Differential susceptibility of subsarcolemmal and intermyofibrillar mitochondria to apoptotic stimuli

Peter J. Adhihetty,2 Vladimir Ljubicic,1 Keir J. Menzies,2 and David A. Hood1,2

1School of Kinesiology and Health Science and 2Department of Biology, York University, Toronto, Ontario, Canada

Submitted 26 January 2005 ; accepted in final form 13 May 2005


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Apoptosis can be evoked by reactive oxygen species (ROS)-induced mitochondrial release of the proapoptotic factors cytochrome c and apoptosis-inducing factor (AIF). Because skeletal muscle is composed of two mitochondrial subfractions that reside in distinct subcellular regions, we investigated the apoptotic susceptibility of subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS and IMF mitochondria exhibited a dose-dependent release of protein in response to H2O2 (0, 25, 50, and 100 µM). However, IMF mitochondria were more sensitive to H2O2 and released a 2.5-fold and 10-fold greater amount of cytochrome c and AIF, respectively, compared with SS mitochondria. This finding coincided with a 44% (P < 0.05) greater rate of opening (maximum rate of absorbance decrease, Vmax) of the protein release channel, the mitochondrial permeability transition pore (mtPTP), in IMF mitochondria. IMF mitochondria also exhibited a 47% (P < 0.05) and 60% (0.05 < P < 0.1) greater expression of the key mtPTP component voltage-dependent anion channel and cyclophilin D, respectively, along with a threefold greater cytochrome c content, but similar levels of AIF compared with SS mitochondria. Despite a lower susceptibility to H2O2-induced release, SS mitochondria possessed a 10-fold greater Bax-to-Bcl-2 ratio (P < 0.05), a 2.7-fold greater rate of ROS production, and an approximately twofold greater membrane potential compared with IMF mitochondria. The expression of the antioxidant enzyme Mn2+-superoxide dismutase was similar between subfractions. Thus the divergent protein composition and function of the mtPTP between SS and IMF mitochondria contributes to a differential release of cytochrome c and AIF in response to ROS. Given the relatively high proportion of IMF mitochondria within a muscle fiber, this subfraction is likely most important in inducing apoptosis when presented with apoptotic stimuli, ultimately leading to myonuclear decay and muscle fiber atrophy.

reactive oxygen species; skeletal muscle; mitochondrial permeability transition pore; cytochrome c; apoptosis


APOPTOSIS, OR PROGRAMMED CELL DEATH, is a systematic dismantling of the cell into discrete packages that are marked for phagocytosis. The defining morphological features of apoptosis include plasma membrane blebbing, nuclear breakdown, and DNA fragmentation. In skeletal muscle, previous studies have shown that apoptosis contributes to muscle degeneration in pathological states such as muscular dystrophies, disuse atrophy, denervation, burn injury, ischemia-reperfusion injury, and mitochondrial myopathies (1, 36, 41, 42). In addition, apoptosis appears to contribute to the aging-induced decrease in muscle mass known as sarcopenia (11, 12).

Apoptosis can be triggered by signal transduction pathways that originate within mitochondria. These organelles are intimately involved in apoptosis because 1) mitochondria contain proapoptotic proteins, and 2) mitochondria are the primary producers of reactive oxygen species (ROS), which can have both direct and indirect effects on apoptosis. The proapoptotic proteins cytochrome c, found on the outer face of the inner mitochondrial membrane, and apoptosis-inducing factor (AIF), located in the intermembrane space, can be released and lead to cell death (30, 43). However, the apoptosis-inducing pathways are different for each of these proteins. Release of cytochrome c into the cytosol induces the formation of an apoptosome complex, which triggers a series of proteolytic cleavages, termed the caspase cascade. The terminal proteolytic cleavage involves caspase-3 activation, which ultimately results in the degradation of DNA and apoptosis (1). In contrast, AIF release from the intermembrane space induces apoptosis independent of caspases and thereby provides a direct molecular conduit between the mitochondria and nuclear breakdown/DNA fragmentation.

Liberation of either AIF or cytochrome c from mitochondria is partially dependent on the formation of a specialized channel within the mitochondrial membrane called the mitochondrial permeability transition pore (mtPTP; Refs. 4, 8). The mtPTP comprises the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane as well as the adenine nucleotide translocase (ANT) and cyclophilin D of the inner mitochondrial membrane (9, 46, 47). Despite some controversy regarding the role of mtPTP in apoptosis, the majority of the literature published to date supports the formation of the mtPTP to facilitate the release of proapoptotic proteins during apoptosis (8, 36, 48). The conformational status of the mtPTP is regulated by the Bcl-2 family of proteins, which are associated with the outer membrane of the mitochondrion. This family comprises both proapoptotic (i.e., Bax, Bak, Bok) and antiapoptotic members (i.e., Bcl-2, Bcl-XL, Bcl-w) that can effectively neutralize or titrate the function of one another by forming heterodimers (39). Thus the relative proportion of pro- and antiapoptotic proteins residing on the outer mitochondrial membrane appears to be an important contributing factor in determining apoptotic susceptibility.

ROS production in muscle can occur via extracellular reactions (35) or via mitochondria within the cell. During normal mitochondrial respiration, electrons are transferred through a succession of oxidation-reduction reactions involving inner membrane proteins. However, the inefficient transfer of electrons can produce a variety of unstable and potentially damaging ROS. Mitochondria are the primary site of ROS production within the cell, and the mitochondrial matrix has 5- to 10-fold higher concentrations of ROS than that of the cytosol (5). ROS can directly interact with mtPTP components to facilitate pore opening and can also induce the dissociation of cytochrome c from the inner mitochondrial membrane (32). ROS can also indirectly influence the apoptotic pathway by activating various redox-sensitive transcription factors involved in the expression of both anti- and proapoptotic gene expression (2, 16).

Within skeletal muscle, there are two morphologically distinct subfractions of mitochondria located in different regions of the fiber. Subsarcolemmal (SS) mitochondria are found immediately underneath the sarcolemmal membrane, and intermyofibrillar (IMF) mitochondria are intermingled within the myofibrils. These mitochondrial subfractions possess different functional (i.e., respiration), compositional (protein and lipid), and biochemical (e.g., protein import) properties, which may contribute to their capacities for adaptation (7, 31, 33, 34, 44). The SS subfraction is more labile than the IMF subfraction, displaying greater adaptive changes during conditions of chronic muscle use or disuse and in disease (20, 21). The purposes of this study were to determine 1) the apoptotic susceptibility of skeletal muscle SS and IMF mitochondria, 2) whether there are any differences in the expression of apoptosis-related proteins in SS and IMF mitochondria, and 3) ROS production in SS and IMF mitochondria.


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Animals. Male Sprague-Dawley rats weighing between 300 and 350 g (n = 31; Charles River Laboratories, St. Constant, QC, Canada) were housed individually and allowed access to food and water ad libitum. Rats were anesthetized with pentobarbital sodium (60 mg/kg), and the quadriceps muscle groups from both hindlimbs were excised and used to isolate mitochondria. The use of animals was approved by the York University Animal Care Committee. Animals were treated in accordance with Canadian Council of Animal Care guidelines.

Mitochondrial isolation. Quadriceps muscles were quickly excised and immediately placed into ice-cold buffer, briefly minced, and homogenized. IMF and SS mitochondria were fractionated by performing differential centrifugation as described previously in detail (7, 31, 44). Mitochondria were resuspended in resuspension medium (100 mM KCl, 10 mM MOPS, and 0.2% BSA). After the isolation procedure, SS and IMF mitochondria were used for analyses of protein release and expression, ROS production, membrane potential, and mtPTP opening.

Protein release assay. Isolated SS and IMF mitochondrial fractions (50 µl) were incubated with mitochondrial resuspension medium containing 50 mM FeSO4 and concentrations of H2O2 ranging from 0 to 100 µM. Incubations occurred for 60 min at 30°C. Reaction mixtures were then centrifuged at 14,000 g (4°C) to pellet mitochondria, and the supernatant was collected for subsequent assessment of AIF and cytochrome c using Western blot analysis. We chose a range of H2O2 concentrations and incubation times previously shown to induce cytochrome c release in cells in tissue culture (14, 45), because to date no literature exists with respect to isolated mitochondria from skeletal muscle.

Mitochondrial permeability transition pore assessment. mtPTP opening is facilitated under conditions of elevated Ca2+ concentration [Ca2+] and exacerbated when combined with oxidative stress (18, 19, 22, 47). mtPTP opening causes massive mitochondrial swelling, outer membrane rupture, and release of proapoptotic factors that can induce apoptosis. mtPTP opening was measured by monitoring the decrease in light scattering associated with mitochondrial swelling at 540 nm. Isolated SS and IMF mitochondria were resuspended (to obtain a final concentration of 1 mg/ml) in a buffer containing 215 mM mannitol, 71 mM sucrose, 3 mM HEPES, and 5 mM succinate (pH 7.4). SS and IMF mitochondria were treated with 400 µM CaCl2 and 75 µM tert-butyl hydroperoxide (t-BuOOH). The decrease in absorbance was monitored for 15 min with a spectrophotometer (Beckman DU-64).

Mitochondrial respiration. Samples of isolated IMF and SS mitochondria were incubated with 2 ml of VO2 buffer (250 mM sucrose, 50 mM KCl, 25 mM Tris·HCl, 10 mM K2HPO4, and 0.2% BSA, pH 7.4) at 30°C in a water-jacketed respiratory chamber with continuous stirring. Respiration rates (n atoms O2·min–1·mg–1) were evaluated in the presence of 10 mM glutamate (state 4 respiration) or 70 mM ADP (state 3 respiration) with the use of a Clark oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) as demonstrated previously (31, 44).

ROS assay and mitochondrial respiration. SS and IMF mitochondria (50 µg) were incubated with 50 µM dichlorodihydrofluorescein diacetate (H2DCFDA) and VO2 buffer at 37°C for 80 min in a 96-well plate. ROS production is directly proportional to fluorescence emission (between 480 and 520 nm) measured using a multidetection microplate reader (Synergy HT; Bio-Tek Instruments, Winooski, VT). Microplate data were compiled and analyzed using KC4 software (version 3.0). To assess ROS production during states 3 and 4 respiration, the addition of 10 mM glutamate and 70 mM ADP, respectively, were combined with the mitochondria immediately before the addition of H2DCFDA.

Immunoblotting. Isolated IMF and SS mitochondrial protein extracts or supernatant aliquots from the protein release assay were separated by performing 12% SDS-PAGE and subsequently electroblotted onto nitrocellulose membranes. After being transferred, membranes were blocked (1 h) with 5% skim milk in 1x TBST solution (Tris-buffered saline with Tween 20, 25 mM Tris·HCl, pH 7.5, 1 mM NaCl, and 0.1% Tween 20). Blots were then incubated in blocking buffer with antibody directed against AIF (1:1,500 dilution), cytochrome c (1:750 dilution), Mn2+-superoxide dismutase (Mn-SOD; 1:2,000 dilution), cyclophilin D (1:400 dilution), Bax (1:500 dilution), Bcl-2 (1:1,000 dilution), VDAC (1:1,000 dilution), or ANT (1:2,000 dilution) overnight at 4°C. After being washed three times for 5 min each with TBST, blots were incubated at room temperature (for 45 min) with the appropriate secondary antibody coupled to horseradish peroxidase and washed again three times for 5 min each with TBST. Antibody-bound protein was revealed using the ECL method. Films were scanned and analyzed using SigmaGel software (Jandel Scientific, San Rafael, CA).

Flow cytometric analysis of IMF and SS mitochondrial membrane potential. Flow cytometry was performed using a three-color FACSCalibur cytometer equipped with a 488-nm argon laser (Becton Dickinson, San Jose, CA) (see Fig. 6). The analysis of IMF and SS mitochondria was performed after the forward-angle light scatter (FSC) and side-angle light scatter (SSC) detectors were set using the {Delta}{Psi}-insensitive mitochondrial dye Mitotracker Green FM, which is similar to the method used by Lecoeur et al. (28). This method allowed for the discrimination of mitochondria from debris. To measure {Delta}{Psi} of the isolated IMF and SS mitochondria, the fluorescent probe 5,5',6,6',-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used. A JC-1 stock solution of 1 mg/ml in DMSO was diluted in mitochondrial resuspension buffer to a final working concentration of 4 µg/ml. JC-1 solutions were vortexed upon dilution to avoid aggregate formation (15). IMF and SS mitochondria were suspended in the JC-1 working solution for 30 min at 37°C in the dark. Samples were then centrifuged and resuspended in fresh mitochondrial resuspension buffer without JC-1 and immediately analyzed using the flow cytometer. JC-1 monomers were collected in the green FL-1 channel (530/30 nm). JC-1 orange fluorescence, caused by the formation of J-aggregates, was measured using the FL-2 channel (585/21 nm). To depolarize the mitochondria, 2,4-dinitrophenol (DNP; 200 µM) was added to the JC-1-stained mitochondria after resuspension, and the samples were then analyzed. All analyzed data were derived from at least 20,000 gated events.



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Fig. 6. Flow cytometric analysis of IMF and SS mitochondria stained with the fluorescent probe 5,5',6,6',-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). A: overlay scatterplot of JC-1 green vs. orange fluorescence of each mitochondrial population. B: JC-1 orange fluorescence overlay histogram of IMF and SS mitochondria. C: JC-1 green fluorescence overlay histogram of IMF and SS mitochondria. D: quantification of the mean geometric orange fluorescence of JC-1-stained IMF, SS, and 2,4-dinitrophenol (DNP) mitochondrial subfractions.

 
Statistical analyses. Data are expressed as means ± SE. Paired Student's t-tests were used for comparison of data obtained from IMF and SS mitochondria. Statistical differences were considered significant at P < 0.05.


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Effect of H2O2 on AIF and cytochrome c release from isolated SS and IMF mitochondria. A progressive, linear increase in cytochrome c and AIF release was evident in SS and IMF mitochondria after 0, 15, 30, and 60 min of H2O2 treatment (data not shown). Thus, using the 60-min incubation time, we examined the dose-dependent release of protein using increasing concentrations of H2O2 at 0, 25, 50, and 100 µM (Fig. 1, A and B). At 0 µM H2O2, IMF mitochondria possessed a 4.1- and 2.3-fold (P < 0.05 and P = 0.058, respectively) greater basal release of cytochrome c and AIF, respectively, than SS mitochondria (Fig. 1C). With increasing concentrations of H2O2, IMF mitochondria exhibited an ~2.5-fold greater release of cytochrome c (Fig. 1A) and a 10-fold greater AIF release (Fig. 1B) compared with SS mitochondria. In addition, H2O2 treatment caused similar release rates (2- to 3-fold) for both cytochrome c and AIF in SS mitochondria but resulted in differential release rates of cytochrome c (5- to 7-fold) and AIF (10- to 15-fold) in IMF mitochondria.



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Fig. 1. A and B: dose-dependent release of cytochrome c (A) and apoptosis-inducing factor (AIF) (B) from subsarcolemmal (SS; {blacksquare}) and intermyofibrillar (IMF; {lozenge}) mitochondria in response to progressive increases in H2O2 concentrations (0, 25, 50, and 100 µM; n = 4–7/concentration) for 60 min at 30°C. C: basal release (in the absence of H2O2 treatment) of cytochrome c and AIF from SS and IMF mitochondria after incubation for 60 min at 30°C (n = 10). Representative Western blot analyses for cytochrome c and AIF released into the supernatant at each H2O2 concentration (A and B) and without H2O2 treatment (C) are shown above the graphs.

 
Function and composition of the mtPTP in SS and IMF mitochondria. We measured SS and IMF mtPTP opening characteristics using Ca2+ and t-BuOOH to assess whether the differences in H2O2-induced protein release from SS and IMF mitochondria could be attributed to functional differences in the mtPTP between mitochondrial subfractions. This treatment specifically targets the mtPTP because preincubation of mitochondria with the pore inhibitor cyclosporin A significantly reduced mtPTP opening (Fig. 2A). IMF mitochondria exhibited a 52% greater time to Vmax and a 44% higher Vmax compared with SS mitochondria (P < 0.05; Fig. 2, B and C). To determine whether these functional differences were related to mtPTP composition, we measured individual components of the pore. VDAC levels were greater by 47% (P < 0.05), while cyclophilin D was 60% higher (0.05 < P < 0.1), compared with SS mitochondria (Fig. 3, B and C). In contrast, there was no difference in ANT levels between the two mitochondrial subfractions (Fig. 3A).



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Fig. 2. Mitochondrial transition pore kinetics from SS and IMF mitochondria (n = 9). Isolated SS and IMF mitochondria were treated with a high concentration of Ca2+ in conjunction with a reactive oxygen species (ROS) agent (400 µM CaCl2 and 75 µM tert-butyl hydroperoxide, t-BuOOH) to specifically target and open the mitochondrial permeability transition pore (mtPTP). A: typical mtPTP pore kinetics in SS mitochondria quantified by measuring the maximal rate of the decrease in absorbance (Vmax) and also the time required to reach Vmax. The specificity of pore opening was verified using the mtPTP inhibitor cyclosporin A (CsA) to suppress the drop in absorbance after treatment with CaCl2 and t-BuOOH [dashed lines, CsA treated; solid line, control (no CsA)]. B: effect of CaCl2 and t-BuOOH treatment on the time to Vmax in SS and IMF mitochondria. IMF mitochondria exhibited a 52% longer time than SS mitochondria to Vmax. *P < 0.05. C: effect of CaCl2 and t-BuOOH treatment on Vmax in SS and IMF mitochondria. Vmax was significantly elevated by 44% in IMF mitochondria compared with SS mitochondria after treatment with CaCl2 and t-BuOOH. *P < 0.05.

 


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Fig. 3. Expression of mtPTP components adenine nucleotide translocase (ANT), voltage-dependent anion channel (VDAC), and cyclophilin D from SS and IMF using Western blot analysis (n = 7–10). A: no difference existed in ANT levels between SS and IMF mitochondria. B: VDAC expression was 47% greater in IMF compared with SS mitochondria. *P < 0.05. C: cyclophilin D expression was 60% higher (0.05 < P < 0.1) in IMF compared with SS mitochondria.

 
Content of apoptotic proteins in SS and IMF mitochondria. We evaluated the content of cytochrome c and AIF in SS and IMF mitochondria to determine whether the mitochondrial subfractions contained inherently different levels of proapoptotic proteins (Fig. 4, AD) . Cytochrome c levels were threefold greater in IMF compared with SS mitochondria. In contrast, AIF expression was similar between the mitochondrial subfractions. Antiapoptotic Bcl-2 expression was not different between SS and IMF mitochondria. In marked contrast, proapoptotic Bax levels were ~10-fold greater in SS compared with IMF mitochondria (P < 0.05). Thus the Bax-to-Bcl-2 ratio was much higher in SS mitochondria compared with the IMF subfraction.



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Fig. 4. Apoptotic protein levels in SS and IMF mitochondria examined using Western blot analysis (n = 3–10/protein). A: cytochrome c levels are approximately threefold higher in IMF than in SS mitochondria. *P < 0.05. B: AIF expression levels are similar between mitochondrial subfractions. C: Bax levels are dramatically greater, by ~10-fold, in SS compared with IMF mitochondria. *P < 0.05. D: Bcl-2 expression does not differ between SS and IMF mitochondria. AU, arbitrary scanner units.

 
Mitochondrial ROS production and Mn-SOD expression. ROS production was greater during resting respiration (state 4) compared with active respiration (state 3) in both SS and IMF mitochondria (Fig. 5A) . However, ROS production in the SS mitochondrial subfraction was ~2.7-fold greater than in IMF mitochondria during both state 3 (ADP stimulated) or state 4 respiration (Fig. 5A). We have previously shown that states 3 and 4 respiration rates in SS mitochondria are ~2.5-fold lower than in IMF mitochondria (7, 31). This indicates that oxygen consumption is inversely related to ROS production. Expression of the antioxidant enzyme Mn-SOD was not different between SS and IMF mitochondria (Fig. 5B).



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Fig. 5. A: ROS production in SS and IMF mitochondria during state 3 (ADP-stimulated) and state 4 (glutamate-stimulated) respiration (n = 12). ROS production is ~2.5-fold greater in SS compared with IMF mitochondria during both states 3 and 4 respiration. AFU, arbitrary fluorescence units. B: Mn2+-superoxide dismutase (Mn-SOD) expression is similar between mitochondrial subfractions (n = 7). AU, arbitrary scanner units.

 
Flow cytometric analysis of SS and IMF membrane potential. Isolated IMF and SS mitochondrial membrane potentials were examined using the lipophilic fluorescent cation dye JC-1. JC-1 can detect variations in {Psi} at the organelle level by reversibly changing its emitted light from green to orange as the membrane potential increases. In the depolarized state, JC-1 maintains a green-emitting monomeric form, which is subject to aggregation with increasing potential, thus causing a shift in the emitted light to orange. A dot plot of JC-1 green and orange fluorescence for isolated IMF and SS mitochondria is shown in Fig. 6A . At the same level of green fluorescence, a twofold higher JC-1 fluorescence was observed in SS compared with IMF mitochondria, indicating a greater {Psi} in SS mitochondria (Fig. 6, B and C). To assess any differences in nonspecific staining between IMF and SS mitochondria, the resultant JC-1 green and orange fluorescent intensities were examined after the addition of the mitochondrial depolarizing agent DNP. As expected, DNP induced a significant decrease in orange fluorescence to values that were not significantly different between IMF and SS mitochondria (Fig. 6D). This indicates that there is no difference in the nonspecific JC-1 staining of these mitochondrial subfractions.


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Apoptosis has convincingly been shown to occur in skeletal muscle, and it contributes to the overall muscle atrophy and degeneration observed during various physiological and pathophysiological conditions (1). At least two initiating pathways are possible. The first involves the activation of cell surface receptors via external stimuli, while the second is mediated internally and leads to the release of proapoptotic proteins from mitochondria. ROS represent one of the primary triggers that evoke mitochondrial protein release. ROS can be derived from mitochondria or, alternatively, via extracellular pathways (5, 35).

In skeletal muscle, mitochondria exist in reticulum-like structures (24) that are localized beneath the sarcolemma (subsarcolemmal or SS mitochondria) as well as between the myofibrils (intermyofibrillar or IMF mitochondria). These pools of mitochondria can be separated during isolation, and numerous studies have revealed that they possess subtle but distinct biochemical and physiological properties (7, 31, 44). This raises the possibility that their contribution to the mitochondrial apoptotic pathway may also differ and that the release of proapoptotic proteins such as cytochrome c and AIF may vary in different cellular compartments of the muscle. The primary purpose of our study was to investigate the potential contributions of both SS and IMF mitochondrial subfractions to apoptosis in skeletal muscle. For this purpose, we developed an in vitro assay to investigate the susceptibility of isolated mitochondria to ROS-induced release of both cytochrome c and AIF. In both subfractions, our assay revealed a dose- and time-dependent (data not shown) release of cytochrome c and AIF. This likely shows the overall potential of the mitochondrial pathway to induce apoptosis in muscle cells. However, the contribution of this mitochondrial pathway to the overall incidence of apoptosis observed in a variety of muscle-linked pathologies (1, 36) remains unknown.

Our data demonstrate that IMF mitochondria were more sensitive to, and also released a greater amount of AIF and cytochrome c in response to, exogenously added H2O2, as well as in the basal state. To determine the reason for this finding, we examined the function and composition of the mtPTP in the two subfractions, because ROS-induced protein release is mediated via the opening of this pore (8, 36). Although the time required to open the mtPTP was somewhat longer for IMF mitochondria, the rate of opening was 44% greater than for SS mitochondria. This greater Vmax of pore opening in IMF mitochondria may be related to differential composition of the mtPTP or to a higher number of functional pores per mitochondrion. The opening of the mtPTP is partially dependent on contact sites between the outer membrane protein, VDAC, and the inner mitochondrial proteins, ANT and cyclophilin D (8, 9). VDAC and cyclophilin D levels are greater in IMF mitochondria than in SS mitochondria, while no difference in ANT levels between the two subfractions was observed. ANT levels may be less critical in determining the kinetics of pore opening, because mitochondria isolated from ANT–/– mice remain capable of opening the mtPTP (25), implying a greater importance for either VDAC and/or cyclophilin D. The role of cyclophilin D is primarily to interact with ANT, transforming it from a selective ADP-ATP antiporter to a nonselective pore component in mtPTP formation (17, 29). Overexpression of cyclophilin D results in a greater susceptibility to mtPTP formation by Ca2+ and oxidative stress (29). Thus, despite the lack of difference in ANT levels between mitochondrial subfractions, our data suggest that the tendency toward a greater expression of cyclophilin D as well as VDAC may be responsible for the enhanced sensitivity to exogenous ROS and the greater rate of mtPTP opening in the IMF mitochondrial subfraction. In addition, we conclude that this greater sensitivity to ROS is also partly responsible for the higher basal release of AIF and cytochrome c in the absence of the exogenous addition of H2O2. Two other factors contribute to this basal release rate. The first is the rate of endogenous mitochondrial ROS production, and the second is the concentration of cytochrome c or AIF within the mitochondrial subfraction. With respect to AIF, the concentration of the protein is identical in the two subfractions (Fig. 4B), but the rate of ROS production is 2.7-fold higher in SS mitochondria (Fig. 5A). This would be expected to lead to a higher rate of AIF release from SS mitochondria. However, the sensitivity of AIF release to ROS is ~10-fold greater in the IMF subfraction. This effectively counteracts the higher endogenous ROS production in SS mitochondria and results in a greater basal release of AIF from the IMF subfraction in the absence of added H2O2. In the case of cytochrome c, the greater sensitivity of the IMF subfraction to exogenous ROS (2.5-fold) should be offset by the higher internal ROS production of the SS mitochondria, a situation that should result in identical cytochrome c release rates under basal conditions. Thus we think that the observed greater basal release rate of cytochrome c in IMF mitochondria is due to the higher endogenous levels of the protein (3- to 4-fold) within this subfraction.

The higher cytochrome c content within the IMF subfraction is in accordance with previous studies showing that IMF mitochondria possess greater oxidative enzyme activities (7, 33, 34) and respiration rates (23, 31, 44) compared with SS mitochondria. Thus the elevated cytochrome c content is an important reason for the enhanced oxidative phosphorylation in IMF mitochondria, but it also represents a larger pool of proapoptotic protein compared with SS mitochondria, and this likely contributes to its greater rate of release from IMF mitochondria under basal and H2O2-induced conditions. Clearly, the difference in the mitochondrial content of AIF cannot account for the ~10-fold higher sensitivity of AIF release to H2O2 concentration, and this difference in release is more likely related to the greater Vmax of mtPTP opening in IMF mitochondria.

The ROS-induced release of proapoptotic proteins appears to be dissociated from the content of Bax and Bcl-2 within skeletal muscle mitochondria. Traditionally, the Bax-to-Bcl-2 ratio has been used as a barometer of apoptotic susceptibility. However, mitochondria isolated from Bax+/+ and Bax–/– cells show no apparent difference in Ca2+-induced mtPTP opening (10). In addition, others have shown that simply increasing the mitochondrial Bax-to-Bcl-2 ratio is insufficient to promote mtPTP formation and/or induce cell death (13). Our data demonstrate a much greater Bax-to-Bcl-2 ratio in SS mitochondria, despite faster protein release rates exhibited by IMF mitochondria. Thus mitochondrial Bax expression may play a lesser role than previously thought in invoking mitochondrially driven apoptosis (27, 39), at least in skeletal muscle. The possibility that other Bcl-2 family members (i.e., t-Bid; Refs. 27, 38, 39) may be differentially expressed in SS and IMF mitochondria to affect mtPTP inducibility remains to be determined.

We wanted to relate our data on ROS-induced apoptotic protein release to endogenous rates of ROS production within each mitochondrial subfraction. Our results confirm the expectation that ROS production is highest in the absence of ADP (i.e., state 4 respiration), when the mitochondrial membrane potential ({Delta}{Psi}) is highest (3, 6, 26). In contrast, in the presence of ADP, proton flux occurs through the F1F0-ATPase, thereby reducing {Delta}{Psi} and diverting electron flux to cytochrome c oxidase, and less to the formation of ROS. Thus ROS production is inversely related to the rate of oxygen consumption, and this was evident in both the SS and IMF mitochondrial subfractions. This inverse relationship is further fortified by the observation that rates of states 3 and 4 respiration are significantly higher in IMF mitochondria (7, 31), while ROS production is lower in this mitochondrial subfraction compared with SS mitochondria. Similar results have been reported previously (40). Our data also demonstrate that the higher overall ROS production in SS mitochondria is associated with greater membrane potential as determined using flow cytometry. This association between ROS production and membrane potential is established (26), and it argues against an artifactual production of ROS because of selective membrane damage in SS mitochondria. Physiologically, the higher ROS production in the SS mitochondrial subfraction may contribute to its greater potential for adaptation during conditions of exercise (20, 21) or disease (37). For example, ROS are known activators of transcription factors such as NF-{kappa}B. The activation of these factors may contribute to altering the expression of nuclear genes encoding mitochondrial proteins, leading to disparate mitochondrial adaptations within different regions of the muscle cell.

Because ROS production differed in the two mitochondrial subfractions, we measured the expression of Mn-SOD, a primary mitochondrial antioxidant enzyme involved in quenching ROS concentrations. We hypothesized that if SS mitochondria had a greater antioxidant enzyme activity, this could serve to offset elevated ROS production and reduce ROS-induced damage within SS mitochondria. However, we found no difference in the expression of Mn-SOD among mitochondrial subfractions. This does not preclude the possibility that other antioxidant enzymes (glutathione peroxidase and catalase) may be expressed differentially in SS and IMF mitochondria.

In conclusion, our data reveal that the composition of the mtPTP differs between SS and IMF mitochondria. This difference in composition results in a greater rate of mtPTP opening within the IMF subfraction in response to ROS and, consequently, a greater rate of cytochrome c and AIF release. However, it is unlikely that the magnitude of this difference in protein release rates is retained in intact cells, because it would be tempered by the two- to threefold higher endogenous ROS production in SS mitochondria over a broad range of respiration rates. However, as discussed above, accounting for this difference does not equalize the rates of AIF and cytochrome c release rates from SS and IMF mitochondria. Taken together with the relatively greater abundance of IMF (~80%) compared with SS (~20%) mitochondria within the muscle cell (21), this evidence points to a potentially dominant role for the IMF subfraction in evoking the mitochondrially driven apoptosis pathway in muscle. Whether this remains during the induction of specific pathological states leading to myonuclear decay, muscle atrophy, and muscle degeneration, or in aging-induced sarcopenia remains to be determined.


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This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant (to D. A. Hood). P. J. Adhihetty is a recipient of a Heart and Stroke Foundation of Canada Doctoral Fellowship. V. Ljubicic is a recipient of an NSERC Postgraduate Scholarship. D. A. Hood holds a Canada Research Chair in Cell Physiology.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Hood, School of Kinesiology and Health Science, York Univ., Farquharson Building, 302, Toronto, ON, Canada M3J 1P3 (e-mail: dhood{at}yorku.ca)

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.


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