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Am J Physiol Heart Circ Physiol 294: H928-H935, 2008. First published December 14, 2007; doi:10.1152/ajpheart.01231.2007
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Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli

Andreas N. Kavazis,1 Joseph M. McClung,1 David A. Hood,2 and Scott K. Powers1

1Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida; and 2School of Kinesiology and Health Science, York University, Toronto, Ontario, Canada

Submitted 23 October 2007 ; accepted in final form 10 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ischemia-reperfusion-induced calcium overload and production of reactive oxygen species can trigger apoptosis by promoting the release of proapoptotic factors via the mitochondrial permeability transition pore. While it is clear that endurance exercise provides cardioprotection against ischemia-reperfusion-induced injury, it is unknown if exercise training directly alters mitochondria phenotype and confers protection against apoptotic stimuli in both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We hypothesized that exercise training increases expression of endogenous antioxidant enzymes and other antiapoptotic proteins, resulting in a SS and IMF mitochondrial phenotype that resists apoptotic stimuli. Mitochondria isolated from hearts of sedentary (n = 8) and exercised-trained (n = 8) adult male rats were studied. Endurance exercise increased the protein levels of primary antioxidant enzymes in both SS and IMF mitochondria. Furthermore, exercise increased the levels of antiapoptotic proteins in the heart, including the apoptosis repressor with a caspase recruitment domain and inducible heat shock protein 70. Importantly, our findings reveal that endurance exercise training attenuates reactive oxygen species-induced cytochrome c release from heart mitochondria. These changes are accompanied by a lower maximal rate of mitochondrial permeability transition pore opening (Vmax) and prolonged time to Vmax in both SS and IMF cardiac mitochondria. These novel findings reveal that endurance exercise promotes biochemical alterations in cardiac SS and IMF mitochondria, resulting in a phenotype that resists apoptotic stimuli. Furthermore, these results are consistent with the concept that exercise-induced mitochondrial adaptations contribute to exercise-induced cardioprotection.

antioxidants; redox balance

CORONARY ARTERY DISEASE REMAINS a major cause of morbidity and mortality around the world (38, 40). The primary pathological expression of coronary artery disease is myocardial injury due to an ischemia-reperfusion (IR) insult. Given the prevalence of IR injury in the heart, developing a strategy that results in a cardioprotective phenotype is important. Numerous approaches to cardioprotection have been investigated, and, to date, the only practical and sustainable countermeasure capable of promoting cardioprotection is regular bouts of endurance exercise. Two lines of evidence strongly support the concept that regular exercise is cardioprotective. First, a wide array of human epidemiological studies indicates that regular exercise reduces the risk of death due to an IR insult [reviewed by Ignarro et al. (20)]. Second, numerous animal studies demonstrate that consecutive bouts of endurance exercise provide cardioprotection against IR injury [reviewed by Powers et al. (32)].

Although it is clear that exercise promotes a cardioprotective phenotype, a detailed understanding of the cellular mechanisms responsible for this cardioprotection remains incomplete. In this regard, it has been hypothesized that one of the health benefits of endurance exercise training is due, at least in part, to mitochondrial adaptations (5, 22, 23, 35). For example, recent evidence reveals that exercise training results in a reduction in mitochondrial oxidant production (23) and enhanced mitochondrial antioxidant enzyme activity (22, 23, 41). This is an attractive hypothesis to explain, at least in part, the exercise-induced cardioprotection. Indeed, mitochondria are vital organelles that can serve as the final arbitrators of life or death during an IR insult, as they are not only required to produce ATP, but can also trigger both necrosis and apoptosis (15, 16). During an IR insult, mitochondrial production of reactive oxygen species (ROS) increases in conjunction with an influx of calcium into both the cardiomyocyte and mitochondria (43). Increased ROS and calcium can interact to induce opening of the mitochondria permeability transition pore (mtPTP), resulting in the release of proapoptotic proteins (i.e., cytochrome c) and subsequent activation of programmed cell death (1–3, 10, 21, 28).

Cardiac muscle contains two morphologically distinct subfractions of mitochondria, located in different regions of the muscle fiber (30, 39). Subsarcolemmal (SS) mitochondria are found immediately beneath the sarcolemmal membrane, whereas intermyofibrillar (IMF) mitochondria are intermingled within the myofibrils. These mitochondrial subfractions possess different functional, compositional, and biochemical properties (2, 30, 39). However, it is unknown whether these two subpopulations of cardiac mitochondria differ in their susceptibilities to apoptotic stimuli, and/or adapt differentially to endurance exercise training. Therefore, we investigated the apoptotic susceptibility of SS and IMF mitochondria isolated from hearts of both sedentary and endurance exercise-trained animals. We hypothesized that endurance exercise training would increase both antiapoptotic proteins and endogenous antioxidant enzymes in cardiac SS and IMF mitochondria, resulting in reduced susceptibility to apoptotic stimuli.


   METHODS
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 METHODS
RESULTS
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Experimental design. These experiments were approved by the University of Florida Animal Care and Use Committee and followed guidelines established by the American Physiological Society for the use of animals in research. Adult Sprague-Dawley (male) rats (4–6 mo old) were randomly assigned to one of two experimental groups: sedentary (Sed) (n = 8) or endurance exercise trained (ExTr) (n = 8). Throughout the experimental period, all animals were housed on a 12:12-h light-dark cycle and were provided food (AIN93 diet) and water ad libitum.

Exercise training protocol. Animals assigned to the ExTr group were habituated to treadmill exercise for 5 consecutive days. This habituation period involved a gradual increase in running time, beginning with 10 min/day and ending with 50 min/day. After 2 days of rest, the animals then performed 5 consecutive days of treadmill exercise for 60 min/day at 30 m/min, 0% grade (estimated work rate of 70% maximum O2 consumption) (8). This exercise training protocol provides cardioprotection against a variety of ischemic insults (i.e., IR-induced oxidant injury, contractile dysfunction, and necrotic/apoptotic cell death) (14, 17, 25–27, 36). All hearts were excised 24 h after the final exercise bout.

Mitochondrial isolation. Differential centrifugation was used to fractionate SS and IMF mitochondria, as described previously (7) with minor modifications. Briefly, cardiac muscle was minced thoroughly in buffer 1 (wt/vol 1:10): 0.22 M mannitol, 0.07 M sucrose, 2 mM Tris base (pH 7.2), 1 mM EDTA, 20 mM HEPES, and 0.2% bovine serum albumin (BSA). Samples were then homogenized for 10 s using polytron at 50% power. The sample was centrifuged at 800 g (10 min) to separate the SS mitochondria from the myofibrils. The supernate containing the SS mitochondria was filtered through cheesecloth, and the mitochondria were pelleted at 9,000 g (10 min). The pellet was washed by resuspension in buffer 1 and was recentrifuged at 9,000 g (10 min). The SS mitochondrial pellet was washed once again in buffer 1 minus BSA and finally suspended in a small volume of resuspension medium 1 or 2 as shown below.

The myofibrillar pellet containing the IMF mitochondria was resuspended, rehomogenized using the polytron, and recentrifuged at 800 g. The supernate was discarded, and the pellet was diluted in buffer 1 (wt/vol 1:10). The samples were exposed to the protease nagarse (1 mg nagarse/g tissue; Sigma, St. Louis, MO) for 5 min on ice. Termination of the action of nagarse was accomplished by the addition of equal volume of buffer 1 and centrifugation at 5,000 g for 5 min. The pellet was suspended in buffer 1 and was centrifuged at 800 g. The supernate was centrifuged at 9,000 g (10 min) to pellet the IMF mitochondria. The mitochondria were first washed in buffer 1, followed by a wash in buffer 1 minus BSA, and finally suspended in a small volume of resuspension medium 1 or 2, as shown below. All centrifuge spins were done at 4°C.

SS and IMF mitochondria were resuspended in medium 1 (100 mM KCl, 10 mM MOPS, 0.2% BSA) for the mitochondrial release assay, or in medium 2 (215 mM mannitol, 71 mM sucrose, 3 mM HEPES, 5 mM succinate) for the mtPTP assay.

Mitochondrial integrity. We evaluated the integrity of mitochondria isolated from Sed animals in two ways. First, we isolated both SS and IMF mitochondria and visually examined their structural integrity via electron microscopy. Furthermore, we assessed the functional integrity of mitochondria by measuring state 3 and 4 oxygen consumption in mitochondria isolated from hearts of Sed animals. Moreover, we computed the respiratory control ratio (RCR) as an indicator of mitochondrial coupling. Briefly, the preparation of mitochondria for electron microscopy involved the following steps. SS and IMF mitochondria were immersed in Trumps fixative and washed with 0.1 M sodium cacodylate. Fixed samples were pelleted and encapsulated in 3% agarose (Sigma type VII), postfixed with 1% buffered osmium tetroxide, and washed with buffer followed by distilled water. The fixed samples were then dehydrated in a graded ethanol series followed by 100% acetone. Dehydrated samples were infiltrated in a graded acetone/Embed 812 resin series and cured in 100% Embed 812 at 60°C for 2 days. Ultrathin sections were collected on 200-mesh copper grids and poststained with 2% uranyl acetate and Reynold's lead citrate. Sections were examined with a Hitachi H-7000 transmission electron microscope, and images were acquired with Soft Imaging Systems MegaView III AnalySIS software.

To measure mitochondrial oxygen consumption, isolated SS and IMF mitochondria from Sed animals were incubated with 1 ml of respiration buffer (100 mM KCl, 5 mM K2HPO4, 1 mM EGTA, 50 mM MOPS, 10 mM MgCl2, and 0.2% BSA, pH 7.4) at 37°C in a water-jacketed respiratory chamber with continuous stirring. For state 3 respiration, 2 mM pyruvate and 2 mM malate were used as substrates in the presence of 0.25 mM ADP, and state 4 respiration was recorded following the phosphorylation of ADP. Respiration rates were evaluated with the use of an oxygen electrode (Hansatech Instruments). RCR was calculated as the ratio of state 3 to state 4 oxygen consumption.

mtPTP assessment. mtPTP opening is facilitated under conditions of elevated calcium concentration and exacerbated when combined with oxidative stress (2). The opening of the mtPTP is associated with mitochondrial swelling, outer membrane rupture, and release of proapoptotic factors that can induce apoptosis. We assessed mtPTP opening by monitoring the decrease in light scattering associated with mitochondrial swelling at 540 nm. Isolated SS and IMF mitochondria were resuspended in medium 2 to a final concentration of 1 mg/ml. SS and IMF mitochondria were then treated with 400 µM CaCl2 and 75 µM tert-butyl hydroperoxide. The decrease in absorbance was monitored for 15 min with a spectrophotometer (SpectraMax 190, Molecular Devices, Union City, CA).

Protein release assay. Isolated SS and IMF mitochondrial fractions (150 µg) from Sed and ExTr cardiac muscle were incubated with mitochondrial resuspension medium 1 containing 50 mM FeSO4 and final concentrations of H2O2 ranging from 0 to 100 µM. Samples were incubated at 30°C for 60 min. Reaction mixtures were then centrifuged at 14,000 g (4°C) to pellet mitochondria, and the supernatant was collected for subsequent assessment of cytochrome c release using Western blot analysis. We chose a range of H2O2 concentrations and incubation times previously shown to induce cytochrome c release from mitochondria myocytes (2).

Immunoblotting. Isolated IMF and SS mitochondrial protein extracts, supernatant aliquots from the protein release assay, or cytosolic cardiac proteins were separated by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently electroblotted onto nitrocellulose membranes. The resulting membranes were then stained with Ponceau S and analyzed to verify equal loading and transfer. Membranes were blocked (2 h) with 5% skim milk in phosphate-buffered saline solution containing 0.1% Tween 20 (PBST). Blots were then incubated in blocking buffer with antibody directed against cytochrome c (sc-8385; 1:750 dilution), apoptosis inducing factor (AIF; sc-9416; 1:750 dilution), voltage-dependent anion channel 1 (VDAC1; sc-8829; 1:1,000 dilution), adenine-nucleotide translocase (ANT; sc-9299; 1:1,000 dilution), cyclophilin D (sc-33068; 1:750 dilution), apoptosis repressor with a caspase recruitment domain (ARC; sc-8241; 1:1,000 dilution), copper zinc superoxide dismutase (CuZnSOD; sc-11407; 1:1,000 dilution), manganese superoxide dismutase (MnSOD; SOD-111; 1:2,000 dilution), heat shock protein 70 (HSP70; SPA-812; 1:2,500 dilution), catalase (ab16731, 1:1,500 dilution), and glutathione peroxidase 1 (GPX1; ab16798; 1:1,500 dilution) overnight at 4°C. Antibodies directed against AIF, cytochrome c, VDAC1, ANT, ARC, and CuZnSOD were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), against MnSOD and HSP70 were purchased from Stressgen (San Diego, CA), and against catalase and GPX1 were purchased from Abcam (Cambridge, MA). After being washed with PBST, blots were incubated at room temperature for 1 h with the appropriate secondary antibody coupled to horseradish peroxidase and washed again with PBST. The membranes were then treated with chemiluminescent reagents (luminol and enhancer; Amersham Biosciences, Pittsburgh, PA) and exposed to light-sensitive film. Images of these films were captured and analyzed using the 440CF Kodak Imaging System (Kodak, New Haven, CT).

Statistical methods. Statistical significance between groups for dependent variables was determined by a one-way analysis of variance. For the apoptotic protein release, data were analyzed by a one-way analysis of variance followed with Tukey's multiple-comparison test. Significance was established at P < 0.05. Results are presented as means ± SE.


   RESULTS
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SS and IMF mitochondrial content and integrity. Exercise training did not alter heart weight (Sed = 1.17 ± 0.02 g vs. ExTr = 1.10 ± 0.02 g) or cellular level of mitochondrial protein in either mitochondrial subpopulation (Fig. 1). Importantly, images of isolated mitochondria obtained with transmission electron microscopy reveal that our isolation procedure produced high levels of intact and pure SS and IMF mitochondria (Fig. 2). Importantly, the integrity of our isolation protocol was verified by measuring oxidative phosphorylation (i.e., states 3 and 4) in SS and IMF mitochondria isolated from Sed animals. RCR from both SS (4.56 ± 0.25) and IMF (11.03 ± 1.15) mitochondria demonstrate that our isolation technique yielded well-coupled mitochondria and agree with published data that IMF mitochondria exhibit higher rates of oxidative phosphorylation compared with SS mitochondria (30, 39). Collectively, these findings indicate that the mitochondria used in our experiments were intact and functionally viable.


Figure 1
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  		Fig. 1. Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial yield from sedentary (Sed) and endurance exercise-trained (ExTr) hearts. Note that 5 days of endurance exercise training did not alter the content of either SS or IMF cardiac mitochondria.

 

Figure 2
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  		Fig. 2. Electron microscopic pictures of SS and IMF mitochondrial obtained from rat hearts. Note that both subfractions show morphological integrity.

 
ROS-mediated cytochrome c release from isolated SS and IMF cardiac mitochondria following endurance exercise training. SS and IMF cardiac mitochondria isolated from Sed and ExTr animals were compared for their ability to release cytochrome c under both control (without ROS) and hydrogen peroxide (H2O2)-treated conditions. Exposure to varying levels of H2O2 (i.e., 25–100 µM) did not promote cytochrome c release in SS mitochondria from Sed or ExTr groups (Fig. 3, A and C). In contrast, treatment of sedentary IMF mitochondria with 100 µM H2O2 caused a significantly higher release of cytochrome c; importantly, this response was attenuated by endurance exercise training (Fig. 3, B and D).


Figure 3
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  		Fig. 3. Dose-dependent release of cytochrome c in Sed animals from SS (A) and IMF (B) cardiac mitochondria in response to progressive increases in H2O2 concentrations ([H2O2]) (0, 25, 50, and 100 µM; n = 7–8/concentration) for 60 min at 30°C. Dose-dependent release of cytochrome c is also shown in ExTr animals from SS (C) and IMF (D) cardiac mitochondria (n = 6/concentration). *P < 0.05 from all other concentrations. Representative Western blots for cytochrome c released at each H2O2 concentration are shown above the graphs.

 
Function and composition of the mtPTP of SS and IMF mitochondria. During exposure to both calcium and ROS, we measured SS and IMF mtPTP opening characteristics [i.e., maximal rate of pore opening (Vmax) and the time to Vmax] to determine whether endurance exercise training alters this important mitochondrial property. Importantly, endurance exercise training resulted in lower Vmax in both SS and IMF mitochondria compared with Sed animals (P < 0.05) (Fig. 4A). Furthermore, SS mitochondria isolated from hearts of exercised animals exhibited a longer time to Vmax (P < 0.05) (Fig. 4B). To determine whether these functional differences were related to mtPTP composition, we measured individual components of the mtPTP. Following exercise training, VDAC1 levels did not change in SS mitochondria [Sed = 52 ± 2 vs. ExTr = 44 ± 3 arbitrary densitometry units (ADU)] or IMF mitochondria (Sed = 12 ± 3 vs. ExTr = 18 ± 6 ADU). However, ANT levels decreased (P < 0.05) in both SS and IMF mitochondria after exercise training (SS-Sed = 399 ± 21; SS-ExTr = 272 ± 17; IMF-Sed = 221 ± 12; IMF-ExTr = 49 ± 11 ADU). Following exercise training, cyclophilin D levels increased (P < 0.05) in SS mitochondria (Sed = 226 ± 45 vs. ExTr = 326 ± 19 ADU), but decreased (P < 0.05) in IMF mitochondria (Sed = 97 ± 39 vs. ExTr = 16 ± 6 ADU).


Figure 4
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  		Fig. 4. Mitochondrial transition pore kinetics from SS and IMF mitochondria after administration of exogenous high concentration of calcium and tert-butyl hydroperoxide (n = 4/group). Endurance exercise training resulted in lower rate of the decrease in absorbance [maximal rate of pore opening (Vmax)] in SS and IMF mitochondria (A) and longer time to reach Vmax in SS mitochondria compared with Sed animals (B). P < 0.05 in *SS-Sed vs. IMF-Sed; {ddagger}SS-Sed vs. SS-ExTr; and §IMF-Sed vs. IMF-ExTr.

 
Endurance exercise training increases mitochondrial antioxidant enzymes. To assess the effects of exercise on the antioxidant capacity of the cardiac muscle, the protein levels of important antioxidant enzymes were evaluated. The antioxidants selected were chosen on the basis that each enzyme is considered to be a primary antioxidant enzyme involved in providing mitochondrial protection against oxidative injury. Endurance exercise training resulted in alterations of mitochondrial levels of both MnSOD and CuZnSOD; each of these SODs can dismutate superoxide anion (O2–) into H2O2. Both mitochondrial MnSOD and CuZnSOD protein levels were similar in SS and IMF mitochondria isolated from Sed animals. Compared with control, MnSOD increased in SS mitochondria (+34.9%; P < 0.05) after exercise training (Fig. 5A). In IMF mitochondria, MnSOD increased (+22.5%) following endurance exercise training, but these results did not reach statistical significance (P = 0.09) (Fig. 5A). Furthermore, endurance exercise training increased CuZnSOD by 27.1% in IMF mitochondria (P < 0.05), but, in contrast, CuZnSOD levels were not altered in SS mitochondria following exercise training (Fig. 5B). In Sed animals, the levels of two antioxidant enzymes that convert H2O2 to H2O (i.e., GPX and catalase) were less in IMF mitochondria compared with SS mitochondria. Importantly, the levels of these two primary antioxidants increased in both SS and IMF mitochondria following exercise training. Specifically, GPX was increased by 35.2 and 50.0% in SS and IMF mitochondria (P < 0.05), respectively (Fig. 6A). Furthermore, exercise training increased mitochondrial levels of catalase in SS and IMF mitochondria by 37.5 and 34.3%, respectively (P < 0.05) (Fig. 6B).


Figure 5
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  		Fig. 5. Manganese superoxide dismutase (MnSOD; A) and copper zinc superoxide dismutase (CuZnSOD; B) protein levels in SS and IMF mitochondria from Sed and ExTr animals (n = 5–6/group). P < 0.05 in {ddagger}SS-Sed vs. SS-ExTr and §IMF-Sed vs. IMF-ExTr. Representative Western blots for mitochondrial MnSOD and CuZnSOD protein levels are shown above the graphs.

 

Figure 6
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  		Fig. 6. Glutathione peroxidase (GPX; A) and catalase protein levels (B) from SS and IMF mitochondria from Sed and ExTr animals (n = 5–6/group). Mitochondrial IMF GPX levels were quantified by summing the duplex band shown in the blot. P < 0.05 in *SS-Sed vs. IMF-Sed; {ddagger}SS-Sed vs. SS-ExTr; §IMF-Sed vs. IMF-ExTr; and #SS-ExTr vs. IMF-ExTr. Representative Western blots for mitochondrial GPX and catalase are shown above the graphs.

 
Effect of endurance exercise training on mitochondrial and cytosolic pro- and antiapoptotic protein expression. We evaluated the levels of cytochrome c and AIF in SS and IMF mitochondria to determine whether the mitochondrial subpopulations contained different levels of proapoptotic proteins and to determine the effect of exercise training on the expression of these proteins. Cytochrome c levels did not differ between the two mitochondrial subfractions (Fig. 7A). In contrast, AIF levels were higher in SS mitochondria compared with IMF mitochondria (P < 0.05) (Fig. 7B). Although exercise training did not alter mitochondrial levels of AIF protein, cytochrome c protein content was significantly increased in both SS and IMF mitochondria after endurance exercise training.


Figure 7
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  		Fig. 7. Cytochrome c (A) and apoptosis inducing factor (AIF; B) protein levels in SS and IMF mitochondria from Sed and ExTr animals (n = 7/group). SS and IMF cardiac mitochondria contain similar levels of cytochrome c, whereas IMF mitochondria contain less AIF than SS mitochondria. ExTr induced a threefold increase of cytochrome c, but no response in AIF. P < 0.05 in {ddagger}SS-Sed vs. SS-ExTr; §IMF-Sed vs. IMF-ExTr; *SS-Sed vs. IMF-Sed; and #SS-ExTr vs. IMF-ExTr. Representative Western blots for mitochondrial cytochrome c and AIF are shown above the graphs.

 
Since ARC and HSP70 are cytosolic proteins that exhibit antiapoptotic properties (13, 37), we assessed the effect of exercise training on their levels in the heart. Exercise training resulted in a threefold increase in cardiac HSP70 levels (P < 0.05) (Fig. 8A), and a 37.5% increase in ARC protein levels (P < 0.05) (Fig. 8B). Finally, exercise training did not alter myocardial levels of cytosolic CuZnSOD (Fig. 8C).


Figure 8
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  		Fig. 8. Endurance exercise training provoked increased cytosolic protein levels of 70-kDa heat shock protein (HSP70; A), apoptosis repressor with a caspase recruitment domain (ARC; B), but not CuZnSOD (C) compared with Sed animals. *P < 0.05 in Sed vs. ExTr. Representative Western blots for cytosolic HSP70, ARC, and CuZnSOD are shown above each graph.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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Overview of principle findings. These experiments tested the hypothesis that exercise training increases the expression of antioxidant enzymes and other antiapoptotic proteins in cardiac SS and IMF mitochondria, resulting in a mitochondrial phenotype that is resistant to apoptotic stimuli. Our findings support this postulate, as numerous antioxidant enzymes were increased in cardiac mitochondria following exercise training, and, importantly, both SS and IMF mitochondria exhibited decreased susceptibility to apoptotic stimuli. To our knowledge, this report provides the first direct evidence that exercise promotes important adaptations to both SS and IMF cardiac mitochondria conferring an apoptotic resistant phenotype. Moreover, these results are consistent with the notion that exercise-induced mitochondrial adaptations significantly contribute to the exercise-induced cardioprotective phenotype. A brief discussion of these and related issues follows.

Cardiac SS and IMF mitochondria possess differential susceptibility to apoptotic stimuli. In Sed animals, exposure to moderate levels of ROS (i.e., 100 µM H2O2) promoted cytochrome c release from IMF mitochondria, but this response did not occur in SS mitochondria, indicating that IMF mitochondria are more sensitive to ROS-induced apoptotic stimuli. To investigate the mechanism for this finding, we examined the function and composition of the mtPTP in the two subfractions of mitochondria, because ROS-induced protein release is mediated via the opening of the mtPTP (9, 35). In Sed animals, although the time to Vmax was longer for IMF compared with SS mitochondria, the Vmax was greater in IMF compared with SS mitochondria. These differences may be related to differential composition of the mtPTP and/or to the number of functional pores per mitochondrion. In this regard, the mtPTP is associated with several proteins (i.e., VDAC, ANT, cyclophilin D) that can connect the mitochondrial matrix to the cytosolic space. Specifically, it has been reported that ANT can be transformed from a selective ADP-ATP antiporter to a nonselective pore component in mtPTP formation (2). In the present study, VDAC, ANT, and cyclophilin D protein levels in Sed animals were higher in SS mitochondria compared with IMF mitochondria. Therefore, since these proteins are part of the biochemical composition of the mtPTP, this higher level of VDAC, ANT, and cyclophilin D proteins in SS mitochondria may contribute to the shorter time to Vmax observed in SS mitochondria compared with IMF mitochondria.

Exercise training evokes a mitochondrial phenotype that is protective against apoptotic stimuli. Cytochrome c is a small heme protein associated with the inner membrane of the mitochondrion. The main function of cytochrome c is in the electron transfer chain, and it has a primary role in oxidative phosphorylation and energy provision. Elevations in cytochrome c following endurance exercise training may be indicative of improvements in mitochondrial electron transport chain function during periods of high-energy demands (i.e., exercise) (1, 19). Moreover, this adaptation may also augment the cellular apoptotic potential, since cytochrome c can initiate cell death upon release from the mitochondria (28). Despite the greater cytochrome c levels in SS and IMF mitochondria obtained from hearts of ExTr animals, ROS-induced release of cytochrome c from IMF mitochondria isolated from ExTr animals was reduced compared with IMF mitochondria obtained from Sed animals.

Furthermore, our data indicate that both subfractions of mitochondria undergo biochemical adaptations in response to endurance exercise, leading to decreased apoptotic susceptibility. Specifically, IMF and SS mitochondria exhibited significantly lower Vmax after endurance exercise training compared with Sed animals. Also, SS mitochondria obtained from hearts of ExTr animals exhibited a longer time to Vmax compared with SS mitochondria isolated from Sed animals. These results suggest that endurance exercise training induced a protective adaptation within mitochondria, resulting in a reduced cytochrome c release after a ROS challenge, despite higher mitochondrial levels of this protein. An unexpected finding is that cyclophilin D increased by ~30% in SS mitochondria following exercise training. However, our results also indicate that cyclophilin D decreased by approximately sixfold in IMF mitochondria and that ANT protein levels decreased significantly in both SS and IMF mitochondria following exercise training. Collectively, these results suggest that ANT may be a contributory factor to the protective phenotype observed in mitochondria isolated from the hearts of exercised animals.

At present, the molecular mechanisms responsible for exercise-induced cardioprotection are not well established. It has been hypothesized that the cardioprotective benefits of exercise may be at least partially due to a reduction in oxidant production and increased myocardial antioxidant capacity, both of which could reduce IR-induced oxidative damage (5, 12, 17, 18, 25, 26, 29, 31–34, 36, 44). Cells contain several naturally occurring mechanisms of protection from ROS injury. Primary enzymatic antioxidant defenses include SOD, GPX, and catalase. Each of these antioxidants is capable of combining with ROS to produce other less reactive species. Both SOD isoforms (i.e., MnSOD and CuZnSOD) promote the dismutation of the superoxide radical to form H2O2 and oxygen. Catalase can then exert protection against oxidative injury by converting H2O2 to water and oxygen. Similarly, GPX utilizes reduced glutathione as a reducing equivalent to reduce H2O2 to form oxidized glutathione and water. Importantly, our laboratory has reported that increased cardiac MnSOD antioxidant activity is important in providing protection against both myocardial necrosis and apoptosis (17, 27, 32, 34). Interestingly, published papers have reported increases, no changes, or even decreases in antioxidant enzymes (i.e., catalase, SOD, and GPX) following endurance exercise training (for a complete review, see Ref. 4). The ambiguity of these findings may be due to a variety of factors, including methodological differences in the assay of the antioxidant activity, variations in the exercise training paradigm, or improper handling of the tissue. Importantly, all of the previous studies have assessed antioxidant activity in crude heart homogenates. In contrast, this is the first study to investigate antioxidant enzyme protein levels following endurance exercise training in isolated mitochondria from cardiac tissue. Considering the small increases (~30%) in mitochondrial antioxidant enzyme levels observed in the present study, it is possible that the exercise-induced increases in mitochondrial antioxidants are masked when crude homogenate is used for this analysis.

Note that SS mitochondria isolated from hearts of Sed animals contained significantly higher catalase and GPX protein abundance compared with IMF mitochondria. Therefore, compared with IMF mitochondria, it appears that SS mitochondria were more capable of resisting the H2O2 challenge used for the proapoptotic (i.e., cytochrome c) protein release because of the higher levels of catalase and GPX in SS mitochondria. Furthermore, following endurance exercise training, catalase and GPX levels were significantly increased in cardiac SS and IMF mitochondria, leading to the attenuation of cytochrome c release in response to the ROS challenge. Collectively, these findings indicate that exercise training promotes a mitochondrial phenotype that is resistant to ROS challenges, thus benefiting heart during exposure to an IR insult.

Consistent with previous studies, exercise training resulted in increased ARC and HSP70 cytosolic protein content in the heart (1, 11, 18, 33, 34, 42). The increase in these proteins is important, since ARC has been shown to specifically inhibit the cytochrome c-mediated apoptotic pathway within the cytosol (24). Furthermore, increased HSP70 levels in the heart may facilitate nuclear-encoded protein importation and assembly in the mitochondrial matrix. Moreover, evidence exists that HSP70 is also capable of inhibiting both the cytochrome c- and AIF-mediated apoptotic signaling pathway (6, 37).

Conclusions and recommendations for future studies. These experiments provide new and important information about the two subpopulations of cardiac mitochondria. First, SS and IMF mitochondria isolated from hearts of Sed animals show a differential susceptibility to apoptotic stimuli. Indeed, compared with IMF mitochondria, SS mitochondria are less susceptible to ROS-induced cytochrome c release. Importantly, our results also reveal that both SS and IMF mitochondrial undergo exercise-induced adaptations that result in a phenotype that is less susceptible to ROS-induced apoptosis. These findings are consistent with the concept that exercise-induced beneficial adaptations in cardiac mitochondria play an important role in the cardioprotection associated with exercise. Future translational studies should determine whether the observed exercise-induced resistance to apoptotic stimuli in isolated SS and IMF mitochondria translates into in vivo protection of mitochondria to a cardiac IR insult. If this is the case, future studies should focus upon investigating which of the observed exercise-induced changes in cardiac mitochondria are essential contributors to cardioprotection.


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This study was supported by National Heart, Lung, and Blood Institute Grant R01HL067855 awarded to S. K. Powers.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. K. Powers, Dept. of Applied Physiology and Kinesiology, Univ. of Florida, Gainesville, FL, 32611 (e-mail: spowers{at}hhp.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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  1. Adhihetty PJ, Ljubicic V, Hood DA. Effect of chronic contractile activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal muscle. Am J Physiol Endocrinol Metab 292: E748–E755, 2007.[Abstract/Free Full Text]
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