA levels. An inverse relationship was found between the protein level of HSP70 and Bax mRNA level in exercise-trained muscles. We also found that Mn-SOD protein content is positively correlated with Bcl-2 transcript content and HSP70 protein content, whereas negative linear correlations exist between Mn-SOD protein content and apoptotic index and caspase-3 activity. Our data are consistent with the hypothesis that HSP70 and Mn-SOD may play a role in modulating the homeostasis of apoptotic factors in skeletal and cardiac muscles.

Single bouts of strenuous exercise have been shown to result in DNA fragmentation and decreased Bcl-2 levels in skeletal muscle. However, to our knowledge this is the first study to investigate the effect of regular moderate exercise on apoptosis in cardiac and skeletal muscle tissues. We demonstrate here that exercise training reduced DNA fragmentation, increased Bcl-2 protein and transcript levels, decreased Bax transcript levels, and decreased Apaf-1 protein levels in rat soleus and cardiac muscles compared with sedentary control animals. Together, the data indicate that regular moderate-intensity exercise is associated with augmented levels of anti-apoptotic genes and reduced levels of proapoptotic genes in postmitotic skeletal and cardiac muscle (Fig. 2 ).

View larger version (12K): [in this window] [in a new window]   Figure 2. Summary of findings leading to the conclusion that anti-apoptotic genes are up-regulated and proapoptotic genes are down-regulated in skeletal and cardiac muscles in response to prolonged endurance exercise.

Reactive oxygen species influences apoptosis mainly through modulation of the mitochondrial-mediated pathway. It has been hypothesized that a high oxidative stress level destabilizes mitochondrial membrane homeostasis and induces the formation of mitochondrial membrane permeability pores and release of proapoptotic factors (e.g., cytochrome c). Bcl-2 family proteins are known to be responsible for the modulation of mitochondrial membrane pore formation, therefore regulating mitochondrial-mediated apoptosis. We have shown that Bcl-2, an anti-apoptotic gene product, was up-regulated and Bax, a proapoptotic gene product, was down-regulated in muscle samples from rats trained for 8 wk. Exercise training of 10 wk has been shown to improve the antioxidant capacity in skeletal and cardiac muscles. A variety of antioxidant enzymes, including Cu/Zn-SOD, catalase, glutathione peroxidase, glutathione reductase, and mitochondrial Mn-SOD, have been suggested to be among the crucial endogenous antioxidant enzymes in biological systems. In the present study, we have demonstrated that the protein content of Mn-SOD increases (by 64% in soleus and 39% in ventricle muscle) in TR animals relative to CON animals after 8 wk endurance training. We also show that Mn-SOD is negatively correlated with apoptotic index and caspase-3 activity, and positively correlated with Bcl-2 and HSP70 transcript activity. Although not causative, these observations suggest that apoptosis attenuation in muscles after exercise training is potentially linked to the muscle’s increased antioxidant capacity and modulated oxidative stress levels resulting from exercise training.

We have demonstrated that apoptosis is reduced and HSP70 is elevated in muscle samples from trained rats and that HSP70 is significantly correlated to Bcl-2 and Bax. Although the molecular mechanism for the training-induced apoptosis reduction is unclear, our data are consistent with the hypothesis that the training-induced decrease in apoptosis is related to the elevation of HSP70.

Apoptosis is not present in high levels in healthy control muscles, but our data suggest that proapoptotic gene products are expressed even in control muscles, which may indicate that muscle cells are poised to rapidly initiate widespread apoptosis given the proper initiation signals. This could be problematic in conditions such as aging, where control muscles are smaller and weaker than their younger counterpart. When animals or humans are subjected even to temporary disuse (e.g., tail suspension in rats, confined bed rest for several weeks in humans), muscles undergo rapid and extensive atrophy and apoptosis, resulting in further loss of muscle mass and further compromised function, especially in the elderly. We speculate that the data in this study support the idea that prior endurance training will down-regulate proapoptotic genes, which may result in reduced levels of cell suicide when muscles are exposed to apoptotic-inducing signals such as disuse.

Together, our data support the hypothesis that exercise training is able to reduce the extent of apoptosis in ventricle and skeletal muscles of young adult animals. Our data show that caspase-sensitive markers of apoptosis decrease with endurance training (Fig. 2) . Additional research is required to determine whether caspase-independent pathways are also sensitive to exercise training in skeletal cardiac muscles.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1291fje; doi: 10.1096/fj.03-1291fje

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