Am J Physiol Cell Physiol 288: C757–C767, 2005.
First published October 20, 2004; doi:10.1152/ajpcell.00281.2004.
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Methods in Cell Physiology
Mitochondrial regular arrangement in muscle cells:
a “crystal-like” pattern
Marko Vendelin,1,2 Nathalie Béraud,1,* Karen Guerrero,1,* Tatiana Andrienko,1,3
Andrey V. Kuznetsov,4 Jose Olivares,1 Laurence Kay,1 and Valdur A. Saks1,5
1
Submitted 11 June 2004; accepted in final form 19 October 2004
Vendelin, Marko, Nathalie Béraud, Karen Guerrero, Tatiana
Andrienko, Andrey V. Kuznetsov, Jose Olivares, Laurence Kay,
and Valdur A. Saks. Mitochondrial regular arrangement in muscle
cells: a “crystal-like” pattern. Am J Physiol Cell Physiol 288: C757–
C767, 2005. First published October 20, 2004; doi:10.1152/ajpcell.
00281.2004.—The aim of this work was to characterize quantitatively
the arrangement of mitochondria in heart and skeletal muscles. We
studied confocal images of mitochondria in nonfixed cardiomyocytes
and fibers from soleus and white gastrocnemius muscles of adult rats.
The arrangement of intermyofibrillar mitochondria was analyzed by
estimating the densities of distribution of mitochondrial centers relative to each other (probability density function). In cardiomyocytes
(1,820 mitochondrial centers marked), neighboring mitochondria are
aligned along a rectangle, with distance between the centers equal to
1.97 ⫾ 0.43 and 1.43 ⫾ 0.43 ␮m in the longitudinal and transverse
directions, respectively. In soleus (1,659 mitochondrial centers
marked) and white gastrocnemius (621 pairs of mitochondria
marked), mitochondria are mainly organized in pairs at the I-band
level. Because of this organization, there are two distances characterizing mitochondrial distribution in the longitudinal direction in these
muscles. The distance between mitochondrial centers in the longitudinal direction within the same I band is 0.91 ⫾ 0.11 and 0.61 ⫾ 0.07
␮m in soleus and white gastrocnemius, respectively. The distance
between mitochondrial centers in different I bands is ⬃3.7 and ⬃3.3
␮m in soleus and gastrocnemius, respectively. In the transverse
direction, the mitochondria are packed considerably closer to each
other in soleus than in white gastrocnemius, with the distance equal to
0.75 ⫾ 0.22 ␮m in soleus and 1.09 ⫾ 0.41 ␮m in gastrocnemius. Our
results show that intermyofibrillar mitochondria are arranged in a
highly ordered crystal-like pattern in a muscle-specific manner with
relatively small deviation in the distances between neighboring mitochondria. This is consistent with the concept of the unitary nature of
the organization of the muscle energy metabolism.
RECENT STUDIES HAVE SHOWN the existence of multiple specific
functional interactions among mitochondria, sarcoplasmic reticulum (SR), and myofibrils in permeabilized muscle fibers (5,
14, 30, 34). Namely, endogenous ATP has been shown to be
more efficient than exogenous ATP in maintaining calcium
uptake into SR (14). In addition, kinetic studies have shown a
direct supply of endogenous ADP from ATPases to mitochondria (30, 34). Such interaction can be explained by the existence of localized intracellular diffusion restrictions (28, 39). A
mild treatment of the fibers with trypsin leads to the removal of
these diffusion restrictions, and at the same time, distribution
of mitochondria in the fiber is changed from regular arrangement in the control to random distribution after the treatment
(28). Similarly, in ischemic hearts, various alterations in mitochondrial function such as the significant decrease in maximal
respiration rate and half-saturation constant for ADP were
observed in parallel with the changes in structural organization
of the cardiac muscle cells (7, 16). These experimental results
suggest that there is a direct link between regulation of muscle
cell energetics and structural organization of the cell (28).
According to ultrastructural electron microscopic studies,
mitochondrial position in the muscle cells is rather regular and
depends on the muscle type. In the heart muscle, mitochondria
are arranged in a longitudinal lattice between the myofibrils
and are located within the limits of the sarcomeres (33, 36).
The correlation between sarcomere and mitochondrial length is
preserved during the heart muscle contraction or when the
muscle is stretched (21). In white and red skeletal muscles,
mitochondria either are arranged in pairs on both sides of the
Z line or form columns in the longitudinal direction in interfilament space (22, 23). Thus the arrangement of mitochondria
is rather repetitive and similar for each sarcomere. According
to studies in which the fluorescence recovery technique was
used, mitochondria are morphologically and functionally heterogeneous in a number of cells (10). In cardiac muscle,
mitochondria present single poorly branching organelles in
normal conditions (3). In hypoxic conditions, cardiac mitochondria can fuse together to form gigantic mitochondria that
are longer than several sarcomeres (37). This is one of the
examples showing that mitochondrial morphology is a dynamic process that is regulated by the balance of fission and
* N. Béraud and K. Guerrero contributed equally to this work.
Address for reprint requests and other correspondence: M. Vendelin, Institute of Cybernetics at Tallinn Technical University, Akadeemia 21, 12618
Tallinn, Estonia (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
confocal microscopy; quantitative analysis; cardiac and skeletal muscles; probability density function; unitary structure of cells
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Group of Quantitative and Structural Bioenergetics, Laboratory of Fundamental and Applied Bioenergetics, Institut
National de la Santé et de la Recherche Médicale E0221, Joseph Fourier University, Grenoble, France; 2Institute of
Cybernetics, Tallinn Technical University, Tallinn, Estonia; 3International Biotechnological Center, Moscow State
University, Moscow, Russia; 4Department of Transplant Surgery, University Hospital Innsbruck, Innsbruck, Austria;
and 5Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia
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MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
CMXRos for 45 min at 4°C in flexiPERM chambers (Vivascience,
Hanau, Germany) in solution A with 5 mM glutamate and 2 mM
malate.
Muscle fibers and cardiomyocytes were mounted in flexiPERM
slides (Vivascience) in the presence of solution A as follows. Muscle
fibers were put on a slide and stretched slightly before the ends of the
muscles were fixed by attaching flexiPERM micro12 to the slide.
Solution A was then added to each chamber of flexiPERM micro12
that contained the fiber. In the case of cardiomyocytes, flexiPERM
micro12 was attached to the slide first, and then solution A and
cardiomyocytes were added to a chamber.
All preparations were imaged using a confocal microscope Leica
DM IRE2 (Leica Microsystems, Heidelberg, Germany) with a ⫻63
water-immersion objective lens (NA 1.2) at 25°C.
Proteolytic Treatment of Isolated Cardiomyocytes With Trypsin
Isolated cardiomyocytes were permeabilized by the addition of 50
␮g/ml saponin under the microscope within the flexiPERM micro12
chamber. After 15 min of permeabilization, 0.2 ␮M trypsin (Sigma,
St. Louis, MO) was added. Proteolytic treatment of trypsin was
stopped after 25 min by the addition of 0.025 mg/ml trypsin inhibitor
(Sigma). As a result of inhibition, the trypsin reaction was effectively
stopped, as judged by the disappearance of movement of mitochondria
as observed using a confocal microscope.
Solutions
METHODS
Animals
Adult Wistar rats (male ⬃300 g and female ⬃240 g) were used in
all experiments. The investigation conforms with the Guide for the
Care and Use of Laboratory Animals published by the National
Institutes of Health (NIH Publication No. 85-23, revised 1985).
Solution A contained (in mM) 2.77 CaK2-EGTA, 7.23 K2-EGTA
(free calcium concentration ⬃0.1 ␮M), 6.56 MgCl2, 0.5 DTT, 53.3
K-MES, 20 imidazole, 20 taurine, 5.3 Na2ATP, and 15 phosphocreatine, pH 7.1 adjusted at 25°C. All reagents were purchased from
Preparation of Skeletal Muscle Fibers
The fibers were prepared from adult female Wistar rat white
gastrocnemius and soleus muscles. Fibers were stored and imaged in
solution A (see below for composition) in the presence of exogenous
substrates (5 mM glutamate and 2 mM malate). The preparation of the
fibers was described previously (29).
Preparation of Isolated Cardiomyocytes
Intact ventricular cardiomyocytes were isolated from adult male
Wistar rat heart as described by Kay et al. (15).
Confocal Microscopy
Two independent methods were used to visualize the mitochondrial
position in the nonpermeabilized muscle fibers and cardiomyocytes.
Imaging of mitochondrial calcium using rhod-2 AM. Rhod-2 AM is
a cell-permeable fluorescent probe for mitochondrial matrix calcium.
Nonpermeabilized muscle fibers were incubated in Multidish 24 wells
(Nunc, Roskilde, Denmark) with 5 ␮M rhod-2 AM in solution A for
⬃1 h at 4°C in the presence of exogenous substrates (5 mM glutamate
and 2 mM malate). Rhod-2 AM has a rhodamine-like fluorophore
whose excitation and emission maxima are 557 and 581 nm, respectively, making it convenient to use with argon and krypton lasers as an
excitation source.
Imaging of mitochondria using MitoTracker Red CMXRos. MitoTracker Red CMXRos is a derivative of x-rosamine and specifically
binds to mitochondria. The fluorescence of this dye was measured
(excitation and emission maxima at 579 and 599 nm, respectively).
Nonpermeabilized muscle fibers were incubated in Multidish 24 wells
(Nunc) with 100 nM MitoTracker Red CMXRos in solution A for 2 h
at 4°C in the presence of exogenous substrates (5 mM glutamate and
2 mM malate). Cardiomyocytes were incubated with MitoTracker Red
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Fig. 1. Schematic presentation of the method of analysis of distances between
mitochondrial centers. First, the centers of mitochondria were marked with
small boxes. Next, the closest neighbors were found for each mitochondrion,
one per sector (the borders of 45° sectors are indicated by dashed lines). In this
scheme, the mitochondrion whose neighbors are sought is highlighted, and the
closest neighbors to this mitochondrion are indicated by arrows. The relative
coordinates of the closest neighbors, i.e., the coordinates relative to those of the
highlighted mitochondrion, were stored and analyzed further.
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fusion, and this balance can be changed by such interventions
as hypoxia (10, 27).
In all of these studies, however, mitochondrial distribution
was not analyzed quantitatively. Such quantitative description
is required to relate the structural organization of the muscle
cells to interactions between mitochondria and intracellular
ATP-consuming systems, and thus to the intracellular energy
cross talk. In addition, fixed preparations have been used in the
cited ultrastructural studies, and depending on the procedures
used in freezing and preparing the fibers, the fixation might
influence the results (26).
The aim of this work was to analyze quantitatively the
arrangement of mitochondria in heart and skeletal muscles. In
this work, confocal images of nonfixed cardiomyocytes and
fibers from soleus and white gastrocnemius muscles were
analyzed using an algorithm for this purpose. The position of
mitochondria relative to each other in different muscles was
described using probability density functions. The results show
ordered (crystal-like) arrangement of mitochondria with different distribution functions in various muscle cells. This approach may be used for analysis of structural and functional
changes in muscle cells in different physiological and pathophysiological states.
MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
Sigma except ATP, which was obtained from Boehringer (Mannheim,
Germany).
Quantitative Analysis of Mitochondrial Positioning
transverse direction and the found local “mitochondrial line” direction. Thus the mitochondrial neighbors were sheared to form the
“mitochondrial line” that was perpendicular to the fiber. The relative
coordinates obtained after such transformation were stored and analyzed statistically.
The statistical analysis was performed by computing the distribution function of the distance between the centers of neighbor mitochondria as well as by computing probability density functions. Both
functions (distribution and probability density) were computed for
each sector separately. Thus integration of the probability density
function over a sector gives a probability equal to one. Probability
density function was calculated by dividing each of the sectors with a
mesh consisting of 0.1 ⫻ 0.1-␮m boxes and counting the amount of
mitochondrial centers within each of the boxes.
In the analysis, the values are expressed as means ⫾ SD. The
programs developed for this analysis were written in Python and are
available on request.
RESULTS
Representative confocal images of nonfixed intact cardiomyocytes and skeletal muscle fibers are shown in Fig. 2.
Cardiomyocytes
A representative confocal image of a nonpermeabilized
cardiomyocyte with marked mitochondria is shown in Fig. 3.
In Fig. 4, the distances between mitochondrial centers taken
from the image in Fig. 3 are analyzed. According to our
analysis (Fig. 4A), the distances between mitochondrial centers
are smallest in the direction transverse to the fiber. The largest
distances are in the diagonal direction (angle 45°). The distribution can be presented in a radial plot, where the average
distance between mitochondrial centers is related to the direction between mitochondria. In this plot, the distances between
the centers are given as the distances from the reference point
(coordinates 0, 0) plotted in the direction corresponding to each
sector (Fig. 4B). From inspection of the radial plot (Fig. 4B), it
is clear that mitochondrial centers are not distributed randomly
but are arranged according to some regular pattern. Indeed, if
Fig. 2. Representative confocal images of nonpermeabilized cardiomyocytes (A), soleus fibers (B), and white gastrocnemius fibers (C). In these images,
cardiomyocytes and the skeletal muscle fibers are oriented almost horizontally. Cardiomyocytes and soleus fibers were preloaded with MitoTracker Red
CMXRos; white gastrocnemius fibers were preloaded with rhod-2. In cardiomyocytes (A), mitochondria are rather regularly spaced, forming a mesh covering
each cardiomyocyte. In soleus and white gastrocnemius (B and C, respectively), mitochondria form rows running across the fiber. These rows are formed by
mitochondrial pairs (arrows in insets in B and C). In some images, “column-forming” mitochondria (22) can be identified (arrowheads in insets in B and C). In
gastrocnemius, long mitochondria running in pairs in the transverse direction were seen occasionally (asterisks in insets in C). Sizes of images: A, 59.5 ⫻ 59.5
␮m; B, 94.3 ⫻ 94.3 ␮m; B, insets, 14.3 ⫻ 7.1 ␮m (bottom left) and 7.8 ⫻ 7.8 ␮m (bottom right); C, 112 ⫻ 112 ␮m; C, insets, 6.9 ⫻ 6.9 ␮m (top left), 6 ⫻
4.55 ␮m (bottom left), and 8.2 ⫻ 7.6 ␮m (bottom right).
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To analyze how mitochondria are positioned relative to each other,
we used confocal images of the skeletal muscle fibers and cardiomyocytes with easily distinguishable mitochondria. Each image was
rotated until the muscle fiber or cell was oriented in a vertical
direction, as judged by eye. Next, the centers of mitochondria were
marked manually. For each mitochondrion, the closest mitochondrial
centers were found among mitochondria in several sectors as shown in
Fig. 1. The relative coordinates of the closest neighbors were then
computed.
Before the analysis was performed, the relative coordinates of the
closest neighbors were corrected as follows. In cardiomyocytes, the
local myofibrillar orientation was estimated and accounted for. Because the contractile apparatus in the cardiac muscle is widely branching, one has to take into account such organization of the muscle
before the distances between any two points in the cell are measured
(36). For each mitochondrial center, we approximated local myofibrillar orientation by fitting with a straight line the positions of the
mitochondrial center and the centers of its two neighbors found in the
sectors aligned along the fiber. The fitting was performed using the
least-squares method. The obtained line was considered a local orientation of myofibrils, and all of the neighboring mitochondria were
rotated to orient the line vertically. The relative coordinates obtained
after rotation were stored and analyzed statistically.
In soleus and gastrocnemius, mitochondria form lines that are not
exactly perpendicular to fiber orientation (see Fig. 2, B and C).
Because the angle between the lines formed by mitochondria and the
fiber orientation was different in different fibers and even in the
different sections of the same fiber, we transformed the relative
mitochondrial positions as follows. First, for each mitochondrial
center, we approximated local orientation of the “mitochondrial line”
by fitting with the straight line the positions of the mitochondrial
center and the centers of its two neighbors found in the sectors aligned
across the fiber. The fitting was performed using the least-squares
method. Second, new relative coordinates of mitochondrial neighbors
were computed by reducing the coordinate value along the fiber by the
factor x 䡠 sin ␣, where x is the relative coordinate of the neighbor
mitochondria in the transverse direction and ␣ is an angle between the
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MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
mitochondria were distributed randomly, the mean distance as
well as the distribution functions would not depend on direction, and in a radial plot, the centers would have been aligned
along a circle.
In the analysis presented in Fig. 4, only the distances
between mitochondrial centers within each sector (see Fig. 1
for definition of the sector) are taken into account. To distinguish the distributions of mitochondrial centers in each of the
sectors, we computed the probability density functions for
mitochondrial centers in each of the sectors (Fig. 5A) using
confocal images (n ⫽ 10) of cardiomyocytes with a total of
1,820 mitochondrial centers marked. According to the computed probability density function, the mitochondrial centers
are packed in certain areas within the sectors. The areas with
the highest probabilities are aligned along a rectangle with the
longer side of the rectangle oriented along the fiber (Fig. 5A).
Because the distances between mitochondrial centers are not
the same in the directions along and perpendicular to the fiber,
a considerable number of mitochondrial centers are located
In skeletal muscle, mitochondrial arrangement is different
from that in cardiomyocytes, with most of the mitochondria
arranged in pairs in soleus (Fig. 2). In our analysis, we marked
only mitochondria that were seen as spots in the image (indicated by arrows in Fig. 2B) and ignored the long mitochondria,
which were connecting the mitochondrial pairs in the longitudinal direction (marked by arrowheads in Fig. 2B). This approach was used because it is impossible to distinguish whether
the connection between pairs of mitochondria is formed by
separate but not resolved mitochondria or by long extensions of
mitochondria within the pairs. An example confocal image of
soleus fiber with marked mitochondrial centers is shown in Fig.
6. Because the arrangement of mitochondria in soleus is
different from that in cardiomyocytes, there are large differences in the distribution of mitochondrial centers relative to
each other in these muscles. These differences were taken into
account as follows.
B
100
75
50%
75%
50
0
45
90
25
0
25%
2
Y,µm
A
Distribution function,%
Fig. 4. Distribution function (A) and a radial
plot (B) showing distribution of the distances
between mitochondrial centers depending on
the direction in the cardiac muscle. These
data are analyses of the confocal image
shown in Fig. 3. In A, the distribution functions of distances between the centers of
neighboring mitochondria along the fiber
(90°), in the cross-fiber direction (0°), and in
the diagonal direction (45°) are shown. In B,
the distances that enclose 25, 50, and 75% of
neighboring mitochondrial centers are
shown in a radial plot. In this plot, the
distance between mitochondrial centers is
given through the distance from the reference point (coordinates 0, 0), and the direction is taken as equal to the middle of the
corresponding sector. Sector borders are indicated by dashed lines.
Soleus
0
1
2
3
4
-2
5
Distance, µm
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0
-2
0
X,µm
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Fig. 3. Representative confocal image of a nonpermeabilized cardiomyocyte.
In this image, the cardiomyocyte is oriented vertically. Cells were preloaded
with MitoTracker Red CMXRos at 4°C. Centers of mitochondria (n ⫽ 414)
were marked with small black boxes, as shown. Image size is 59.5 ⫻ 59.5 ␮m.
near the borders of 45° sectors (Fig. 5A). Taking into account
that the distance between mitochondrial centers in the diagonal
direction is usually larger than that in the longitudinal or
transverse direction, the mitochondrial centers in the diagonal
direction are not accounted for by our algorithm if the center is
positioned in a different sector. To avoid such interaction of
mitochondria in different sectors, the sectors were adjusted as
follows: the sectors in the longitudinal direction were taken as
equal to 30°, those in the transverse direction as 60°, and those
in the diagonal direction as 45°. The probability density functions corresponding to adjusted partitioning of the neighboring
mitochondria to the sectors are shown in Fig. 5, B and C.
The regular (crystal-like) arrangement of mitochondrial centers is clear from the mean positions of mitochondrial centers
(Fig. 5C). The mean positions are aligned along a rectangle,
with longer distances between mitochondrial centers along the
fiber. Using the same partitioning of the sectors as in Fig. 5, B
and C, the following distances between mitochondrial centers
were found: 1.97 ⫾ 0.43 and 1.43 ⫾ 0.43 ␮m in the longitudinal and transverse directions, respectively. These distances
are significantly different (Welch test, P ⬍ 0.001), with the
difference in the mean distances in the longitudinal and transverse directions equal to 0.52– 0.59 ␮m (95% confidence
interval).
MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
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To determine how the neighboring mitochondria should be
partitioned to represent the spatial distribution of mitochondria
in soleus, we analyzed in detail the distribution of all marked
mitochondria shown in Fig. 6. As a first step of our analysis,
we found, for each marked center, the neighboring mitochondria that were positioned within 5 ␮m in the longitudinal and
transverse directions, i.e., within a 5 ⫻ 5-␮m box. The size of
the box was selected to cover more than one sarcomere length
in the muscle. Next, the probability density function describing
all neighbor mitochondria was computed (Fig. 7A). Because
this probability density function describes not only the closest
mitochondrial neighbors (as in Fig. 5) but also all neighbors
that lie in the 5 ⫻ 5-␮m box, there are several maxima of
probability density function in each direction. Because these
maxima are distributed quite symmetrically relative to the
reference point (cross in Fig. 7), it is sufficient to recognize the
origin of the maxima located in one-fourth of the plot only. The
origin of marked maxima in the positive transverse and longitudinal directions is indicated in the scheme in Fig. 7B. For
example, maximum 5 in Fig. 7A corresponds to the neighboring
mitochondria in the transverse directions. Because both mitochondria within the pair usually have neighbors in the transverse direction, this maximum is considerably larger than
maximum 6 in Fig. 7. Other maxima produced by both mitochondria within the pair are maxima 3 and 8. Note that these
maxima are considerably larger (presented by brighter spots in
Fig. 7A) than the others (compare with maxima 2, 4, 6, 7, and
9) in the map (Fig. 7A). In the longitudinal direction, the
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maxima (maxima 1–4) are clearly separated from each other
because of the small relative variation in the distances between
mitochondrial centers in this direction. In the transverse direction, the maxima corresponding to the closest neighbors (maxima 5–9) are distinguishable, but not as clearly as in the
longitudinal direction. The maxima corresponding to the
neighboring mitochondria that are further away in the transverse direction from the center are fused together, leading to
the formation of lines in Fig. 7A. This indicates that the relative
variation of distances in the transverse direction is considerably
larger than in the longitudinal direction.
To describe the distribution of mitochondria in soleus, we
determined the distribution of mitochondrial neighbors corresponding to maxima 1, 2, 5, 6, and 7 in Fig. 7 and their
symmetric reflections. Indeed, mitochondria positioned near
these maxima determine the characteristic distance in the
transverse direction (distance 5 in Fig. 7B) as well as both
characteristic distances in the longitudinal direction (distances
1 and 2 in Fig. 7B). The probability density function corresponding to such partitioning of neighbors into sectors is
shown in Fig. 8. The probability density function was found by
using n ⫽ 10 confocal images with a total of 1,659 mitochondrial centers marked. Note that the variance of the distances
was considerably larger in the transverse than in the longitudinal direction (Fig. 8A). The distances between centers of
neighboring mitochondria were as follows: 0.91 ⫾ 0.11,
2.83 ⫾ 0.65, and 0.75 ⫾ 0.22 ␮m, corresponding to distances
1, 2, and 5 in Fig. 7B.
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Fig. 5. Distribution of centers of neighboring mitochondria in the cardiomyocytes was
analyzed by taking into account not only the
distance but a precise direction as well. Images (n ⫽ 10) were analyzed with 1,820
mitochondrial centers marked. In A and B,
probability density function is indicated by
colors; the correspondence between values
and color is indicated on the bar at right of B.
In each sector (sector borders are indicated
by dashed lines in C), mitochondria are not
distributed evenly but are packed in certain
areas. Thus there is a maximum of probability density surrounded by an area without
any significant number of mitochondrial centers. In A and B, different partitioning of the
sectors was used: in A, the space was partitioned into sectors of the same size, whereas
in B, the partitioning was performed with
sectors that were adjusted for analysis of
cardiac muscle cell. Note that after such
adjustment, the regions with a high density
of mitochondrial centers are adrift from the
borders of the sectors. In C, the mean positions of neighboring mitochondrial centers
are indicated by solid dots for each of the
sectors. The area containing 50% of the centers in the sector closest to each mean position is surrounded by a solid line. A similar
area corresponding to 75% of the centers is
enclosed by a dashed line.
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MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
Fig. 6. Representative confocal image of a nonpermeabilized soleus fiber. In
this image, the fiber is oriented almost diagonally, with sarcomeres oriented
from the top left corner to the bottom right corner of the image. The fiber was
preloaded with MitoTracker Red CMXRos at 4°C. Centers of mitochondria
(n ⫽ 434) were marked with small black boxes, as shown. Image size is 29.8 ⫻
29.8 ␮m.
White Gastrocnemius
In white gastrocnemius, most of the mitochondria are arranged in pairs, similar to soleus (Fig. 2). As for soleus, we
marked only mitochondria that were seen as spots in the image
(indicated by arrows in Fig. 2C) and ignored the long mitochondria, which were connecting the pairs in either the longitudinal direction (marked by arrowheads in Fig. 2C) or the
transverse direction (marked by asterisk in Fig. 2C).
Because the distances between mitochondria within each of
the pairs is considerably smaller than in soleus (see below), we
were not able to distinguish the mitochondria within the pairs
in most of our images. For this reason, we analyzed the
distances between pairs of mitochondria, marking the center of
each pair as shown in Fig. 9. The probability density function
and the partitioning of neighboring pairs to the sectors are
shown in Fig. 10. As in the case of oxidative muscles analyzed
earlier, the mitochondrial pairs are positioned rather regularly
Alternation of Mitochondrial Organization by
Proteolytic Treatment
Mitochondrial arrangement in the cell can be changed dramatically by rather selective proteolytic treatment of permeabilized cardiac muscle cells (28, 30). After the addition of
trypsin into solution with permeabilized cardiomyocytes, several rather drastic changes occur in the structure of the cells.
After a period of time required for proteolytic challenge of the
cell, cardiomyocytes contract very abruptly, but then the volume of the cell starts to increase slowly, leading to complete
digestion of the cell if trypsin is not inhibited (results not
shown). A representative confocal image after 25-min exposure of the permeabilized cardiomyocyte to 0.2 ␮M trypsin is
shown in Fig. 11.
From a visual comparison of the mitochondrial distribution
in the control (Fig. 3) and after trypsin addition (Fig. 11), the
difference is evident: instead of the highly ordered pattern in
the control, mitochondria are seen as disorganized clusters
after trypsin treatment. Intriguingly, these mitochondrial clusters are still firmly fixed to some intracellular structures. To
quantify changes in the distribution of mitochondria within the
cell, we computed probability density functions corresponding
to the situation after trypsin treatment. When computing probability density function based on the images of cardiomyocytes
subjected to prolonged treatment with trypsin, we were not
Fig. 7. Probability density function describing the distribution of mitochondria in soleus
fiber corresponding to the image shown in
Fig. 6. In A, the probability density function
describes the distribution of all mitochondrial centers within a 5 ⫻ 5-␮m box from
each other. The cross in the center of the
image in A marks the reference point (coordinates 0, 0). Note that there are several
maxima of the probability density function.
The origin of maxima 1–9 (marked by circles
on the probability density map) is explained
in B, where distances 1–9 corresponding to
the marked maxima are indicated by arrows
with an index corresponding to the maxima.
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in white gastrocnemius. The distances between the pairs of
mitochondria were as follows: 3.33 ⫾ 0.96 and 1.09 ⫾ 0.41
␮m in the longitudinal and transverse directions, respectively.
According to the Welch test, the mean distance obtained for the
transverse direction is considerably larger (P ⬍ 0.001) than the
same distance in soleus (0.75 ⫾ 0.22 ␮m, distance 5 in Fig.
7B). The difference between the mean transversal distances in
soleus and white gastrocnemius was 0.31– 0.38 ␮m (95%
confidence interval).
In some confocal images, we were able to distinguish
mitochondria within the pairs. According to our data (10
images with 85 distances found), the distance between the
mitochondrial centers in pairs was 0.61 ⫾ 0.07 ␮m. Note that
the mean value of this distance was considerably smaller
(Welch test, P ⬍ 0.001) than that found for soleus (0.91 ⫾ 0.11
␮m, distance 1 in Fig. 7B). The difference between the mean
distances between mitochondria in pairs in white gastrocnemius and soleus was 0.28 – 0.31 ␮m (95% confidence interval).
MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
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Fig. 8. Distribution of mitochondria in soleus fiber. Images (n ⫽ 10) were analyzed
with a total of 1,659 marked mitochondria.
In A, the probability density function is
shown. In B, the mean positions of neighboring mitochondrial centers are indicated by
solid dots for each of the sectors. The areas
containing 50% of the centers in the sector
closest to each mean position is surrounded
by a solid line. A similar area corresponding
to 75% of the centers is enclosed by a dashed
line.
DISCUSSION
In this work, the arrangement of mitochondria within the
muscle was assessed by analyzing quantitatively the distances
between mitochondrial centers. To our knowledge, this is the
first time the positions of neighboring mitochondria have been
Fig. 9. Representative confocal image of nonpermeabilized white gastrocnemius fiber. In this image, the fiber is oriented vertically. The fiber was
preloaded with rhod-2 at 4°C. Because the distances between mitochondria
within each of the pairs are considerably smaller than in soleus (see RESULTS),
we were not able to distinguish the mitochondria within the pairs in most of our
images. Therefore, centers of mitochondrial pairs (n ⫽ 137) were marked with
small black boxes, as shown. Image size is 25.4 ⫻ 25.4 ␮m.
AJP-Cell Physiol • VOL
characterized by using probability density functions in muscle
cells. The analysis of confocal images of nonfixed cardiomyocytes and skeletal muscle fibers by using this method reveals
that intermyofibrillar mitochondria are arranged in a highly
ordered pattern (crystal-like) with relatively small deviation in
the distances between neighboring mitochondria in cardiac and
skeletal muscles. Moreover, this arrangement is muscle specific.
According to ultrastructural studies of cardiac muscle, mitochondria occupy ⬃30 –35% of myocardial cell volume (6,
19, 32, 36) and are distributed everywhere in the cell (36).
Intermyofibrillar mitochondria usually do not violate the limits
of sarcomeres and are located between the zone demarcated by
two Z lines (20, 33, 35, 36). Thus, in the longitudinal direction,
the distance between the centers of two neighboring mitochondria is expected to be similar to the sarcomere length. From our
analysis, this distance is ⬃2 ␮m and is close to the measured
sarcomere length value in the relaxed state (36). In the transverse direction, it has been suggested that all contractile material is within 0.5 ␮m of mitochondria in cardiac muscle (36).
By taking the maximal diameter of a myofibril as equal to 1
␮m, we determined that the distance between the centers of
neighboring mitochondria in the transverse direction should be
larger than 1 ␮m by the diameter of a mitochondrion. The
mean distance found in our study (⬃1.5 ␮m) suggests that the
diameter of mitochondria is at least ⬃0.5 ␮m in nonfixed
preparations.
In skeletal muscle, intermyofibrillar mitochondria are arranged in register with the sarcomere. In red fibers, mitochondria form either pairs on both sides of the Z line or columns on
the A-band level (22, 23). In white fibers, most mitochondria
are organized in pairs next to the Z line, and rather few are
located between Z lines in the form of thin columns (22, 23).
The same pattern of mitochondrial arrangement is evident from
confocal images of skeletal muscles obtained using specific
fluorescent markers (Figs. 2 and 6). In some images, thin links
between the bright spots can be identified (arrowheads in Fig.
2, B and C, insets), corresponding to the “column-forming”
mitochondria of Ogata and Yamasaki (22). In addition, long
pairs of mitochondria running along the I band (see mitochondria marked by asterisks in Fig. 2C) were observed in white
gastrocnemius, in agreement with earlier studies (22, 23). In
our analysis, we did not consider the thin columns or pairs of
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able to relate the distances between mitochondrial centers to
local fiber orientation, because this orientation was not visible
(Fig. 11). For reference, the cell border was used instead. As is
clear from the computed probability distribution functions
(Fig. 12), the regular arrangement of mitochondrial centers
disappears after applied proteolytic treatment. Indeed, the main
parameter determining the distribution of mitochondrial centers relative to each other seems to be the distance between the
centers, leading to a probability density function that is almost
radially symmetrical (Fig. 12).
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MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
Fig. 10. Distribution of mitochondrial pairs
in white gastrocnemius fiber. Images (n ⫽ 8)
were analyzed with a total of 621 mitochondrial pairs marked. In A, the probability density function is shown. In B, the mean positions of neighboring pairs are indicated by
solid dots for each of the sectors. The area
containing 50% of the centers in the sector
closest to each mean position is surrounded
by a solid line. A similar area corresponding
to 75% of the centers is enclosed by a dashed
line.
Fig. 11. Representative confocal image of a permeabilized cardiomyocyte
after proteolytic treatment with trypsin. Cardiomyocytes were preloaded with
MitoTracker Red CMXRos. Note that after this treatment with trypsin, mitochondria formed clusters. Centers of mitochondria (n ⫽ 83) within 2 such
clusters were marked with small black boxes, as shown. Image size is 35.3 ⫻
35.3 ␮m.
AJP-Cell Physiol • VOL
regardless of the differences in the participation of mitochondria in energy cross talk in these two muscle types, mitochondria are arranged in a similar and highly ordered manner. In the
longitudinal direction, the distances between two pairs of
mitochondria are similar for soleus (sum of the distances
corresponding to maxima 1 and 2 in Fig. 7, ⬃3.7 ␮m) and
white gastrocnemius (distance between the pairs, ⬃3.3 ␮m)
and, according to the ultrastructural studies, correspond to the
distances between two Z lines. Because we stretched the
skeletal muscle fibers when mounting them on a microscopic
slide (see METHODS), the sarcomere length is somewhat larger
than that attributed to physiological conditions (9) and corresponds to the descending limb in the force-length relationship
(24). The main difference between the soleus and white gastrocnemius is in the distances between mitochondrial centers
within the same I band. Namely, in the longitudinal direction,
mitochondrial centers are considerably closer to each other in
white gastrocnemius muscle than in soleus, leading to the
reduced width of “mitochondrial band” covering each Z line in
white gastrocnemius. In the transverse direction, the distances
between mitochondrial centers are considerably smaller in
soleus than in white gastrocnemius, pointing to the higher
density of mitochondria in soleus.
Most of the ultrastructural studies of mitochondrial arrangement in muscle cells have been performed on fixed and frozen
material. Depending on the procedures used in freezing and
preparing the fibers, the results of cellular volume assessments
can vary ⬎50% (26). In our study, we were able to analyze the
mitochondrial distribution in nonfixed fibers surrounded with
solution containing substrates. The fibers or cells were isolated
to investigate them using confocal microscopy, but the intracellular environment was kept, similar to the recent studies on
nuclear patterning in living muscle cells in the intact animal
(8). Because there is no fixation involved, our technique allows
us to study quantitatively alteration in mitochondrial distribution in response to changes in the physiological state of the
same fiber or cell, i.e., with fully functional mitochondria.
There is an important difference between mitochondrial
distributions in cardiac muscle cells observed using electron
and confocal microscopy. Usually, the images obtained using
electron microscopy from the thin sections of cardiac muscle
show larger amounts of irregularities in the position of intermyofibrillar mitochondria than the images obtained using con-
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mitochondria (marked by arrowheads and asterisks in Fig. 2)
but marked only the centers of mitochondria within the pairs
along the I band. This method was used because it is hard to
distinguish whether these long structures are formed by separate mitochondria or long extensions of mitochondria (12)
within the marked bright spots in I band-limited mitochondria.
Thus the distances analyzed in skeletal muscle represent the
distances between the mitochondria forming pairs on both
sides of the Z line.
On the basis of our images, we conclude that the pattern of
mitochondrial arrangement is similar in soleus (mainly oxidative muscle) and white gastrocnemius (mainly glycolytic muscle). This is in contrast to the differences in the regulation of
respiration by exogenous ADP: in oxidative skeletal muscles,
the affinity for ADP is very low (apparent Km is very high),
whereas in glycolytic muscle, the mitochondrial affinities for
ADP are very high in vivo and in vitro (17, 38). Thus,
MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
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Fig. 12. Distribution of mitochondria in cardiomyocytes after proteolytic treatment with
trypsin. Images (n ⫽ 6) were analyzed with
a total of 582 mitochondria marked. In A, the
probability density function is shown. In B,
the mean position of neighboring mitochondrial centers is indicated by solid dots for
each of the sectors. The area containing 50%
of the centers in the sector closest to each
mean position is surrounded by a solid line.
A similar area corresponding to 75% of the
centers is enclosed by a dashed line. Note
that the distances between mitochondrial
centers do not depend on direction and that
there are no crystal-like patterns in this case.
AJP-Cell Physiol • VOL
cell, followed by relaxation (not shown). These structural
changes are most probably related to successive destruction of
cytoskeletal components responsible for precise arrangement
of mitochondria in the cells. The precise nature of these
components is still unknown. Other cardiac challenges, such as
ischemia and hypoxia, represent areas of high interest in which
the method described in this work can be applied.
Our study has an important limitation. Namely, we used only
one section in our analysis and not the series of sections
covering a three-dimensional bulk of tissue. In confocal images, the fluorescent signal is detected from a rather thick
section, ⬎0.3 ␮m (given for emission at 488 nm), because of
the limitation in resolution. Thus the distances that we computed are projections from three-dimensional space to our
image plane, leading to some underestimation of these distances. To overcome this limitation, the series of images must
be acquired covering the three-dimensional area of interest and
analyzed to reveal three-dimensional organization of mitochondria in the cells. Such an extension of our method is under
development.
The regular arrangement of mitochondria in the heart muscle
cells is in accordance with the recently described functional
interactions between mitochondria and myofibrillar and SR
ATPases (5, 14, 30, 34). In the 1960s, the even distribution of
mitochondria in the cross sections of heart muscles led to the
following suggestion: “Each mitochondrion serves only a very
limited area of the myofilament mass immediately surrounding
it” (12). As pointed out by Sommer and Jennings (36), the
close association of mitochondria with other intracellular structures must not be taken as prime evidence for specific functional interaction, because the amount of mitochondria is very
large in the cardiac cells. However, there is kinetic evidence of
such interactions from the experiments on permeabilized cardiac fibers: 1) it is impossible to inhibit completely mitochondrial respiration when stimulated by endogenous ATPases
using an externally added ADP-trapping system as long as the
intracellular structures are kept intact (30, 34); 2) calcium
uptake can be enhanced by endogenous rephosphorylation of
ADP to ATP by using either endogenous creatine kinase or
oxidative phosphorylation compared with exogenous ATP
(14). The interaction between mitochondria and ATP-consuming systems can be explained by local intracellular diffusion
restrictions that divide the cell into unitary structures containing a mitochondrion and enzymatic systems consuming ATP
next to the mitochondrion, such as SR and actomyosin
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focal microscopy. For example, it is common that there are
several mitochondria packed together between sarcomeres (12,
13, 36). The confocal images of cardiac muscle cells show
remarkable regularity in arrangement of mitochondria between
myofibrils (11). The regular arrangement of mitochondria in
confocal images of cardiac muscle has been demonstrated by
cross-correlating the fluorescent images of flavoprotein
(autofluorescence) and tetramethylrhodamine ethyl ester (mitochondrial inner membrane potential-sensitive dye) (25).
Namely, such a cross-correlation map has several peaks that
are regularly spaced in the longitudinal and transverse directions. If a central peak in the cross-correlation map represents
the coincidence of both images, then peripheral peaks observed
in the map indicate the periodicity of the packing of mitochondria along myofibrils (25). Recently, Aon et al. (2) used the
regularity of mitochondrial positioning in the cardiac cell by
dividing the image of the cell with the grid filled with small
squares (⬃2⫻2 ␮m) and analyzing the fluorescence within
each of the squares. With the use of such an approach, usually
one to two mitochondria were observed in each square of the
grid (2). The reasons for such a difference between mitochondrial distribution assessed using electron microscopy and confocal microscopy are not clear. One of the possible explanations is that when thin sections are analyzed, as in electron
microscopy, a mitochondrion either can be left out of the
section by a very small margin or can have several membrane
invasions that split a mitochondrion into several mitochondria
in the image plane. In addition, fixation procedures used in
electron microscopy may play a role here. Alternatively, several mitochondria could be represented by a single bright spot
in confocal images because of the resolution limits of confocal
microscopy. Whether any of these explanations is correct is not
clear and requires further investigation.
Organization of mitochondria in the cardiac muscle cells can
be influenced by several challenges. As an example, we followed proteolytic treatment of trypsin, which leads to disarrangement of mitochondria in permeabilized cardiac muscle
fibers (28, 30). By using the probability density function
describing the distribution of mitochondria, it is possible to
analyze quantitatively the transition between a highly ordered
pattern in the control and a rather random pattern after prolonged treatment with trypsin. According to our recordings, the
transition between a crystal-like pattern (control, Fig. 5) and a
radially symmetric one (prolonged treatment with trypsin, Fig.
12) is rather abrupt and goes first through contraction of the
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MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
ACKNOWLEDGMENTS
We thank Joëlle Demaison and Serge Soyez (Joseph Fourier University,
Grenoble, France) for technical assistance and Dr. Frédéric de Oliveira for
discussions and the kind gift of MitoTracker Red CMXRos.
GRANTS
This work was supported in part by Marie Curie Fellowship of the European
Community Programme “Improving Human Research Potential and the Socioeconomic Knowledge Base” contract no. HPMF-CT-2002-01914 (to M. Vendelin).
REFERENCES
1. Aon MA, Cortassa S, Marban E, and O’Rourke B. Synchronized whole
cell oscillations in mitochondrial metabolism triggered by a local release
of reactive oxygen species in cardiac myocytes. J Biol Chem 278:
44735– 44744, 2003.
2. Aon MA, Cortassa S, and O’Rourke B. Percolation and criticality in a
mitochondrial network. Proc Natl Acad Sci USA 101: 4447– 4452, 2004.
3. Bakeeva LE, Chentsov YS, and Skulachev VP. Intermitochondrial
contacts in myocardiocytes. J Mol Cell Cardiol 15: 413– 420, 1983.
4. Bereiter-Hahn J and Voth M. Dynamics of mitochondria in living cells:
shape changes, dislocations, fusion, and fission of mitochondria. Microsc
Res Tech 27: 198 –219, 1994.
5. Birkedal R and Gesser H. Regulation of mitochondrial energy production in cardiac cells of rainbow trout (Oncorhynchus mykiss). J Comp
Physiol [B] 174: 255–262, 2004.
6. Bossen EH, Sommer JR, and Waugh RA. Comparative stereology of the
mouse and finch left ventricle. Tissue Cell 10: 773–784, 1978.
AJP-Cell Physiol • VOL
7. Boudina S, Laclau MN, Tariosse L, Daret D, Gouverneur G, BonoronAdele S, Saks VA, and Dos Santos P. Alteration of mitochondrial
function in a model of chronic ischemia in vivo in rat heart. Am J Physiol
Heart Circ Physiol 282: H821–H831, 2002.
8. Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K.
Number and spatial distribution of nuclei in the muscle fibres of normal
mice studied in vivo. J Physiol 551: 467– 478, 2003.
9. Burkholder TJ and Lieber RL. Sarcomere length operating range of
vertebrate muscles during movement. J Exp Biol 204: 1529 –1536, 2001.
10. Collins TJ, Berridge MJ, Lipp P, and Bootman MD. Mitochondria are
morphologically and functionally heterogeneous within cells. EMBO J 21:
1616 –1627, 2002.
11. Duchen MR. Contributions of mitochondria to animal physiology: from
homeostatic sensor to calcium signalling and cell death. J Physiol 516:
1–17, 1999.
12. Fawcett DW and McNutt NS. The ultrastructure of the cat myocardium.
I. Ventricular papillary muscle. J Cell Biol 42: 1– 45, 1969.
13. Forbes MS and Sperilakis N. Ultrastructure of mammalian cardiac
muscle. In: Physiology and Pathophysiology of the Heart, edited by
Sperilakis N. Boston, MA: Nijhoff, 1984, p. 3– 42.
14. Kaasik A, Veksler V, Boehm E, Novotova M, Minajeva A, and
Ventura-Clapier R. Energetic crosstalk between organelles: architectural
integration of energy production and utilization. Circ Res 89: 153–159,
2001.
15. Kay L, Li Z, Mericskay M, Olivares J, Tranqui L, Fontaine E, Tiivel
T, Sikk P, Kaambre T, Samuel JL, Rappaport L, Usson Y, Leverve X,
Paulin D, and Saks VA. Study of regulation of mitochondrial respiration
in vivo. An analysis of influence of ADP diffusion and possible role of
cytoskeleton. Biochim Biophys Acta 1322: 41–59, 1997.
16. Kay L, Saks VA, and Rossi A. Early alteration of the control of
mitochondrial function in myocardial ischemia. J Mol Cell Cardiol 29:
3399 –3411, 1997.
17. Kuznetsov AV, Tiivel T, Sikk P, Kaambre T, Kay L, Daneshrad Z,
Rossi A, Kadaja L, Peet N, Seppet E, and Saks VA. Striking
differences between the kinetics of regulation of respiration by ADP in
slow-twitch and fast-twitch muscles in vivo. Eur J Biochem 241:
909 –915, 1996.
18. Leterrier JF, Rusakov DA, Nelson BD, and Linden M. Interactions
between brain mitochondria and cytoskeleton: evidence for specialized
outer membrane domains involved in the association of cytoskeletonassociated proteins to mitochondria in situ and in vitro. Microsc Res Tech
27: 233–261, 1994.
19. McCallister LP and Page E. Effects of thyroxin on ultrastructure of rat
myocardial cells: a stereological study. J Ultrastruct Res 42: 136 –155,
1973.
20. Muir AR. The effects of divalent cations on the ultrastructure of the
perfused rat heart. J Anat 101: 239 –261, 1967.
21. Nozaki T, Kagaya Y, Ishide N, Kitada S, Miura M, Nawata J, Ohno
I, Watanabe J, and Shirato K. Interaction between sarcomere and
mitochondrial length in normoxic and hypoxic rat ventricular papillary
muscles. Cardiovasc Pathol 10: 125–132, 2001.
22. Ogata T and Yamasaki Y. Scanning electron-microscopic studies on the
three-dimensional structure of mitochondria in the mammalian red, white
and intermediate muscle fibers. Cell Tissue Res 241: 251–256, 1985.
23. Ogata T and Yamasaki Y. Ultra-high-resolution scanning electron microscopy of mitochondria and sarcoplasmic reticulum arrangement in
human red, white, and intermediate muscle fibers. Anat Rec 248: 214 –223,
1997.
24. Rassier DE, MacIntosh BR, and Herzog W. Length dependence of
active force production in skeletal muscle. J Appl Physiol 86: 1445–1457,
1999.
25. Romashko DN, Marban E, and O’Rourke B. Subcellular metabolic
transients and mitochondrial redox waves in heart cells. Proc Natl Acad
Sci USA 95: 1618 –1623, 1998.
26. Roy RR, Pierotti DJ, and Edgerton VR. Skeletal muscle fiber crosssectional area: effects of freezing procedures. Acta Anat (Basel) 155:
131–135, 1996.
27. Rube DA and van der Bliek AM. Mitochondrial morphology is dynamic
and varied. Mol Cell Biochem 256 –257: 331–339, 2004.
28. Saks V, Kuznetsov A, Andrienko T, Usson Y, Appaix F, Guerrero K,
Kaambre T, Sikk P, Lemba M, and Vendelin M. Heterogeneity of ADP
288 • MARCH 2005 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 14, 2017
ATPases (28, 30, 39). The crystal-like arrangement seen in our
study is consistent with this hypothesis and suggests the unitary
structure of the studied muscle cells. Implications of such a
unitary structure in the regulation of oxidative phosphorylation
within units as well as synchronization of events in these units
within the cell are not clear and are the subject of active
research (1, 2, 31, 40).
Although in the cardiac cells the very precise, crystal-like
arrangement of mitochondria is clearly important for regulation
of their function and is related to the rigid and precise organization of cytoskeletal structures and sarcomeric proteins, in
many other cells the situation is very different and much more
dynamic. Randomly organized and changing mitochondrial
networks have been described for many types of cells, but
always the mitochondrial position is dependent on their connection with the cytoskeleton, notably with the microtubular
network and intermediate filaments (4, 27). In addition, cytoskeletal changes that disrupt the mitochondrial distribution
can cause cell death (41, 42). The mitochondrial movement is
particularly interesting in the neurons, where axonal mitochondrial transport directed by molecular motors can occur along
both microtubular or actin filaments to the sites of high ATP
demand (18, 41, 42). It may be assumed that, in these cells, the
formation of the intracellular energetic units is of a dynamic
nature and occurs in a spatially and temporally regulated
manner dependent on local energy demand.
In conclusion, our results show that mitochondria, which are
situated between the myofibrils, are arranged in a highly
ordered crystal-like pattern in a muscle-specific manner with
relatively small deviation in the distances between neighboring
mitochondria. This is consistent with the concept of the unitary
nature of the spatial organization of the muscle energy metabolism. The developed method may be used for relating structural and functional changes in muscle cells in several physiological and pathophysiological states.
MITOCHONDRIAL ARRANGEMENT IN MUSCLE CELLS
29.
30.
31.
32.
33.
AJP-Cell Physiol • VOL
35. Shimada T, Horita K, Murakami M, and Ogura R. Morphological
studies of different mitochondrial populations in monkey myocardial cells.
Cell Tissue Res 238: 577–582, 1984.
36. Sommer JR and Jennings RB. Ultrastructure of cardiac muscle. In: The
Heart and Cardiovascular System, edited by Fozzard HA, Haber E,
Jennings RB, Katz AM, and Morgan HE. New York: Raven, 1986, p. 61–100.
37. Sun CN, Dhalla NS, and Olson RE. Formation of gigantic mitochondria
in hypoxic isolated perfused rat hearts. Experientia 25: 763–764, 1969.
38. Veksler VI, Kuznetsov AV, Anflous K, Mateo P, van Deursen J,
Wieringa B, and Ventura-Clapier R. Muscle creatine kinase-deficient
mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of
the mitochondrial function. J Biol Chem 270: 19921–19929, 1995.
39. Vendelin M, Eimre M, Seppet E, Peet N, Andrienko T, Lemba M,
Engelbrecht J, Seppet EK, and Saks VA. Intracellular diffusion of
adenosine phosphates is locally restricted in cardiac muscle. Mol Cell
Biochem 256 –257: 229 –241, 2004.
40. Vendelin M, Kongas O, and Saks V. Regulation of mitochondrial
respiration in heart cells analyzed by reaction-diffusion model of energy
transfer. Am J Physiol Cell Physiol 278: C747–C764, 2000.
41. Wagner OI, Lifshitz J, Janmey PA, Linden M, McIntosh TK, and
Leterrier JF. Mechanisms of mitochondria-neurofilament interactions.
J Neurosci 23: 9046 –9058, 2003.
42. Yaffe MP. The machinery of mitochondrial inheritance and behavior.
Science 283: 1493–1497, 1999.
288 • MARCH 2005 •
www.ajpcell.org
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on June 14, 2017
34.
diffusion and regulation of respiration in cardiac cells. Biophys J 84:
3436 –3456, 2003.
Saks V, Dos Santos P, Gellerich FN, and Diolez P. Quantitative
studies of enzyme-substrate compartmentation, functional coupling
and metabolic channelling in muscle cells. Mol Cell Biochem 184:
291–307, 1998.
Saks VA, Kaambre T, Sikk P, Eimre M, Orlova E, Paju K, Piirsoo A,
Appaix F, Kay L, Regitz-Zagrosek V, Fleck E, and Seppet E. Intracellular energetic units in red muscle cells. Biochem J 356: 643– 657,
2001.
Saks VA, Kuznetsov AV, Vendelin M, Guerrero K, Kay L, and Seppet
EK. Functional coupling as a basic mechanism of feedback regulation of
cardiac energy metabolism. Mol Cell Biochem 256 –257: 185–199, 2004.
Schaper J, Meiser E, and Stammler G. Ultrastructural morphometric
analysis of myocardium from dogs, rats, hamsters, mice, and from human
hearts. Circ Res 56: 377–391, 1985.
Segretain D, Rambourg A, and Clermont Y. Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle
fiber of the rat. Anat Rec 200: 139 –151, 1981.
Seppet EK, Kaambre T, Sikk P, Tiivel T, Vija H, Tonkonogi M, Sahlin
K, Kay L, Appaix F, Braun U, Eimre M, and Saks VA. Functional
complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells. Biochim Biophys Acta 1504: 379 –395, 2001.
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