Free
Eye Movements, Strabismus, Amblyopia and Neuro-ophthalmology  |   January 2009
Lower Respiratory Capacity in Extraocular Muscle Mitochondria: Evidence for Intrinsic Differences in Mitochondrial Composition and Function
Author Affiliations
  • Samir P. Patel
    From the Spinal Cord and Brain Injury Research Center and the
    Department of Physiology, University of Kentucky, Lexington, Kentucky.
  • Jorge L. Gamboa
    Department of Physiology, University of Kentucky, Lexington, Kentucky.
  • Colleen A. McMullen
    Department of Physiology, University of Kentucky, Lexington, Kentucky.
  • Alexander Rabchevsky
    From the Spinal Cord and Brain Injury Research Center and the
    Department of Physiology, University of Kentucky, Lexington, Kentucky.
  • Francisco H. Andrade
    Department of Physiology, University of Kentucky, Lexington, Kentucky.
Investigative Ophthalmology & Visual Science January 2009, Vol.50, 180-186. doi:10.1167/iovs.08-1911
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Samir P. Patel, Jorge L. Gamboa, Colleen A. McMullen, Alexander Rabchevsky, Francisco H. Andrade; Lower Respiratory Capacity in Extraocular Muscle Mitochondria: Evidence for Intrinsic Differences in Mitochondrial Composition and Function. Invest. Ophthalmol. Vis. Sci. 2009;50(1):180-186. doi: 10.1167/iovs.08-1911.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

purpose. The constant activity of the extraocular muscles is supported by abundant mitochondria. These organelles may enhance energy production by increasing the content of respiratory complexes. The authors tested the hypothesis that extraocular muscle mitochondria respire faster than do mitochondria from limb muscles because of the higher content of respiratory complexes.

methods. Inner mitochondrial membrane density was determined by stereological analysis of triceps surae (a limb muscle) and extraocular muscles of adult male Sprague-Dawley rats. The authors measured respiration rates of isolated mitochondria using a Clark-type electrode. The activity of respiratory complexes I, II, and IV was determined by spectrophotometry. The content of respiratory complexes was estimated by Western blot.

results. States 3, 4, and 5 respiration rates in extraocular muscle mitochondria were 40% to 60% lower than in limb muscle mitochondria. Extraocular muscle inner mitochondrial membrane density was similar to that of other skeletal muscles. Activity of complexes I and IV was lower in extraocular muscle mitochondria (approximately 50% the activity in triceps), but their content was approximately 15% to 30% higher. There was no difference in complex II content or activity or complex III content. Finally, complex V was less abundant in extraocular muscle mitochondria.

conclusions. The results demonstrate that extraocular muscle mitochondria respire at slower rates than mitochondria from limb muscles, despite similar mitochondrial ultrastructure. Instead, differences were found in the activity (I, IV) and content (I, IV, V) of electron transport chain complexes. The discrepancy between activity and content of some complexes is suggestive of alternative subunit isoform expression in the extraocular muscles compared with limb muscles.

Voluntary and reflexive eye movements initiated by the ocular motor system rely on the fast and constant activity of the extraocular muscles. These small skeletal muscles have one of the highest mitochondrial contents of mammalian skeletal muscles. 1 This has been considered to reflect the metabolic demands imposed by their activation patterns. Recently, we showed that mitochondria also serve as Ca2+ sinks and influence the kinetics of cytosolic free Ca2+ concentration during the activation of extraocular muscle fibers, increasing the dynamic response range for this muscle group. 2  
Aerobic capacity is typically measured by mitochondrial volume density (percentage of muscle fiber occupied by mitochondria). In general, mitochondrial volume density is well matched to the metabolic needs of skeletal muscle, and it scales almost linearly with maximal oxygen uptake among muscles and across mammalian species. 3 4 In other words, the consensus is that changes in the oxidative (aerobic) capacity of mammalian skeletal muscles are met by corresponding increases or decreases in mitochondrial volume density. Because the mitochondrial content and the activity of respiratory complexes and enzymes of mitochondrial metabolic pathways change in parallel, enzymatic activities are used as indices of mitochondrial content and aerobic capacity. Highly aerobic muscle groups in mammals have abundant capillaries and elevated mitochondrial volume density. 5 6 That is clearly not the case in some extremely aerobic nonmammalian skeletal muscles in which the increase in oxidative capacity is achieved by combining high mitochondrial content with modified mitochondrial ultrastructure and composition. For example, the mitochondrial cristae in hummingbird flight muscles are packed tightly, increasing their ability to use oxygen by more than 50% over the maximal limit in mammalian muscle mitochondria. 7  
Given that the extraocular muscles have arguably the highest mitochondrial content of all mammalian skeletal muscles, it seemed likely their mitochondrial density would be high enough to require changes in their composition, function, or both to maximize aerobic capacity without restricting contractile performance. We anticipated that, to minimize the mitochondrial volume needed to sustain the high activation patterns of the extraocular muscles, mitochondrial respiratory capacity would be enhanced by increasing protein content. Therefore, the purpose of this study was to test the hypothesis that extraocular muscle mitochondria respire at faster rates than those from limb muscles because of a higher content of respiratory complexes. We measured the in vitro respiration rates of mitochondria isolated from extraocular and limb muscles. Then we determined whether the activities and relative content of mitochondrial respiratory complexes correlated with differences in respiration between mitochondria isolated from these two muscle groups. 
Materials and Methods
Animals
The use of experimental animals was approved by the Institutional Animal Care and Use Committee at the University of Kentucky and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult male Sprague-Dawley rats (300–350 g; Harlan, Indianapolis, IN) were anesthetized with ketamine hydrochloride/xylazine hydrochloride (100 mg/8 mg/kg body weight injected intraperitoneally). For electron microscopy, rats were perfused transcardially with phosphate-buffered saline (pH 7.4) and then with 2% paraformaldehyde/4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and 130 mM NaCl. For biochemistry, rats were killed by exsanguination after medial thoracotomy; then extraocular muscles (eight rectus and two superior oblique muscles pooled per animal), and midbelly samples of triceps surae (a mixed fiber-type limb muscle) were dissected and used immediately to isolate mitochondria. 
Morphometry
Perfusion-fixed extraocular muscle samples were postfixed in 1% osmium tetroxide, stained en bloc in uranyl acetate, dehydrated in a methanol series and propylene oxide, and embedded in epoxy resin. Thin (70-nm) sections were stained with uranyl acetate and lead citrate and were photographed with a transmission electron microscope (Tecnai 12; Philips, Eindhoven, Netherlands). Four extraocular muscles from two rats were studied. In each muscle, five fibers were selected randomly; then four mitochondria per fiber were imaged at a magnification of 120,000×. The surface density of inner mitochondrial membranes (cristae) was determined from digital image files using a standard point-counting method (144-point square-grid) and assuming randomly oriented structures. 8 9  
Mitochondrial Isolation
On dissection, muscles were placed in 4 mL ice-cold isolation buffer with 1 mM EGTA (215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin [BSA], 20 mM HEPES, 1 mM EGTA; pH adjusted to 7.2 with KOH). Nagarse or trypsin (0.25 mg/mL isolation buffer) was added, and the tissue was minced with scissors and homogenized in ice with a motor-driven (Potter-Elvehjem; Kimble/Kontes, Vineland, NJ) homogenizer. A protease inhibitor cocktail (40 μL/4 mL homogenate; P8340; Sigma, St. Louis, MO) was added to the homogenates with trypsin. The homogenates were centrifuged at 600g for 5 minutes at 4°C, and the supernatant was decanted and centrifuged at 5000g for 10 minutes at 4°C. The resultant mitochondrial pellets were resuspended in isolation buffer at a concentration of approximately 12 to 15 mg/mL and were stored on ice until subsequent use. We confirmed that this method resulted in a pure mitochondrial preparation by following the disappearance of transaminase (cytosolic enzyme, monoclonal antibody; Developmental Studies Hybridoma Bank, University of Iowa) and the enrichment of mitochondrial proteins from the initial homogenate to the final mitochondrial pellet in Western blots. Figure 1shows that transaminase was present in the cytosolic fraction (lanes labeled c) in both muscles and that it disappeared in the mitochondrial pellet (lanes labeled m). In contrast, complexes IV and V went from barely detectable in the cytosolic fraction to abundant in the mitochondrial pellet. 
Mitochondrial Respiration
Mitochondrial respiration was measured with a miniature Clark-type electrode (Hansatech Instruments, Norfolk, UK) in a sealed, thermostatically controlled chamber at 37°C, as described previously. 10 Briefly, mitochondria were added to the chamber containing respiration buffer (215 mM mannitol, 75 mM sucrose, 2 mM MgCl2, 2.5 mM inorganic phosphates, 0.1% BSA, 20 mM HEPES, pH 7.2) to yield a mitochondrial protein concentration of approximately 200 to 300 μg/mL in 250 μL (final volume). Measurement of respiration was started with the addition of pyruvate (5 mM) and maleate (2.5 mM) and was designated as state 2 respiration, followed by the addition of 0.75 μM adenosine diphosphate (ADP; state 3 respiration). 11 12 Oligomycin (1 μM, an adenosine triphosphate [ATP] synthase inhibitor) was added to induce state 4 respiration, a measure of mitochondrial uncoupling activity. 11 12 13 State 5 respiration was induced with the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 μM) to get pyruvate + maleate-driven (complex I) maximum uncoupled electron transport. The complex I inhibitor rotenone (0.8 μM) was then added to the buffer, followed by succinate (10 mM), to allow for quantification of complex II-driven maximum electron transport (state 5 succinate). Respiration states were calculated from the oxygen consumption slopes as nanomoles of oxygen consumed per minute per milligram of mitochondrial protein. The respiratory control ratio (RCR) was calculated by dividing the slope of the response of isolated mitochondria to state 3 respiration (presence of ADP) by slope of the response to oligomycin-induced state 4 respiration. 
Activity of Mitochondrial Respiratory Complexes
The activities of selected respiratory complexes were measured (Multi-detection Microplate Reader; Bio-Tek Instruments, Winooski, VT) and were normalized to milligram of mitochondrial protein. First, mitochondria were diluted in 10 mM phosphate buffer to a concentration of 1 mg/mL, freeze thawed, and sonicated three times. Complex I activity was measured as the rotenone-sensitive decrease in NADH absorption at 340 nm with ubiquinone-1 as the final acceptor, as previously described, 14 with slight modifications. The assay was performed in 25 mM KPO4 buffer (pH 7.2) containing mitochondrial protein (6 μg), 5 mM MgCl2, 1 mM KCN, 1 mg/mL BSA, and 150 μM NADH. The reaction was preincubated for 2 minutes at 30°C and was initiated by the addition of ubiquinone-1 (50 μM). NADH fluorescence was monitored (340 nm excitation, >450 nm emission) over time. The assay was also performed in the presence of rotenone (10 μM) to determine the rotenone-insensitive activity. Rotenone-sensitive complex I activity was calculated by subtracting the rotenone-insensitive activity from the total activity. 
The complex II (succinate dehydrogenase) assay was performed in 100 mM KPO4 buffer, 20 mM succinate, 10 μM EDTA, 0.01% Triton, 1 μg/100 μL coenzyme Q2 containing mitochondrial protein (6 μg) at 30°C. The reaction was initiated by the addition of 0.07% 2,6-dichloroindophenol, and the rate of reduction of coenzyme Q by succinate was determined by following the reduction of 2,6-ichloroindophenol at 600 nm. 15  
Complex IV (cytochrome c oxidase) assay was carried out in 10 mM KPO4 buffer and 50 μM reduced cytochrome c. The reaction was initiated by the addition of 6 μg mitochondrial protein. The rate of oxidation of cytochrome c was measured at 30°C by following the decrease in absorbance of reduced cytochrome c at 550 nm. 16 17  
Content of Mitochondrial Respiratory Complexes
We used Western blots to compare the differences in the content of representative subunits of the respiratory complexes between mitochondria isolated from extraocular muscle and triceps surae. Only mitochondria isolated with trypsin were used because nagarse is not compatible with the sample buffer used for SDS-PAGE and leads to almost complete proteolysis (results not shown and Wilson et al. 18 ). Mitochondrial proteins (15 μg/lane) were resolved electrophoretically in 10% to 20% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) and were transferred to polyvinylidene difluoride membranes (Immobilon-FL; Millipore, Billerica, MA). Equivalent protein loading was confirmed by total protein stain with Ponceau S. This strategy avoids the biases potentially introduced by the use of “housekeeping” proteins as loading controls. 19 20 Membranes were blocked for 1 hour at room temperature and were incubated overnight with mouse monoclonal antibodies (Invitrogen, Carlsbad, CA) against the following subunits of mitochondrial respiratory complexes: α subcomplex of complex I (39 kDa), iron-sulfur protein of complex II (30 kDa), core I of complex III (51.6 kDa), subunit VIb of complex IV (10 kDa), and subunit α of complex V (F1F0-ATPase subunit a, 59.6 kDa). After the membranes were washed with phosphate-buffered saline and 0.1% Tween, they were incubated for 1 hour with Alexa Fluor 680-conjugated goat anti-mouse secondary antibody (1:7500; Invitrogen) and were washed again with PBS and 0.1% Tween. Membranes were finally rinsed with PBS and scanned (Odyssey Infrared Imaging System; LI-COR Biosciences, Lincoln, NE). Density of the resultant bands was quantified using NIH Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). 21  
Data Analysis
All results are presented as mean ± SE of n observations, unless otherwise noted. Mitochondrial volume density, respiration, and enzymatic activities were compared with the use of Student’s t tests. The significance level for rejection of the null hypothesis was set at P ≤ 0.05 for all comparisons. 
Results
Mitochondrial Bioenergetics
The first step in this study was to compare oxygen consumption by mitochondria isolated from triceps surae and extraocular muscles. Typical oxygen consumption traces for mitochondria isolated from triceps and extraocular muscles are shown in Figure 2 . Compared with triceps, oxygen consumption was slower in mitochondria from extraocular muscles throughout most of the protocol. Respiration rates were calculated in terms of nanomoles of oxygen consumed per minute per milligram of mitochondrial protein. We started the study using nagarse to isolate muscle mitochondria. To check that the enzyme was not responsible for the observed differences, we also measured oxygen consumption rates in mitochondria isolated with trypsin. RCRs after isolation with nagarse and trypsin were similar and in a range consistent with organelle integrity: 7.4 ± 0.3 and 7.8 ± 0.4 in extraocular muscle and 9.1 ± 0.3 and 8.7 ± 0.4 in triceps surae, nagarse, and trypsin. Therefore, data from both enzymes were pooled. Significant differences were noted between mitochondria from triceps and extraocular muscles: the respiration states 3, 4, 5, and 5+ succinate in mitochondria from extraocular muscles were only 45.6% ± 1.8%, 55.2% ± 3.7%, 38.1% ± 3.0%, and 57.6% ± 4.8% of the rates measured in mitochondria from triceps (Fig. 3) . No significant difference was noted for state 2 respiration in mitochondria from extraocular muscle (84.3% ± 11.5% of state 2 in mitochondria from triceps). Importantly, RCR was high for mitochondrial preparations from both muscles: 8.9 ± 0.2 for triceps and 7.5 ± 0.2 for extraocular muscle. Although the difference was statistically significant (P < 0.001), both values were in the range that demonstrated mitochondrial integrity after the isolation procedure. 
Mitochondrial Morphology
The decreased in oxygen consumption rates in extraocular muscle mitochondria could have been caused by the smaller inner mitochondrial membrane (cristae) surface area in these muscles. As exemplified in Figures 4A and 4B , extraocular muscles have one of the highest mitochondrial volume densities (percentage of muscle fiber occupied by mitochondria) of mammalian skeletal muscles. 1 2 The mitochondria in extraocular muscle appears to have a typical internal architecture with densely packed cristae (inner mitochondrial membrane; Fig. 4D ). The estimate of inner mitochondrial membrane surface density (surface area per volume of mitochondria) was 40.9 ± 9.7 m2 cm−3 for extraocular muscle mitochondria, well within the range reported for a variety of mammalian skeletal muscles. 4 8  
Content and Activity of Respiratory Complexes
Given the major differences in the capacity of extraocular muscle mitochondria to consume oxygen, we measured the isolated activities of some of the respiratory complexes (Fig. 5) . The activity of complex I (NADH oxidoreductase) in mitochondria from extraocular muscles was only 55.1% ± 4.5% of the activity measured in triceps (P < 0.001). A similar difference was found for complex IV (cytochrome c oxidase). Its activity in extraocular muscle mitochondria was 47.9% ± 4.9% of the level obtained in triceps (P < 0.001). That was not the case for complex II (succinate dehydrogenase) because we did not detect a significant difference in its activity between the mitochondria from the two muscles; extraocular muscles had 88.6% ± 7.8% of the activity of measured triceps mitochondria. 
To follow up on the enzymatic activity measurements, we estimated respiratory complex content by immunodetection of individual subunits for each complex. Surprisingly, we observed differences in respiratory complex content that did not correspond with enzymatic activity (Fig. 6) . The content of complexes I and IV was significantly higher in extraocular muscle mitochondria (P < 0.05): 116% ± 4.1% and 132.9% ± 7.3% of the content measured in triceps mitochondria, respectively (P < 0.05). In contrast, the content of complex V in extraocular muscle mitochondria was only 42.6% ± 6.2% of triceps mitochondria. Finally, the content of complexes II and III was not significantly different in extraocular muscle mitochondria (79.2% ± 2.5% and 103.1% ± 5.2% of triceps, respectively). 
Discussion
To our surprise, the results did not support our original hypothesis that extraocular muscle mitochondria have faster respiratory rates than mitochondria from limb muscles. Instead, the opposite conclusion seems more likely: mitochondria from extraocular muscles have lower content or lower activity of some enzyme complexes of the electron transport system, causing them to respire at slower rates. These findings were not explained by differences in the ultrastructure of extraocular muscle mitochondria: the surface area of their inner membrane was comparable to values reported for other skeletal muscle. 8 Furthermore, the differences were not generalized or systematic. Complex II content and activity and complex III content were similar in mitochondria from triceps surae and extraocular muscle. Complexes I and IV gave the most puzzling results: their activities were lower but their content was higher in the extraocular muscle mitochondria. These are multimeric protein complexes, and differential expression of isoforms of some subunits has been described in skeletal muscle and other tissues. 22 Therefore, the content of some electron transport chain complexes (I, IV, and V) and the subunit composition of some others (I and IV) may not be the same in extraocular muscles compared with limb muscles. Others and we 23 24 25 26 have already reported evidence of divergence between extraocular and limb muscles in the relative importance of major metabolic pathways, including an alternative mitochondrial biogenesis program in the extraocular muscles. The present study demonstrates that metabolic divergence between extraocular and limb muscles includes major differences in the composition and basic function of their respective mitochondrial populations. 
Mitochondrial Bioenergetics in Extraocular Muscle
The main finding in our study is that the respiration rates of extraocular muscle mitochondria are substantially lower than those in mitochondria from limb muscles. This applies particularly to faster substrate- and ADP-driven (state 3) and uncoupled (state 5) respiration rates that are normalized to mitochondrial protein (Figs. 2 3) , indicating that the activity or content of one or more of the respiratory complexes is lower in the mitochondria from extraocular muscles. Our findings confirmed this notion: the lower activities of complex I and IV and the lower complex V content in extraocular muscle mitochondria compared with triceps surae mitochondria were fully consistent with the observed differences in respiration rates (Figs. 5 6) . To complicate matters, we detected significantly higher content of complexes I and IV in extraocular muscle mitochondria. Because there is no evidence of differences in mitochondrial complex subunit stoichiometry between tissues, we propose that differences in the complex subunit isoforms expressed in extraocular and limb muscle mitochondria will explain the apparent discrepancy between the activity and content of complexes I and IV. Others 27 have reported that mitochondrial composition can vary because of tissue-specific reliance on certain mitochondrial pathways and differential expression of respiratory complex subunit isoforms. For example, cytochrome c oxidase (complex IV) subunits IV, VIa, VIIa, and VIII are known to have tissue-specific expression in mammalian mitochondria. 27 28 29 One gene for each subunit isoform is transcribed only in striated muscle (H isoform), and the other (L isoform) is present in most tissues thus far examined, including at low levels in heart and muscle. 30 31 32 33 The molecular diversity observed for subunit VIa isoforms was proposed to be responsible for tissue-specific regulation of the efficiency of energy transduction in cytochrome c oxidase of heart/muscle mitochondria. 34 Moreover, isoforms of the nuclear-encoded subunits of cytochrome c oxidase affect the activity of the whole complex and are regulated by environmental and developmental signals and probably allow tissues to adjust their energy production to different energy demands 35  
Matching Metabolic Capacity to Contractile Function
The primary role of mitochondria is to generate ATP. Although our results demonstrate that extraocular muscle mitochondria have a lower intrinsic respiratory capacity than mitochondria from limb muscles, we do not know whether this is enough to limit oxygen consumption in vivo. There is evidence of greater blood flow (per muscle mass) to the extraocular muscles. 36 37 However, we are unaware of any data on maximal oxygen uptake (VO2max) by the extraocular muscles in situ that would permit comparison with other skeletal muscles; this is an issue that will require a novel technical approach. Moreover, the content of respiratory complexes is just one parameter behind tissue variations in mitochondrial respiration, and some argue that it is not particularly relevant for metabolic control. 38 Under experimental conditions, mitochondrial respiration in skeletal muscle and heart is regulated at the level of the respiratory chain, whereas in liver, kidney, and brain, it is controlled mainly at the phosphorylation level by ATP synthase (complex V) and phosphate carrier. 38 39 That may not be the case in vivo, when different parameters such as cellular steady state, energy demand, and energy supply of the tissue may also regulate mitochondrial respiration. 39 For extraocular muscles, allosteric regulation of respiratory complexes may combine with changing metabolite concentrations to maintain mitochondrial respiration closer to its theoretical maximum. 38 40 For example, a mechanism to enhance energy production in extraocular muscle is mitochondrial calcium influx during contractile activity. 2 Calcium influx into mitochondria coordinates ATP demand by the contractile apparatus with ATP supply by aerobic metabolism. 41 For extraocular muscles, rapid mitochondrial calcium uptake during contractions would couple metabolic supply to demand. Increases in mitochondrial calcium stimulate the activity of enzyme systems that exert strong control on substrate oxidation: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase and glycerol 3-phosphate dehydrogenase. 42 The combined activity of these enzymes maintains a high NADH/NAD+ ratio and increases the driving force for oxidative phosphorylation. ATP synthase (complex V) and adenine nucleotide translocator may also be activated by calcium. 43  
Metabolic Integration in the Extraocular Muscles
High mitochondrial content is one of the hallmarks of the extraocular muscles (Fig. 4and Porter and Baker 44 ). Recent evidence is beginning to put this fact into the more global perspective of extraocular muscle aerobic metabolism. First, glycogen content is low, and the glycogenolysis pathway seems to be downregulated in the extraocular muscles. 23 24 Second, creatine kinase activity and content, including the mitochondrial isoform present in striated muscles (sarcomeric creatine kinase), are significantly lower in extraocular muscles. 25 45 Moreover, inhibition of creatine kinase activity does not change the fatigue resistance of the extraocular muscles in vitro, indicating that phosphocreatine may be a less important temporal and spatial ATP buffer in these muscles. 45 In other words, mitochondrial ATP production may be sufficiently high and close to cellular sinks as to obviate the need for energy buffers in the form of phosphocreatine or glycogen. Third, extraocular muscles can use lactate as an oxidizable substrate because of the presence of a lactate dehydrogenase isoform that catalyzes the conversion of lactate to pyruvate that then goes to Krebs cycle. 46 These facts indicate that mitochondrial metabolism is the predominant source of ATP in the extraocular muscles. The results of this study demonstrate that these mitochondria are intrinsically different, and their control may depend on processes particular to the extraocular muscles. 
Finally, an intriguing possibility is that extraocular muscle mitochondria could optimize their respiratory capacity by reducing their proton leak, the inverse of the mechanism of thermogenesis in brown fat. Adaptive thermogenesis, the dissipation of energy in the form of heat in response to external stimuli, has been implicated in the regulation of energy balance and body temperature. 47 Brown fat can increase its thermogenic activity by decreasing the efficiency of coupling between respiration and ATP production with uncoupling protein 1 (UCP1; increases the magnitude of the proton leak), leading to the generation of heat. 48 Its homologues UCP2 and UCP3 are expressed skeletal muscle (and other tissues) and may regulate energy metabolism. 47 49 50 Oligomycin-induced state 4 respiration is a measure of mitochondrial uncoupling activity and was significantly lower in extraocular muscle mitochondria (Fig. 3) . Thus, the extraocular muscles may have a lower content of these proteins than other skeletal muscles, allowing for more efficient use of the generated proton gradients. This is an intriguing possibility that we are actively pursuing. 
Conclusions and Future Directions
Our results clearly demonstrate that the respiratory capacity of extraocular muscle mitochondria is significantly lower than that of limb muscle mitochondria because of a combination of decreased respiratory complex activity and content. In addition, these data add weight to the concept that the metabolic organization of the extraocular muscles is unique. An interesting question not addressed by this study is why the extraocular muscles have to maintain such a high mitochondrial volume density. In other words, what functions do the extraocular muscles gain by accumulating mitochondria with a built-in handicap? 
 
Figure 1.
 
Mitochondrial isolation minimizes cytosolic contamination. Cytoplasmic (c) and mitochondrial (m) fractions from triceps surae (TS) and extraocular muscles (E) were evaluated by Western blots of transaminase (cytosolic protein), complex IV (subunit Vb), and complex V (a subunit). Transaminase is present in the cytosolic fractions in triceps and extraocular muscle and is absent in the mitochondrial fractions. Signals for complexes IV and V are faint in the cytosolic fractions but become strongly positive in the mitochondrial preparations.
Figure 1.
 
Mitochondrial isolation minimizes cytosolic contamination. Cytoplasmic (c) and mitochondrial (m) fractions from triceps surae (TS) and extraocular muscles (E) were evaluated by Western blots of transaminase (cytosolic protein), complex IV (subunit Vb), and complex V (a subunit). Transaminase is present in the cytosolic fractions in triceps and extraocular muscle and is absent in the mitochondrial fractions. Signals for complexes IV and V are faint in the cytosolic fractions but become strongly positive in the mitochondrial preparations.
Figure 2.
 
Reduced oxygen consumption in mitochondria from extraocular muscles. Representative tracings of oxygen consumption by mitochondria isolated from rat triceps surae (solid line) and extraocular muscles (dashed line). Respiration was initiated by addition of the substrates pyruvate + maleate (state 2 respiration), followed by ADP (state 3 respiration). Oligomycin was then added to determine state 4 respiration. The mitochondrial uncoupler FCCP was added to measure complex I-driven maximum respiration (state 5). Finally, rotenone (complex I inhibitor) and succinate were added to determine state 5-succinate respiration.
Figure 2.
 
Reduced oxygen consumption in mitochondria from extraocular muscles. Representative tracings of oxygen consumption by mitochondria isolated from rat triceps surae (solid line) and extraocular muscles (dashed line). Respiration was initiated by addition of the substrates pyruvate + maleate (state 2 respiration), followed by ADP (state 3 respiration). Oligomycin was then added to determine state 4 respiration. The mitochondrial uncoupler FCCP was added to measure complex I-driven maximum respiration (state 5). Finally, rotenone (complex I inhibitor) and succinate were added to determine state 5-succinate respiration.
Figure 3.
 
Respiration rates were lower in mitochondria from extraocular muscles. Respiration rates of mitochondria isolated from triceps surae (black bars) and extraocular muscles (hatched bars). Respiration was calculated as nanomoles of oxygen consumed per minute per milligram of protein. All respiration rates except those for state 2 were significantly lower in mitochondria from extraocular muscle than in mitochondria from triceps. *P < 0.001 for all comparisons. Data are mean ± SEM; n = 10 rats.
Figure 3.
 
Respiration rates were lower in mitochondria from extraocular muscles. Respiration rates of mitochondria isolated from triceps surae (black bars) and extraocular muscles (hatched bars). Respiration was calculated as nanomoles of oxygen consumed per minute per milligram of protein. All respiration rates except those for state 2 were significantly lower in mitochondria from extraocular muscle than in mitochondria from triceps. *P < 0.001 for all comparisons. Data are mean ± SEM; n = 10 rats.
Figure 4.
 
Electron microscopy of triceps and extraocular muscle mitochondria. (AC) Mitochondria in triceps surae muscle fibers showing scattered mitochondria, some clustered close to the sarcolemma (A, bottom). (BD) Mitochondria in extraocular muscle fibers showing numerous mitochondria throughout the sarcoplasm. Scale bars: (A, B) 2 μm; (C) 1 μm; (D) 0.5 μm.
Figure 4.
 
Electron microscopy of triceps and extraocular muscle mitochondria. (AC) Mitochondria in triceps surae muscle fibers showing scattered mitochondria, some clustered close to the sarcolemma (A, bottom). (BD) Mitochondria in extraocular muscle fibers showing numerous mitochondria throughout the sarcoplasm. Scale bars: (A, B) 2 μm; (C) 1 μm; (D) 0.5 μm.
Figure 5.
 
Lower activities of respiratory complexes in extraocular muscle mitochondria. Enzymatic activities of complexes I, II, and IV in mitochondria from triceps surae (TS) and extraocular muscles (E). The activities of complexes I and IV were low in the mitochondria from extraocular muscle compared with triceps. Bars represent means ± SEM; n = 6 samples per group. *P < 0.05 compared with triceps.
Figure 5.
 
Lower activities of respiratory complexes in extraocular muscle mitochondria. Enzymatic activities of complexes I, II, and IV in mitochondria from triceps surae (TS) and extraocular muscles (E). The activities of complexes I and IV were low in the mitochondria from extraocular muscle compared with triceps. Bars represent means ± SEM; n = 6 samples per group. *P < 0.05 compared with triceps.
Figure 6.
 
Differences in the content of respiratory complexes in extraocular muscle mitochondria. Mean relative content of the five mitochondrial respiratory complexes in triceps surae (TS) and extraocular muscle (E); n = 6 samples/group. Complexes I and IV were significantly more abundant in mitochondria from extraocular muscles than from triceps (P < 0.05). Complex V content was lower in extraocular muscle mitochondria (P < 0.05). The content of complexes II and III was not significantly different between the mitochondria from triceps and extraocular muscles. (Inset) Representative Western blots showing the relative abundance of respiratory complexes (rows marked I–V) in mitochondrial samples from triceps surae (TS) and extraocular muscle (E).
Figure 6.
 
Differences in the content of respiratory complexes in extraocular muscle mitochondria. Mean relative content of the five mitochondrial respiratory complexes in triceps surae (TS) and extraocular muscle (E); n = 6 samples/group. Complexes I and IV were significantly more abundant in mitochondria from extraocular muscles than from triceps (P < 0.05). Complex V content was lower in extraocular muscle mitochondria (P < 0.05). The content of complexes II and III was not significantly different between the mitochondria from triceps and extraocular muscles. (Inset) Representative Western blots showing the relative abundance of respiratory complexes (rows marked I–V) in mitochondrial samples from triceps surae (TS) and extraocular muscle (E).
MayrR. Structure and distribution of fibre types in the external eye muscles of the rat. Tissue Cell. 1971;3:433–462. [CrossRef] [PubMed]
AndradeFH, McMullenCA, RumbautRE. Mitochondria are fast Ca2+ sinks in rat extraocular muscles: a novel regulatory influence on contractile function and metabolism. Invest Ophthalmol Vis Sci. 2005;46:4541–4547. [CrossRef] [PubMed]
MathieuO, KrauerR, HoppelerH, et al. Design of the mammalian respiratory system, VII: scaling mitochondrial volume in skeletal muscle to body mass. Respir Physiol. 1981;44:113–128. [CrossRef] [PubMed]
SchwerzmannK, HoppelerH, KayarSR, WeibelER. Oxidative capacity of muscle and mitochondria: correlation of physiological, biochemical, and morphometric characteristics. Proc Natl Acad Sci U S A. 1989;86:1583–1587. [CrossRef] [PubMed]
ConleyKE, KayarSR, RöslerK, HoppelerH, WeibelER, TaylorCR. Adaptive variation in the mammalian respiratory system in relation to energetic demand, IV: capillaries and their relationship to oxidative capacity. Respir Physiol. 1987;69:47–64. [CrossRef]
Mathieu-CostelloO, SzewczakJM, LogemannRB, AgeyPJ. Geometry of blood-tissue exchange in bat flight muscle compared with bat hindlimb and rat soleus muscle. Am J Physiol. 1992;262:R955–R965. [PubMed]
SuarezRK, LightonJRB, BrownGS, Mathieu-CostelloO. Mitochondrial respiration in hummingbird flight muscles. Proc Natl Acad Sci U S A. 1991;88:4870–4873. [CrossRef] [PubMed]
HoppelerH, MathieuO, KrauerR, ClaassenH, ArmstrongRB, WeibelER. Design of the mammalian respiratory system, VI: distribution of mitochondria and capillaries in various muscles. Respir Physiol. 1981;44:87–111. [CrossRef] [PubMed]
WeibelER. Stereological Methods. 1979;Academic Press London.
SullivanPG, DubéC, DorenbosK, StewardO, BaramTZ. Mitochondrial uncoupling protein-2 protects the immature brain from excitotoxic neuronal death. Ann Neurol. 2003;53:711–717. [CrossRef] [PubMed]
AndersonEJ, NeuferPD. Type II skeletal myofibers possess unique properties that potentiate mitochondrial H2O2 generation. Am J Physiol Cell Physiol. 2006;290:C844–C851. [PubMed]
Kim-HanJS, KoppSJ, DuganLL, DiringerMN. Perihematomal mitochondrial dysfunction after intracerebral hemorrhage. Stroke. 2006;37:2457–2462. [CrossRef] [PubMed]
AndrewsZB, DianoS, HorvathTL. Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Nat Rev Neurosci. 2005;6:829–840. [CrossRef] [PubMed]
SullivanPG, RabchevskyAG, KellerJN, et al. Intrinsic differences in brain and spinal cord mitochondria: implications for therapeutic interventions. J Comp Neurol. 2004;474:524–534. [CrossRef] [PubMed]
ZieglerD, RieskeJS. Preparation and properties of succinate dehydrogenase. Methods Enzymol. 1967;10:231–235.
SmithL. Spectrophotometric assay of cytochrome c oxidase. Methods Biochem Anal. 1955;2:427–434. [PubMed]
WhartonDC, TzagoloffA. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol. 1967;10:245–250.
WilsonEJ. Should nagarse be used during the isolation of brain mitochondria?. Neurochem Res. 1987;12:831–834. [CrossRef] [PubMed]
AldridgeGM, PodrebaracDM, GreenoughWT, WeilerIJ. The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. J Neurosci Methods. 2008;172:250–254. [CrossRef] [PubMed]
FergusonRE, CarrollHP, HarrisA, MaherER, SelbyPJ, BanksRE. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics. 2005;5:566–571. [CrossRef] [PubMed]
AbramoffMD, MagelhaesPJ, RamSJ. Image processing with ImageJ. Biophotonics Int. 2004;11:36–42.
SheehanT, KumarPA, HoodDA. Tissue-specific regulation of cytochrome c oxidase subunit expression by thyroid hormone. Am J Physiol Endocrinol Metab. 2004;286:E968–E974. [CrossRef] [PubMed]
FischerMD, GorospeJR, FelderE, et al. Expression profiling reveals metabolic and structural components of extraocular muscles. Physiol Genomics. 2002;9:71–84. [CrossRef] [PubMed]
PorterJD, KhannaS, KaminskiHJ, et al. Extraocular muscle is defined by a fundamentally distinct gene expression profile. Proc Natl Acad Sci U S A. 2001;98:12062–12067. [CrossRef] [PubMed]
AndradeFH, MerriamAP, GuoW, et al. Paradoxical absence of M lines and downregulation of creatine kinase in mouse extraocular muscle. J Appl Physiol. 2003;95:692–699. [CrossRef] [PubMed]
AndradeFH, HatalaDA, McMullenCA. Carbonic anhydrase isoform expression and functional role in rodent extraocular muscle. Pflügers Arch. 2004;448:547–551. [PubMed]
KunzWS. Different metabolic properties of mitochondrial oxidative phosphorylation in different cell types-important implications for mitochondrial cytopathies. Exp Physiol. 2003;88:149–154. [CrossRef] [PubMed]
GrossmanLI, LomaxMI. Nuclear genes for cytochrome c oxidase. Biochim Biophys Acta. 1997;1352:174–192. [CrossRef] [PubMed]
HüttemannM, KadenbachB, GrossmanLI. Mammalian subunit IV isoforms of cytochrome c oxidase. Gene. 2001;267:111–123. [CrossRef] [PubMed]
ArnaudoE, HiranoM, SeelanRS, et al. Tissue-specific expression and chromosome assignment of gene specifying two isoforms of subunit VIIa of human cytochrome c oxidase. Gene. 1992;119:299–305. [CrossRef] [PubMed]
CapaldiRA. Mitochondrial myopathies and respiratory chain proteins. Trends Biochem Sci. 1988;13:144–148. [CrossRef] [PubMed]
LomaxMI, GrossmanLI. Tissue-specific genes for respiratory proteins. Trends Biochem Sci. 1989;14:501–503. [CrossRef] [PubMed]
SchlerfA, DrosteM, WinterM, KadenbachB. Characterization of two different genes (cDNA) for cytochrome c oxidase subunit VIa from heart and liver of the rat. EMBO J. 1988;7:2387–2391. [PubMed]
FrankV, KadenbachB. Regulation of the H+/e stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios. FEBS Lett. 1996;382:121–124. [CrossRef] [PubMed]
PoytonRO, McEwenJE. Crosstalk between nuclear and mitochondrial genomes. Annu Rev Biochem. 1996;65:563–607. [CrossRef] [PubMed]
WootenGF, ReissDJ. Blood flow in extraocular muscle of cat. Arch Neurol. 1972;26:350–352. [CrossRef] [PubMed]
WilcoxLM, Jr, KeoughEM, ConnollyRJ, HotteCE. Comparative extraocular muscle blood flow. J Exp Zool. 1981;215:87–90. [CrossRef] [PubMed]
FellDA. Metabolic control analysis: a survey of its theoretical and experimental development. Biochem J. 1992;286:313–330. [PubMed]
RossignolR, LetellierT, MalgatM, RocherC, MazatJP. Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. Biochem J. 2000;347:45–53. [CrossRef] [PubMed]
FellDA. Increasing the flux in metabolic pathways: a metabolic control analysis perspective. Biotechnol Bioeng. 1998;58:121–124. [CrossRef] [PubMed]
HansfordRG. Role of calcium in respiratory control. Med Sci Sports Exerc. 1994;26:44–51. [PubMed]
KavanaghNI, AinscowEK, BrandMD. Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria. Biochim Biophys Acta. 2000;1457:57–70. [CrossRef] [PubMed]
TerritoPR, MoothaVK, FrenchSA, BalabanRS. Ca2+ activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase. Am J Physiol Cell Physiol. 2000;278:C423–C435. [PubMed]
PorterJD, BakerRS. Muscles of a different “color”: the unusual properties or the extraocular muscles may predispose or protect them in neurogenic and myogenic disease. Neurology. 1996;46:30–37. [CrossRef] [PubMed]
McMullenCA, HayessK, AndradeFH. Fatigue resistance of rat extraocular muscles does not depend on creatine kinase activity. BMC Physiol. 2005;5:12. [CrossRef] [PubMed]
AndradeFH, McMullenCA. Lactate is a metabolic substrate that sustains extraocular muscle function. Pflügers Archiv. 2006;452:102–108. [CrossRef] [PubMed]
SchrauwenP, HesselinkM. UCP2 and UCP3 in muscle controlling body metabolism. J Exp Biol. 2002;205:2275–2285. [PubMed]
NichollsDG, BernsonVS, HeatonGM. The identification of the component in the inner membrane of brown adipose tissue mitochondria responsible for regulating energy dissipation. Experientia. 1978;32:89–93. [PubMed]
ArgyropoulosG, HarperM-E. Uncoupling proteins and thermoregulation. J Appl Physiol. 2002;92:2187–2198. [CrossRef] [PubMed]
NedergaardJ, CannonB. The ‘novel’ ‘uncoupling’ proteins UCP2 and UCP3: what do they really do? pros and cons for suggested functions. Exp Physiol. 2003;88:65–84. [CrossRef] [PubMed]
Figure 1.
 
Mitochondrial isolation minimizes cytosolic contamination. Cytoplasmic (c) and mitochondrial (m) fractions from triceps surae (TS) and extraocular muscles (E) were evaluated by Western blots of transaminase (cytosolic protein), complex IV (subunit Vb), and complex V (a subunit). Transaminase is present in the cytosolic fractions in triceps and extraocular muscle and is absent in the mitochondrial fractions. Signals for complexes IV and V are faint in the cytosolic fractions but become strongly positive in the mitochondrial preparations.
Figure 1.
 
Mitochondrial isolation minimizes cytosolic contamination. Cytoplasmic (c) and mitochondrial (m) fractions from triceps surae (TS) and extraocular muscles (E) were evaluated by Western blots of transaminase (cytosolic protein), complex IV (subunit Vb), and complex V (a subunit). Transaminase is present in the cytosolic fractions in triceps and extraocular muscle and is absent in the mitochondrial fractions. Signals for complexes IV and V are faint in the cytosolic fractions but become strongly positive in the mitochondrial preparations.
Figure 2.
 
Reduced oxygen consumption in mitochondria from extraocular muscles. Representative tracings of oxygen consumption by mitochondria isolated from rat triceps surae (solid line) and extraocular muscles (dashed line). Respiration was initiated by addition of the substrates pyruvate + maleate (state 2 respiration), followed by ADP (state 3 respiration). Oligomycin was then added to determine state 4 respiration. The mitochondrial uncoupler FCCP was added to measure complex I-driven maximum respiration (state 5). Finally, rotenone (complex I inhibitor) and succinate were added to determine state 5-succinate respiration.
Figure 2.
 
Reduced oxygen consumption in mitochondria from extraocular muscles. Representative tracings of oxygen consumption by mitochondria isolated from rat triceps surae (solid line) and extraocular muscles (dashed line). Respiration was initiated by addition of the substrates pyruvate + maleate (state 2 respiration), followed by ADP (state 3 respiration). Oligomycin was then added to determine state 4 respiration. The mitochondrial uncoupler FCCP was added to measure complex I-driven maximum respiration (state 5). Finally, rotenone (complex I inhibitor) and succinate were added to determine state 5-succinate respiration.
Figure 3.
 
Respiration rates were lower in mitochondria from extraocular muscles. Respiration rates of mitochondria isolated from triceps surae (black bars) and extraocular muscles (hatched bars). Respiration was calculated as nanomoles of oxygen consumed per minute per milligram of protein. All respiration rates except those for state 2 were significantly lower in mitochondria from extraocular muscle than in mitochondria from triceps. *P < 0.001 for all comparisons. Data are mean ± SEM; n = 10 rats.
Figure 3.
 
Respiration rates were lower in mitochondria from extraocular muscles. Respiration rates of mitochondria isolated from triceps surae (black bars) and extraocular muscles (hatched bars). Respiration was calculated as nanomoles of oxygen consumed per minute per milligram of protein. All respiration rates except those for state 2 were significantly lower in mitochondria from extraocular muscle than in mitochondria from triceps. *P < 0.001 for all comparisons. Data are mean ± SEM; n = 10 rats.
Figure 4.
 
Electron microscopy of triceps and extraocular muscle mitochondria. (AC) Mitochondria in triceps surae muscle fibers showing scattered mitochondria, some clustered close to the sarcolemma (A, bottom). (BD) Mitochondria in extraocular muscle fibers showing numerous mitochondria throughout the sarcoplasm. Scale bars: (A, B) 2 μm; (C) 1 μm; (D) 0.5 μm.
Figure 4.
 
Electron microscopy of triceps and extraocular muscle mitochondria. (AC) Mitochondria in triceps surae muscle fibers showing scattered mitochondria, some clustered close to the sarcolemma (A, bottom). (BD) Mitochondria in extraocular muscle fibers showing numerous mitochondria throughout the sarcoplasm. Scale bars: (A, B) 2 μm; (C) 1 μm; (D) 0.5 μm.
Figure 5.
 
Lower activities of respiratory complexes in extraocular muscle mitochondria. Enzymatic activities of complexes I, II, and IV in mitochondria from triceps surae (TS) and extraocular muscles (E). The activities of complexes I and IV were low in the mitochondria from extraocular muscle compared with triceps. Bars represent means ± SEM; n = 6 samples per group. *P < 0.05 compared with triceps.
Figure 5.
 
Lower activities of respiratory complexes in extraocular muscle mitochondria. Enzymatic activities of complexes I, II, and IV in mitochondria from triceps surae (TS) and extraocular muscles (E). The activities of complexes I and IV were low in the mitochondria from extraocular muscle compared with triceps. Bars represent means ± SEM; n = 6 samples per group. *P < 0.05 compared with triceps.
Figure 6.
 
Differences in the content of respiratory complexes in extraocular muscle mitochondria. Mean relative content of the five mitochondrial respiratory complexes in triceps surae (TS) and extraocular muscle (E); n = 6 samples/group. Complexes I and IV were significantly more abundant in mitochondria from extraocular muscles than from triceps (P < 0.05). Complex V content was lower in extraocular muscle mitochondria (P < 0.05). The content of complexes II and III was not significantly different between the mitochondria from triceps and extraocular muscles. (Inset) Representative Western blots showing the relative abundance of respiratory complexes (rows marked I–V) in mitochondrial samples from triceps surae (TS) and extraocular muscle (E).
Figure 6.
 
Differences in the content of respiratory complexes in extraocular muscle mitochondria. Mean relative content of the five mitochondrial respiratory complexes in triceps surae (TS) and extraocular muscle (E); n = 6 samples/group. Complexes I and IV were significantly more abundant in mitochondria from extraocular muscles than from triceps (P < 0.05). Complex V content was lower in extraocular muscle mitochondria (P < 0.05). The content of complexes II and III was not significantly different between the mitochondria from triceps and extraocular muscles. (Inset) Representative Western blots showing the relative abundance of respiratory complexes (rows marked I–V) in mitochondrial samples from triceps surae (TS) and extraocular muscle (E).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×