IOVS AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2005;46:4541-4547.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0809
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Andrade, F. H.
Right arrow Articles by Rumbaut, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andrade, F. H.
Right arrow Articles by Rumbaut, R. E.

Mitochondria Are Fast Ca2+ Sinks in Rat Extraocular Muscles: A Novel Regulatory Influence on Contractile Function and Metabolism

Francisco H. Andrade,1 Colleen A. McMullen,1 and Rolando E. Rumbaut2,3,4

1From the Department of Physiology, University of Kentucky, Lexington, Kentucky; the 2Departments of Medicine and 3Pediatrics, Baylor College of Medicine, Houston, Texas; and the 4Michael E. DeBakey VA Medical Center, Houston, Texas.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The ultrafast extraocular muscles necessitate tight regulation of free cytosolic Ca2+ concentration ([Ca2+]i). Mitochondrial Ca2+ influx may be fast enough for this role. In the present study, three hypotheses were tested: (1) Mitochondrial Ca2+ uptake regulates [Ca2+]i and production of force in extraocular muscle; (2) mitochondrial content correlates with their use as Ca2+ sinks; and (3) mitochondrial content in extraocular muscle is determined by the transcription factors and coactivators that initiate muscle adaptation to aerobic exercise.

METHODS. Extraocular and extensor digitorum longus (EDL) muscles from adult Sprague-Dawley rats were used to examine how the Ca2+ release agonists caffeine and 4-chloro-3-ethylphenol (CEP), calcimycin (a Ca2+ ionophore) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP; a mitochondrial uncoupler) alter [Ca2+]i and force transients. Mitochondrial volume density and capillary density were analyzed by stereology and citrate synthase and cytochrome c oxidase by biochemical assays. Real-time PCR measured mRNAs of genes involved in mitochondrial biogenesis.

RESULTS. Caffeine, CEP, and calcimycin increased resting [Ca2+]i to a greater extent in EDL. Peak tetanic [Ca2+]i increased in extraocular muscle with caffeine and CEP. CCCP augmented peak tetanic and submaximum [Ca2+]i and force significantly more in extraocular muscles. Mitochondrial volume density and capillary density were three times greater, and citrate synthase and cytochrome c oxidase were only ~2-fold higher in extraocular muscle. Calcineurin A{alpha}, calcineurin B, and peroxisome proliferator activated receptor (PPAR){gamma} were more abundant in extraocular muscle.

CONCLUSIONS. These data support the hypothesis that mitochondria serve as Ca2+ sinks in extraocular muscles. The high mitochondrial content of these muscles may partly reflect this additional function. It is likely that mitochondrial Ca2+ influx increases the dynamic response range of the extraocular muscles and matches metabolic demand to supply.

The extraocular muscles are the final effector arm of the ocular motor system. These small and constantly active skeletal muscles are categorized as "ultrafast, " mostly based on their extremely short isometric contraction time and associated right-shift in their force frequency relationship (high stimulation frequencies required to obtain maximum force).1 In skeletal muscle, the duration of a contraction is determined mostly by changes in the cytosolic free Ca2+ concentration ([Ca2+]i), which rapidly increases three to four orders of magnitude from the resting state to the start of a contraction. The sarcoplasmic reticulum (SR) is the foremost locus for the coupling of excitation and contraction of the muscle fibers: It contains the channels that release Ca2+ into the cytosol in response to depolarization and the Ca2+-adenosine triphosphatases (ATPases; Ca2+ pumps) that transfer the cytosolic Ca2+ back into its lumen and terminate the contraction. Nevertheless, other organelles may serve as effective Ca2+ sinks. In particular, the kinetics of Ca2+ flux into mitochondria appear to be sufficiently rapid to influence [Ca2+]i during rapid events such as muscle contraction and neurotransmitter release.2 3 4 5 Therefore, mitochondria can modulate the amplitude of [Ca2+]i transients during muscle contraction, and it is likely that the magnitude of this effect is proportional to mitochondrial content. The extraocular muscles are reported to have one of the highest mitochondrial contents of mammalian skeletal muscles.6 This high mitochondrial content has been considered to reflect the metabolic demands imposed by their fast and constant activity. However, mitochondria could also be important in regulating [Ca2+]i kinetics during the activation of extraocular muscle fibers, influencing force production and increasing the dynamic response range for this muscle group. This study addressed three hypotheses to examine the relevance of this novel mitochondrial property on extraocular muscle function. First, we tested whether mitochondrial Ca2+ uptake is an important regulator of [Ca2+]i and force during contraction of rat extraocular muscles. Second, we explored whether mitochondrial content in extraocular muscles correlates with their use as Ca2+ sinks; in other words, mitochondrial content could be greater than oxidative capacity in this muscle group. Third, we examined whether the high mitochondrial content of the extraocular muscles depends on the same group of transcription factors and coactivators that trigger mitochondrial biogenesis in limb muscles adapted to aerobic exercise.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Use of experimental animals was approved by the local Institutional Animal Care and Use Committee 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 and xylazine hydrochloride (100 and 8 mg per kg body weight, respectively, injected intraperitoneally) and killed by exsanguination after a medial thoracotomy. Intact extraocular muscles and EDL muscle bundles (a representative fast-twitch limb muscle) were dissected for in vitro function. For mRNA expression and biochemical studies, extraocular muscles and midbelly samples of EDL were quickly excised, frozen in liquid nitrogen, and stored at –80°C until used. For histochemistry, orbital contents and EDL muscles were frozen in 2-methylbutane cooled to its freezing point in liquid nitrogen.

Mitochondrial Volume Density
Rats were anesthetized with thiobutabarbital (130 mg/kg body weight, injected intraperitoneally) and then perfused transcardially with phosphate-buffered saline (pH 7.4), followed by 2% paraformaldehyde-4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and 130 mM NaCl. Perfusion-fixed 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. One-micrometer cross-sections of whole muscles were stained with toluidine blue and used to determine capillarity with a 100-point eyepiece grid at 400x with a light microscope.7 Thin (80 nm) sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (model 1200EX; JEOL, Inc., Peabody, MA). Mitochondrial volume density (% of muscle fiber volume occupied by mitochondria) was determined from scanned negatives of 154 extraocular muscle fibers (sampled from global and orbital layers) and 61 EDL fibers using a standard point-counting method (144-point grid) with systematic sampling.8

Histochemistry
Ten-micrometer-thick sections of orbital contents and EDL muscles were processed concurrently for cytochrome c oxidase activity in phosphate buffer (pH 7.4) containing (in mg/mL) 1 cytochrome c, 0.5 4,3,3'-diaminobenzidine, and 0.02 catalase. Slides were dehydrated in an ethanol series, cleared with xylene, mounted (Permount), and viewed with a microscope (model E600; Nikon Inc., Melville, NY). Images were captured with a digital camera (Spot RT; Diagnostic Instruments, Inc., Sterling Heights, MI) and transmitted to a computer (PowerMac G4; Apple Computer Inc., Cupertino, CA, equipped with Spot RT software, ver. 4.0; Diagnostic Instruments, Inc.).

Mitochondrial Enzymes
EDL and extraocular muscle samples were homogenized (1:20 wt/vol) in 26 mM Tris, 30 mM dithiothreitol, 0.3 M sucrose, and 1% Triton X-100 (pH 8.0), and extracted on ice for 1 hour. The crude homogenates were centrifuged at 10,000g at 4°C for 30 minutes to pellet the cellular debris. Protein content of the supernatant was determined by the Bradford method using bovine serum albumin as the standard.9 Citrate synthase activity was measured in triplicate by observing the coupling of coenzyme A, released by citrate synthase, to 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB).10 Units were defined as the amount of protein that catalyzes the synthesis of 1 micromole of citrate per minute at 25°C, and calculated per milligram of protein. Cytochrome c oxidase activity was measured by observing the oxidation of cytochrome c.11

Mitochondrial Biogenesis Program
The abundance of mRNAs for transcription factors known to influence mitochondrial content in skeletal muscles was determined by real-time quantitative PCR (qPCR). Muscles were pulverized in liquid nitrogen and total RNA was isolated (TRIzol; Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Muscles from four animals were combined into each RNA sample to lessen the effect of intersubject variability. Reverse transcription was performed with reverse transcriptase (Superscript II RNase H–; Invitrogen) with random hexamers. Primer pairs for genes of interest were designed on computer (Primer Express, ver. 1.5; Applied Biosystems, Foster City, CA) from GenBank nucleotide sequences (Table 1) . cDNA samples (2 µg each) were analyzed in triplicate with a sequence-detection system (model Prism 7700; Applied Biosystems) using SYBR green and ß-actin as the calibrator housekeeping gene. The relative abundance of target mRNAs in the extraocular and EDL muscles was determined with the comparative cycle threshold method.12 13


View this table:
[in this window]
[in a new window]
  		TABLE 1. qPCR Analysis of Transcription Factors and Coactivators Involved in Mitochondrial Biogenesis in Skeletal Muscle

 
Force and [Ca2+]i Transients
Isometric contractile properties of extraocular muscles (superior rectus) and EDL muscle bundles (~10%–15% of total muscle mass) were studied in vitro as described previously.14 Briefly, muscles were placed in a muscle bath with platinum field electrodes and filled with a physiological salt solution (in mM): 137 NaCl, 5 KCl, 2.0 CaCl2, 1.0 MgSO4, 1.0 Na2HPO4, 24 NaHCO3, 11 glucose, and 0.026 D-tubocurarine, bubbled with 95% O2-5% CO2 to maintain pH at 7.4 at 25°C. The distal tendon was attached to a micropositioner and the proximal bone fragment to a force transducer (Akers 801; SensoNor, Horten, Norway, or ELG-H; Entran, Fairfield, NJ) and stretched to the length giving maximum force in response to electrical stimulation (optimal length). Muscles were stimulated with 0.5-second trains (15 V, 0.5-ms pulses) of variable pulse frequency (1–300 Hz) delivered by a stimulator (model S48; Grass Telefactor Instruments, Braintree, MA). Force–frequency relationships were determined for all muscles. Response to treatments was measured with submaximum contractions (at a stimulation frequency giving 50% of maximum force) or full tetanic contractions. At the end of the study, the length of muscle fibers at L0 was measured and bone and tendons removed. The muscles were blotted dry and weighed. Force measurements (in Newtons) were normalized to muscle cross-sectional area (in square centimeters). For the simultaneous measurement of force and [Ca2+]i transients, muscles were loaded with indo-1 acetoxymethyl ester, a fluorescent Ca2+ indicator (Molecular Probes, Eugene, OR). Indo-1 fluorescence was measured with a system consisting of a light source, a high-speed wavelength selector, an electronic shutter, and two photomultiplier tubes (M-40 RatioMaster; Photon Technology International, Monmouth Junction, NJ) attached to a microscope (model TE200; Nikon Inc.). Muscles were illuminated with excitation light set to 360 nm. The emitted light was directed to the photomultiplier tubes, and intensity at 405 and 495 nm was measured. The ratio (R) of the light emitted at 405 nm to that at 495 nm was used as an index of [Ca2+]i. The Ca2+ ionophore calcimycin (100 nM) and the SR Ca2+ release agonists caffeine (5 mM) and 4-chloro-3-ethylphenol (CEP, 50 µM) were prepared as concentrated stocks and added to the bathing solution as needed. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 10 µM) was used to dissipate the mitochondrial proton gradient and inhibit mitochondrial Ca2+ influx.3 4

Data Analysis
All results are presented as the mean ± SE of n observations, unless otherwise noted. Mitochondrial volume density, enzymatic activities and qPCR results were compared with Student’s t-tests. The treatment effects in the functional studies were determined by analysis of variance, and group differences were evaluated by Student-Newman-Keuls tests. The significance level for rejection of the null hypothesis was set at P ≤ 0.05 for all comparisons.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Our first step was to test whether the resting [Ca2+]i of EDL and extraocular muscles would respond differently to caffeine and CEP (which stimulate SR Ca2+ release) and the Ca2+ ionophore calcimycin. Overall, the extraocular muscles tolerated the exposure to caffeine, CEP and calcimycin better than EDL. Figure 1A shows that the increase in resting [Ca2+]i was much greater in EDL than in extraocular muscles when incubated with caffeine, CEP, or calcimycin. To demonstrate that the extraocular muscles are indeed responsive to the SR Ca2+ release agonists caffeine and CEP, Figure 1B shows original recordings of force and [Ca2+]i transients during a maximum tetanic contraction by an extraocular muscle before (Control) and during incubation with caffeine (5 mM). Caffeine increased peak [Ca2+]i significantly (~170%), whereas force increased by 2.8%; CEP had similar effects (not shown). These changes were typical of the effect of caffeine and CEP seen in other skeletal muscles.15 Therefore, the small increase in extraocular muscle resting [Ca2+]i with the SR agonists and the Ca2+ ionophore was not due to low SR Ca2+ content.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 1. Resting [Ca2+]i in extraocular muscle was resistant to Ca2+ release agonists. (A) Increase in resting [Ca2+]i in response to caffeine, CEP, and calcimycin; dashed reference line marks the control level. EDL muscle bundles incubated with caffeine, CEP, or calcimycin had two- to threefold increases in resting [Ca2+]i (*P < 0.01 each treatment versus untreated state, n = 6 muscles per treatment). The effect of caffeine and calcimycin on extraocular muscle (EOM) resting [Ca2+]i was an order of magnitude smaller (**P < 0.05 versus untreated state and treated EDL, n = 6 muscles per treatment). (B) Original records demonstrating the effect of caffeine on maximum tetanic contractions (300 Hz) by an extraocular muscle. Peak tetanic [Ca2+]i (top) increased from 1.4 (Control) to 3.8 µM (Caffeine). Force (bottom) increased from 7.1 (Control) to 7.3 Newtons/cm2 (Caffeine).

 
We used CCCP, a protonophore that uncouples mitochondria, to investigate whether fast mitochondrial Ca2+ influx influences [Ca2+]i transients during tetanic contractions. CCCP increased peak tetanic [Ca2+]i to a greater extent in extraocular muscle than in EDL (by 72% in extraocular muscle versus 14% in EDL; Fig. 2A ). Because nearly all, if not all, the muscle fibers are fully activated during maximum tetani under the control conditions, the increases in tetanic [Ca2+]i cannot increase force much more. Two extraocular muscles and three EDL bundles increased peak tetanic force by up to 5% in response to CCCP, but the rest of the muscles (four extraocular muscles, three EDL bundles) did not present any changes in force, but the increase in peak tetanic [Ca2+]i during incubation with CCCP was a robust and consistent finding. All the responses of the extraocular muscles were greater than for EDL (Fig. 2B) .



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. Mitochondrial uncoupling greatly increased peak tetanic [Ca2+]I in extraocular muscle. (A) Original records showing the effect of CCCP on peak tetanic [Ca2+]i in EDL and extraocular muscle. CCCP increased peak [Ca2+]i in EDL (200 Hz tetani, top) from 1.22 to 1.39 µM. The effect on extraocular muscle (EOM, 300 Hz tetani, bottom) was more intense: [Ca2+]i increased from 1.9 to 3.27 µM. (B) The mitochondrial uncoupler CCCP induced consistent increases in peak tetanic [Ca2+]i in EDL and extraocular muscles (EOM); dashed line: untreated control level. The magnitude of the effect was significantly greater in extraocular muscle. *P < 0.05 versus control (pretreatment); **P < 0.05 versus control (pretreatment) and treated EDL, n = 8 muscles per treatment group.

 
To demonstrate that mitochondrial Ca2+ uptake is fast enough to influence contractile function, we measured force and [Ca2+]i transients during submaximum tetani (~50% of maximal force) under control conditions and in the presence of CCCP. Figure 3A presents the typical response of a 50-Hz submaximum contraction by extraocular muscle on incubation with CCCP: peak tetanic [Ca2+]i increased by ~19% and force by 21%. Although CCCP also altered [Ca2+]i and force during submaximum contractions of EDL bundles, the magnitude of this response was significantly less than in the extraocular muscles (Fig. 3B) .



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 3. Mitochondrial uncoupling had a greater effect on submaximum force in extraocular muscle. (A) Force and [Ca2+]i transients during 50-Hz submaximum tetani recorded from an extraocular muscle before (Control) and during incubation with CCCP. The mitochondrial uncoupler increased peak [Ca2+]i from 0.58 to 0.69 µM and force from 2.8 to 3.4 Newtons/cm2. (B) Changes in submaximum force and [Ca2+]i in EDL and extraocular muscle (EOM) in response to CCCP; dashed line: untreated control level. The magnitude of the changes was greater in the extraocular muscles compared to EDL (*P < 0.05 versus control EDL; **P < 0.05 versus control EOM and treated EDL, n = 8 muscles per treatment group).

 
Using stereological analysis, we confirmed that extraocular muscles have higher mitochondrial content (expressed as mitochondrial volume density) than EDL, a fast-twitch limb muscle. The extraocular muscles have ~3-fold higher mitochondrial volume density than EDL (exemplified in Fig. 4A , left). The mitochondrial volume density range for extraocular muscle was 7% to 39% of fiber volume, and for EDL 2% to 8% (Fig. 4B , left). Therefore, even the least oxidative fibers of the extraocular muscles have approximately the same mitochondrial volume density as the most oxidative fibers in EDL. In limb skeletal muscle, the enzymes citrate synthase and cytochrome c oxidase are used as biochemical correlates of mitochondrial content. We found that citrate synthase activity in extraocular muscle was ~217% higher than in EDL, just over two thirds as great as expected, based on the difference in mitochondrial volume density. The same was true for cytochrome c oxidase: enzyme activity in the extraocular muscles was 1.9 times higher than in EDL (Fig. 4B , center). The difference in cytochrome c oxidase activity was easily detectable by histochemistry (Fig. 4A , center). Capillaries were also very abundant in extraocular muscle (Fig. 4A , right). Capillary density, another morphologic correlate of aerobic capacity, was markedly higher in the extraocular muscles than in the EDL (Fig. 4B , right), and corresponded to the mitochondrial volume density measured for each muscle type.



View larger version (87K):
[in this window]
[in a new window]
  		FIGURE 4. Extraocular muscles showed high oxidative capacity. (A) Indicators of aerobic capacity in extraocular (EOM) and EDL muscles. Left: electron micrographs demonstrating the difference in mitochondrial volume density in EDL (top) and EOM (bottom). Center: micrographs of cytochrome c oxidase activity illustrating that the histochemical reaction was stronger in EOM (bottom) than in EDL (top). Right, representative micrographs of EDL (top) and EOM (bottom) toluidine blue–stained sections illustrating the difference in capillary density between the two muscles. Scale bar: (left) 1 µm; (middle and right), 50 µm. (B) Mitochondrial volume density (left), mitochondrial enzyme activity (middle) and capillary density (right) in EDL and extraocular muscle (EOM). The extraocular muscles had significantly higher mitochondrial density than did the EDL (*P < 0.01). The activity of the mitochondrial enzymes citrate synthase and cytochrome c oxidase was significantly greater in extraocular muscle (*P < 0.05; n = 8 EOM and 7 EDL; independent replicate experiments). The number of capillaries per square millimeter was three times higher in the extraocular muscles, similar to the difference in mitochondrial volume density (*P < 0.05).

 
It is possible that the high mitochondrial volume density of the extraocular muscles reflects the sustained expression of genes normally associated with mitochondrial biogenesis in response to aerobic exercise. Using qPCR, we compared mRNA levels of key genes known to control mitochondrial content and oxidative capacity in skeletal muscle (Table 1) . The only mRNAs found at higher levels in extraocular muscle were calcineurin A{alpha}, calcineurin B, and peroxisome proliferator activated receptor {gamma} (PPAR{gamma}). The expression of Tfam (transcription factor A, mitochondrial) was not significantly different in the two muscles. mRNAs for other regulators of mitochondrial biogenesis were unexpectedly found at lower levels than in the EDL. Of note, mRNAs for Cain, calcineurin Aß, Nrf1 (nuclear respiratory factor-1), and PGC-1 (PPAR{gamma} co-activator 1) were significantly lower in the extraocular muscles. Hif1 (hypoxia-inducible factor 1) and Mef2 (myocyte enhancing factor 2) were not always detectable in extraocular muscle. It could therefore be inferred that mRNAs for these transcription factors were less abundant in extraocular muscle but no relative comparison could be calculated. AMP-dependent protein kinase (AMPK) was also downregulated. Message for its two active site isoforms (Prkaa1 and Prkaa2) was found at very low levels in the extraocular muscle (–631 ± 2.7 and –5352 ± 2, respectively).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The results from this study support the hypothesis that mitochondrial uptake of Ca2+ regulates the contractile properties of rat extraocular muscles. The high mitochondrial volume density of extraocular muscle fibers correlated with their use as Ca2+ sinks, and there was evidence of disparity between Ca2+ sequestering and oxidative capacity. Our data indicated that mitochondrial content in the extraocular muscles is regulated by a transcriptional program different from the one used by limb muscles in response to aerobic exercise.

Use of Mitochondria as Fast Ca2+ Sinks
The extraocular muscles are considered "ultrafast" based on their extremely short contraction time; however, their actual speed of shortening may not be that different.1 16 17 This suggests that regulation of [Ca2+]i kinetics is relatively more important for extraocular muscle fibers in determining their contraction amplitude to a given stimulation frequency. In principle, the extraocular muscles have the profile of very efficient Ca2+ handling capacity: an extensive and well-developed SR and the expression of fast Ca2+ ATPase isoforms.18 19 20 Moreover, the extraocular muscles contain parvalbumin, a low-weight Ca2+-binding protein that serves as a temporary buffer to accelerate the removal of Ca2+ from its binding sites on the myofilaments and facilitates muscle relaxation.21 22 In the present study, the mitochondria were also important in the regulation of [Ca2+]i during contraction. Others had already shown that the kinetics of Ca2+ flux into mitochondria are fast enough to influence very rapid events such as neurotransmitter release from motor nerve terminals.3 The specific inhibition of mitochondrial Ca2+ transport slows the relaxation of mitochondria-rich skeletal muscles.5 These data and the results of this study indicate that the mitochondria are physiological regulators of the [Ca2+]i transients during skeletal muscle contraction.

There are at least two ways in which mitochondria can serve as effective Ca2+ sinks in extraocular muscles. First, the fraction of extraocular muscle fiber volume occupied by mitochondria is three times greater than in EDL. Assuming that mitochondria are functionally the same in both muscle groups, there is a larger Ca2+ sink volume in the extraocular muscles. The second alternative starts with the assumption that mitochondria in extraocular muscles may be functionally different, a novel concept that was superficially addressed in this study. Mitochondrial content in the extraocular muscles is not due to the same mitochondrial biogenesis program used by limb muscles in response to exercise (Table 1) . Also, the ~2-fold difference in the activity of citrate synthase and cytochrome c oxidase, typical indices of mitochondrial content, was not equal to the ~3-fold difference in mitochondrial content between EDL and extraocular muscles. Combined, these data suggest that extraocular muscle mitochondria have a unique functional profile that may include the ability to handle Ca2+ at faster rates.

The Genetic Program Determining the Mitochondrial Content of Extraocular Muscles
Even the most cursory inspection of an electron micrograph of extraocular muscles reveals their typical abundance of mitochondria (see Fig. 4A , left). Functionally, fatigue resistance is roughly proportional to the oxidative capacity of a muscle (i.e., its mitochondrial content).23 Recent studies have begun to elucidate the regulatory steps that control mitochondrial content in skeletal muscles.24 For example, Nrf1 and PGC-1 have been shown to participate in the control of mitochondrial biogenesis.24 25 26 27 Surprisingly, the expression of PGC-1, a novel mitochondrial biogenesis regulator, was actually lower in the extraocular muscles than in the EDL. PGC-1 was originally described as involved in the formation of slow muscle fibers.28 The predominantly fast-fiber extraocular muscles may rely on a different pattern of transcription factors to regulate their mitochondrial content.

Recent reports point to extensive differences between extraocular and limb muscles in the relative importance of major metabolic pathways.14 29 30 31 This degree of metabolic divergence may explain the need for an alternative mitochondrial biogenesis program in the extraocular muscles. The role of cell type and initial stimulus in mitochondrial biogenesis is exemplified by the pattern of expression of transcription factors and coactivators in brown fat.32

One of the few activators of mitochondrial biogenesis upregulated in extraocular muscle was the Ca2+-dependent phosphatase calcineurin. Its presence may reflect another function for [Ca2+]i transients in extraocular muscle. It is possible that constant Ca2+ cycling is the signal to establish and sustain the mitochondrial volume density in the extraocular muscles, as seen in cell culture systems.33 Other studies support the concept of Ca2+ as the trigger of mitochondrial biogenesis, but the experimental designs did not come close to the extreme physiological characteristics of the extraocular muscles.34 35

The differences in the activity of citrate synthase and cytochrome c oxidase relative to mitochondrial volume density were not totally unexpected. This has been shown in other highly aerobic systems.36 Mitochondrial volume density was well-matched to capillary density, at least in terms of comparing the differences between mitochondria-poor EDL and mitochondrial-rich extraocular muscle. Moreover, mitochondrial content correlated well with the effect on [Ca2+]i transients and contractile function. Although the lower than expected citrate synthase and cytochrome c oxidase activities are suggestive, the question of potential intrinsic differences in mitochondrial function between EDL and extraocular muscles remains unresolved.

Matching of Contractile Function to Metabolism by Mitochondrial Ca2+
The primary role of mitochondria is to generate adenosine triphosphate (ATP). Emerging functions, such as behaving as Ca2+ sinks, must allow the organelles to fulfill the energy demands of the cell. With this premise in mind, it is not surprising that Ca2+ influx coordinates the demand for ATP by the contractile apparatus with the supply of ATP by aerobic metabolism.37 To accomplish this, the Ca2+ sensitivity of these disparate processes must be similar.38 For the extraocular muscles, rapid mitochondrial Ca2+ uptake appears to serve three complementary functions. First, it couples metabolic supply to demand; increases in mitochondrial Ca2+ 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.39 The combined activity of these enzymes sustains NADH/NAD+ and maximizes the driving force for oxidative phosphorylation. ATP synthase and adenine nucleotide translocator may also be activated by Ca2+.40 In addition, Ca2+ cycling across the mitochondrial membrane increases proton leak.39 Second, by limiting the [Ca2+]i increase during contractions in response to submaximum stimulation frequencies, mitochondria widen the dynamic range of the extraocular muscles. As shown in Figure 3A , the amplitude of the [Ca2+]i and force transients during a submaximum tetanus increased when Ca2+ flux into mitochondria was impeded with CCCP. Therefore, the capacity of extraocular muscles to produce force is spread over a wider stimulation frequency range, increasing the fine control of the effector arm of the ocular motor system. Coincidentally, this may explain why the force-Ca2+ sensitivity of mechanically skinned extraocular muscle fibers is not different from that in limb muscle fibers.41 The third role for mitochondrial Ca2+ influx is to serve as a positive feedback signal and sustain mitochondrial volume density as presented herein.33 The first two roles serve to match metabolic demand and supply instantaneously. This last proposed function for mitochondrial Ca2+ would maintain the highly aerobic phenotype characteristic of the extraocular muscles.


   Footnotes
 
Supported by Grants EY12998 and EY13724 from the National Eye Institute (FHA) and Grant HL64721 from the National Heart, Lung, and Blood Institute (RER).

Submitted for publication June 25, 2005; revised July 27, 2005; accepted September 22, 2005.

Disclosure: F.H. Andrade, None; C.A. McMullen, None; R.E. Rumbaut, None

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

Corresponding author: Francisco H. Andrade, MS508 UKMC, 800 Rose Street, Lexington, KY 40536-0298; paco.andrade{at}uky.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Close RI, Luff AR. Dynamic properties of inferior rectus muscle of the rat. J Physiol. 1974;236:259–270.[Web of Science][Medline][Order article via Infotrieve]
  2. Sparagna GC, Gunter KK, Sheu S-S, Gunter TE. Mitochondrial calcium uptake from physiological-type pulses of calcium. J Biol Chem. 1995;270:27510–27515.[Abstract/Free Full Text]
  3. David G, Barrett JN, Barrett EF. Evidence that mitochondria buffer physiological Ca2+ loads in lizard motor nerve terminals. J Physiol. 1998;509:59–65.[Abstract/Free Full Text]
  4. Landolfi B, Curci S, Debellis L, Pozzan T, Hofer AM. Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured in situ in intact cells. J Cell Biol. 1998;142:1235–1243.[Abstract/Free Full Text]
  5. Gillis JM. Inhibition of mitochondrial calcium uptake slows down relaxation in mitochondrial-rich skeletal muscles. J Muscle Res Cell Motil. 1997;18:473–483.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  6. Mayr R. Structure and distribution of fibre types in the external eye muscles of the rat. Tissue Cell. 1971;3:433–462.
  7. Mathieu-Costello O, Szewczak JM, Logemann RB, Agey PJ. Geometry of blood-tissue exchange in bat flight muscle compared with bat hindlimb and rat soleus muscle. Am J Physiol. 1992;262:R955–R965.
  8. Weibel ER. Stereological Methods. 1979; Academic Press London.
  9. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  10. Shepherd D, Garland PB. Citrate synthase from rat liver. Methods Enzymol. 1969;13:11–16.
  11. Storrie B, Madden EA. Isolation of subcellular organelles. Methods Enzymol. 1990;182:203–225.[Web of Science][Medline][Order article via Infotrieve]
  12. Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods. 2001;25:386–401.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  13. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2{Delta}{Delta}Ct method. Methods. 2001;25:402–408.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  14. Andrade FH, Hatala DA, McMullen CA. Carbonic anhydrase isoform expression and functional role in rodent extraocular muscle. Pflugers Arch. 2004;448:547–551.[Web of Science][Medline][Order article via Infotrieve]
  15. Westerblad H, Andrade FH, Islam MS. Effects of ryanodine receptor agonist 4-chloro-m-cresol on myoplasmic free Ca2+ concentration and force of contraction in mouse skeletal muscle. Cell Calcium. 1998;24:105–115.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  16. Asmussen G, Beckers-Bleukx G, Maréchal G. The force-velocity relation of the rabbit inferior oblique muscle; influence of temperature. Pflugers Arch. 1994;426:542–547.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  17. Luff AR. Dynamic properties of the inferior rectus, extensor digitorum longus, diaphragm and soleus muscles of the mouse. J Physiol. 1981;313:161–171.[Abstract/Free Full Text]
  18. Porter JD, Karathanasis P. The development of extraocular muscle calcium homeostasis parallels visuomotor system maturation. Biochem Biophys Res Commun. 1999;257:678–683.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  19. Porter JD, Karathanasis P. Extraocular muscle in merosin-deficient muscular dystrophy: cation homeostasis is maintained but is not mechanistic in muscle sparing. Cell Tissue Res. 1998;292:495–501.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  20. Jacoby J, Ko K. Sarcoplasmic reticulum fast Ca2+-pump and myosin heavy chain expression in extraocular muscles. Invest Ophthalmol Vis Sci. 1993;34:2848–2858.[Abstract/Free Full Text]
  21. Heizmann CW, Berchtold MW, Rowlerson AM. Correlation of parvalbumin concentration with relaxation speed in mammalian muscles. Proc Natl Acad Sci USA. 1982;79:7243–7247.[Abstract/Free Full Text]
  22. Müntener M, Rowlerson AM, Berchtold MW, Heizmann CW. Changes in the concentration of the calcium-binding parvalbumin in cross-reinnervated rat muscles. J Biol Chem. 1987;262:465–469.[Abstract/Free Full Text]
  23. Fitts RH. Cellular mechanisms of muscle fatigue. Physiol Rev. 1994;74:49–94.[Abstract/Free Full Text]
  24. Hood DA. Contractile activity-induced mitochondrial biogenesis in skeletal muscle. J Appl Physiol. 2001;90:1137–1157.[Abstract/Free Full Text]
  25. Duguez S, Féasson L, Denis C, Freyssenet D. Mitochondrial biogenesis during skeletal muscle regeneration. Am J Physiol Endocrinol Metab. 2002;282:E802–E809.[Abstract/Free Full Text]
  26. Scarpulla RC. Nuclear control of respiratory chain expression in mammalian cells. J Bioenerg Biomembr. 1997;29:109–119.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  27. Scarpulla RC. Nuclear activators and coactivators in mammalian mitochondrial biogenesis. Biochim Biophys Acta. 2002;1576:1–14.[Medline][Order article via Infotrieve]
  28. Lin J, Wu H, Tan PT, et al. Transcriptional co-activator PGC-1{alpha} drives the formation of slow-twitch muscle fibres. Nature. 2002;418:797–801.[CrossRef][Medline][Order article via Infotrieve]
  29. Fischer MD, Gorospe JR, Felder E, et al. Expression profiling reveals metabolic and structural components of extraocular muscles. Physiol Genomics. 2002;9:71–84.[Abstract/Free Full Text]
  30. Porter JD, Khanna S, Kaminski HJ, et al. Extraocular muscle is defined by a fundamentally distinct gene expression profile. Proc Natl Acad Sci USA. 2001;98:12062–12067.[Abstract/Free Full Text]
  31. Andrade FH, Merriam AP, Guo W, et al. Paradoxical absence of M lines and downregulation of creatine kinase in mouse extraocular muscle. J Appl Physiol. 2003;95:692–699.[Abstract/Free Full Text]
  32. Villena JA, Carmona MC, Rodriguez de la Concepción M, et al. Mitochondrial biogenesis in brown adipose tissue is associated with differential expression of transcription regulatory factors. Cell Mol Life Sci. 2002;59:1934–1944.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  33. Ojuka EO, Jones TE, Han D-H, et al. Intermittent increases in cytosolic Ca2+ stimulate mitochondrial biogenesis in muscle cells. Am J Physiol. 2002;283:E1040–E1045.[Web of Science]
  34. Carroll S, Nicotera P, Pette D. Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat. Am J Physiol. 1999;277:C1122–C1129.
  35. Duncan CJ, Smith JL. Action of caffeine in initiating myofilament degradation and subdivision of mitochondria in mammalian skeletal muscle. Comp Biochem Physiol. 1980;65:143–145.[CrossRef]
  36. Suarez RK. Upper limits to mass-specific metabolic rates. Annu Rev Physiol. 1996;58:583–605.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  37. Hansford RG. Role of calcium in respiratory control. Med Sci Sports Exerc. 1994;26:44–51.[Web of Science][Medline][Order article via Infotrieve]
  38. Balaban RS. Cardiac energy metabolism homeostasis: role of cytosolic calcium. J Mol Cell Cardiol. 2002;34:1259–1271.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  39. Kavanagh NI, Ainscow EK, Brand MD. Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria. Biochim Biophys Acta. 2000;1457:57–70.[Medline][Order article via Infotrieve]
  40. Territo PR, Mootha VK, French SA, Balaban RS. Ca2+ activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase. Am J Physiol. 2000;278:C423–C435.
  41. Lynch GS, Frueh BR, Williams DA. Contractile properties of single skinned fibres from the extraocular muscles, the levator and superior rectus, of the rabbit. J Physiol. 1994;475:337–346.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 IOVSHome page
S. P. Patel, J. L. Gamboa, C. A. McMullen, A. Rabchevsky, and F. H. Andrade
Lower Respiratory Capacity in Extraocular Muscle Mitochondria: Evidence for Intrinsic Differences in Mitochondrial Composition and Function
Invest. Ophthalmol. Vis. Sci., January 1, 2009; 50(1): 180 - 186.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. Amthor, R. Macharia, R. Navarrete, M. Schuelke, S. C. Brown, A. Otto, T. Voit, F. Muntoni, G. Vrbova, T. Partridge, et al.
Lack of myostatin results in excessive muscle growth but impaired force generation
PNAS, February 6, 2007; 104(6): 1835 - 1840.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Andrade, F. H.
Right arrow Articles by Rumbaut, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andrade, F. H.
Right arrow Articles by Rumbaut, R. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS