HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:1211-1218 (2006)
© 2006 The Gerontological Society of America

Capillary Electrophoresis Reveals Changes in Individual Mitochondrial Particles Associated With Skeletal Muscle Fiber Type and Age

Hossein Ahmadzadeh, Dmitry Andreyev, Edgar A. Arriaga and LaDora V. Thompson

1 Department of Chemistry, California State Polytechnic University, Pomona.
2 Departments of Chemistry and 3 Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis.

Address correspondence to LaDora V. Thompson, PhD, University of Minnesota, 420 Delaware Street, S.E., Minneapolis, MN 55455. E-mail: thomp067{at}umn.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Capillary electrophoresis (CE) with postcolumn laser-induced fluorescence detection (LIF) was used to analyze single skeletal muscle fibers from young and old rats. Due to selective labeling of mitochondria with 10-N-nonyl acridine orange, the zeptomole (10–21 mole) sensitivity, and the high separation power, three properties of individual mitochondrial particles were revealed: the number, the distributions of cardiolipin, and their electrophoretic mobilities. Type I fibers had more mitochondrial particles and cardiolipin per particle than did type IIb fibers from rats of similar age. Individual fibers of the same fiber type from young rats contained more mitochondrial particles and cardiolipin per particle than did fibers from old rats. There were fiber type-specific and age-specific differences in the electrophoretic mobility of individual mitochondrial particles. The CE-LIF results of individual mitochondrial particles are the first of their kind in that they reveal fiber type-specific and age-specific differences that are not obviously noticed in bulk measurements of heterogeneous tissues.

AGING is associated with a progressive decline in skeletal muscle performance (1,2). This decline in performance is muscle-specific and fiber type-specific (3). Particularly, the performance of muscles with a high proportion of Type II fibers (e.g., gastrocnemius) declines more rapidly with aging than does that of skeletal muscles composed predominantly of Type I fibers (e.g., soleus) (3–5). Furthermore, the individual Type II fibers show a preferential decline in contractile properties compared to Type I fibers with age (3–5).

One of the aspects of muscle performance affected by aging is endurance capacity, which is highly dependent on mitochondrial oxidative phosphorylation. It would be expected that, with aging, the reduction in skeletal muscle endurance capacity would be paralleled by a reduction in the oxidative capacity of muscle mitochondria. Although age-associated decline in oxidative capacity of mitochondria has been reported (6–8), others have reported that there are no age-related changes of this property (9–11). In these studies, the effects of age on oxidative capacity are confounded because the measurements are from heterogeneous skeletal muscle tissue (Type I and Type II skeletal muscle fibers). The availability of analytical techniques that (i) take into account tissue heterogeneity (i.e., ability to investigate a single fiber type) and (ii) reveal whether the reduction in oxidative capacity is associated with fewer mitochondria, an average decrease in mitochondrial function, or both of these factors may be useful to tease out the effects of aging and complement the previous studies.

Individual organelle analysis by capillary electrophoresis with postcolumn laser-induced fluorescence detection (CE-LIF) was introduced by our group and used in the analysis of mitochondria isolated from cultured cells (12). Recently, we applied this technique for the analysis of mitochondria that were directly sampled from skeletal muscle serial cross-sections (13). Because the inner diameter (i.e., 50 µm) of the capillary is smaller than the average cross-sectional area of a single skeletal muscle fiber, micropositioning of the capillary tip on top of the muscle serial cross-section surface, aided by microscopy, allows for sampling a desired individual fiber among the many muscle fibers. After sampling by suction (c.a. a few nanoliters) and tagging with a mitochondrion-selective fluorescent label (10-N-nonyl acridine orange [NAO]), the capillary tip is placed in a vial with a conductive buffer, and high voltage (e.g., –16,000 V) is applied to generate an electric field along the capillary. Mitochondria being negatively charged at the buffer pH (i.e., 7.4) (14) migrate towards the other end of the capillary where the LIF detector is found. As each mitochondrial particle reaches the LIF detector, a spike is detected. The collection of detected spikes (electropherogram) is analyzed to obtain (i) the number of mitochondrial particles sampled from the single fiber, (ii) the fluorescence intensity of each detected particle, and (iii) the electrophoretic mobility of each detected particle.

In the present investigation, our overall goal was to describe the use of CE-LIF to compare mitochondrial particles from individual skeletal muscle fibers from the soleus (Type I) and gastrocnemius (Type IIb) muscles, from young (11- to 12-month-old) and old (33- to 36-month-old) Fischer 344 rats. Comparisons made in this investigation include the number of particles, their relative cardiolipin content, and their electrophoretic mobility. Our findings indicate that this technique (CE-LIF) detects differences in the number of mitochondrial particles from the sampled fibers. The detected differences are in agreement with the metabolic profile of each fiber type and demonstrate a decline of oxidative capacity with aging. The relative abundance in cardiolipin content per individual particle, represented as fluorescence intensity of each particle, suggests a fiber-type associated cardiolipin content and an age-related decline of this phospholipid at the organelle level. Because cardiolipin is an important phospholipid in mitochondrial function (e.g., complex III and IV activity and metabolite transport) (15–17), an age-related decline is also supported by this finding. Lastly, we observed fiber type–specific and age-specific differences in the electrophoretic mobilities of individual mitochondrial particles. Because this unexploited property is a function of the surface composition and the morphology of mitochondria (12,14), the observed differences may become useful in separating mitochondrial subpopulations and subsequently understanding physiological and biochemical properties of mitochondria associated with fiber type, aging, and muscle conditioning.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Fluorescein and NAO were purchased from Molecular Probes (Eugene, OR). Trypsin and N-[2-hydroxyethyl]piperazine-N-[ethanesulfonic acid] (HEPES) were purchased from Sigma (St. Louis, MO). KOH was purchased from Aldrich (Milwaukee, WI). Sucrose, dimethyl sulfoxide (DMSO), and ethanol were purchased from Fisher Scientific (Pittsburg, PA). Capillary electrophoresis buffer (CE buffer) contained 10 mM HEPES and 250 mM sucrose, adjusted to pH 7.4 with KOH. All buffers were made using Milli-Q deionized water and were filtered using a 0.22 µm filter before being used. Stock solutions of 1 mM fluorescein and 1 mM NAO were made in ethanol and DMSO, respectively. Dilutions of these solutions were prepared immediately prior to use.

Animal and Tissue Preparation
Skeletal muscle serial cross-sections were obtained from 11- to 12- and 33- to 36-month-old Fischer 344 rats. The superficial region of the gastrocnemius muscles (composed primarily of Type IIb fibers) and the soleus muscles (composed primarily of Type I fibers) were excised, placed on corks in tissue-embedding medium and flash frozen in isopentane over liquid nitrogen (18). Specimens were stored at –80°C until serial muscle cross-sections were cut. Serial muscle cross-sections were taken from midbelly, sectioned at 10 µm in a cryostat (Leica Microsystems, Nussloch, Germany) at –25°C, and placed on gelatinized slides. Several muscle cross-sections were stained by using the routine myofibrillar adenosine triphosphatase (ATPase) histochemical techniques for fiber type identification (19). The remaining muscle cross-sections were stored at –20°C until analyzed by CE-LIF. The day of the analysis, the muscle cross-section was brought to room temperature and then used for direct sampling.

Capillary Electrophoresis
The custom-built CE-LIF system used in this work has been previously described (13). The fluorescence excitation source was from the 488-nm line of an argon–ion laser (Melles Griot, Irvine, CA), and NAO emission was measured in the range of 522–552 nm (535DF35; Omega Optical, Brattleboro, VT). The data were collected at 100 Hz with a PCI-MIO-16E-50 I/O board controlled by Labview software (National Instruments, Austin, TX) and stored as binary files.

The capillary electrophoretic separations were performed in CE buffer (250 mM sucrose, 10 mM HEPES, pH 7.4) at –200 Vcm–1 in a 50 cm long, 50-µm i.d., 150 µm o.d., poly(acrylaminopropanol)-coated capillary. The buffer composition prevents dissolution of the sampled organelles; the capillary coating reduces adsorption of mitochondria to the capillary walls. After alignment, the signal-to-noise ratio (S/N) of the LIF detector was 900 ± 30 for 1 attomole (10–18) fluorescein, a fluorescent compound commonly used to calibrate LIF detectors. The response of the LIF detector to particle detection was assessed with 1 µm-diameter fluorescently labeled polystyrene beads (Fluoresbrite; Polyscience, Inc., Warrington, PA), which had a S/N = 380 ± 40. After analysis, the capillary was reconditioned by rinsing it in a series of water, methanol, water, and CE buffer (1 minute per solution).

Sampling of Mitochondria
Sampling of mitochondria or introduction of reagents into the capillary has been previously described (20), and it is outlined in Figure 1. Briefly, the capillary injection end is micromanipulated until it is positioned just above the region to be sampled or immersed into a drop of a reagent solution. This process is monitored by bright field imaging using an inverted microscope (Nikon Eclipse TE300; Huntley, IL). After the capillary is positioned, a 1-second pulse of negative pressure (110 cm of water) is applied at the other end of the capillary leading to sampling or loading of reagents. Sequentially, the following are loaded or sampled: (i) 5 µM NAO (Figure 1A), (ii) a single muscle fiber in a serial cross-section that was previously treated with 1 µL of (0.5 mg/mL) trypsin (Figure 1B), and (iii) a second volume of 5 µM NAO (Figure 1C). This sequence of injections results in the sample sandwiched between the two plugs of the mitochondrion-selective NAO solution, which then are allowed to mix due to diffusion for 5 minutes at room temperature (Figure 1D). Upon completion of the incubation period, with the injection end immersed in a vial containing CE buffer, high voltage is applied to generate an electric field of –200 Vcm–1 (Figure 1E). This procedure leads to the electrophoretic separation of mitochondria. When fluorescently labeled mitochondria reach the LIF detector, they are detected as narrow individual events (particles).


Figure 01
View larger version (21K):
[in this window]
[in a new window]

  		Figure 1. Sampling and 10-N-nonyl acridine orange (NAO) labeling of mitochondria. A, NAO is introduced into the injection end of the capillary. B, Capillary is brought in contact with a fiber, and tissue sample is taken. C, Second plug of NAO is introduced into the injection end of the capillary. D, Labeling of mitochondria with NAO caused by diffusional mixing. E, Application of high voltage and electrophoresis of mitochondria particles towards the detector

 
Data Analysis
Igor Pro software (Wavemetrics, Lake Oswego, OR) was used for plotting electropherograms (fluorescence intensity vs time) and for data analysis. Tabulation of peak intensities and migration times (i.e., travel time from the injection end to the detector end of the capillary) for individual particles was performed using a custom written Igor procedure (available upon request). Briefly, the program selects only the particles with intensities larger than a threshold that is equal to five times the standard deviation of the background of the electropherogram. The program then calculates individual peak intensities, electrophoretic mobilities, and the number of detected particles. Most of the particles that are identified in this manner fall within a well-defined migration time window. The electrophoretic mobility (µ) of each particle is then calculated as µ = L2/VtM, where L is the capillary length (cm), V is the voltage applied across the capillary (Volts), and tM is the migration time of a detected particle. Unless otherwise noted, three individual fibers were analyzed for each muscle type and age group.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
CE-LIF Analysis of Mitochondria
Bright field imaging was used to position the capillary on the serial muscle cross-section and to remove a small sample by applying a 1-second negative pressure pulse to the opposite end of the capillary. After sampling, the capillary was removed. Observation of the serial muscle cross-section, shown in Figure 2A, illustrates the region that was captured (arrow pointing to inner circle ~ 50 µm diameter) and the contact area of the capillary (larger circle, ~ 150 µm diameter). This figure elegantly shows that only a small region of the serial muscle cross-section (single fiber) was sampled.


Figure 02
View larger version (27K):
[in this window]
[in a new window]

  		Figure 2. Capillary electrophoresis with postcolumn laser-induced fluorescence detection (CE-LIF) analysis of mitochondrial particles from a muscle cross-section. A, Bright field image of a muscle cross-section (old soleus) after a sample has been taken. White arrow: individual fiber that was sampled. B, Detection of an individual particle (spike). C, Complete electropherogram of the sample taken in (A) (upper trace) and the control unlabeled mitochondria (lower trace). D, Complete electropherogram of a sample taken from a muscle cross-section (young soleus). The separations were performed using a 50-cm acrylaminopropanol (AAP)-coated capillary with the application of –200 V/cm and using 10 mM HEPES, 250 mM sucrose, pH 7.4 as the separation buffer. Other conditions are described in Materials and Methods

 
As described in Materials and Methods, the sample taken into the capillary was then subjected to fluorescence labeling with the mitochondrion-selective probe, NAO. Subsequently, an electric field was applied and labeled mitochondria migrated towards the LIF detector where they were detected as individual spikes (e.g., Figure 2B). The entire collection of spikes is referred to as an electropherogram (e.g., Figure 2C, upper trace). In the electropherogram, each individual particle (spike) has a different migration time (x-axis), resulting from the unique electrophoretic mobility of each particle. Although zooming on each spike (e.g., Figure 2B) would be needed to manually determine the properties of each one of the spikes detected in the electropherogram (e.g., Figure 2C), the data analysis software automatically determined the spikes' properties. For example, in Figure 2C, which corresponds to a tissue sampling taken from an old soleus (fiber Type I), the software determined the properties of 110 spikes with S/N > 5 (confirmed manually). Similarly, the software determined the properties of 830 spikes with S/N > 5 (confirmed manually) in the electropherogram of a tissue sample taken from a young soleus (Figure 2D). These examples illustrate that the software can handle the analysis of electropherograms having hundreds of spikes that otherwise would appear unresolved (migration time scale) or absent (fluorescence intensity scale).

Critical to the interpretation of this experiment is the mitochondrion-selective nature of NAO. NAO binds specifically to cardiolipin in the mitochondrial inner membrane, which provides the rationale to identify these particles as mitochondria (21). Therefore, in the absence of this labeling reagent, only a minimal number of peaks (likely caused by scattering or autofluorescence of large tissue aggregates) are detected (Figure 2C, lower trace). The interpretation of this experiment must also take into consideration the possibility of detecting more than one particle at the same time, as it would erroneously suggest a higher NAO (i.e., cardiolipin) content per particle. As previously reported, the probability of simultaneously detecting more than one event is low (i.e., 0.07) and increases with the number of events detected in a given electropherogram (22). Using the same approach, the probabilities of simultaneously detecting two or three particles in the most event-dense region in Figure 2D (i.e., 430–490 seconds) are 0.08 and 0.002, respectively. These estimates predict that ~92% of the detected spikes correspond to individual particles. Thus, the bias in peak intensity measurements affects only a tolerable fraction of events and does not impact the interpretation of the data presented below.

Number of Mitochondrial Particles
As stated above, the number of mitochondrial particles sampled from a single fiber within a muscle serial cross-section was determined by counting the spikes (S/N > 5) in the electropherograms. Table 1 summarizes the average particle number sampled from Type I and Type IIb fibers in young and old rats. Assuming that equal volumes are taken in each fiber sampling, Type I fibers have more mitochondrial particles than Type IIb fibers have. There are fewer mitochondrial particles in single fibers from muscle samples of old rats than in single fibers from the muscle samples of young rats, regardless of fiber type. The more dramatic difference in the number of mitochondrial particles is between single Type I fibers of the two ages (an average of 740 particles in Type I fibers from the young rats compared to an average of 80 particles in Type I fibers from the old rats).


View this table:
[in this window]
[in a new window]

  		Table 1. Number of Mitochondrial Particles in Single Skeletal Muscle Fibers.

 
Individual Fluorescence Intensities
The intensity of each spike detected in a given electropherogram is associated with its NAO content. Using 5 µM NAO in the labeling procedure, NAO and cardiolipin have a defined 1:1 molecular relationship, and the fluorescence intensity may be used for quantification of cardiolipin (23). In this study, the fluorescence intensity of each particle was used to indicate the relative cardiolipin content of the corresponding particle. (The absolute quantification of this phospholipid was not determined.)

Figure 3 summarizes and compares the fluorescence intensities of all the particles that were sampled from the single fibers. To compare the relative cardiolipin content between fiber type and age, the particles with similar fluorescence intensities were grouped, and then the average fluorescence intensity of the group (y-axis) was plotted versus the accumulated fraction of particles (x-axis). Figure 3 shows that, at any given fraction of particles, the individual particle fluorescence intensity (relative cardiolipin content) is greater for single muscle fibers from young rats than those single fibers taken from old rats. Similarly, the fiber type–specific comparison in young rats also indicates that there is a difference (in the y-axis log scale) between the individual particle fluorescence intensity over the entire range of the fraction of particles (Type I greater than Type IIb). Interestingly, the results of fiber type-specific comparison in old rats appear more complex because, below the 0.60 fraction of particles higher fluorescence intensity levels for the Type IIb fibers are noted, whereas there is a reversal in the fluorescence intensity above the 0.6 fraction.


Figure 03
View larger version (15K):
[in this window]
[in a new window]

  		Figure 3. Comparison of normalized fluorescence intensities. For a given fiber type and age, individual intensities of three single fibers (three different serial muscle cross-sections) were grouped, and then the grouped fluorescence intensity was plotted as a function of the cumulative fraction of particles. Young rat, Type I fiber: Y(I); young Type IIb fiber: Y(IIb); old Type I fiber: O(I); old Type IIb fiber: (IIb). Capillary electrophoresis with postcolumn laser-induced fluorescence detection (CE-LIF) analysis conditions are described in
Figure 2

 
As shown in Table 2, the medians of the individual fluorescence intensity values and the total fluorescence intensities (i.e., summation of the individual intensities) confirm the observations seen for relative cardiolipin content in Figure 3. That is, the fluorescence intensity per particle (or total) is greater in the single fibers from young rats than in the single fibers from old rats. Furthermore, the fluorescence intensity per particle (or total) is greater in the Type I fibers than in Type IIb fibers. Surprisingly, the average fluorescence intensity per particle (total fluorescence intensity divided by total number of mitochondrial particles) shows that particles associated with Type I fibers are less fluorescent than those of Type IIb fibers at a given age.


View this table:
[in this window]
[in a new window]

  		Table 2. NAO Fluorescence Intensity (i.e., Cardiolipin) of Individual Particles.

 
Mitochondria Mobility Distributions
The electrophoretic mobility of individual mitochondrial particles is believed to be associated with surface properties of the organelle (e.g., modifications to the phospholipids or the proteins on the surface, or changes in their morphology). The electrophoretic mobility of each detected particle was calculated as described in Data Analysis in the Materials and Methods section. Table 3 summarizes and compares the medians and means of the electrophoretic behavior of mitochondria. When using this comparison, we found that the Type IIb fibers from old rats have a different median and mean electrophoretic mobility (–2.27 and –2.5 x 104cm2V–1s–1, respectively) compared to the other groups.


View this table:
[in this window]
[in a new window]

  		Table 3. Electrophoretic Mobility of Individual Particles.

 
Interestingly, graphing the electrophoretic mobility data in histogram form reveals several other differences between the two fiber types and age groups that are not discernable when comparing medians and means of the experimental groups. To obtain an averaged histogram for each sample, similar individual electrophoretic mobilities were grouped and normalized by the number of total particles. Figure 4 shows the averaged electrophoretic mobility histograms for the two fiber types and two age groups. The histograms for Type IIb fibers, young and old, are narrower (electrophoretic mobility range) than the histograms of Type I fibers, suggesting that mitochondria from Type IIb fibers are more electrophoretically homogeneous. In addition to the observed differences in mobility ranges, the electrophoretic mobilities of the individual mitochondrial particles appear to be overall more negative for young than for old rats; this finding was observed in the median and mean data. Lastly, the abundance of mitochondrial particles (relative number of particles) for distinct mobilities (cm2V–1s–1) is characteristic to fiber type and age.


Figure 04
View larger version (15K):
[in this window]
[in a new window]

  		Figure 4. Electrophoretic mobility distributions of individual mitochondrial particles. The y-axis is the normalized number of detected mitochondria (N/Nt), and the x-axis is electrophoretic mobility (cm2V–1s–1). For each group, the thick bar is the average of three histograms; the thin bar is the standard deviation. Young rat, Type I fiber: Y(I); young Type IIb fiber: Y(IIb); old Type I fiber: O(I); old Type IIb fiber: (IIb). Capillary electrophoresis with postcolumn laser-induced fluorescence detection (CE-LIF) analysis conditions are described in
Figure 2

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The CE-LIF analysis of individual mitochondrial particles from single fibers (sampled from serial muscle cross-sections of Fischer 344 rats) shows marked differences between the two distinct fiber types, Type I (soleus) and Type IIb (gastrocnemius) and between the two distinct age groups, 11 to 12 months old and 33 to 36 months old. These differences are demonstrated in the number of particles, the individual fluorescence intensities (i.e., relative cardiolipin content), and the individual electrophoretic mobilities. The number of mitochondrial particles in both fiber types (I and IIb) is reduced in the individual fibers from the old rats (Table 1). Overall, mitochondrial particles from a given skeletal muscle fiber type isolated from young rats contain more cardiolipin (i.e., are more fluorescent) than are particles from the corresponding skeletal muscle fiber type isolated from older rats (Table 2, Figure 3). However, the differences between cardiolipin content of Type I and Type IIb particles are more complex because, on average, there is more cardiolipin per particle in Type IIb fibers than in Type I fibers (Table 2). The electrophoretic mobilities of the individual mitochondrial particles are more homogeneous for Type IIb fibers and appear to be overall more negative for young than for old rats (Table 3, Figure 4).

The mitochondria particles detected in this study correspond to the two types of mitochondria that are found in skeletal muscle: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) (24). Whereas SSM are clustered beneath the sarcolemmal membrane of the muscle fibers, IFM reside between the myofibrils of the muscle fiber. Whereas SSM can be easily removed from the tissue, IFM require exposure to a protease such as trypsin or nagarase to release them from the tissue (25). As previously reported (13) and described in Materials and Methods, tissue exposure to trypsin prior to sampling causes release of IFM. Therefore, the detected mitochondrial particles most likely originate from both SSM and IFM.

Because the muscle is frozen at –80°C before sectioning and then at –20°C before CE-LIF analysis, this process may cause ice crystal formation with subsequent mitochondrial fragmentation (i.e., formation of more mitochondrial particles). Previous studies suggested that mitochondria (i.e., their electrophoretic mobility) are affected by cryogenic preservation (26). However, these studies used isolated liver mitochondria and added 10% DMSO as a cryoprotectant, which may affect the mitochondrial membranes and the organelles' electrophoretic mobility while maintaining intact organelles. Moreover, electron microscopy did not reveal any apparent damage in these mitochondria, suggesting that they have not been fragmented as a result of the freezing–thawing process. Thus, it is not expected that in this study mitochondria were significantly fragmented during the freezing–thawing of the tissue.

Mitochondria may also fragment when the tissue (or cells) are subject to harsh disruption procedures that use mechanical disruption (e.g., using a Dounce homogenizer) or nitrogen cavitation (26). In the current study, mitochondria release is accomplished through tissue sectioning in the microtome, trypsin digestion of structural proteins, and suctioning of the sample into the capillary. The microtome blade most likely affects only a small fraction of mitochondria found in the surface of 5 µm-thick cross-sections. Trypsin treatment does not cause mitochondrial disruption. The pulling forces experienced by mitochondria during sampling are much less intense than forces experienced during harsh disruption methods (e.g., mechanical homogenization). Therefore, we expect that direct sampling from tissue cross-sections affects only a small fraction of mitochondria. Altogether, these observations suggest that most of the detected mitochondrial particles are not disrupted mitochondria and that their properties are adequate to compare different tissue types and age groups.

The increased numbers of mitochondrial particles detected after sampling Type I fibers compared to Type IIb fibers from serial muscle cross-sections of young rats are in agreement with their respective muscle function. It is well known that the soleus muscle (c.f. 740 ± 120 mitochondrial particles, Table 1) relies more on mitochondrial oxidative phosphorylation than does the gastrocnemius muscle (c.f. 30 ± 12 mitochondrial particles, Table 1) for energy. In the fibers taken from the muscle cross-sections of old rats, the number of mitochondrial particles of both fiber types (80 ± 30 in Type I and 16 ± 6 in Type IIb; c.f. Table 1) is lower than in the corresponding fiber types from young rats. These findings (the number of mitochondrial particles) are consistent with a decline in contractile performance of both muscle types with age (1–5). In other words, a decline in endurance capacity would be associated with lower numbers of mitochondria per volume. In the current study there are two possible explanations for the lower number of mitochondrial particles: (i) fewer mitochondria or (ii) the presence of less cardiolipin per mitochondrion particle. That is, less cardiolipin per mitochondrion would alter the labeling with NAO. For example, fewer mitochondrial particles would lead to fewer oxidative phosphorylation sites for ATP production. In contrast, due to the many biological roles of cardiolipin in the membrane, less cardiolipin per mitochondrion implies changes in ATP production efficiency.

Changes in cardiolipin abundance in aging muscle have the potential to impact function, as phospholipids have multiple functions in mitochondria. For example, cardiolipin is associated with carnitine and acylcarnitine transport across the inner membrane (17), is needed for optimal activity of complex III and complex IV of the electron transport chain in mitochondria (15,24), and serves as anchoring molecule for cytochrome c, a key player in apoptotic cascades (27). To our knowledge, cardiolipin levels of individual fibers, Type I and Type II, from young and old rats have not been reported. In contrast, cardiolipin content of mitochondria from heart muscle has been reported. Paradies and colleagues (28) and McMillin and colleagues (17) report that there is a decrease in cardiolipin content of mitochondria of heart mitochondria, whereas Moghaddas and colleagues (29) and Hoppel and colleagues (24) report no change in mitochondria from rat cardiac tissue. Cardiac muscle is composed predominantly of myocytes that depend on oxidative phosphorylation. The results from the current study shown in Figure 3 and Table 2 indicate that, in both fiber types sampled (Type I and Type IIb), the single fibers from the old rats have lower cardiolipin content per mitochondrial particle and lower total cardiolipin content (i.e., total of all the individual cardiolipin contents) than in single fibers from the young rats. These results would be consistent with the speculation that mitochondrial function is impaired in aged skeletal muscle. Thus, the use of individual CE-LIF measurements of cardiolipin could be a sensitive strategy for exploring age-related changes in mitochondria and muscle function.

Bulk measurements of electrophoretic mobility of mitochondria date back to the early 1970s (14). This property, resulting from the balance of forces associated with the electrical charge on the surface of organelles and their movement through an aqueous medium, was investigated as an approach to purify organelles. More recently, we demonstrated that CE-LIF can be used to determine the electrophoretic mobility of individual organelles, including mitochondria (12). The current study elegantly shows there are fiber type-specific and age-specific changes in the electrophoretic mobility of individual mitochondrial particles. These changes are believed to be associated with changes in the surface properties of the organelles (e.g., modifications to the phospholipids or the proteins on the surface) or in their movement (e.g., change in morphology). Therefore, the changes in electrophoretic mobility distributions are an indication that, in the single muscle fibers from old rats, alterations to either or both of these properties have taken effect. The use of electrophoretic mobility-based separations to isolate subdistributions of mitochondria could then be analyzed by other techniques (e.g., proteomics for posttranslational modifications) in order to identify changes in organelle morphology and biochemistry.

Conclusion
In this study CE-LIF was used to (i) count individual mitochondrial particles from single skeletal muscle fibers of serial muscle cross-sections, specifically Type I and Type IIb fibers of Fischer 344 rats at two different ages, (ii) compare the fluorescence intensities of individual particles, which are associated with the cardiolipin content of the particle, and (iii) compare the individual electrophoretic mobilities. These three measurements clearly show fiber type-specific and age-specific differences that could be easily exploited in future studies on developmental stages of, diseases of, and aging of skeletal muscle. These differences are in agreement with fewer, less active mitochondria in aging skeletal muscle.


   Acknowledgments
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by National Institutes of Health grant R01-AG20866. E. A. is supported by 1K02-AG21453. L. T. is supported by AG-17768 and AG-21626.

We thank Janice Shoeman at the Department of Physical Medicine and Rehabilitation of the University of Minnesota for preparing the muscle cross-sections.


   Footnotes
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor:Charlotte Peterson, PhD

Received February 28, 2006

Accepted May 1, 2006


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Carmeli E, Reznick AZ. The physiology and biochemistry of skeletal muscle atrophy as a function of age. Proc Soc Exp Biol Med. 1994;206:103-113.[Medline]
  2. Larsson L, Grimby G, Karlsson J. Muscle strength and speed of movement in relation to age and muscle morphology. J Appl Physiol. 1979;46:451-456.[Abstract/Free Full Text]
  3. Thompson LV. Skeletal muscle adaptations with age, inactivity, and therapeutic exercise. J Orthop Sports Phys Ther. 2002;32:44-57.[Medline]
  4. Holloszy JO, Chen M, Cartee GD, Young JC. Skeletal muscle atrophy in old rats: differential changes in the three fiber types. Mech Ageing Dev. 1991;60:199-213.[Medline]
  5. Sandmann ME, Shoeman JA, Thompson LV. The fiber-type-specific effect of inactivity and intermittent weight-bearing on the gastrocnemius muscle of 30-month-old rats. Arch Phys Med Rehabil. 1998;79:658-662.[Medline]
  6. Coggan AR, Abduljalil AM, Swanson SC, et al. Muscle metabolism during exercise in young and older untrained and endurance-trained men. J Appl Physiol. 1993;75:2125-2133.[Abstract/Free Full Text]
  7. Coggan AR, Spina RJ, King DS, et al. Histochemical and enzymatic comparison of the gastrocnemius muscle of young and elderly men and women. J Gerontol. 1992;47:B71-B76.[Abstract]
  8. Conley KE, Jubrias SA, Esselman PC. Oxidative capacity and ageing in human muscle. J Physiol. 2000;526:(pt 1): 203-210.[Abstract/Free Full Text]
  9. Kent-Braun JA, Ng AV. Skeletal muscle oxidative capacity in young and older women and men. J Appl Physiol. 2000;89:1072-1078.[Abstract/Free Full Text]
  10. Chilibeck PD, McCreary CR, Marsh GD, et al. Evaluation of muscle oxidative potential by 31P-MRS during incremental exercise in old and young humans. Eur J Appl Physiol Occup Physiol. 1998;78:460-465.[Medline]
  11. Schunk K, Pitton M, Duber C, Kersjes W, Schadmand-Fischer S, Thelen M. Dynamic phosphorus-31 magnetic resonance spectroscopy of the quadriceps muscle: effects of age and sex on spectroscopic results. Invest Radiol. 1999;34:116-125.[Medline]
  12. Duffy CF, Fuller KM, Malvey MW, O'Kennedy R, Arriaga EA. Determination of electrophoretic mobility distributions through the analysis of individual mitochondrial events by capillary electrophoresis with laser-induced fluorescence detection. Anal Chem. 2002;74:171-176.[Medline]
  13. Ahmadzadeh H, Johnson RD, Thompson L, Arriaga EA. Direct sampling from muscle cross sections for electrophoretic analysis of individual mitochondria. Anal Chem. 2004;76:315-321.[Medline]
  14. Heidrich HG, Stahn R, Hannig K. Surface charge of rat liver mitochondria and their membranes. Clarification of some controversies concerning mitochondrial structure. J Cell Biol. 1970;46:137-150.[Abstract/Free Full Text]
  15. Zhang M, Mileykovskaya E, Dowhan W. Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria. J Biol Chem. 2005;280:29403-29408.[Abstract/Free Full Text]
  16. Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci U S A. 1994;91:10771-10778.[Abstract/Free Full Text]
  17. McMillin JB, Taffet GE, Taegtmeyer H, Hudson EK, Tate CA. Mitochondrial metabolism and substrate competition in the aging Fischer rat heart. Cardiovasc Res. 1993;27:2222-2228.[Abstract/Free Full Text]
  18. Snow LM, Sanchez OA, McLoon LK, Serfass RC, Thompson LV. Effect of endurance exercise on myosin heavy chain isoform expression in diabetic rats with peripheral neuropathy. Am J Phys Med Rehabil. 2005;84:770-779.[Medline]
  19. Fox J, Garber P, Hoffman M, et al. Morphological characteristics of skeletal muscles in relation to gender. Aging Clin Exp Res. 2003;15:264-269.[Medline]
  20. Ahmadzadeh H, Thompson LV, Arriaga EA. On-column labeling for capillary electrophoretic analysis of individual mitochondria directly sampled from tissue cross sections. Anal Bioanal Chem. 2006;384:169-174.[Medline]
  21. Petit JM, Maftah A, Ratinaud MH, Julien R. 10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria. Eur J Biochem. 1992;209:267-273.[Medline]
  22. Ahmadzadeh H, Dua R, Presley AD, Arriaga EA. Automated analysis of individual particles using a commercial capillary electrophoresis system. J Chromatogr A. 2005;1064:107-114.
  23. Fuller KM, Duffy CF, Arriaga EA. Determination of the cardiolipin content of individual mitochondria by capillary electrophoresis with laser-induced fluorescence detection. Electrophoresis. 2002;23:1571-1576.[Medline]
  24. Hoppel CL, Moghaddas S, Lesnefsky EJ. Interfibrillar cardiac mitochondrial complex III defects in the aging rat heart. Biogerontology. 2002;3:41-44.[Medline]
  25. Manneschi L, Federico A. Polarographic analyses of subsarcolemmal and intermyofibrillar mitochondria from rat skeletal and cardiac muscle. J Neurol Sci. 1995;128:151-156.[Medline]
  26. Fuller KM, Arriaga EA. Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations. J Chromatogr B Analyt Technol Biomed Life Sci. 2004;806:151-159.[Medline]
  27. Pfeiffer K, Gohil V, Stuart RA, et al. Cardiolipin stabilizes respiratory chain supercomplexes. J Biol Chem. 2003;278:52873-52880.[Abstract/Free Full Text]
  28. Paradies G, Ruggiero FM, Petrosillo G, Quagliariello E. Age-dependent decline in the cytochrome c oxidase activity in rat heart mitochondria: role of cardiolipin. FEBS Lett. 1997;406:136-138.[Medline]
  29. Moghaddas S, Stoll MS, Minkler PE, Salomon RG, Hoppel CL, Lesnefsky EJ. Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria. J Gerontol Biol Sci. 2002;57A:B22-B28.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS