Mitochondrial apoptosis and apoptotic signaling modulations by aerobic training were studied in cardiac and skeletal muscles of obese Zucker rats (OZR), a rodent model of metabolic syndrome. Comparisons were made between left ventricle, soleus, and gastrocnemius muscles from OZR (n = 16) and aged-matched lean Zucker rats (LZR; n = 16) that were untrained (n = 8) or aerobically trained on a treadmill for 9 wk (n = 8). Cardiac Bcl-2 protein expression levels were ∼50% lower in the OZR compared with the LZR, with no difference in either of the skeletal muscles. Bax protein expression levels were similar in skeletal muscles of the OZR compared with the LZR. Furthermore, mitochondrial apoptotic signaling was not different in skeletal muscles of OZR and LZR groups. However, there was an approximate sevenfold increase in the Bax protein accumulation in the myocardial mitochondrial-rich protein fraction of the OZR compared with the LZR. Additionally, there was an increase in cytosolic cytochrome c released from the mitochondria, caspase-9 and caspase-3 activity, with a corresponding elevation in DNA fragmentation in the cardiac muscles of the OZR compared with the LZR. Exercise training reduced cardiac Bax protein levels, the mitochondrial localization of Bax, cytosolic cytochrome c, caspase activity, and DNA fragmentation in cardiac muscles of the OZR after exercise, with no change in the skeletal muscles. These data show that mitochondrial apoptosis is elevated in the cardiac but not skeletal muscles of the OZR, but aerobic exercise training was effective in reducing cardiac mitochondrial apoptotic signaling.
- insulin resistance
- metabolic syndrome signaling
- muscle wasting
apoptosis is a highly organized and tightly coordinated biological process that plays a vital role in monitoring a variety of nonpathological cellular events (e.g., tissue turnover). Aberrant regulation of apoptosis has been implicated in conditions leading to muscle atrophy, including Duchenne and Facioscapulohumeral muscular dystrophies (36), aging (14), muscle wasting (39, 40), and sarcopenia (3). The molecular events resulting in the activation and subsequent execution of the apoptotic program is principally controlled by the balance between pro- and anti-apoptotic signaling and this is primarily determined by specific apoptotic regulatory proteins.
The B-cell leukemia/lymphoma-2 (Bcl-2) family of upstream regulators of apoptosis includes crucial intracellular checkpoint proteins in the apoptotic signaling pathway (12, 30). Bcl-2 family members, Bcl-2 associated X (Bax) protein and Bcl-2, have been identified as putative key proteins involved in the formation of mitochondrial apoptotic channels and also in the regulation of mitochondria permeability and mitochondrial-associated apoptotic signaling (13). Bax has been shown to translocate to the mitochondria and expose its NH2 terminus via a conformational change on induction of apoptosis (6, 30). This conformational change permits the Bax-Bax-oligomerization and insertion of Bax into the outer mitochondrial membrane (4, 45), which is followed rapidly by the release of the apoptogenic factors (e.g., cytochrome c) from the mitochondrial intermembrane space (9, 10). Collectively, Bax oligomerization is thought to be critical for mitochondrial membrane permeabilization, whereas Bcl-2 opposes the pro-apoptotic activity of Bax by preventing Bax-Bax oligomerization (4, 30, 45). Although the precise mechanism of Bax-mediated apoptogenic factor release from the mitochondrial intermembrane space is still under active investigation, it is indisputable that Bax plays an important role in promoting the activation of apoptotic signaling cascades.
The obese Zucker (fa/fa) rat (OZR) is used as an animal model for metabolic syndrome because it exhibits severe skeletal muscle insulin resistance, hyperglycemia, and hyperlipidemia (47). Recently, mitochondrial-associated apoptosis has been demonstrated to be elevated in the myocardium of the OZR model of metabolic syndrome (21, 26). While the skeletal muscles of the OZR are smaller than the lean Zucker rats (LZR), it is not known if mitochondrial apoptotic signaling might be an important regulator of muscle mass in this model. Although metabolic syndrome has not been linked to skeletal muscle apoptosis, it has been linked to skeletal muscle atrophy (5), and metabolic syndrome also has been associated with increased apoptosis in cardiac tissue (8, 21, 26). Mitochondrial-associated apoptotic signaling, including an elevation in the Bax/Bcl-2 ratio, has been implicated in muscle wasting and aging-associated losses of skeletal muscle (2). Although, skeletal muscle apoptosis has not been studied in conjunction with metabolic syndrome, there is a strong correlation between skeletal muscle atrophy and metabolic syndrome (5, 33). It is possible that an increased Bax/Bcl-2 ratio in metabolic syndrome contributes to increased susceptibility to apoptosis in cardiac muscles and has a role in regulating apoptotic-induced atrophy in skeletal muscles.
The first purpose of this study was to confirm if obesity increased mitochondrial apoptotic signaling of cardiac muscles in the OZR model of metabolic resistance compared with the LZR. The second purpose was to determine if mitochondrial apoptotic signaling is elevated in the skeletal muscles of the OZR compared with the lean phenotype. Furthermore, our lab previously demonstrated that aerobic exercise training can reduce apoptotic signaling in both skeletal and cardiac muscles of nonobese rodents (38). However, the effects of exercise on mitochondrial apoptotic signaling, in a model of metabolic syndrome, have not been examined in either cardiac or skeletal muscles. Therefore, the final purpose of this study was to determine if aerobic exercise training would reduce mitochondrial apoptotic signaling in the cardiac and/or skeletal muscles of the OZR model of metabolic syndrome.
MATERIALS AND METHODS
OZR (n = 16) and LZR (n = 16; Harlan, Indianapolis, IN) were housed in pathogen-free conditions, at 20–22°C with a reversed 12:12-h light/dark cycle, and fed rat chow and water ad libitum throughout the study period. The Institutional Animal Use and Care Committee from West Virginia University School of Medicine approved all experiments. The animal care standards were followed by adhering to the recommendations for the care of laboratory animals as advocated by the American Association for Accreditation of Laboratory Animal Care and fully conformed to the Animal Welfare Act of the U.S. Department of Health and Human Services.
At 16 wk of age, after an overnight fast, rats were anesthetized with injections of pentobarbital sodium (50 mg/kg ip) and received tracheal intubation to facilitate maintenance of a patent airway. In all rats, a carotid artery and an external jugular vein were cannulated for determination of arterial pressure.
Both the OZR and LZR were randomly assigned into equal groups of sedentary (OZR, n = 8; LZR, n = 8) or exercise trained (OZR-ET, n = 8; LZR-ET, n = 8). The exercise-trained animals ran on a motorized rodent treadmill (Columbus Instruments, Columbus, OH) 5 days weekly for 9 wk at 0% grade. During the first 3 wk of training, the animals began running for 15 min at a speed of 10 m/min. This was gradually increased to 55 min/day at 20 m/min for the OZR-ET and 24 m/min for the LZR-ET. This pace was maintained for the remainder of the study. Because the OZR animals are heavier, the same running pace would result in more total work than the lighter LZR. The higher training intensity for the LZR was used to approximate the training intensity of the LZR-ET to the much heavier OZR-ET (∼40% increase in body wt). We previously showed that this training protocol stimulates a moderate endurance training effect with a similar ∼25% increase in citrate synthase activity in both the OZR-ET and LZR-ET (18).
Muscles from control and exercise-trained animals were harvested after anesthetizing the animals with 2% isoflurane. The gastrocnemius and soleus muscles from each limb were dissected from the surrounding connective tissues, and 4–5 mm of the left ventricle were quickly removed and frozen immediately in liquid nitrogen and stored at −80°C until further analysis.
Total protein homogenates were prepared in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% SDS) with the addition of Protease inhibitor cocktail (PIC; Sigma). Separate mitochondria-enriched and mitochondria-free protein fractions were also prepared according to methods previously performed in our lab (39). Briefly, muscles were homogenized in mitochondrial isolation buffer (in mM: 20 HEPES pH 7.5, 10 KCl, 1.5 MgCl2, 1 EGTA, 1 EDTA, 1 dithiothreitol, 250 sucrose) with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The homogenates were centrifuged at 800 g to remove nuclei and cell debris followed by centrifugation at 16,000 g for 20 min at 4°C to pellet the mitochondria. The supernatants were used as mitochondria-free cytosolic fraction. The mitochondrial-rich pellet was washed twice then resuspended in total protein lysis buffer for analysis of Bax associated with the mitochondrial membrane.
Protein contents were quantified in duplicate by using bicinchoninic acid reagents (Pierce, Rockford, IL) and bovine serum albumin (BSA) standards. Soluble protein (50 μg) was boiled for 4 min at 100°C in Laemmli buffer and separated on a 4–12% gradient polyacrylamide gel (Invitrogen, Carlsbad, CA). The gels were blotted to nitrocellulose membranes (Bio-Rad, Hercules, CA) and stained with Ponceau red (Sigma Chemical, St Louis, MO) to confirm equal loading and transferring of proteins to the membrane in each lane.
The membranes were blocked in 5% nonfat milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T) and probed with anti-Bcl-2 or Bax antibodies (Santa Cruz, CA). Additionally, as a second means of confirming equal loading, the membranes were also probed for β-tubulin (Abcam). Secondary antibodies were conjugated to horseradish peroxidase (Chemicon) and the signals were developed by chemiluminescence (ECL advance, Amersham Biosciences). The signals were visualized by exposing the membranes to X-ray films (BioMax MS-1, Eastman Kodak, Rochester, NY), and digital records of the films were captured with a Kodak 290 camera. The resulting bands were quantified as optical density (OD) x band area by a one-dimensional image analysis system (Eastman Kodak) and expressed in arbitrary units normalized to β-tubulin.
Cytosolic cytochrome c assay.
The concentration of cytosolic cytochrome c in the essentially mitochondrial-free cytosolic protein fraction was measured with an ELISA kit (Medical and Biological Laboratories). The change in color was monitored at a wavelength of 450 nm using a Dynex MRX plate reader. Measurements were performed in duplicate with all comparisons performed with the same assay. The cytosolic cytochrome c content was expressed as OD450 per milligram of protein and reported as relative to LZR control values.
Fluorometric caspase activity assay.
To determine whether changes in Bax or Bcl-2 resulted in apoptotic events that were downstream of the mitochondria, the activity of caspase-3 and caspase-9 were analyzed by a commercial caspase assay kit (APO-54A-019-KI01, Apotech, Switzerland) according to the manufacturer's procedure and as previously performed in our laboratory (38). Briefly, 50 mg of muscle samples were gently homogenized with a Teflon pestle on ice in 1 ml of nuclear isolation lysis buffer (10 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, pH 7.4, 20% glycerol, 0.1% Triton X-100, and 1 mM dithiothreitol). The homogenates were centrifuged at 1,200 g for 1 min at 4°C. The supernatants contained the cytoplasmic protein fraction and were collected. The cytoplasmic protein fraction was then used for the caspase activity assays and also for the cell death ELISA.
Samples were mixed in equal parts assay buffer (50 mM PIPES, 0.1 mM EDTA, 10% glycerol, 10 mM DTT, pH 7.2) and then 100 μM of the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-conjugated substrate (Ac-DEVD-AFC, Alexis, San Diego, CA) was added at 37°C for 2 h. The change in fluorescence was measured on a spectrofluorometer with an excitation wavelength of 390/20 nm and an emission wavelength of 530/25 nm (CytoFluor; Applied Biosystems, Foster City, CA) before and after the 2-h incubation. Caspase activity was estimated as the change in arbitrary fluorescence units normalized to milligrams of protein used in the assay and data were reported relative to LZR. All measurements were performed in triplicate.
Cell death ELISA.
A cell death detection ELISA kit (Roche Applied Science, Indianapolis, IN) was used to quantitatively determine the apoptotic DNA fragmentation by measuring the cytosolic histone-associated mono- and oligonucleosomes. The change in color was measured at a wavelength of 450 nm by using a Dynex MRX plate reader controlled through PC software (Revelation; Dynatech Laboratories). Measurements were performed in duplicate, with samples from OZR and LZR analyzed at the same time. The OD450 reading was then normalized to the milligrams of protein used in the assay.
In situ TdT-mediated dUTP nick end labeling staining.
The nuclei with DNA strand breaks were assessed using a fluorometric TdT-mediated dUTP nick end labeling (TUNEL) detection kit according to the manufacturer's instructions (1684795; Roche Applied Science). Frozen cross sections (10-μm thick) were cut in a freezing cryostat at −20°C. Tissue sections were air dried at room temperature, fixed in 4% paraformaldehyde in PBS, pH 7.4, at room temperature for 20 min, permeabilized with 0.2% Triton X-100 in 0.1% sodium citrate at 4°C for 2 min, and incubated with fluorescein-conjugated TUNEL reaction mixture in a humidified chamber at 37°C for 1 h in the dark. Negative control experiments were done by omitting the TdT enzyme in the TUNEL reaction mixture on the tissue sections. After TUNEL labeling, the muscle sections were incubated with an anti-myosin heavy chain monoclonal antibody (MF20; Hybridoma Bank) followed by an anti-mouse Cy3 conjugated antibody (C2181, Sigma Aldrich) and mounted with DAPI (Vectashield mounting medium). TUNEL-positive and DAPI-positive nuclei were examined under a fluorescence microscope (Nikon SE 800; Nikon Instruments, Melville, NY). The images were captured and stacked using a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI), and SPOT RT software (Universal Imaging, Downingtown, PA), respectively. The number of TUNEL-positive and DAPI-positive nuclei was counted from six random, nonoverlapping fields at an objective magnification of ×40. Data were expressed as the frequency of TUNEL-positive nuclei per 100 nuclei.
Statistical analyses were performed using the SYSTAT 11.0 software package. A one-way ANOVA was performed on the difference in all measured variables. Statistical significance was accepted at P < 0.05. All data are given as means ± SE.
The animals were 16 wk of age at the end of the study. Fasting blood glucose, insulin, body weight, citrate synthase activity, and mean arterial blood pressure have been reported previously (18). Prior to 12–13 wk of age, these animals were normotensive (∼100 mmHg). At the end of the study, the obese Zucker rats had developed a moderate elevated blood pressure in both control (131 ± 6 mmHg) and exercise-trained animals (125 ± 6 mmHg). The lean Zucker rats remained normotensive in both sedentary (102 ± 5 mmHg) and exercise-trained groups (101 ± 6 mmHg). These data indicate that the OZR used in our study were obese, hyperinsulinemic, hyperglycemic, and hyperlipidemic. These data are consistent with previous data in this animal model as reported in our group by Frisbee and colleagues (18) and other laboratories (5, 47).
Nine weeks of treadmill exercise reduced body mass by ∼23%, reduced fasting insulin by ∼38%, and reduced plasma triglycerides by ∼34% in OZR-ET vs. control OZR (18). Additionally, the 9 wk of treadmill exercise used in this study increased citrate synthase activity ∼40% in both phenotypes (18). The mass of the heart, gastrocnemius, and soleus muscles are reported in Table 1.
Bax and Bcl-2 protein expression.
The ability for anti-apoptotic Bcl-2 to inhibit the activity of pro-apoptotic Bax is dependent not only on the expression levels of these proteins, but also on the ratio of Bax to Bcl-2 (1, 24). When Bax is in excess, Bcl-2 cannot bind and sequester all of the Bax protein, thus allowing the free Bax protein to oligomerize and act on the mitochondrial membrane. Nonetheless, there is considerable evidence that oligomeric Bax/Bax, Bax/Bak, and/or Bak/Bak are putative components of the mitochondrial apoptosis-induced channel that is located in the outer membrane (13, 23). This mitochondrial channel directly provides a pathway for the release of cytochrome c from the intermembrane space to the cytosolic space. Thus we would anticipate that greater levels of Bax would potentially facilitate formation of additional permeability channels thereby increasing the susceptibility to mitochondrial permeability.
Skeletal and cardiac Bax protein expression levels were similar in control muscles of OZR and LZR animals, respectively. However, aerobic exercise reduced Bax levels by 35% in the cardiac muscles of the OZR (Fig. 1A). On the other hand, no differences were observed in Bax protein content between OZR and LZR for either gastrocnemius or soleus muscles.
Bcl-2 protein expression in the myocardium of the OZR was about one-half of that found in the LZR. However, Bcl-2 protein expression was increased ∼25% in the OZR-ET compared with the OZR controls (Fig. 1B). On the other hand, no differences were observed in Bcl-2 content in either the gastrocnemius or soleus muscles among the four groups.
The myocardial Bax/Bcl-2 ratio was significantly higher in the OZR compared with LZR (Fig. 1C). Aerobic exercise had a strong trend toward a decrease in the Bax/Bcl2 ratio OZR-ET vs. control OZR (P = 0.072). No differences were observed in the Bax/Bcl-2 ratio in either the soleus or the gastrocnemius muscles among the groups.
Mitochondrial Bax accumulation.
The mitochondrial-rich membrane fraction was isolated to determine if the decrease in Bcl-2 protein expression resulted in a greater accumulation of Bax in the mitochondria from cardiac or skeletal muscles of the OZR compared with the LZR. There was a large accumulation of Bax in the myocardial mitochondrial protein fraction of OZR compared with LZR (Fig. 2A). The amount of Bax associated with the mitochondrial protein fraction was reduced in the OZR-ET, although there was still a significant accumulation compared with the LZR. On the other hand there was no observable accumulation of Bax in the isolated mitochondrial protein fraction from either the soleus or gastrocnemius muscles regardless of phenotype (data not shown). We cannot fully rule out the possibility that there was not cross-contamination of Bax in mitochondrial-rich and essentially mitochondrial-free protein fractions. However, this possibility seems low, because the quality of the mitochondrial-rich and essentially mitochondrial-free fractions was high, as confirmed by Western blots against manganese superoxide dismutase (MnSOD) and copper zinc superoxide dismutase (CuZnSOD). The data (Fig. 2B) show that the mitochondrial-rich fraction had high levels of mitochondrial specific MnSOD, but only low levels of CuZnSOD, which resides primarily in the cytosolic protein fraction, although it is also present in small quantities in the outer mitochondrial membrane. Furthermore, as expected, the essentially mitochondrial-free fraction had low levels of mitochondrial specific MnSOD.
Cytosolic cytochrome c ELISA.
Increased permeability of mitochondrial membranes and opening of the mitochondrial permeability pore and mitochondrial apoptotic channels results in cytosolic cytochrome c release from the mitochondria to the cytosol (23). Increased cytosolic cytochrome c in part regulates caspase-dependent apoptosis (10). The essentially mitochondrial-free cytosolic protein fractions of cardiac muscles from the OZR had a significant, ∼30%, increase in cytosolic cytochrome c compared with the LZR (Fig. 3). Aerobic training abolished this increase in cytosolic cytochrome c accumulation in the cytosol in cardiac muscles samples of the OZR-ET when compared with the LZR. No differences were noted in the amount of cytosolic cytochrome c in the mitochondria-free cytosolic protein fraction between the OZR and LZR in either the gastrocnemius muscles or soleus muscles.
Caspase activity and DNA fragmentation.
Caspase-3 and caspase-9 activities were examined to confirm the functional importance of the upstream increases in the Bax/Bcl-2 ratio in the cardiac muscle samples. When the mitochondrial membrane is permeabilized and cytochrome c is released, cytochrome c in turn activates caspase-9. When cleaved, caspase-9 activates and cleaves caspase-3, an effector caspase, which causes DNA fragmentation and apoptosis. In this study, both caspase-3 and caspase-9 were elevated the cardiac muscle samples of the OZR compared with LZR (Fig. 4) with a corresponding increase in DNA fragmentation (Fig. 5). Exercise training significantly reduced caspase activity and DNA fragmentation in OZR-ET compared with the control OZR. There were no changes in caspase activity or DNA fragmentation in either the gastrocnemius or soleus muscles among the four groups. Additionally, TUNEL staining of cardiac cross sections was performed. Similar to findings of others (25), we did not observe a significant number of TUNEL-positive nuclei in our control or exercise-trained LZR (<0.1% of observed nuclei). There was a measurable amount of TUNEL-positive nuclei in the control OZR but exercise training significantly reduced the presence of TUNEL-positive nuclei (Fig. 5).
The important finding of this study is that mitochondrial apoptotic signaling is elevated in the cardiac but not skeletal muscles of the OZR, a rodent model of metabolic syndrome. During the preparation of this manuscript, Lu and colleagues (26) reported a decrease in the Bcl-2 protein expression and an increase in mitochondrial apoptosis in cardiac muscles of OZR, supporting many of the current findings, although Bax was not investigated in their study. However, our current study adds to the findings of Lu et al. (26) by examining the role of Bax in mitochondria-associated apoptotic signaling and the effects of exercise on mitochondrial apoptosis in metabolic syndrome. Furthermore, our study provides novel data pertaining to the localization of the pro-apoptotic signaling protein Bax and the relationship of Bax to Bcl-2 in muscles of the OZR. We observed an accumulation of Bax protein expression in the mitochondrial-rich protein fraction in the cardiac muscles of the OZR. This is consistent with a greater translocation of Bax to the mitochondria, which is part of the activation events in mitochondrial-associated apoptotic signaling. Furthermore, exercise training reduced the Bax protein expression in the myocardium, indicating that this may be a primary factor in the reduction of mitochondrial apoptotic signaling in the heart observed after exercise.
Moreover, the current study provided the novel and surprising finding that although elevated mitochondrial apoptotic signaling occurred in cardiac muscles, it was absent in skeletal muscles of the OZR. This was unexpected because the skeletal muscles of the OZR are significantly smaller than the aged-matched lean control, and we hypothesized that myonuclear apoptosis may contribute to this decrease in muscle mass. This apparent paradox between muscle mass and apoptosis in skeletal muscle could be explained if basal levels of nuclear apoptosis were at a slightly elevated rate in the skeletal muscles of OZR animals compared with LZR animals, but this level was below the detection limits of our assays. If this were the case, we would anticipate that muscle mass in OZR would not be significantly reduced compared with LZR, unless the muscles were examined over an extended period of time of weeks or months. This may not be the case in cardiac muscle, where hypertrophy and apoptosis can occur simultaneously especially during heart failure (29, 46).
It is estimated that the prevalence of metabolic syndrome exceeds 20% of the population in the Unites States (16). Similar to subjects with metabolic syndrome, the OZR suffers from hyperglycemia, dyslipidemia, and hyperinsulinemia (18, 47). Diabetic conditions, including hyperglycemia, dyslipidemia, and hyperinsulinemia, have been demonstrated to cause mitochondrial membrane hyperpolarization and the formation of reactive oxygen species and apoptosis in cardiac (8, 44) and smooth muscles (43). Although apoptosis has not been previously examined in diabetic skeletal muscle, we hypothesized that apoptotic signaling would be similar in skeletal muscle and cardiac muscle and that apoptosis would explain in part, the reduced muscle mass that we have found in OZR compared with LZR (Table 1). Since apoptosis is elevated in response to various diabetic conditions, we expected to see increased presence of the mitochondrial apoptotic markers Bax, Bcl-2, and cytosolic cytochrome c in cardiac and skeletal muscles of the OZR compared with LZR. Furthermore, because endurance exercise has been shown to attenuate apoptotic signaling (38, 42), we anticipated a reduction in apoptotic signaling after 9 wk of treadmill training.
Bax and Bcl-2.
The ratio of Bcl-2 to Bax is indicative of mitochondrial induced apoptotic potential as Bcl-2 binds and opposes the pro-apoptotic activity of Bax by preventing Bax conformational change or translocation to the mitochondria (11, 45). Once incorporated into the mitochondria, Bax is believed to form a pore in the mitochondria membrane that releases proteins (e.g., cytochrome c) and activates the downstream apoptotic signaling caspase cascade (e.g., caspases-9 and caspases-3; Ref. 4). In this study we observed an increase in the Bax/Bcl-2 ratio and an increased Bax protein in the mitochondrial protein fraction of the cardiac but not skeletal muscles of the OZR. Furthermore, there was an increase in the accumulation of cytosolic cytochrome c in cardiac muscle of the OZR. Additionally, apoptotic signaling downstream of the mitochondria including caspase-3 and caspase-9 activities were also elevated in cardiac muscle samples of the OZR compared with the LZR. This indicates that mitochondrial permeability and apoptotic signaling was increased in the cardiac muscle cells of the OZR.
Exercise improves apoptotic signaling in the myocardium of lean and obese rats. Similar to skeletal muscle, mitochondria appear to play a critical role in regulation of apoptosis in cardiac muscle cells. This includes mobilization of pro-apoptotic proteins to the mitochondria (e.g., Bax) permeabilization of the mitochondrial membrane resulting in a release of mitochondrial housed factors (e.g., cytosolic cytochrome c; Ref. 27, 28) to the cytosol and initiation of downstream apoptotic signaling. The importance of mitochondrial permeability is highlighted in recent work by Fang et al. (15), who showed that blocking the mitochondria permeability pore reduces the degree of apoptosis after myocardial infarction. This is consistent with the idea that reduction in Bax or the Bax/Bcl-2 ratio could reduce apoptosis by minimizing mitochondria permeability.
Several differences between the patterns of apoptosis exist in skeletal and cardiac muscle. For example, a sharp elevation in myocardial apoptosis has been reported during acute myocardial ischemia and particularly after reperfusion (41) and in heart failure (27, 28). Furthermore, cardiac hypertrophy frequently occurs concurrently with apoptosis in the myocardium, and this leads to heart failure (29, 46). This differs somewhat from skeletal muscle, where except for disease or pathology, apoptosis occurs at low rates and only impacts muscle function or mass when it occurs for a long period of time over the life span of an animal and contributes to sarcopenia (3). In addition, nuclear apoptosis typically accompanies loss of skeletal muscle (3, 37). Nevertheless, there are some similarities in apoptotic signaling in cardiac and skeletal muscle. For example, even low levels of myocardial apoptosis may make important contributions to myocardial dysfunction and heart failure (7, 20). This is because basal levels of myocardial apoptosis are likely low at any one point in time in young animals, but apoptosis that occurs among nondividing myocytes, even at low rates over many months or years, could be substantial (27) and contribute to myocardial dysfunction or heart failure in old age (7, 20). This is the same general pattern as seen in aging-associated apoptosis and sarcopenia in skeletal muscle (3, 34).
Oxidative stress has been implicated as an important initiator of apoptosis in myocardial cells, especially following reperfusion of ischemic tissue (27). Basal levels of oxidative stress also increase with aging and may contribute to an elevation of apoptosis signaling in the myocardium of aged animals. Exercise has been shown to be effective in reducing apoptosis and apoptotic signaling in response to acute myocardial insults (22, 35). Although the mechanism whereby exercise acts as a countermeasure against myocardial apoptosis has not been fully elucidated, recent work by French and coworkers (17) suggests that this may be due, at least in part, to improvements in antioxidant enzyme activity in the myocardium including MnSOD activity. This is an important observation that highlights the importance of exercise therapy for improving antioxidant signaling as a means to prevent apoptosis in response to acute myocardial ischemia/reperfusion. Exercise also appears to reduce apoptotic signaling under nonischemic conditions (24, 38) and aging (24) where oxidative stress would be expected to be much less severe than under conditions of ischemia/reperfusion. Additional studies will be required to determine if changes in Bax or Bax/Bcl-2 may also partially explain the increase in susceptibility to myocardial apoptosis subsequent to ischemia-reperfusion or other acute insults in metabolic syndrome (8, 19).
Mitochondria appear to be an important determinant of myocardial apoptotic signaling at least in pathological conditions (32). However, mitochondrial signaling is also important in determining apoptotic signaling in nonischemic myocardium. Our laboratory previously showed that DNA fragmentation was reduced and both mRNA and protein levels of Bcl-2 were increased in the myocardium of endurance-trained healthy animals compared with sedentary control animals (38). Nevertheless, the effects of exercise on the myocardium of obese rats have not been previously examined in detail. In this study we were interested in determining if Bax and the ratio of Bax to Blc-2 might be also be reduced by exercise, thereby minimizing mitochondrial permeability in the myocardium of obese animals under resting (i.e., nonischemic) conditions. Our data show a tread toward a decreased Bax/Bcl-2 ratio (P = 0.072) with 9 wk of treadmill exercise in the cardiac muscle samples of the OZR, and this is consistent with observations that endurance exercise reduced the Bax/Bcl-2 ratio in the myocardium of old rats (24). In addition, our novel data show a significant decrease in the amount of Bax in the mitochondria-enriched protein fraction (∼50% reduction) of the cardiac muscle samples of the OZR after exercise training. Furthermore, we observed a reduction in both caspase-9 and caspase-3 activities in the myocardium of the OZR with 9 wk of treadmill exercise. Together, these data indicate that aerobic exercise provides a strong stimulus for reducing mitochondrial apoptotic signaling in cardiac muscles of animals with metabolic syndrome, as is the case after a myocardial infarction (17, 22) in young healthy animals (38) and in aging (24). Although we cannot fully rule out the possibility that some mitochondrial associated Bax could have been isolated with the cytosolic Bax fraction, or vice versa, this seems unlikely because the MnSOD and CuZnSOD data shown in Fig. 2B indicate that the protein fraction was largely free from contamination.
The Bax/Bcl-2 ratio was similar in skeletal muscle samples of LZR and OZR, and aerobic exercise did not improve this ratio. These data suggest that skeletal muscle may be more resistant to changes in apoptotic signaling than cardiac muscle in metabolic syndrome. Because endurance exercise induces improved antioxidant enzyme levels and activity in both heart and skeletal muscle (17, 38), the improved blunting of myocardial apoptosis could be explained if the relative contribution of oxidative stress to initiating apoptosis in metabolic syndrome was greater in the myocardium than in skeletal muscle. Nevertheless, we cannot rule out the possibility that more intense or longer exercise training parameters may have improved apoptotic signaling in skeletal muscles of the LZR or the OZR animals. Previous training studies in our lab were performed with a treadmill speed of 28 m/min, compared with 24 for the LZR and 20 for the OZR (the OZR would not comply with faster pace). Nevertheless, the exercise parameters in this study were sufficient to induce improvements in citrate synthase activity (18) and mitochondrial cytosolic cytochrome c oxidase (data not shown).
The experiments in this study provide new and important information concerning the role of exercise in increasing the apoptotic resistance of myocardial mitochondria in a model of metabolic syndrome. In particular, we report a ∼50% decrease in the amount of Bax in the mitochondrial-enriched myocardial protein fraction of OZR after exercise training. The reduced Bax and Bax/Bcl-2 levels in the myocardium after endurance training in obese animals should improve mitochondrial membrane stability. This would be expected to reduce subsequent mitochondrial pore formation and downstream apoptotic signaling by caspase-9 and caspase-3, thereby leading to reduced nuclear fragmentation. Together, these data suggest that exercise provides a powerful tool for not only suppressing myocardial apoptosis by improving the antioxidant capacity of the myocardium (17, 38), but it also reduces the potential for Bax to elevate mitochondria permeability, thereby reducing mitochondrial apoptotic signaling in cardiac muscles of animals with metabolic syndrome. Future studies should focus on investigating which of the observed exercise-induced changes in cardiac mitochondria (e.g., control of oxidative stress as an initiator or control of mitochondrial stability via Bax or some other protein) are essential contributors to cardioprotection against apoptosis in metabolic syndrome. This is particularly important in aging, which accelerates the effects of metabolic syndrome (31).
This study was supported by National Institutes of Health: National Institute on Aging Grant R01-AG-021530 to S. E. Alway.
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- Copyright © 2008 the American Physiological Society